european parasitoids of the cherry bark tortrix : assessing
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EUROPEAN PARASITOIDS OF THE CHERRY BARK TORTRIX: ASSESSING THE ICHNEUMONID, CAMPOPLEX DUBITATOR, AS A POTENTIAL
CLASSICAL BIOLOGICAL CONTROL AGENT FOR NORTH AMERICA
Wade H. Jenner BSc, Augustana University College 1999
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
In the Department
of Biological Sciences
0 Wade H. Jenner 2003 SIMON FRASER UNIVERSITY
November, 200 3
All rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other means, without permission of the author.
APPROVAL
Name:
Degree:
Wade Harley Jenner
Master of Science
Title of Thesis:
European parasitoids of the cherry bark tortrix: assessing the ichneumonid, Campoplex dubitator, as a potential classical biological control agent for North America.
Examining Committee:
Chair: Dr. R.C. Ydenberg
- Dr. B.D.Roitberg. Professor, Senior Supervisor Department of Biological Sciences, S.F.U.
Dr. U. Kuhlmann, ~ e a d , ~gric;ltural Pest Research CAB1 Bioscience Centre
DV! J.E. Cossentine, Research Scientist Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada
Department bf Biological ~kiences, S.F.U.
a
Dr. J] Myers, Professor Dep rtment of Zoology and Plant Science, U.B.C. P d ic Examiner
- /
/ ,{,iL" ,/, ( .; ,- 3 .? '-, i - L 1 a 4 ? s
Date Approved
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Title of ThesislProjectlExtended Essay
European parasitoids of the cherry bark tortrix: assessing the ichneumonid, Campoplex dubitator, as a potential classical biological control agent for North America.
Author: (signature)
Wade Harley Jenner (name>
(date)
Abstract
The cherry bark tortrix (=CBT), Enarmonia formosana Scopoli (Lepidoptera: Tortricidae), is
poorly known in its native Palaearctic range. However, since its recent introduction into North
America, this species poses a threat to the nursery and orchard industries in British Columbia,
Washington State, and Oregon State. As part of a classical biological control approach to
managing this bark-boring pest, the objective of this thesis research was to find European
parasitoids of the CBT for release in North America. In total, 13 parasitoid species were reared
from CBT larvae and pupae collected in Europe between 2000 and 2002. However, 12 of these
species were collected only very rarely, suggesting weak associations with the CBT, while the
larval parasitoid, Campoplex dubitator Horstmann (Hymenoptera: Ichneumonidae), was
responsible for 99% of the larval parasitism and 85% of the larval and pupal parasitism
combined. Hence, despite a wide distribution of parasitism throughout the southern Rhine Valley,
Black Forest, and northern Jura Mountains, C. dubitator appeared to be the only parasitoid having
a substantial impact on CBT populations. Campoplex dubitator was therefore selected for a more
thorough evaluation of its role as a biological control agent. A method was developed for small-
scale rearing of C. dubitator using host larvae of all but the first instar, and information on the
parasitoid's reproductive biology was obtained. In olfactometers, females were shown to respond
to volatile cues emitted from cherry bark and host frass, but not from the host larvae themselves.
When the attractiveness of uninfested cherry bark and host frass was compared, parasitoids
demonstrated a strong preference for host frass. This observation supports the philosophy that
foragers should respond more strongly to stimuli that are more directly linked to their target hosts,
since those cues provide more reliable information regarding host availability and location. This
knowledge of C. dubitator's foraging strategy may also indicate the types of habitats or hosts this
species might be most likely to encounter, which could be valuable in the selection of non-target
species to use in future host-range testing, Finally, in a patch time allocation experiment, C.
dubitator females invested a greater search effort on patches of higher host density. The
observation from this experiment that C. dubitator could not accurately discriminate against
previously parasitised hosts, or effectively distinguish between empty and occupied frass tubes,
may be useful in explaining the inverse density dependence that was observed in the field.
Acknowledgements
First and foremost, I thank my advisory committee, which has been very valuable in helping me
get my project off the ground, securing project funding, and providing sound guidance each step
of the way. Our collaboration with the CABI Bioscience Centre in Switzerland was a key element
in conducting the European field studies. I was also able to prolong my data collection by
maintaining experimental insects in SFU's Global Forest Quarantine Facility, construction of
which was completed through financial support from Global Forest (GF-18-2000-SFU-2). We
depended heavily on the taxonomic assistance provided by Dr. Klaus Horstmann and Dr. Hannes
Baur, which was a crucial component of this programme. The speedy preparation of meridic diet
by Linda Jensen - whenever we needed it - was never taken for granted. I owe thanks to the SFU
Department of Biological Sciences for financial support and to John Mathies (Cannor Nurseries
Ltd.) whose donation made it possible for me to obtain a Graduate Engineering and Technology
Scholarship through the Science Council of British Columbia. I would like to give special thanks
to Manfred Grossrieder, Erik Osborn, and other colleagues on the Swiss CABI Bioscience team
who took part in field excursions, as well to those who were with me when I learned that, while
not glamorous, washing one million Petri dishes can actually be fun! I would like to acknowledge
Emma Hunt and the rest of the CABI clan (2000 to 2002) who also played a major role in helping
me to unwind at the end of a long day. If it were not for them, I may have never experienced all
the joys that Switzerland has to offer. Finally, I am grateful for the patience and relaxed manner
of the many farmers who caught us carving our signatures into the bark of their cherry trees. One
quickly learns to speak German or French when one must explain why one's chisel is sticking out
the side of an orchard cherry tree!
Table of Contents . . Approval ..................................................................................................................................... 11 ... Abstract .................................................................................................................................. 111
Acknowledgements .................................................................................................................... iv ......................................................................................................................... Table of Contents v ... List of Figures .......................................................................................................................... VIII
List of Tables ............................................................................................................................... x
.................................................................................................. CHAPTER 1 1 ............................................ General Introduction and Literature Review 1
........................................................................................................................................ Abstract 1 .................................................................. 1.2 Biology and Ecology of the Cherry Bark Tortrix 2
........................................................................................................ 1.3 Assessment of Pest Risk 5 1.4 Research Objectives ............................................................................................................... 7
1.4.1 Survey of Parasitoid Community .................................................................................... 7 ...................................................................................... 1.4.2 Evaluation of a Potential Agent 8
............................................................................................................................ 1.5 References 1 1
CHAPTER 2 ................................................................................................ 14 Distribution. phenology. and field parasitism of the cherry bark tortrix ....................................................................................................................... 14
................................................................................................................................... Abstract 1 4 2.1 Introduction .......................................................................................................................... 15 2.2 Materials and Methods ......................................................................................................... 16
........................................................................................ 2.2.1 CBT Parasitoid Associations 17 2.2.2 Spatial Distribution of the CBT and its Parasitoids ...................................................... 17
....................................................................................................... 2.2.2.1 Regional Scale 18 ....................................................................................................... 2.2.2.2 Orchard Scale 1 8
2.2.2.3 Tree Scale .............................................................................................................. 19 2.2.3 CBT Phenology ............................................................................................................ 20
.................................................................................. 2.2.4 Temporal Analysis of Parasitism 20 2.3 Results .................................................................................................................................. 21
........................................................................................ 2.3.1 CBT Parasitoid Associations 21 ...................................................... 2.3.2 Spatial Distribution of the CBT and its Parasitoids 22
....................................................................................................... 2.3.2.1 Regional Scale 22 ........................................................................................................ 2.3.2.2 Orchard Scale 22
.............................................................................................................. 2.3.2.3 Tree Scale 23 2.3.3 CBT Phenology ............................................................................................................ 24
.................................................................................. 2.3.4 Temporal Analysis of Parasitism 25 ............................................................................................................................ 2.4 Discussion 26
........................................................................................ 2.4.1 CBT Parasitoid Associations 26 ...................................................... 2.4.2 Spatial Distribution of the CBT and its Parasitoids 29
2.4.3 CBT Phenology ............................................................................................................ 33 .................................................................................. 2.4.4 Temporal Analysis of Parasitism 34
........................................................................................................................... 2.5 Conclusion 35 ........................................................................................................................... 2.6 References -36
CHAPTER 3 ................................................................................................ 62 Biology and Rearing of Campoplex dubitator ........................................ 62
Abstract ...................................................................................................................................... 62 3.1 Introduction .......................................................................................................................... 63 3.2 Methods ............................................................................................................................... 64
3.2.1 Host Rearing ................................................................................................................. 64 3.2.2 Biology and Rearing of Campoplex dubitator .............................................................. 65
3.3 Results and Discussion ........................................................................................................ 66 3.4 Conclusion ........................................................................................................................... 70
CHAPTER 4 ................................................................................................ 81 Response of the parasitoid Campoplex dubitator to host- and habitat-related odours in an olfactometer ................................................ 81
Abstract ................................................................................................................................. 8 1 4.1 Introduction .......................................................................................................................... 82
......................................................................................................... 4.2 Materials and Methods 83 ........................................................................................................... 4.2.1 Study Organisms 83
4.2.2 Experiments 1-3: Parasitoid Response to Volatile Cues ............................................... 84 .................................................................................................... 4.2.2.1 Materials Tested 84
4.2.2.2 Experiment 1 ......................................................................................................... 84 ......................................................................................................... 4.2.2.3 Experiment 2 85 ......................................................................................................... 4.2.2.4 Experiment 3 85
................................................................. 4.2.2.5 Static Chamber Olfactometer Bioassay 85 4.2.3 Experiment 4: Parasitoid Preference for Volatile Cues ................................................ 87
.................................................................................................. 4.2.3.1 Materials Tested 87 .............................................................................. 4.2.3.2 Y-tube Olfactometer Bioassay 88
4.3 Results .................................................................................................................................. 89 ............................................... 4.3.1 Experiments 1-3 : Parasitoid Response to Volatile Cues 89 ................................................ 4.3.2 Experiment 4: Parasitoid Preference for Volatile Cues 90
............................................................................................................................ 4.4 Discussion 90 4.5 References ............................................................................................................................ 95
CHAPTER 5 ............................................................................................. 108 Oviposition behaviour and patch time allocation of
.................................................................................. Campoplex dubitator 108 ................................................................................................................................. Abstract 1 0 8
........................................................................................................................ 5.1 Introduction 109 5.2 Materials and Methods ....................................................................................................... 112
5.2.1 Study Organisms ......................................................................................................... 1 12 5.2.2 Experiment I: Description of Oviposition Behaviour ................................................. 1 13
....................................................................... 5.2.3 Experiment 11: Patch Residence Time 1 15
....................................................................... 5.2.3.1 The Proportional Hazards Model 1 17 ................................................................................................................................ 5.3 Results 1 18
................................................. 5.3.1 Experiment I: Description of Oviposition Behaviour 1 18 5.3.2 Experiment 11: Patch Residence Time ........................................................................ 119
5.4 Discussion .......................................................................................................................... 121 5.4.1 Experiment I: Description of Oviposition Behaviour ................................................. 121 5.4.2 Experiment 11: Patch Residence Time ........................................................................ 122
.......................................................................................................................... 5.5 References 128
CHAPTER 6 .............................................................................................. 144 .............................................................. Conclusions and Final Remarks 144
6.1 Conclusion ......................................................................................................................... 144 .......................................................................................................................... 6.2 References 148
vii
List of Figures
Figure 2- 1 Parasitoid community structure associated with the larval and pupal stages of Enarmonia formosana. Arrows connect host stages attacked and killed by each of the four parasitoid guilds: (1) larval, (2) larval - pupal, (3) late larval ecto, and (4) pupal. Arrow width indicates the relative abundance of each parasitoid species interacting with the host. Arrows drawn through the host stage bubble represent endoparasitism while those not
............................................................. passing through the bubble represent ectoparasitism 40
Figure 2- 2 Association between Enarmonia formosana density and tree trunk circumference. (a) Number of total CBT (healthy and parasitised) per 1000 cm2 at the tree base (r2 = 0.001; P = 0.449). (b) Number of only parasitised CBT per 10002 at the tree base (y = 0 .006~ + 0.444; r2 = 0.03, P = 0.013) .................................................................................................... 42
Figure 2- 3 Comparison of within-tree distributions of Enarmonia formosana frass tubes (hSE) on the trunks of cherry trees in central Europe and Canada. The European within-tree distribution of frass tubes differs significantly from that of Vancouver (chi-square test for heterogeneity: ~2 = 70.596; df = 5; P < 0.001). ..................................................................... 44
Figure 2- 4 Spatial distribution of healthy and parasitised Enarmonia formosana on the trunks of cherry trees in central Europe. Error bars indicate 1 standard error. Significantly more healthy (P < 0.001) and parasitised (P < 0.001) E. formosana specimens were collected from the bottom section than the middle and top sections of trees. ............................................... 46
Figure 2- 5 Parasitism response of Campoplex dubitator to host density per tree, based on the collection and rearing of all host (late instar) Enarmonia formosana from selected trees, between 19 April to 14 June (y = -1 5.8Ln(x) + 67.89; 3 = 0.57, P < 0.001). ....................... 48
Figure 2- 6 Change in the relative abundance of Enarmonia formosana pupae (columns) and the mean head capsule width (points) of field-collected E. formosana larvae (* SE) over the 2001 and 2002 field seasons. ................................................................................................. 50
Figure 2- 7 Frequency distribution of measurements of 2133 Enarmonia formosana larval head capsule widths collected during the summer of 2002 in central Europe. Columns with different fill patterns show the head capsule size ranges of seven instars. Horizontal lines above columns indicate the head capsule size ranges of the five instars described by Roediger (1956). .................................................................................................................... 52
Figure 3-1 Relationship between Enarmonia formosana host size at oviposition and hind right tibia length of eclosing adult Campoplex dubitator. (r2 = 0.004; P = 0.730) ......................... 73
Figure 4-1 Illustration of the static chamber olfactometer used in identifling sources of attractive odours in experiment 1. Gauze bags were suspended >5 cm away from all walls of the arena to reduce the chance of accidental discovery of treatment and control substrates by the
................................................................................................................................ parasitoid 98
Figure 4-2 Response of Campoplex dubitator females to odours presented in a static chamber olfactometer. These figures show the response of parasitoids to cherry bark (2 * 0.2 g) (experiment 1: ~2 = 6.025; P = 0.014 ), host frass (0.20 h 0.05 g) (experiment 2: x2 = 1.667; P = 0.197), and three naked Enarmonia formosana host larvae representing the third, fourth,
... V l l l
and fifth instars (experiment 3: ~2 = 0.404; P = 0.525). A choice was determined based on a comparison of the total time spent on the treatment and control halves of the arena. ......... 100
Figure 4-3 Response of Campoplex dubitator females to odours released from host frass (2.5 g) versus uninfested cherry bark (2.5 g) in a Y-tube olfactometer (experiment 4). A "choice" was made when parasitoids walked to within 5 cm of the distal end of either arm. Parasitoids demonstrated a strong preference for frass-related volatiles ( ~ 2 = 5.580; P =
Figure 5- 1 Ethogram of Campoplex dubitator pre-oviposition and post-oviposition behaviour when attacking Enarmonia formosana hosts. Width of each solid line is associated with the transitional probability of the indicated behavioural event (n = 28). Following Check behaviour, the transitions between Rest, Probe, Search, Groom, and Walk become much less predictable. See Appendices 5-A and 5-B for actual values of transitional probabilities between behaviours. See text for definition of individual behaviours ................................. 13 1
Figure 5-2 Mean (+ SE) patch residence times of Campoplex dubitator on patches containing ............................................................. different densities of Enarmonia formosana hosts. 134
Figure 5-3 Proportion of Enarmonia formosana hosts parasitised by Campoplex dubitator versus host density. Percent parasitism is calculated based on the number of ovipositions observed during the patch visit (trials with zero ovipositions are kept in data set) ( r2 = 0.045; P =
0.222). .................................................................................................................................. 136
List of Tables
Table 2- 1 List of identified parasitoid species emerging from pupal and larval Enarmonia formosana hosts collected in central Europe between 2000 and 2002. Regions of discovery
............................................................... and relative abundance are given for each species. 54
Table 2- 2 Comparison of overall percent parasitism of Enarmonia formosana by region during ....... 200 1 and 2002. Parasitism rates are further divided into larval and pupal host groups. 56
Table 2- 3 Association of host tree features with the probability and density of Enarmonia formosana infestations on cherry trees in central Europe (2001-2002). ................................ 58
Table 2- 4 Association of host tree features with the probability and density of parasitised ............................... Enarmonia formosana on cherry trees in central Europe (200 1-2002). .60
Table 3-1 Comparison of the results of parasitism on the five Enarmonia formosana instars and of the effect of single vs multiple and brief vs prolonged ovipositions. ................................ 75
Table 3-2 Comparison of female and male parasitoid development time within three age classes of Enarmonia formosana hosts .............................................................................................. 77
Table 3-3 Ovariole and mature egg counts from dissections of Campoplex dubitator ovaries. ... 79
Table 4-1 Summary of olfactory experiments investigating the response of Campoplex dubitator to volatiles associated with the host and host habitat. ......................................................... 104
Table 4-2 Effect of mating status on the response of foraging female Campoplex dubitator to ................................................................................................... stimuli in an olfactometer. 106
Table 5-1. Average transitional probabilities between behaviours displayed by foraging ................... Campoplex dubitator females from beginning of search to oviposition event. 138
Table 5-2. Average transitional probabilities between behaviours displayed by foraging .............................. Campoplex dubitator females from oviposition to departure from host 140
Table 5-3. (a) Estimated regression coefficients (P), standard errors (SE), and hazard ratio (exp(j3)) for only those covariates that had an effect on the patch leaving tendency of Campoplex dubitator females. (b) Estimated regression coefficient (P), standard error (SE), and hazard ratio (exp(P)) for the single covariate that had an effect on the giving up time of C. dubitator females. ........................................................................................................... 142
CHAPTER 1 General Introduction and Literature Review
Abstract
The cherry bark tortrix (CBT), Enarmonia formosana Scopoli (Lepidoptera: Tortricidae), was
first discovered in North America in 1989, when specimens were collected in Richmond, British
Columbia. Since its appearance in the Pacific Northwest region, the CBT has spread as far east as
Armstrong, British Columbia and as far south as Portland, Oregon. This pest is known to attack
many rosaceous plants, but is most commonly associated with cherry trees. The larvae feed
solitarily on the phloem tissues beneath the outer bark and may ultimately reduce tree vigour,
with symptoms including foliage dieback or even tree death. In its native range of Europe,
temperate Asia, and North Africa, the CBT has only infrequently been considered a major
orchard pest. In contrast, infestation levels on ornamental cherry trees in North America are
substantially greater. Several active control measures have been applied against the CBT in
Europe and North America including the application of insecticides, creosote and tar oil, the
removal of vegetation from the bases of trees, and pheromone-based mating disruption. Classical
biological control was only recently considered as an IPM tool for dealing with this pest. The
initial work of this Masters thesis project included a survey of the parasitoid community of the
CBT in central Europe. Larval and pupal parasitoids were collected, reared, and identified, with
the aim of isolating potential biological control agents. Once one candidate species was selected,
further studies of its biology and ecology were conducted. These investigations included
development rates, host detection, host instar suitability, and response to host density.
1.1 Introduction Cherry trees have been cultivated for both the fruit and wood for over 2000 years (Godwin,
1975). In western North America, ornamental and agricultural cherry trees are grown throughout
British Columbia, Washington, Oregon, Utah, Idaho, Montana, and California. Along the Pacific
coast, these trees and related rosaceous species are particularly valued for their ornamental use. In
the Vancouver area alone, thousands of flowering cherries have been planted along streets for
enhanced aesthetics. Unfortunately, these trees often suffer a variety of health problems. Declines
in cherry tree health have been associated with bacterial canker, nematode attack, malnutrition,
low soil pH, and winter injury (Melakeberhan et al., 1993). Cherry trees may also be stressed
through defoliation by insect herbivores like the cherry ermine moth, Yponomeutapadellus
(Lepidoptera: Yponomeutidae), and fruit production may be hindered by pests like the black
cherry aphid, Myzus cerasi (Fabricius) (Homoptera: Aphididae) (Alford, 1984). Recently, an
additional insect pest, which feeds in the bark of cherry trees, was accidentally introduced into
North America.
In 1989, specimens of a bark-boring insect were collected in Richmond, British Columbia,
and were identified as the cherry bark tortrix (hereafter referred to as CBT), Enarmonia
formosana (Scopoli) (Lepidoptera: Tortricidae) (Dang and Parker, 1990). This was the first report
of CBT in North America. In 1991, the Washington State Department of Agriculture reported a
collection of CBT larvae from an ornamental cherry tree in Blaine, Whatcom county, Washington
(Klaus, 1991). Continued monitoring of the spread of this insect suggests that its distribution now
extends as far east in Canada as Armstrong, British Columbia and as far south as Portland,
Oregon (Cossentine, pers. comm.).
1.2 Biology and Ecology of the Cherry Bark Tortrix
The CBT occurs throughout Europe, temperate Asia, and North Africa (Balachowsky, 1966).
Female specimens collected at the eastern edge of its distribution have smaller body dimensions
than European specimens, but there is currently no sufficient explanation for this trend
(Kuznetsov, 1988). A single species group encompasses all specimens collected to date. The CBT
belongs to the family Tortricidae, one of the most damaging groups of Lepidoptera (Klaus, 199 1).
However, it has not had a history as a widespread damaging pest as some of its relatives,
including the codling moth (Cydiapomonella), the fruit tree tortrix moth (Archips podana), and
the carnation tortrix moth (Cacoecimorphapronubana) have (Alford, 1984). The CBT has been
described in scientific literature since at least 1776 (Roediger, 1956)' but since then has only
appeared in sporadic discussions resulting from short-term, local outbreaks. The published
material dealing specifically with the CBT in Eurasia has focused largely on the identification of
this tortricid pest and subsequent use of pesticides for controlling outbreaks (Shapovalov, 1959;
Winfield, 1964; Lyalyuts'ka, 1965; Savkovskii and Lyalyutskaya, 1967; Dickler, 1970; Minks et
al., 1976; Smol'yannikov, 1979; KoSlinska et al., 1980). Additional papers emerging from
Europe in the last 30 years have described the effectiveness of sex pheromone mixtures for
monitoring pest population levels (Minks et al., 1976; Kratancsik, 1982; Sziraki, 1984). For the
most part, the CBT has been considered a pest of only minor importance in its native range,
where outbreaks are normally rare (Massee, 1954). During the field surveys for this study, cherry
growers encountered between 2000 and 2002 in the southern Rhine Valley and Black Forest in
Germany were commonly unaware of the existence of the CBT on their trees (pers. obs.).
The CBT was first described as a pest of almond and stone-fruit trees in Europe in the early
1800's (Winfield, 1964). Its known host range in Europe and North America now includes
Prunus (cherry, plum, almond, apricot, nectarine, and peach), Malus (apple), Cydonia (quince),
Pyrus (pear), Pyracantha (firethorn), Crataegus (hawthorn) and Sorbus (moutain ash) (Dang and
Parker, 1990; Tanigoshi et al., 1998). On both continents, the preferred host plants of the CBT are
those comprising the genus Prunus (Roediger, 1965) and within some species, a strong variety
preference may exist (Tanigoshi et al., 1998). Older trees appear to be most susceptible to attack
by the CBT. Such mature trees are more likely to possess wounds from years of pruning and
weather damage; pest densities in Washington were greatest on trees with high numbers of
wounds or other opportunities for larval entry into the bark (Tanigoshi et al., 1998).
CBT adults typically fly from early-May until mid-September. Pheromone-based trapping of
males in both Europe and the United States has revealed a bimodal flight pattern, with the first
peak of activity occurring in May or June and the second in lateJuly or August, depending on
seasonal temperatures (Roediger, 1956; Winfield, 1964; Sermann and Zahn, 1986; Tanigoshi et
al., 1998). The phenomenon of two predictable peaks in adult activity has led previous
researchers to believe that the CBT is a bivoltine moth (Theobald, 1909; Samal, 1926). Only upon
closer inspection of flight data and comparison with larval development observations, have
scientists begun to suggest a single, staggered generation per year (Roediger, 1956; Savkovskii
and Lyalyutskaya, 1967; Sermann and Zahn, 1986).
CBT eggs are deposited singly or in small clusters on the trunk of host trees. The ovipositing
females apparently prefer sites where the bark has been damaged by previous feeding or pruning
(Roediger, 1956). Following hatching, first instar larvae immediately seek shelter, and if the site
is suitable, penetrate the bark to feed. Old tunnels often provide excellent locations for larvae to
establish new feeding galleries, as long as the surrounding nutritive bark tissues are not
completely excavated or desiccated. First instar larvae feed exclusively in the outer part of the
phloem, while later instars may colonise the entire space between the cork and cambium. The
cambium itself is rarely reached by the tunneling larvae and the central wood is never damaged
by feeding (Alford, 1984). Throughout the course of development, a larva may abandon its
original tunnel to create a new feeding gallery at a more profitable location on the tree. This
behaviour is common among early instars. The CBT overwinter in the larval stage. Sermann and
Zahn (1986) have found larval-feeding activity ceases completely once the temperature has
dropped below a threshold of approximately 7" C. Later instar larvae, with deeper tunnels, are
presumably better equipped to survive the winter season.
The larvae of the CBT live solitarily in complete concealment beneath the bark. Aggregation
of feeding tunnels around a site of bark damage is quite common however and aggressive
behaviour is typical during interactions between conspecific larvae. The larvae therefore maintain
their physical concealment by closing any opening in the tunnel with a web of silken threads.
This silk is also used in the construction of a capsule-like structure at the entrance to the feeding
tunnel. Larvae routinely deposit their faeces on the exterior of this shelter, thereby enlarging it
over time. This faecal pouch, or "frass tube", probably plays a very important role for natural
enemies and extension service staff, as it is the only visual cue revealing the presence of these
insects.
The proper description of the larval instars of the CBT has been even more perplexing than
the aforementioned discussion on the number of generations per year. Because CBT larvae
develop beneath bark, they cannot easily be subjected to daily monitoring. Using measurements
of larval head capsule widths from field-collected CBT, Roediger (1956) concluded that the
larvae pass through five instars before pupating. More than one decade later, Savkovskii and
Lyalyutskaya (1967) published their description of four CBT larval stages while Dickler (1972)
was unable to discern clear instar stages at all, based on head capsule measurements. This
inconsistency in reported developmental patterns may arise from variance in either the actual
number of instars or the head capsule sizes for each larval stage (Russell and Bouzouane, 1989;
Savopoulou-Soultani and Tzanakakis, 1990; Gold et al., 1999). Such reported variations in
developmental patterns or instar sizes can result from differences in rearing conditions, including
temperature and diet (Russell and Bouzouane, 1989; Savopoulou-Soultani and Tzanakakis, 1990;
Gold et al., 1999).
1.3 Assessment of Pest Risk
The magnitude of the threat that the CBT poses to the nursery and orchard industries is not
known. Its geographic spread in North America may be limited by a number of factors. For
example, the CBT is associated with relatively high humidity, which might reduce the risk of
contamination of cherry orchards more inland, such as the arid Okanagan growing region.
Additionally, the moths are suspected not to have a high dispersal rate since Roediger (1956)
suggested that females have a tendency to oviposit on the trees in which they had developed.
Tree loss has often been attributed to infestation by the CBT. However, it is still not yet
certain that the CBT is the sole factor causing the death of trees. In a pest risk assessment of CBT,
Orr (1991) suggests that the CBT can generate three types of damage: direct damage by larval
feeding, resulting in the dieback of shoots; indirect damage due to the attraction of secondary
pests such as scolytids and fungi; and indirect damage through increased vulnerability to frost and
other unfavourable weather conditions. Many of the infested trees in Whatcom county,
Washington State, USA and Vancouver, British Columbia, Canada, for instance, have also shown
symptoms of bacterial canker caused by Pseudomonas syringae (Klaus, 1992). This plant disease
is common in the coastal area, due to the favourably cool and wet weather, and flowering cherry
trees are perhaps its most vulnerable hosts (Orr, 1991). Pseudomonas syringae, however, is
considered to be a weak, opportunistic pathogen, which will invade a host that has first been
weakened by some predisposing condition (Moore, 1988), such as larval feeding beneath the
outer bark. Cankers created by bacterial infection, in turn, provide attractive oviposition sites for
the adult tortricids. It may be a relationship of this sort between these organisms, which
ultimately leads to tree mortality.
The presence of a small number of larvae will not noticeably lower the health of a tree.
However, tree survival may be threatened by sustained, high-density feeding over successive
generations (Roediger, 1956; Winfield, 1964; Balachowsky, 1966). If left unchecked, CBT tree
kills could jeopardise part of the multi-million dollar nursery industry in British Columbia,
Washington, and Oregon. Since its arrival in the Pacific Northwest, the CBT has thrived on
various host plants with no apparent controls on its spreading population. Densities of larvae on
trees in North America seem to be much higher than levels reported in Europe. It is very possible
that an important regulating force, which exists in Europe, is not present in the North American
ecoregion colonised by the CBT. Whether this controlling factor is biotic, abiotic, or even exists
at all, remains to be ascertained.
Active control measures applied to date against the CBT in Europe include the application of
insecticides, creosote and tar oil, mechanical removal of dead and peeling bark, thinning of tree
canopy, reduction of orchard density (Roediger, 1956), and removal of vegetation from the bases
of trees (Dickler and Zimmerman, 1972). Within North America, attempts have been made to
regulate CBT densities through pyrethroid or organophosphate application (Murray et al., 1998),
pheromone-based mating disruption (McNair et al., 1999), entomopathogenic nematode
application (McNair, pers. comm.), and biological control with the egg parasitoid, Trichogramma
cacoeciae Marchal (Hymenoptera: Trichogrammatidae) (Tanigoshi, 2002). Many of these
management approaches have caused a reduction in local pest densities. However, most are not
consistently reliable or feasible. Success may be a function of the host tree, the tree patch, or local
pest phenology. The extended flight season of the CBT, for instance, can make chemical control
very difficult or impractical (Klaus, 199 1).
In spite of the numerous studies on the biology and distribution of the CBT, little attention
has been given to the natural enemies of this pest. Only three reliable examples from the last 90
years have been found. In his thorough description of CBT biology and ecology, Roediger (1956)
listed four parasitoid species (2 ichneumonids, 1 braconid, and 1 tachinid) reared from field-
collected caterpillars. As no parasitism rates were provided, the importance of Roediger's four
parasitic species is not known. Schuetze and Roman (193 1) identified one ichneumonid parasitoid
that attacked the CBT. Boldyrev and Dobroserdov (1981) highlighted the common raphidiid,
AguIla xanthostigma (Schummel) (Neuroptera: Raphidiidae), as an important predator of the
young tortricid larvae. Consequently, during the early risk assessment of the CBT in North
America, the lack of literature on natural enemies of this pest in Europe was interpreted to mean
that biological control showed little promise. It was suspected that the pest's protective
environment was an obstacle that would hinder biocontrol attempts (Om, 1991).
In the late 199OYs, members of the Washington State Department of Agriculture, Oregon
Department of Agriculture, and Washington State University launched a survey of indigenous
parasitoids attacking the immature stages of the CBT. This investigation, conducted in
northwestern Washington, found four species of larval parasitoids believed to be associated with
the CBT. These species, including one ichneumonid, one braconid, one eupelmid, and one
eurytomid, had a very low total combined parasitsm rate of 1.7% in 1997 and 2.7% in 1998
(Tanigoshi et al., 1998). This finding stimulated interest in an exploration of the parasitoid
community attacking the CBT in its native range in Europe. In 1998, in collaboration with Dr.
Ulrich Kuhlmann at the CABI Bioscience Switzerland Centre, Dr. Lynell Tanigoshi (Washington
State University) performed a search for parasitoid enemies of the CBT in northern Switzerland
and the Rhine Valley of Germany. They found evidence of higher levels of parasitism in Europe
than in North America, coinciding with overall lower CBT densities, which strengthened support
for the idea of a classical biological control approach in North America (Tanigoshi et al., 1998).
1.4 Research Objectives
1.4.1 Survey of Parasitoid Community
The first objective of my research was to conduct a formal survey of the parasitoid
community attacking the larvae and pupae of the CBT in central Europe. This work, carried out in
cooperation with Dr. Joan Cossentine (Agriculture and Agri-Food Canada, Summerland) and Dr.
Ulrich Kuhlmann (CABI Bioscience Centre Switzerland), was a continuation of the earlier
assessment of potential candidates for classical biological control. The field study was important
for the correct identification of parasitoids associated with the CBT, as its natural enemy complex
had not previously been studied in any detail. Despite a long and scattered history of recurring
problems caused by the CBT, little effort had ever been made to understand the top-down control
imposed on it by predators, pathogens, or parasitoids.
Chapter 2 provides a discussion of biological and ecological aspects of both the pest and its
parasitoid complex, based on intensive field surveys between 2000-2002. Sampling from natural
populations of the CBT in the Rhine Valley, Black Forest, and Jura Mountains was a laborious,
but effective, way to examine the interaction between the CBT and its natural enemies. With
respect to the moth pest, one specific aim was to describe its phenology and distribution patterns
at multiple spatial scales. Phenological data were then used to verify the number of CBT
generations per year and the number of larval instars during development. The investigation of
the parasitoids included an analysis of field parasitism rates on host larvae and pupae, with a
comparison of species composition and overall parasitoid impact between the surveyed regions of
Europe. Monitored laboratory rearing of field-collected host specimens provided additional
information on development times and sex ratios of the CBT and its parasitoid enemies. plus,
some insight into the guild structure of the parasitoid community could be ascertained based on
host stages attacked by the various parasitoid species.
1.4.2 Evaluation of a Potential Agent
By 200 1, a larval parasitoid species, Campoplex cf. dubitator Horstmann (Hymenoptera:
Ichneumonidae), was selected for an evaluation of its role as a regulator of CBT populations. The
decision to use C. dubitator was based on observations such as its contribution to host mortality,
a possible high host-specificity, and feasibility of rearing. In completion of part of the
comprehensive assessment, I studied the foraging behaviour and life history traits of this
parasitoid.
To obtain sufficient numbers of C. dubitator for study, it was necessary to establish a
laboratory culture of the parasitoids. This was the first documented attempt at rearing this species.
I therefore recorded several aspects of the rearing process, which are potentially important for
future production of C. dubitator. Chapter 3 provides a description of this information, including
notes on the development rates of immature C. dubitator, adult nutrition and longevity, courtship,
egg load, CBT host instar suitability, and determination of progeny sex.
Once potential biological control agents are identified, they must undergo extensive testing to
ensure their efficacy, reliability, and safety (ie. host specificity) in a pest management program.
Many authors have attempted to define the characteristics that make natural enemies effective for
biological control. However, as Stiling (1993) points out, it may be difficult to attribute success to
just one biological feature. In contrast, it should be easier to link failure with a single trait.
Several factors have been found to cause the breakdown of some previous biological control
programmes. These include: (1) poor climate matching, (2) unpredictable, detrimental weather,
(3) lack of synchronisation between natural enemy and pest, (4) wrong species or strain of natural
enemy, (5) differences in habitat preferences, (6) host refuge, (7) competition with other natural
enemies, (8) predation or hyperparasitism of released natural enemies, (9) lack of alternative
hosts, (10) lack of adult food, (1 1) low rate of increase, and (12) too few natural enemies released
(Stiling, 1993). While a couple of the above problems could be avoided with more careful
planning of the release strategy, most of them result from an inadequate understanding of the
biological and ecological attributes of the natural enemies. This therefore highlights the need for a
thorough analysis of potential biological control candidates.
Another major objective of this project was to investigate the role and importance of odours
associated with the host and host habitat during foraging by C. dubitator females. Since CBT
larvae live and feed in complete concealment, with only their faeces visible on the outside of the
bark, it was suspected that specific volatile compounds play a large part in the host detection
process. Godfray (1994) states that chemical cues are very commonly used by parasitoids in their
hunt for hosts. Preliminary laboratory observations of interactions between C. dubitator and the
cherry bark pest were also suggestive of the use of odours by the searching parasitoids. Females
would typically move towards a host-related substrate while walking or flying, and often from a
considerable distance. In many cases when exposed host larvae were presented to parasitoids, the
wasps did not appear to recognise their hosts, even after direct antennation. Hence, no oviposition
behaviour was instigated. However, when these parasitoids came into contact with the frass of
their hosts, oviposition behaviour was almost always observed.
The observations described above indicated that foraging C, dubitator females might rely
heavily upon indirect cues (of which, chemical stimuli are the most likely candidates) for both
long- and short-range detection of their target hosts. Chapter 4 describes a test of the hypothesis
that these foraging parasitoids show a chemotactic response to certain host plant substrates but
not to host larvae themselves. This experiment was executed in 2001 using a static-air box
olfactometer, in which materials of interest were tested against inert control materials to eliminate
visual effects. Once substrates releasing attractive odours were identified, I predicted that the
more closely-related a particular material was to a host larva, the stronger its attractiveness would
be to C. dubitator. Parasitoid females should find some volatile compounds more attractive than
others if the former are a more reliable cue indicating the presence of hosts. For example,
foraging females were expected to be more strongly attracted to host frass (digested bark) than to
healthy bark. In 2002, a Y-tube olfactometer was employed to compare the relative attractiveness
of those substances found to release stimulating odours.
The final chief objective was to continue the evaluation of C. dubitator by assessing its
specific foraging behaviour. Information on host-finding and attack behaviour can have
considerable practical importance in biological control. It may help to explain observed patterns
of attack in field situations (Lauzibre et al., 2000). An understanding of the mechanisms by which
a parasitoid locates and accepts hosts might also enable pest management teams to enhance a
parasitoid's foraging efficiency through manipulation of host densities or numbers of parasitoids
released (Howard et al., 1998; Loke et al., 1983). Finally, as explained by Nurindah et al. (1 ggg),
this type of information can be used to monitor oviposition performance in future laboratory-
reared wasps (eg. C. dubitator) to ensure that there is no change in behaviour due to in vitro
culture or the use of alternate hosts for rearing.
Presently, no literature exists that depicts the behavioural interaction between C. dubitator
and the CBT. The first goal of Chapter 5, therefore, was to describe the pattern of behaviours
displayed by female parasitoids leading up to, during, and immediately following an attack on a
CBT host larva. Based on direct observations of ovipositions, an ethogram was constructed to
illustrate probable transitions between behaviours. This analysis provided a comprehension of
certain aspects of the parasitoid's foraging strategy, including a description of behaviours
associated with successful parasitism and an estimate of host handling time during oviposition.
The second aim of this section was to investigate the foraging patterns of C. dubitator females
that encounter patches harbouring different numbers of aggregated hosts, as would occur in high
density infestations. This experiment was conducted to learn more about the parasitoid's response
to changes in host densities and its overall search efficiency. Previous work on patch foraging by
many parasitoid species has led to the identification of certain inherent "leaving rules" used to
optimise the time spent searching for hosts in a patch of a given quality. A simple description of
optimal patch leaving theory is as follows: A parasitoid enters a patch and estimates the quality
(host density) of the patch by some means, such as kairomone concentration (Waage, 1979). Over
time the responsiveness of the parasitoid to the kairomone "cocktail" decreases until it falls below
a critical value, at which time the female exits the patch. If a host is encountered, the
responsiveness can either increase ("incremental mechanism"; Waage, 1979) or decrease ("count
down mechanism"; Driessen et al., 1995). Both systems of patch leaving decisions have been
shown to occur among different parasitoid species (van Alphen et al., 2003).
The surveys and experiments described above were intended to contribute to our
understanding of the composition and importance of the European parasitoid community
attacking the CBT. This research fulfilled the exploratory phase of the classical biological control
programme against this pest and also included a partial assessment of a potential agent. The
investigation of the foraging behaviour and reproductive biology of C. dubitator was considered
to be important for the rearing of the parasitoid, but may also be valuable for its evaluation, which
highlights the utility of linking behavioural and basic biological studies with biological control.
1.5 References
Alford, D.V. 1984. A Colour Atlas of Fruit Pests: their recognition, biology and control. Wolfe Publishing Ltd, Glasgow, Scotland 320 pp.
Balachowsky, A.S. 1966. Entomologie Applique'e a I'Agriculture. Tome I1 - Lepidopteres. Masson et Cie (Eds.), Paris, France. 1057 pp.
Boldyrev, M.I. and S.G. Dobroserdov. 198 1. [The raphidiid - an active predator of insects.] Zashchita Rastenii 9: 29
Dang, P.T. and D.J. Parker. 1990. First records of Enarmonia formosana (Scopoli) in North America (Lepidoptera: Tortricidae). Journal of the Entomological Society of British Columbia 87: 3-6
Dickler, E. 1972. [Investigations on the biology and population dynamics of the bark Tortricid Enarmonia formosana Scop. (Leptd., Tortr.)] Mitteilungen aus der Biologischen fur Land- und Forstwirtschaft, Berlin-Dahlem, pp. 85-106
Dickler, E. 1970. [The injuriousness of the bark borer Enarmonia formosana Scop. (Lepid., Tortr.).] Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 22: 170- 172
Dickler, E. and H. Zirnmerman. 1972. [Investigations on the control of the bark Tortricid Enarmonia formosana Scop. (Lepid., Tortr.)] Mitteilungen aus der Biologischen fur Land- und Forstwirtschaft, Berlin-Dahlem, 144: 143- 150
Driessen, G., C. Bernstein, J.J.M. van Alphen, and A. Kacelnik. 1995. A count-down mechanism for host search in the parasitoid Venturia canescens. Journal of Animal Ecology 64: 1 17-1 25
Godfray, H.C .J. 1994. Parasitoids: Behavioural and Evolutionary Ecology. Princeton University Press, Princeton 473 pp.
Godwin, H. 1975. The History of the British Flora: a factual basis forphytogeography. Cambridge University Press, Cambridge 541 pp.
Gold, C.S., P.S. Nemeye, and R. Coe. 1999. Recognition and duration of the larval instars of banana weevil, Cosmopolites sordidus Germar (Coleoptera: Curculionidae), in Uganda. Affican Entomology 7: 49-62
Howard, R.W., M. Charlton, and R.E. Charlton. 1998. Host-finding, host-recognition, and host- acceptance behaviour of Cephalonomia tarsalis (Hymenoptera: Bethylidae). Annals of the EntomoIogical Society of America 91: 879-889
Klaus, M.W. 1992. Cherry Bark Tortrix Survey Report. Washington State Department of Agriculture, Yakima, Washington
Klaus, M.W. 1991. Cherry Bark Tortrix in Washington State. Proceedings of the Washington State Horticultural Association, pg. 241-243
KoSlinska, M., B. Iwanek, H. Papynow, and Z. Szczepanik. 1980. [The control of the bark tortricid (Enarmonia formosana Scop., Lep.: Tortricidae) and the apple clearwing
(Synanthedon myopformis Borkh., Lep.: Aegeriidae).] Prace Instytutu Sadewnietwa w Skierniewicach, A. 20: 229-240.
Kratancsik, L. 1982. [The forecasting of the cherry bark tortrix moth (Enarmonia formosana Scopoli) by the use of sex traps (in Hungary).] Novknyvkdelem 18: 507-509
Kuznetsov, V.I. 1988. Leaf-rollers (Lepidoptera, Tortricidae) of the southern part of the Soviet Far East and their seasonal cycles. In: Lepidopterous Fauna ofthe USSR and Adjacent Countries (ed. O.L. Kryzhanovskii), Oxonian, New Dehli, pp.57-249.
Lauziere, I., G. Perez-Lachaud, and J. Brodeur. 2000. Behaviour and activity pattern of Cephalonomia stephanoderis (Hymenoptera: Bethylidae) attacking the coffee berry borer, Hypothenemus hampei (Coleoptera: Scolytidae). Journal of Insect Behaviour 13: 375-395
Loke, W.H., T.R. Ashley, and R.I. Sailer. 1983. Influence of fall armyworm, Spodoptera fiugiperda (Lepidoptera: Noctuidae) larvae and corn plant damage on host finding in Apanteles marginiventris (Hymenoptera: Braconidae). Environmental Entomology 12: 91 1- 915.
Massee, A.M. 1954. The Pests of Fruits and Hops. Crosky Lockwood and Son Ltd., London, 325 PP.
McNair, C., G. Gries, and M. Sidney. 1999. Toward pheromone-based mating disruption of Enarmonia formosana (Lepidoptera: Tortricidae) on ornamental cherry trees. The Canadian Entomologist 131: 97- 105
Melakeberhan, H., A.L. Jones, P. Sobiczewski, and G.W. Bird. 1993. Factors associated with the decline of sweet cherry trees in Michigan: nematodes, bacterial canker, nutrition, soil pH, and winter injury. Plant Disease 77: 266-27 1.
Minks, A.K., S. Voerman, and M. van de Vrie. 1976. A sex attractant for the cherry-bark tortrix moth, Enarmonia formosana. Entomologia Experimentalis et Applicata 19: 30 1-3 02
Moore, L.D. 1988. Pseudomonas syringae: Disease and ice nucleation activity. Ornamentals Northwest Newsletter 12: 4- 16
Murray, T.A., L.K. Tanigoshi, B. Bai, and E. LaGasa. 1998. Cherry bark tortrix, Enarmonia formosana (Scopoli), bionomics, natural enemy survey and control research project, 1997-98. Washington State University Report
Nurindah, B.W. Cribb, and G. Gordh.1999. Effects of physiological condition and experience on oviposition behaviour of Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) on eggs of Helicoverpa armigera Huebner (Lepidoptera: Noctuidae). Australian Journal of Entomology 38: 104- 1 14
Orr, R.L. 199 1. Pest Risk Assessment on Cherry Bark Tortrix. USDA, APHIS, PPD, PRAS. Hyattsville, Maryland.
Roediger, H. 19%. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) Zeitschrift fur Angewandte Entomologie 38: 195-321
Russell, D.A. and R. Bouzouane. 1989. The effect of diet, temperature, and diapause on the number and identification of larval instars in the oriental fruit moth, Grapholita molesto Busck (Lepidoptera: Tortricidae). Agronomic 9:9 19-926
Samal, J. 1926. Grapholitha woeberiana, Schiff. Bull. Czechost. Acad. Agric. 2: 98-100
Savkovskii, P.P. and E.I. Lyalyutskaya. 1976. [The bark-boring Tortricid and measures for its control.] Sadovodstvo, pt. 6, pp.23-34, Kiev
Savopoulou-Soultani, M. and M.E. Tzanakakis. 1990. Head-capsule width of Lobesia botrana (Lepidoptera: Tortricidae) larvae reared on three different diets. Annals of the Entomological Society of America 83: 555-558
Schuetze, K.T. and A. Roman. 193 1. Schlupfwespen. Isis Budissina 12
Serrnann, H. and H. Zahn. 1986. [Studies on the autecology of the cherry bark tortrix moth (E. form. Scopoli)] Nachrichtenblatt fur den Pflanzenschutz in der DDR 40: 128-132
Shapovalov, A.A. 1959. [Laspeyresia woeheriana Schiff. - a pest of cherry in the Kamennaya steppe.] 2001. Zh., pt. 38, 284-285.
Smol'yannikov, V.V. 1979. [Pests of wood, cambium, and bark.] Zashchita Rastenii No.2: 52-53
Stiling, P. 1993. Why do natural enemies fail in classical biological control programs? American Entomologist spring: 3 1-37
Sziraki, G. 1984. Dispersion and movement activity of some important moth pests living on stone fruits. Acta Phytopathologica Academiae Scientiarum Hungaricae 19: 5 1-64
Tanigoshi, L.K. 2002. Conservation and classical biological control of the cherry bark tortrix in the pacific northwest. Final Project Report 2002, Department of Entomology, WSU Vancouver Research and Extension Unit, Vancouver, Washington
Tanigoshi, L.K., B.B. Bai, and T.A. Murray. 1998. Biology and Control of the Exotic Cherry Bark Tortrix, Enarmonia formosana. Oregon Department of Agriculture Interim Project Report, 1998
Theobald, F.V. 1909 Insect Pests of Fruit. E.W. Classey, London pp. 188-192
van Alphen, J.J.M., Bernstein, C., Driessen, G. 2003. Information acquisition and time allocation in insect parasitoids. Trends in Ecology and Evolution 18: 8 1-87.
Waage, J.K. 1979. Foraging for patchily-distributed hosts by the parasitoid, Nemeritis canescens. Journal of Animal Ecology 48: 3 53-3 7 1
Winfield, A.L. 1964. The biology and control of the cherry-bark tortric moth. Plant Pathology 13: 115-1 20
CHAPTER 2 Distribution, phenology, and field parasitism of the
cherry bark tortrix
Abstract
The aim of the current study was to investigate the previously unsurveyed parasitoid
community attacking the cherry bark tortrix (CBT), Enarmonia formosana Scopoli (Lepidoptera:
Tortricidae) in central Europe. The CBT was found on cherry trees throughout the surveyed
regions of France, Germany, and Switzerland. Similarly, parasitism was recorded from each
region. However, only one of the 13 identified parasitoid species, Campoplex dubitator
Horstmann (Hymenoptera: Ichneumonidae), was collected repeatedly from all regions and in each
year. The bark-boring CBT showed a skewed within-tree distribution, with most larvae
aggregated near the base of trees. Densities were highest on trees with moss growing on the bark,
which is believed to indicate something of the tortrix's preference for, or enhanced survival in,
humid environments. The presence or absence of moss was not associated with the degree of
parasitism. By calculating percent parasitism per tree, the dominant parasitoid, C. dubitator, was
shown to exhibit inverse density dependence. The CBT had a single generation per year, which
overwinters in the larval stage. A frequency distribution of larval head capsule sizes from field-
collected specimens shows seven possible larval instars. Although all of the 13 parasitoid species
emerge from late instar or pupal hosts, C. dubitator is known to attack all, except perhaps the
first, of the five larval instar stages. Due to the staggered nature of CBT phenology, various larval
instars are present in cherry trees throughout the summer. Accordingly, C. dubitator adults are
known to be active over the entire summer.
2.1 Introduction
The cherry bark tortrix (hereafter referred to as CBT), Enarmonia formosana Scopo1i
(Lepidoptera: Tortricidae), has a history of minor to moderate importance as a pest in Europe. At
its worst, this bark-boring pest of cherry and other rosaceous trees has caused sporadic, local
outbreaks in European orchards (Massee, 1954; Smol'yannikov, 1979; Dobroserdov, 198 1).
There are few, if any, reports in recent literature of severe infestations of the CBT in Europe.
Since its recent accidental introduction into British Columbia, Canada (Dang and Parker, 1990),
however, the CBT has established itself as a key pest of ornamental cherries along the Pacific
coast (Klaus, 1992). The first instar larvae of the CBT feed exclusively in the outer part of the
phloem, while later instars may colonise the entire space between the cork and cambium. The
damaged areas of bark extend with each successive generation, sometimes resulting in the death
of large branches or even entire trees (Winfield, 1964).
Although the CBT is of European origin, it is attacked by natural enemies in North America.
However, field sampling in Washington State in 1997 and 1998 showed that the rate of
indigenous parasitism of the CBT was low, only 1.7% and 2. I%, respectively (Tanigoshi et al.,
1998). Similarly, field percent parasitism in the Fraser Valley, British Columbia, in 2001 and
2002 was 2.1% (unpublished). Based on observations made by Roediger (1956), it was suspected
that Europe had parasitoid species that have evolved specialised interactions with the CBT. Such
specialist parasitoid species would most likely have the highest potential as effective and safe
biological control agents in Canada and the United States (Waage, 1990). A parasitoid survey was
therefore conducted in central Europe, in collaboration with the CAB1 Bioscience Centre in
DelCmont, Switzerland. The preliminary study in 1998 found evidence of moderate levels of
parasitism in central Europe, coinciding with overall lower CBT densities (Kuhlmann et al.,
1998). These combined observations strengthened support for the idea of a classical biological
control programme against the CBT in North America.
The objective of this study was to closely investigate the biological and ecological aspects of
both the CBT and its parasitoid community in their native habitat. With respect to the CBT,
specific goals were to describe its distribution patterns at three spatial scales (region, orchard, and
tree) determine the severity of infestations in central Europe, elucidate which host tree or habitat
features might be correlated with pest densities, and clarify the number of generations per year
and the number of lanai instars of this species. A concurrent investigation of the CBT parasitoid
community included an analysis of field parasitism rates on host larvae and pupae, with a
comparison of species composition and overall parasitoid impact on CBT mortality between the
surveyed regions of Europe.
2.2 Materials and Methods
The study was conducted from 2000 to 2002. Field sampling was conducted in cherry
orchards of various management strategies throughout central Europe, including sites in the
Alsace of France, the southern Rhine Valley and Black Forest of Germany, and in the Jura and
other rural areas of north-central Switzerland. In all years, the majority of the sampling effort was
applied to the southern Rhine Valley and Black Forest because these regions contained the largest
and most numerous cherry orchards. Identification of field sites involved the selection of a
geographic region for study and then the use of large-scale maps and visitation to specific areas to
locate parcels of land bearing cherry trees. While it is known that the CBT infests several other
iosaceoiis species io va~yilig degrees (Ruedigel, i956j, ci~erry irtxs are ihe preferred host pimi.
During the current survey, only Prunus avium (L.) (ornamental and naturalised sweet cherry) was
sampled to avoid variability caused by different species of host tree.
The CBT is difficult for surveyors to detect. The frass tubes built at the entrance of the larval
feeding tunnels were the only visible signs that revealed the locations of larvae and pupae. This
indicator was often misleading because larvae of this species have a propensity to migrate to new
feeding sites in the tree bark before completing pupation inside the final feeding gallery. When a
fresh frass tube was discovered (colour was a rough determinant of faeces age), the bark
immediately adjacent to the tunnel entrance was lifted with a wood chisel and the exposed larva
or pupa was gently moved with a paintbrush into a Petri dish. In 2001 and 2002, search effort per
tree was standardised through the collection of all detectable immature CBT from each tree
sampled.
All field-collected host larvae and pupae were taken to the CAB1 Bioscience laboratory and
reared in 5.5 cm Petri dishes to obtain parasitoids. Each host larva had its head capsule width
measured, was labelled, and was placed individually into a Petri dish with food for daily
monitoring of its development. Mid to late instar larvae were reared on a meridic diet modified
from the recipe of Shorey and Hale (1965). Major constituents of this diet included ground pinto
beans, brewer's yeast, and cellulose powder. The earliest instars were kept on small sections of
c h e w bark untii large enough to accept the meridic diet. Pupae brought into the laboratory were
similarly labelled and placed individually into empty plastic vials (7 x 1.5 cm) with perforated
lids to allow gas exchange. These vials were then placed collectively into larger sealed containers
each with a small, wet cotton wick to maintain the humidity of the system. During 2000 and 2001
all immature CBT were kept at 20" C under a 16L:8D photoperiod. In 2002 the larvae and pupae
were reared at slightly warmer temperatures (23 * 2" C) to increase the developmental rate. The
sex of all eclosing CBT adults was determined and adults were allowed to mate for propagation
of a laboratory host culture.
2.2.1 CBT Parasitoid Associations
Parasitoid larvae emerging from the field-collected host larvae or pupae were allowed to form
cocoons and pupate, and were maintained in vials under the conditions described above for host
pupae. Eclosing parasitoid adults were pinned and sent for identification to Dr. Klaus Horstmann
(University of Wuerzburg, Germany - Ichneumonidae), Dr. Erich Diller (Zoologische
Staatssamml~~ng Miinchen, Germany - Ichnei~mnnidae), and Dr. Hannes Ram (Natural Histnry
Museum, Berne, Switzerland - Chalcidoidea). Parasitoid eclosion from field-collected host
specimens was used to calculate field parasitism rates. Percent parasitism was determined using
only those hosts that either survived to the adult moth stage or lived long enough for parasitism to
be recognised without dissection. The contribution of each parasitoid species to total parasitism
was assessed.
There are several ways in which a parasitoid species can exploit its host and these can be
characterised as parasitoid niches. All species that occupy a particular niche constitute a
parasitoid guild, which is defined by the host stage attacked, the mode of parasitism
(endoparasitic or ectoparasitic), and the form of parasitoid development (continuous or
protracted) (Mills, 1992). The developmental niches occupied by CBT parasitoids in this study
were described based on collection and eclosion data.
2.2.2 Spatial Distribution of the CBT and its Parasitoids
The distribution of the CBT and its parasitoid natural enemies in central Europe was studied
at three spatial scales: regional, orchard, and individual tree. Field sampling was conducted
between late April and mid September of each year of the study. A collection site was defined as
one continuous patch of cherry trees and one site was surveyed on each field trip. The motivation
behind this analysis was that, in order to identify productive collection sites and efficient
collection strategies for future parasitoid study and propagation, it was necessary to understand
the trends in and factors governing host and parasitoid distributions in the field.
2.2.2.1 Regional Scale
The three geographic regions in which numerous sites were surveyed to determine the degree
of CBT infestation on cherry trees and to quantify the impact of larval and pupal parasitoids on
the pest populations included the southern Rhine Valley (France and Germany), the Black Forest
(Germany), and Baselland (northwestern Switzerland). The analysis of host distribution and
corresponding parasitism rates relies on field data collected from 103 collection trips made to 62
different sampling locations (33 trips to 24 sites in the southern Rhine Valley; 32 trips to 4 sites in
the Black Forest; 39 trips to 34 sites in Baselland) during the study period. Certain large patches
were visited more than once, although individual trees were never sampled twice in the same
season.
To obtain sufficiently large sample sizes for the estimation of percent parasitism; it ~ B S
necessary to pool data from several collections. Low numbers of CBT hosts for each collection
resulted from 3 phenomena: (1) extraction of larvae and pupae from tree bark was time
consuming and daily collections rarely exceeded 70 healthy insects, (2) 28% of all insects were
fatally injured during the removal from the bark, and (3) approximately 24% of all CBT
specimens returned to the lab died for unknown reasons when reared on the meridic diet.
2.2.2.2 Orchard Scale
Employing the same data set as that used for the regional survey, a comparison of pest
densities and parasitism rates was made between tree patches with different spatial organisations.
Three classes were identified that describe the patch structure of cherry trees sampled: cluster (a
group of trees planted in multiple rows, eg. 3-dimensional arrangement), row (a single row of
trees, eg. 2-dimensional arrangement), and wild (trees occurring at a forest edge and therefore
mixed with unrelated species). This analysis evaluated the effect of patch organisation on pest
and parasitoid distributions, but was not designed to identify specific biotic and abiotic features
that might cause the observed patterns.
If a small patch of trees was selected for the survey, usually all available trees in that patch
were checked for CBT. In larger stands of cherry trees, one tree (typically near the patch edge)
was randomly selected for sampling. Subsequent collecting was conducted on trees adjacent to
the initial one sampled, or running in a transect line with the first tree sampled as a starting point.
In total between 2001 and 2002, we made 60 trips to 29 cluster sites, 12 trips to 12 row sites, and
five trips to five wild sites.
2.2.2.3 Tree Scale
Field sampling in 2001 and 2002 involved an additional level of complexity in that collectors
recorded the approximate within-tree location for each larva sampled. The trunk of each surveyed
tree was divided into six sections, based on north and south positioning and height above ground
("bottom" = 0 to 40 cm above ground, = 40 cm to tree crown, and "top" = tree crown).
With this partitioning system, every collected specimen was allocated to one of the six specific
regions. The within-tree distributions of CBT in Europe and North America were quantitatively
assessed by counting the number of fresh frass tubes in each of the six sections described above.
For this survey, 50 trees in Europe (25 from the Black Forest and 25 from Baselland) and 50 trees
in V~ncnilver~ RC; Canada were arbitrarily selected and assessed.
Additional notes were kept regarding individual trees. The relative sizetage of each tree was
recorded by measuring the trunk circumference at 80 cm above the ground. The vegetation
covering the ground at the immediate base of the tree was graded from 1 to 3, where "1" = little
or no vegetation, "3" = dense vegetation standing at least 40 cm in height, and "2" = an
intermediate degree of base cover. Finally, the presence or absence of moss on the bark of trees
surveyed was recorded as an indicator of the relative humidity close to the tree trunk. Analyses of
tree size and surrounding vegetation were run to test for any correlation between these
microhabitat features and pest and parasitoid numbers.
Parasitism by the dominant larval parasitoid, Campoplex cf. dubitator Horstrnann
(Hymenoptera: Ichneumonidae), relative to host density, was investigated by comparing
parasitism rates for individual trees with varying numbers of hosts. To reduce the possible effect
of parasitism variation among larval instars, only data collected between 19 April and 14 June in
2001 and 2002 were used. This ensures that only mid to late instars are considered, since only
these stages overwinter. Trees from only a single collection site (Feuerbach, Germany) were used
in this analysis to avoid the possible confounding effect of sampling from several locations with
different host and parasitoid populations. Each surveyed tree was included in this analysis of
density dependence if it met two criteria: (1) 50% or more of the CBT collected from the tree
survived to adalthood or a point at which parasitism could be identified in the laboratory and (2)
at least one host from the tree was parasitised, indicating an event of visitation by parasitoids
prior to sampling. Arcsine transformation was used to normalise the percent parasitism data.
2.2.3 CBT Phenology
The development of immature CBT was monitored through the periodic sampling of field
specimens between late April and mid September. Collected CBT larvae were evaluated to aid in
the identification and clarification of larval instars and phenology of this species. Within 24 hours
of collection from the field, the head capsule widths of all CBT larvae were measured. Mean head
capsule widths were plotted over time to determine the developmental progression of each
generation.
As an additional approach to identifying the CBT larval instars, we closely observed several
lawae through~l-!t: their e ~ t i r e ?.eve!qmel.rt cfi merirlic ?.jet i_n_ !he ! ; ? ~ n ~ ~ t ~ q . Fc!!owing Kishi
(1971), we recorded the number of moults of each specimen and several hours after each moult,
the new head capsule width was measured. Moults were identified by the presence of a shed head
capsule, which was then removed from the rearing container.
2.2.4 Temporal Analysis of Parasitism
Repeated sampling (2 collections per week) throughout the summer provided a means to
study changes in parasitism pressure on the CBT over time. A major concern, however, was that
regional or even local differences in ambient conditions would cause an undesirable level of
variation in parasitoid abundance and activity, giving misleading results. To address this problem,
two reliable, large cherry orchard locations were selected for weekly collection visits in 2002.
The first site was a northwest-facing, continuous orchard harbouring over 450 mature trees near
Feuerbach in Germany's Black Forest region (N 47" 44' 28.3" E 07" 33' 12.7"). Sampling at this
location began on 19 April and finished on 12 September. The second survey area consisted of a
more fragmented expanse of cherry orchards stretching along the northwest-facing side of a
valley between Flueh and Aesch in the Baselland canton of Switzerland (N 47" 28' 22.5" E 07"
35' 6.1"). Despite the fragmentation of orchards in this valley, multiple rows of cherry trees were
very common landscape features. Only one discrete orchard was sampled per visit, and
collections were conducted between 7 May and 13 September.
2.3 Results
2.3.1 CBT Parasitoid Associations
Over the three years of study, a total of 13 primary and secondary parasitoid species were
reared from CBT host larvae and pupae collected in central Europe. Table 2-1 presents a list of
these hymenopteran parasitoids, including information on the regions from which each species
was collected and the relative abundance of each. Of the 13 species, 8 were collected from the
southern Rhine Valley, 4 from the Black Forest, and 4 from Baselland. Only one parasitoid, C.
dubitator, was commonly collected from all four of the surveyed regions in every year of the
study. This wasp contributed to approximately 85% of all field parasitism, while no other single
species contributed to more than 5% during the survey period. Hence, most parasitoid species
reared from the CBT were relatively rare, several having only one record of association with its
bark-boring host.
Figure 2-1 illustrates the guild structure within the CBT parasitoid community. Despite the
existence of four guilds and 12 primary parasitoids, the three larval guilds each consist of only a
single species. Only C. dubitator, Isadelphus inimicus (Gravenhorst) (Hymenoptera:
Ichneumonidae), and Lissonota sp. (Hyrnenoptera: Ichneumonidae) have been reared from host
larvae collected in the field. The remaining primary parasitoids appear to attack CBT pupae. The
majority of these parasitic wasps have endoparasitic and solitary development, but the mode of
parasitisation for most is not known. One hyperparasitoid, Theroscopus hemipteron (Riche)
(Hymenoptera: Ichneumonidae), was reared on four occasions in 2000 and 2001 from what are
suspected to be C. dubitator cocoons. Finally, three additional species, Liotryphon crassiseta
(Thomson) (Hyrnenoptera: Ichneumonidae), a second Liotryphon sp. (Hymenoptera:
Ichneumonidae), as well as a second Campoplex sp. (Hymenoptera: Ichneumonidae), were reared
from parasitoid cocoons collected from CBT feeding galleries. Despite having been collected on
12 occasions, L. crassiseta was not included in the assessment of the CBT parasitoid community
due to the uncertainty of its association with the bark-boring pest. The same judgement was given
to Liotryphon sp. and Campoplex sp., for each of which only one specimen was collected.
Most, if not all, of the larval instars of the CBT are vulnerable to attack in the field by one
parasitoid species or another. Using Roediger's (1956) five-instar description, the first instar was
the only stage from which a parasitoid was not reared, although it must be noted that relatively
few of the field-collected neonates were successtitlly reared in the laboratory. When parasitism is
studied for each of the five instars individually, there is an evident trend for older field-collected
CBT larvae to be more likely to yield parasitoids (first instar: 0%, n = 12; second instar: 1.0%, n
= 97; third instar: 5.8%, n = 224; fourth instar: 10.6%, n = 1 16 1 ; fifth instar: 15.0%, n = 547).
2.3.2 Spatial Distribution of the CBT and its Parasitoids
2.3.2.1 Regional Scale
The CBT was found in all survey regions during the study. Despite the ubiquity of
infestations, densities of this pest were rarely high enough to threaten the health of mature cheny
trees. With all surveyed trees combined from 2001 and 2002, there were, on average, 7.6 * 0.4 SE
(range = 0 to 91) immature CBT per tree (n = 484).
A comparison of CBT density between regions indicates that there were 33% more larvae per
tree in the Black Forest (9.6 * 0.8 SE, n = 174) than in either the southern Rhine Valley (6.2 % 0.7
SE: n = 82) or the Baselland reugion (6 5 * 0 6 CF, n = 22X) This difference is st&lsticl!ly
significant (ANOVA: F = 7.16, df = 2, P < 0.001).
Due to high variation in parasitism levels between sample sites and from one year to the next,
the three field seasons were analysed separately for rates of parasitism and the three major
regions are discussed independently. Table 2-2 lists the overall percent parasitism for each region
in 2001 and 2002. When parasitism of CBT larvae is separated from that of the pupae, there is a
trend for parasitism to be slightly greater in the pupal stage.
2.3.2.2 Orchard Scale
By dividing the sampled sites into three categories based on organisation and density, it can
be shown that the CBT is more likely to occur on trees in larger, 3-dimensional clusters than in
more sparse spatial arrangements. Within clusters, 74% of the cherry trees surveyed were
attacked by the CBT (n = 561), while in single rows and wild stands, 42% (n = 134) and 11% (n =
44), respectively, of the trees were infested. When the CBT was present, densities per tree did not
differ significantly between the three types of organisation (cluster = 7.7; row = 6.0; wild = 5.6)
(ANOVA: F = 1.206, df = 2, P = 0.300). Failure to detect any differences may have been due to
the low power of the analysis (P = 0.70), which resulted from the general absence of the CBT on
wild trees.
Based on parasitoid emergence from field-collected CBT, 41%, 32%, and 20% of infested
trees from clusters, rows, and wild stands, respectively, contained at least one parasitised host.
There was no statistical difference between the overall parasitism rates for cluster (15%) and row
(17%) organisations, but again, the analytical power was very weak (ANOVA: F = 0.156, df = 2,
P = 0.856, P = 0.90). Because only two wild sites had evidence of parasitism, this category could
not be included in the analysis.
2.3.2.3 Tree Scale
Variation in infestation by a pest can be considered in two ways: (1) the likelihood of attack
and (2) the degree of attack when it occurs. Some characteristics of cherry trees were associated
with the probability and level of attack by CBT and subsequently by parasitoids. The likelihood
of attack by the CBT was not related to the density of vegetation at the tree base. However,
attacks appeared to be more common on trees with moss growing on the trunk (Table 2-3).
When considering only trees that were attacked: CRT larvae were also fnund in higher
numbers per tree when moss was present. Trees with intermediate densities of surrounding
vegetation had significantly fewer pests per tree than trees with very sparse or very dense
vegetation (Table 2-3). Finally, tree size was not correlated with pest densities (ANOVA: F =
0.575, df = I, P = 0.449)(Figure 2-2 a).
The presence of moss or other surrounding vegetation was not correlated with the probability
that a tree harboured parasitised CBT hosts or with the number of parasitised hosts when at least
one was present (Table 2-4). In contrast, among the trees that held parasitised CBT hosts, larger
trees tended to have significantly more parasitised hosts per 1000 cm2 (ANOVA: F = 6.360, df =
1, P = 0.013)(Figure 2-2 b).
A comparison of the within-tree distribution of the CBT between Vancouver, British
Columbia and central Europe reveals a more even distribution oLer the trunks of cherry trees in
Vancouver (Figure 2-3). A chi-square test for heterogeneity shows that this difference in CBT
distributions is highly significant ( X 2 = 70.596, df = 5, P < 0.001).
The distribution of immature CBT, based on extraction of larvae and pupae, within a single
tree in central Europe was heavily skewed toward the lower section of the bole (ANOVA: F =
675.6, df = 5, P < 0.001) (Figure 2-4). Approximately 95% of all CBT larvae and pupae were
collected from the trunk within 40 cm of the ground. No difference was detected between the
middle and top sections. In general, there were proportionally more CBT on the north-facing side
(0.52) than the south-facing side (0.45) of tree trunks. While this difference was not substantial, it
was statistically significant (Student's t-test: t = 2.307, df = 1 170, P = 0.02 1). The distribution of
parasitised hosts within a tree in central Europe followed a similar pattern to that of healthy hosts
(Figure 2-4). Significantly more parasitised CBT were retrieved from the bottom sections of the
trunk (ANOVA: F = 121.1, df = 5 , P < 0.001). It is worth noting, however, that the parasitism
rate, calculated by pooling the trees surveyed at each site, was actually higher in the middle
section (24.5%) than in either the top or bottom sections (12.4% and 14.7%, respectively)
(ANOVA: F = 4.148, df = 2, P = 0.0 17).
Parasitism by the dominant larval parasitoid, C. dubitator, appeared to be inversely density
dependent. Using a single tree as the spatial unit for assessing density dependence, there was a
strong tendency for the parasitism rate to decline as host abundance increased (ANOVA: F =
4 8 3 , df = 1, P < O.I_)C)!) (Figure 2-51,
2.3.3 CBT Phenology
Phenological data from 200 1 and 2002 consistently showed a single CBT generation per year
based on larval head capsule width measurements (Figure 2-6). Despite the general shift from
late-instar to early-instar larvae in June and July, healthy CBT pupae were collected from trees as
early as 10 May in 2001 and 25 April in 2002 and as late into the season as 24 August in 2001
and 22 August in 2002. The final field samples were taken in mid-September of both 2001 and
2002, when larval activity would have begun to decline, due to reduced temperatures
(approximately 14'C on average). By this point, most larvae collected were late-instar, with very
few specimens in the first and second instar. The same trend was observed during the early-
season collections. First, second, and third instar larvae were not collected until June and July, a
period following the peak in adult moth flight. This suggests that early instar larvae do not
overwinter. Larvae hatching late in the summer may be forced to feed later into the fall in an
attempt to reach a larger, hardier stage. Ultimately, any larvae that are still in the early instar
stages during the winter months will likely perish.
Once CBT larvae were taken to the CAB1 Bioscience laboratory, the duration of rearing was
dependent on the larval stage at the time of collection. The mean head capsule width of larvae
collected in 2001 (0.892 mm) was slightly, yet significantly, greater than that in 2002 (0.868 mm)
(Student's t-test: t = -2.088, df = 3457, P = 0.037). For 3001, the mean development time betwsen
collection and adult eclosion was 38.3 0.7 SE days. For 2002, this mean time was 36.0 It 0.4 SE
days. This difference in overall rearing times between 2001 and 2002 was statistically significant
(Student's t-test: t = 3.197, df = 1297, P = 0.001). Hence, despite being, on average, at a later
stage of development, the CBT larvae developed more slowly in 200 1. This difference is most
likely attributable to the increased rearing temperature in 2002. There was no difference in the
development time of male and female moths for either year (Student's t:test: 2001: t = 0.923, df =
424, P = 0.357, /3 = 0.85; 2002: t = 0.3 15, df = 871, P = 0.753, P = 0.94). The mean development
time for pupae at 20" C was 14 days, with all adults emerging within 10-15 days. The
female:male sex ratio of field-collected CBT was 0.50 : 0.50 (n = 612) in 2001 and 0.53 : 0.47 (n
= 1262) in 2002.
Figure 2-7 depicts the frequency distribution of measurements of 2133 CBT larval head
capsule widths. These measurements were taken from freshly collected larvae during the summer
of 2002 in the central European survey sites. The ahsence of very distinct peak sl?.oV:ing i n t ~
size ranges indicates the variance within and overlap between instars. (This distinction is not
resolved when the sexes are graphed separately.) From the current field data, there appear to be
seven size categories of larvae. Table 2-5 provides a description of larval instars by Roediger
(1956) and a current explanation of instars based on the data shown in Figure 2-6.
Rearing CBT larvae from first to final instar on the meridic diet in the laboratory showed
larvae moulting up to 1 1 times before pupating (Table 2-5). Results from this trial imply that
there are many more than the five or six larval instars defined by head capsule measurements
from field-collected specimens.
2.3.4 Temporal Analysis of Parasitism
In an analysis of seasonal parasitoid activity, only the first three instars were used to estimate
parasitism rates for different periods of the summer. Since the development time of the first three
instars is relatively quick (less than four weeks, based on laboratory rearing), any parasitised first,
second, or third instar specimens taken from the field could only have been attacked within the
four weeks before the survey. In contrast, it is difficult to determine whether parasitised late
instars had been attacked shortly before the collection, much earlier in the season, or even during
the previous summer. Unfortunately, exclusion of the fourth and fifth instars greatly reduced the
available sample size and, as a result, no data on parasitism in April and May are available.
Nonetheless, one can make an informal comparison of early instar parasitism by combining the
months of June and July (peak adult flight time) with August and September (post-peak) of 2002.
In June and July, percent parasitism on early instars was 3.8% (n = 133), while in August and
September it was 5.5% (n = 127). Campoplex dubitator was the parasitising agent in all of these
cases and there is no reason to believe that parasitoid preference for early or late instars should
change within these four months. Hence, the parasitism rate appears to remain fairly constant for
the univoltine CBT in the final two-thirds of the season. The frequent collection of viable
parasitoid cocoons of C. dubitator from late April through to mid-August suggests that parasitoid
activity continues steadily throughout the entire summer.
2.4 Discussion
2.4.1 CBT Parasitoid Associations
Only three publications, since 1913 and prior to this classical biological control programme,
discuss the natural enemies of the CBT in Eurasia. Schuetze and Roman (193 1) identified one
ichneumonid parasitoid, Lissonota versicoior ~o imgren , however, the host was most iikeiy
misidentified as the CBT (E. formosana) (Horstmann, pers. comm.). Later, in his thorough
description of CBT biology and ecology, Roediger (1956) listed four parasitoid species reared
from field-collected caterpillars. These were Campoplex mutabilis Holmgren (syn. C. difformis
(Gmelin)) (Hymenoptera: Ichneumonidae), Hemiteles inimicus Gravenhorst (syn. Isadelphus
inimicus (Gravenhorst)) (Hymenoptera: Ichneumonidae), Dolichogenidea laeviagata Ratzeburg
(Hymenoptera: Ichneumonidae), and Leskia aurea Fallen (Diptera: Tachinidae). Finally,
Boldyrev and Dobroserdov (1 98 1) highlighted the common raphidiid, Agulla xanthostigma
(Schummel) (Neuroptera: Raphidiidae), as an important predator of young tortricid larvae. Since
no parasitism rate or predator impact information was provided regarding any of these natural
enemies, their importance in the suppression of CBT populations is not known.
Of the five parasitoids previously described to attack the CBT, only one species, I. inimicus,
also appears in Table 2-1. But even this host-parasitoid association is regarded with scepticism
since related Isadelphus species are known parasitoids of wood-breeding Aculeata, including
Apidae, Sphecidae, and Eumenidae (Horstmann, pers. comm.). It must also be noted that the C.
mutabilis description (Roediger, 1956) is believed to have been a misidentification of C.
dubitator since this species, a close relative of C. mutabilis, was only described in 1985.
Therefore, it was not possible to separate the two species at the time of Roediger's 1956
publication (Horstmann, pers. comm.).
Mills (1992) described 1 1 distinct parasitoid guilds of tortricoid hosts (Lepidoptera:
Tortricoidea). These include one egg parasitoid guild, eight larval parasitoid guilds (egg-larval,
early larval, larval, mid larval, larval-pupal, late larval ecto, late larval endo, and larval-cocoon)
and two pupal parasitoid guilds (cocoon and pupal). This study aimed to identify the member
species of each guild, minus the egg parasitoids, within the CBT parasitoid community.
Excluding the hyperparasitoid, Theroscopus hemipteron (Riche) (Hymenoptera: Ichneumonidae),
the parasitoids listed in Table 2-1 fall into just four of the niches described by Mills (1992) (see
Figure 2-1).
Using data from the primary literature cited by Herting (1975), Mills (1994a) calculated that
Palaearctic stem-boring tortricid species are attacked by, on average, seven parasitoid species.
The current survey of CBT parasitoids uncovered a community of larval and pupal parasitoids
- -r 9 -- -:*- U-.*~--~PI. nn l x r r\np I-anrn l n~rscitnid r &hitator; and three ppa] CUIlSISLILIg Ul I L ap&lba. l r v v v v v v * , ~ ~ ~ f i ~ , -- r- .----- --, -. -. .. -
parasitoids, Tycherus vagus, Pimpla spuria, and Dibrachys afinis, were collected on more than
one occasion and thus represent 95% of all the parasitoids emerging from larval and pupal CBT
hosts. A conservative description of the parasitoid community would likely exclude the remaining
eight species, which are probably more commonly associated with other host species that may
occur elsewhere on cherry trees. Why does the CBT support a parasitoid community consisting of
only two guilds represented by four species, of which only one was collected with any reliability?
Two possible explanations for this phenomenon may be the endophytic lifestyle of the CBT or its
low abundance. According to Hawkins (1988), stem and wood-boring hosts benefit from both
low visibility and physical protection, resulting in species poor parasitoid complexes. The
galleries of CBT larvae clearly provide substantial protection. The sensitivity to vibration and the
high mobility of CBT larvae within their tunnels also enables them to retreat deeper into their
galleries, out of reach of the ovipositors of C. dubitator females. Mills (19943) also argued that
the predictability of host abundance could influence natural parasitoid communities. The low
densities of a host species, such as the CBT, could limit colonisation by parasitoids, resulting in a
very simple parasitoid community.
Of the 12 primary parasitoid species collected from the CBT, only two are deemed potential
candidates for classical biological control in North America. These are C. dubitator and Tycherus
vagus Bertoumieu (Hymenoptera: Ichneumonidae). The method adopted from Mills (1994~) to
illustrate the structure of the CBT parasitoid community is an effec,tive tool for predicting the
type of interaction that occurs between these two parasitoid species. For instance, there is no
overlap between the larval and pupal parasitoid guilds occupied by C. dubitator and T. v a w ,
respectively. These species would not experience any direct competition with one another,
suggesting that their combined effect might be additive. However, only the substantially more
common parasitoid, C. dubitator, was selected from these for further evaluation for biological
control since too few specimens of T. vagus were collected to establish a laboratory culture. The
decision to specifically investigate C. dubitator was based on observations such as its
contribution to host mortality, a possible high host-specificity, and feasibility of rearing. Further
study of this wasp's chemical ecology (Chapter 4) and foraging strategy (Chapter 5) was
conducted at the CAB1 Bioscience laboratory in 2001 and 2002.
In general, CBT larvae collected in the final instar stage were at least three times more likely
to yield parasitoids than larvae collected as second or third instars. The explanation for this is still
unkmm. It m y be a case of host instar preference by the dominant larval endnparasitnid (I
dubitator. Larger hosts are typically perceived as being of higher quality (Wang and Shi, 200 1 ;
Hebert and Cloutier, 1990) and may therefore be accepted for oviposition more often than smaller
individuals. In a laboratory environment-, C. dubitator is known to readily attack all CBT instars
presented to it (Chapter 3), but under natural conditions with a greater selection of hosts, it may
be that this parasitoid is more discriminating against small larvae. A second possible explanation
may be that late instars are more easily located, due to their larger size. The frass tubes of late
instars, for instance, can be many times larger than those of first and second instars. For a
parasitoid like C. dubitator, which uses these accumulations of faeces to pinpoint its hosts, a
larger beacon would conceivably be easier to find. It might also be possible that late instars suffer
higher parasitism due to temporal synchrony with their natural enemies. In the case of the CBT
and its parasitoid, C. dubitator, this is a less likely scenario since there is evidence that these
wasps are present in the field throughout the entire summer season. A fourth and very probable
potential reason for this trend in parasitism is that later instar larvae have simply had a longer
period of exposure to parasitoids prior to collection. The actual parasitism pressure exerted on the
different instars could not be determined from these field-collections, due to the koinobiotic
development of the dominant parasitoid, C. dubitator. To effectively analyse this, it would be
necessary to expose an assortment of known instars to parasitoids for brief periods under natural
conditions in the field.
2.4.2 Spatial Distribution of the CBT and its Parasitoids
The collection of the CBT from nearly every site surveyed supports all previous work
describing a wide distribution of the pest across central Europe (Balachowsky, 1966). The
differences in pest densities among the southern Rhine Valley, Black Forest, and Baselland
regions, while statistically significant, were not of any agricultural importance. This lack of
biological significance was due to the very low densities of the CBT in Europe. If average pest
densities had been closer to an economic or biological threshold, as they tend to be in North
America, a 33% increase in the number of larvae per tree could have biologically important
consequences.
The CBT parasitoids were much less reliably encountered in the field than their hosts. The
low rate of parasitism and high degree of patchiness in parasitoid distribution indicate that some
methnds nf surveying paraitclid activity, such as sentine! infested cherry !ng ~!acemer.ts, m2y be
ineffective due to the low encounter rate by the parasitoid species of interest. Also, to obtain high
numbers of parasitised hosts, it would be necessary to focus collection efforts on sites known to
have high parasitism rates.
Although the number of CBT larvae per tree did not differ between tree organisations of
varying density, the likelihood of a tree being infested was correlated with tree density. To
increase efficiency while collecting CBT specimens, surveyors should therefore focus their
searching efforts on larger stands of cherry trees (ie. continuous orchards). Since the rates of
parasitism do not vary substantially between tree patches of varying densities, it would again be
advisable for surveyors to concentrate their effort in larger patches because less time would be
wasted searching for infested trees. Previous studies have demonstrated that parasitoids may
locate their hosts by using cues from the host habitat (Vinson, 1976; Godfray, 1994). For
example, Elzen et al. (1983) showed the behavioural response of the ichneumonid Campoletis
sonorensis (Cameron) to cotton odours and Read et al. (1970) revealed the attraction of the
braconid wasp, Diaeretiella rapae (McIntosh), to mustard oil volatiles. In such cases, denser
patches of the host plants should theoretically attract more natural enemies due to the greater
concentrations of cues. Whether parasitoids of the CBT are drawn to orchards based on habitat
features alone is uncertain, but the presence of slightly more CBT larvae parasitised by C.
dubitator on larger trees is suggestive of such a phenomenon.
While CBT densities did not vary on trees with trunk circumferences in the range of 50-200
cm, preliminary observations made it evident that extremely young cherry trees (trunk
circumference 10-50 cm) rarely contained CBT larvae. For this reason, the field survey thus
included only the more mature trees with trunk diameters greater than 50 cm. The greater
probability of infestation on more mature trees may be due to the elevated attractiveness of those
trees resulting from bark damage over time (Winfield, 1964; Tanigoshi et al., 1998), a richer food
supply associated with the thicker bark layers, a more appropriate microclimate provided by a
larger shading canopy (Roediger, 1956), or simply a greater probability of encounter due to do a
larger surface area. A fifth argument, that larger trees can support more hosts, is not appropriate
since there was no evidence of overcrowding by larvae, which have been observed to co-exist at
high densities in bark in the laboratory.
Dickler and Zimmerman (1 972) proposed that remnving a l l the vegetation si-~rmnnding the
trunk base would reduce CBT infestations. The current study, however, does not give clear
support for this. Only the presence of moss on the bark was clearly associated with higher pest
numbers. In answering why moss is linked with larger infestations, one must consider the
developmental needs of a CBT larva. Roediger (1956) showed that eggs and early instars will not
survive temperatures above 32' C and the larval feeding galleries are susceptible to desiccation.
The presence of moss is an excellent indicator of cooler temperatures and elevated relative
humidity due to reduced sun exposure. These conditions are believed to be important for creating
an ideal microclimate for CBT development. Following the suggestion of Dickler and
Zimmerman (1972), other herbaceous plants would also contribute to shading the trunks of cherry
trees. This is likely only the case for isolated trees since the continuous foliar canopy of dense
orchards typically provides substantial shade. Thus, in large orchards, removing small plants and
grasses may negatively impact natural enemies more than the CBT due to the destruction of
potentially important refuges for parasitoids and predators (Denno et al., 2002; Finke and Denno,
2002).
The concentration of most feeding sites at the bases of tree trunks was a consistent feature
among virtually all the sites surveyed in France, Germany, and Switzerland. A highly skewed
spatial distribution within a tree is common for lepidopteran herbivores (Weakley et al., 1990;
Lewis, 1992; Mo et al., 1997). One suggested, and often supported, explanation for non-random
distributions of insects on a tree has been the response to sun-exposure (Mo et al., 1997), or more
specifically, temperature (Wellington, 1950; Carroll and Luck, 1984; Hedstroem, 1992) and
microhabitat quality (Weakley et al, 1990). It was suspected that the CBT aggregates at the bases
of trees to escape excessive sun exposure since larvae require that their feeding galleries remain
moist. The relative humidity would be higher near the ground compared to areas further up on the
trunk, due to shading and reduced wind-speeds. The tunnels of CBT larvae are also often
observed to extend below ground level, which would further assist in preventing desiccation of
the exposed phloem tissues.
There is currently no explanation for the fact that the within-tree distribution of CBT is more
even, if not reversed, on ornamental cheny trees in Vancouver. No formal study has been made to
compare attributes of the Vancouver trees with the orchard cherry trees in central Europe. Local
arborists in Vancouver, however, have made independent observations that the heaviest CBT
infestations occur at graft sites in the crowns of the trees. These calloused areas on the trunk may
provide easier access to the food layers for neonate larvae or may re!ease greater concentrtiom
of volatiles from lesions in the bark, thereby attracting more ovipositing adult moths. The
observed difference may also result from the fact that the trees sampled in Europe were of the
species P. avium whereas sampling was conducted on ornamental cheny trees, including P.
serrulata and P. yadoensis, in Vancouver. It is conceivable that slight morphological or
physiological differences between these related tree species could alter the oviposition behaviour
of CBT adults or the survival of larvae.
Whether the difference in within-tree distributions between Europe and North America is of
any importance to a classical biological control program depends upon the parasitoid agents and
their search capabilities. If, for instance, a European parasitoid species has evolved a fixed spatial
foraging pattern that restricts it to the lower recesses of a tree, it would be heavily constrained
when foraging for hosts that exist in the upper strata of a tree. Gradients in parasitism rates within
a tree have been demonstrated for other parasitoids in the past (Carroll and Luck, 1984;
McAuslane et al., 1993). Parasitism by C. dubitator was shown to occur in all sections of the
trunk, with the highest rates of attack in the middle portion of the trunk. This parasitoid should
therefore have little difficulty in exploiting CBT hosts that tend to aggregate at the tree crown, as
is the case in North America.
The functional response of a parasitoid, often regarded as a critical component in the
selection of optimal biological control agents (Fernandez-Arhex and Corky, 2003), depends upon
various characteristics of the host-parasitoid interaction (Hassell et al., 1985; Lessells, 1985).
Direct density dependence occurs when parasitoids remain longer and parasitise proportionally
more hosts in patches with higher host densities. Early models hinted that optimally foraging
parasitoids would show this form of density dependence (Charnov, 1976; Cook and Hubbard,
1977; Comins and Hassell, 1979; Murdoch and Briggs, 1996). Direct density dependence was
traditionally considered to be the most desirable response from a biological control agent because
it enabled natural enemy populations to increase in synchrony with pest populations while
maintaining appropriate population stability (Hassell 1982). However, as shown by Murdoch and
Stewart-Oaten (1989), the modelling framework applied to this theory may provide opposing
conclusions. Also, as evidenced by Murdoch et al. (1984), effective control of high pest densities
may not always be explained by any sort of response to host density, even when examined at
multiple scales. Ensuing studies have also shown that parasitoid species responding with direct
density dependence may not be as common as once expected (Stiling, 1987; Fernandez-Arhex
and Ccr!ey, 2003) since inverse density dependent parasitism may hetter wit parasitoids in some
host environments (Hassell et al., 1985; Lessells, 1985). Figure 2-5 shows an inverse density
dependent response to host density by C. dubitator in the field. This study was conservative in
that the density response was estimated from only a single site, however it does suggest that
parasitoid impact may generally be greatest at low host densities. Similar inverse patterns of
density dependence were found in approximately half of the cases reviewed by Walde and
Murdoch (1988). Caution must be taken when analysing density dependence from field
parasitism data because the final calculation of parasitism may not accurately reflect parasitoid
effort and impact (Chesson, 1982). Although parasitism may not always result in a healthy
parasitoid progeny, hosts that are attacked may suffer reduced fitness or even early death due to
the parasitism. The current survey did not have the capacity to measure these additional parasitoid
mortality effects.
The mechanism behind the inverse density dependent response of C. dubitator is currently
unknown, although Morrison and Strong (1981) provided a number of hypotheses why such a
pattern might occur. They suggested, for instance, that parasitism rates might decline with
increasing host abundance due to energy and time constraints, egg depletion, or restrictively
lengthy host handling times. Lessells (1985) suggested that imperfect information on patch
quality, a potentially common feature among parasitoids of the CBT, could also lead to this type
of density response. Laboratory-based studies are necessary to fully understand what drives the
inverse density dependence of C. dubitator observed in the field. Chapter 5 includes an
experiment to investigate the behavioural response of this wasp to increasing host densities in the
laboratory.
2.4.3 CBT Phenology
Pheromone-baited traps for monitoring adult CBT flight have been used to determine whether
this pest is univoltine or bivoltine (Winfield, 1964; Tanigoshi et al., 1998). Due to the frequent
occurrence of two peaks in flight activity through the summer, there was suspicion that the CBT
had two generations per year. As witnessed from continuous field surveys over a number of
years, there is a high degree of variation in developmental stages of the CBT between locations,
between trees, or, quite often, within a single tree. Nonetheless, with a large sample size, it was
possible to measure the development of CBT larvae over the duration of the summer. The sudden
decline in head capsule sizes, which occurs between late June and late July (Figure 2-6), closely
rohcides with the ~ e a k In adult flight observed in Europe (Winfield, 1964) and North America
(Tanigoshi et al., 1998). This single major shift in head capsule widths implies there is only one
generation per season, with a major phase of egg-laying occurring between early June and mid-
July.
Determining the number of instars a CBT larva passes through before pupating proved to be
difficult and the results presented here, depending on their interpretation, do not necessarily
support those from previous studies on CBT biology (Roediger, 1956; Savkovskii and
Lyalyutskaya, 1967; Dickler, 1972). Roediger's (1 956) description of CBT larval instars using a
frequency distribution of head capsule widths is astonishingly straightforward considering his
limited sample size and the degree of variance that was noted during the current study. Larval
head capsule measurements taken from over 2100 specimens in 2002 do not clearly indicate the
larval instar stages of the CBT. The way one interprets Figure 2-7 influences whether these
results agree or disagree with those given by Roediger (1 956). The problem arises from the
presence of two peaks in the frequency distribution at 0.91-0.95 mm and 1.01-1 .O5 mm. If each of
these peaks in the graph is interpreted as a separate instar, then the current study finds two more
larval instars than Roediger (1956). If, however, those peaks are simply added to the fourth instar
group, then the current data fit very closely with those of Roediger (1 956).
Although commonly used, the frequency distribution of head capsule to describe larval stages
may be inaccurate due to size variation within each instar (Kishi, 1971). Mizell and Nebeker
(1979) showed temporal variance in head capsule sizes and McNeil(1978) described gender-
related differences in those measurements. The use of this approach was questionable in the
current study as well. Undoubtedly, the most reliable method for determining the number of
larval stages is to count the moulted skins left by a developing larva prior to pupation Kishi
(1971). Unfortunately, this method is not feasible for field study of the CBT because each shed
exuvia is removed from the feeding gallery by the larva and larvae often migrate to new feeding
sites. But under laboratory conditions with meridic diet it was possible to count the moults of
individual larvae. These developing larvae, however, were shown to provide up to seven more
moults than was expected, based on previous literature (Roediger, 1956; Savkovskii and
Lyalyutskaya, 1967). It may be that the meridic diet is the cause of this abnormal development.
The currently used diet has been linked with poor survival of early instars and larvae reared
exclusively on diet do not seem to attain the maximum head capsule widths observed from field-
collected specimens. The phenomenon of environmental factors (including diet) affecting the
number of moults before pupation has previously been dncurnented in other tortricids (Ruse!!
and Bouzouane, 1989; Savopoulou-Soultani and Tzanakakis, 1990; Gold et al., 1999).
2.4.4 Temporal Analysis of Parasitism
There is a trend for the calculated total percent parasitism of the CBT (all host instars
combined) to increase over the summer. But one should expect this pattern to arise even if
parasitism is equal across all host stages. This is due to the accumulation of parasitised late instars
just prior to the peak flight period, resulting from a lengthy exposure to parasitoids (Weseloh and
Adreadis, 1982) and the delayed development of immature parasitoids. Since the larvae
parasitised by C. dubitator are not killed and fully consumed until they have become pre-pupae,
percent parasitism in late June, for example, may reflect the cumulative parasitism from April,
May, and June. Therefore, total parasitism based on field-collected hosts is not the most accurate
method for calculating parasitoid activity for a specific time interval. A major portion of the field
data was excluded (all fourth and fifth instars) to ensure that trends in parasitism were not
confounded by the koinobiotic development of the dominant larval endoparasitoid, C. dubitator.
This reduction of the data set may have created a poor representation of the natural CBT
population, resulting in the failure to detect temporal differences in the parasitism rate. Only
approximately 3% of field-collected early instar CBT yielded parasitoids. It is difficult to
determine whether this represents a significant fraction of total parasitoid effort (and is therefore a
good measure of activity). Additionally, the use of only early instar larvae inevitably excludes the
impact of pupal parasitoids, which, although not extremely abundant, form the most diverse
parasitoid guild. More controlled field or laboratory experiments (ie. sentinel log placement) are
required to properly address the question of temporal patterns in parasitism of the CBT.
2.5 Conclusion
CBT larvae and pupae are parasitised by several hymenopteran species. The importance of
most of these species in the regulation of CBT populations is questionable. Only the larval
endoparasitoid, C. dubitator, was consistently recovered from field-collected hosts. An early
field-based assessment of this wasp indicates only a modest contribution to host mortality, with
an inversely density-dependent response to host density. Nonetheless, C. dubitator appears not to
be seasonally limited, as the adults are active year-round and can successfully parasitise all but
the first instar of the CBT. There may also be additional parasitoid-induced mortality, not
detectable through the collection of living host specimens (van Driesche, 1983). For instance,
whiie CBT first instars are known to be unsuitabie hosts for C. dub~tator, the parasitoids stiii
attack these neonates, which normally die from the trauma of parasitoid oviposition (Chapter 3).
Further studies of C. dubitator biology were carried out at the Switzerland CAB1 Bioscience
Centre in 2001 and 2002 (Chapters 3 ,4 , and 5). An assessment of this parasitoid's host range will
commence in 2003.
For future collections of the CBT and its parasitoids, sampling at specific times in the
summer and in recognised reliable sites will lead to a more efficient use of resources. Preferred
survey sites in central Europe should be large, shaded orchards with mature trees and moss
present on the trunk bases. From this study, we know that C. dubitator collections will be most
productive from early May until late June when CBT larvae are in the final instars. The window
of opportunity for efficient collection of pupal parasitoids is much more brief since most CBT
will pupate between mid June and late July. As the maintenance of high numbers of CBT larvae
on cherry bark is unrealistic, it is necessary to use a meridic diet. Work is currently underway to
identify a diet formula that supports the rapid development of the CBT.
2.6 References
Balachowsky, A S . 1966. Entomologie Applique'e a I 'Agriculture. Tome I1 - Lepidoptbres. Masson et Cie (Eds.), Paris, France. 1057 pp.
Boldyrev, M.I. and S.G. Dobroserdov. 1981. The raphidiid - an active predator of insects. Zashchita Rastenii 9: 29
Carroll, D.P. and R.F. Luck. 1984. Within-tree distribution of California red scale, Aonidiella aurantii (Maskell) (Homoptera: Diaspididae), and its parasitoid Comperiella bifasciata Howard (Hymenoptera: Encyrtidae) on orange trees in the San Joaquin Valley. Environmental Entomology 13: 179-1 83
Charnov, E.L. 1976. Optimal foraging: the marginal value theorem. Theoretical Population Biology 9: 129- 136
Chesson, J. 1982. Estimation and analysis of parasitoid search and attack parameters from field data. Environmental Entomology 11: 53 1-537
Comins, H.N. and M.P. Hassell. 1979. The dynamics of optimally foraging predators and parasitoids. Journal of Animal Ecology 48: 335-35 1
Cook, R.M. and S.F. Hubbard. 1977. Adaptive searching strategies in insect parasites. Journal of Animal Ecology 46: 1 15- 125
Dang, P.T. and D.J. Parker. 1990. First records of Enarmonia formosana (Scopoli) in North America (Lepidoptera: Tortricidae). Journal of the Entomological Society of British Columbia 87: 3-6
Denno, R.F., C. Gratton, M.A. Peterson, G.A. Langellotto, D.L. Finke, and A.F. Hubert-. 2002. Bottom-up forces mediate natural-enemy impact in a phytophagous insect community. Ecology 83: 1443- 1458
Dickler, E. 1972. [Investigations on the biology and population dynamics of the bark Tortricid Enarmonia formosana Scop. (Leptd., Tortr.)] Mitteilungen aus der Biologischen fur Land- und Forstwirtschaft, Berlin-Dahlem, pp. 85-106
Dickler, E. and H. Zimmerman. 1972. [Investigations on the control of the bark Tortricid Enarmonia formosana Scop. (Lepid., Tortr.)] Mitteilungen aus der Biologischen fur Land- und Forstwirtschaft, Berlin-Dahlem, 144: 143- 150
Dobroserdov, S.G. 198 1. Control of the apple clearwing and the bark-boring tortrix. Zashchita Rastenii No.1: 59
Elzen, G.W., H.J. Williams, and S.B. Vinson. 1983. Response by the parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) to chemical synomones in plants: implications for host habitat location. Environmental Entomology 12: 1872- 1876
Fernindez-Arhex, V. and J.C. Corley. 2003. The functional response of parasitoids and its implications for biological control. Biocontrol Science and Technology 13: 403-413
Finke, D.L. and R.F. Denno. 2002. Intraguild predation diminished in complex-structured vegetation: implications for prey suppression. Ecology 83: 643-652
Godfray, H.C.J. 1994. Parasitoids: Behavioural and Evolutionary Ecology. Princeton University Press, Princeton 473 pp.
Gold, C.S., P.S. Nemeye, and R. Coe. 1999. Recognition and duration of the larval instars of banana weevil, Cosmopolites sordidus Germar (Coleoptera: Curculionidae), in Uganda. African Entomology 7: 49-62
Hassell, M.P. 1982. Patterns of parasitism by insect parasitoids in patchy environments. Ecological Entomology 7: 365-377
Hassell, M.P., C.M Lessells, and G.C. McGavin. 1985. Inverse density dependent parasitism in a patchy environment: a laboratory system. Ecological Entomology 10: 393-402
Hawkins, B.A. 1988. Species diversity in the third and fourth trophic levels: patterns and mechanisms. Journal of Animal Ecology 57: 13 7- 162
Hebert, C. and C. Cloutier. 1990. Host instar as a determinant of preference and suitability for two parasitoids attacking late instars of the spruce budworm (Lepidoptera: Tortricidae). Annals of the Entomological Society ofAmerica 83: 734-741
Hedstroem, I. 1992. Why to duava fruit flies, Anastrepha striata (Tephritidae), avoid the upper canopy of host trees? Tropical Pest Management 38: 1 3 6- 143
Herting, B. 1975. A catalogue of the parasites and predators of terrestrial arthropods. Section A: Host or preylenemy. Volume VI Lepidoptera, Part I. Commonwealth Agricultural Bureaux, Farnham Royal
Kishi, Y. 1971. Reconsideration of the method to measure the larval instars by use of the frequency distribution of head-capsule widths or lengths. Canadian Entomologist 103: 101 1- 1015
Klaus, M. 1992. 1992 Cherry bark tortrix survey report. Washington State Department of Agriculture, Yakima, WA
Kuhlmann, U., J. Otani, H. White, N. Lauro, L. Reimer, E. Hunt, S. Epple, S. Micheletti, B. Klander, and B. Jahn. 1998. Cherry bark tortrix (Enarmonia formosana) Summary Report - Progress in 1998 Agricultural Pest Research, CAB1 Bioscience Switzerland Centre 10-1 1
Lessells, C.M. 1985. Parasitoid foraging: should parasitism be density dependent? Journal of Animal Ecology 54: 27-41
Lewis, V.R. 1992. Within-tree distribution of acorns infested by Curculio occidentis (Coleoptera: Curculionidae) and Cydia Iatferreanal (Lepidoptera: Tortricidae) on the coast live oak. Environmental Entomology 21: 975-982
Massee, A.M. 1954. The Pests of Fruits and Hops. Crosky Lockwood and Son Ltd., London, 325 PP.
McAuslane, H.J., F.A. Johnson, D.A. Knauft, and D.L. Colvin. 1993. Seasonal abundance and within-plant distribution of parasitoids of Benisia tabaci (Homoptera: Aleyrodidae) in peanuts. Environmental Entomology 22: 1043- 1050
McNeil, J.N. 1978. The number of larval stages of Thymelicus lineola (Lepidoptera: Hesperiidae) in eastern Canada. The Canadian Entomologist 110: 1293-1295
Mills, N.J. 1994a. Parasitoid guilds: a comparative analysis of the parasitoid communities of tortricids and weevils. Pg.30-46 In: Hawkins, B.A. and W. Sheehan (Eds.) Parasitoid Community Ecology. Oxford University Press, Oxford
Mills, N.J. 1994b. The structure and complexity of parasitoid communities in relation to biological control. Pg. 397-417 In: Hawkins, B.A. and W. Sheehan (Eds.) Parasitoid Community Ecology. Oxford University Press, Oxford
Mills, N.J. 1992. Parasitoid guilds, life-styles, and host ranges in the parasitoid complexes of tortricoid hosts (Lepidoptera: Tortricoidea). Environmental Entomology 21: 230-239
Mizell, R.F. and T.E. Nebeker. 1979. Number of instars of the southern pine h&!e (Co!qtera: Scolytidae) and some comparisons of head capsule widths. Annals of the Entomological Society of America 72: 3 13-3 16
Mo, J., M.T. Tanton, and F.L. Bygrave. 1997. Within-tree distribution of attack by Hypsipyla robusta Moore (Lepidoptera: Pyralidae) in Australian red cedar (Toona australis (F. Muell.) Harmes). Forest Ecology and Management 96: 1 47- 1 54
Morrison, G. and D.R. Strong, Jr. 198 1. Spatial variations in egg density and the intensity of parasitism in a neotropical chrysomelid (Cephaloleia consanguinea). Ecological Entomology 6: 55-61
Murdoch, W.W. and C.J. Briggs. 1996. Theory for biological control: recent developments. Ecology 77: 200 1-20 13
Murdoch, W.W., J.D. Reeve, C.B. Huffaker, and C.E. Kennett. 1984. Biological control of olive scale and its relevance to ecological theory. American Naturalist 123: 371-392
Murdoch, W.W. and A. Stewart-Oaten. 1989. Aggregation by parasitoids and predators: effects on equilibrium and stability. American Naturalist 134: 288-3 10
Read, D.P., P.P. Feeny, and R.B. Root. 1970. Habitat selection by the aphid parasite Diaeretiella rapae and its hyperparasitoid, Charips brassicae. Canadian Entomologist 102: 1567- 1578
Roediger, H. 1956. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) Zeitschrift fur Angewandte Entomologie 38: 195-32 1
Russell, D.A. and R. Bouzouane. 1989. The effect of diet, temperature, and diapause on the number and identification of larval instars in the oriental fruit moth, Grapholita molesta Busck (Lepidoptera: Tortricidae). Agronornie 9:9 19-926
Savkovskii, P.P. and E.I. Lyalyutskaya. 1976. [The bark-boring Tortricid and measures for its control.] Sadovodstvo, pt. 6, pp.23-34, Kiev
Savopoulou-Soultani, M. and M.E. Tzanakakis. 1990. Head-capsule width of Lobesia botrana (Lepidoptera: Tortricidae) larvae reared on three different diets. Annals of the Entomological Society ofAmerica 83: 555-558
Schuetze, K.T. and A. Roman. 193 1. Schlupfwespen. Isis Budissina 12
Shorey, H.H. and R.L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58:522-524.
Smol'yannikov, V.V. 1979. Pests of wood, cambium and bark. Zashchita Rastenii No. 2: 52-53
Tanigoshi, L.K., B.B. Bai, and T.A. Murray. 1998. Biology and Control of the Exotic Cherry Bark Tortrix, Enarmonia formosana. Oregon Department of Agriculture Interim Project Report, 1997 and 1998
van Driesche, R.G. 1983. Meaning of "percent parasitism" in studies of insect parasitoids. Environmental Entomology 12: 16 1 1 - 1622
Vinson, S.B. 1976. Host selection by insect parasitoids. Annual Review of Entomology 21: 109- 133
Waage, J. 1990. Ecological theory and the selection of biological control agents. In: Mackauer, M., L. E. Ehler and J. Roland (Eds.) Critical Issues in Biological Control. Intercept Ltd., Andover
Walde, S.J. and W.W. Murdoch. 1988. Spatial density dependence in parasitoids. Annual Review of Entomology 33: 44 1-466
Wang, Q. and G. Shi. 2001. Host preference and sex allocation of three Hymenopteran parasitoid species (Ichneumonidae and Braconidae) of a longicorn pest, Oemona hirta (Fabr.) (Coleoptera: Cerambycidae). Journal of Applied Entomology 125: 463-467
Weakley, C.V., P.A. Kirsch, and F.G. Zalom. 1990. Within-orchard and within-tree distributions of peach twig borer (Lepidoptera: Gelechiidae) damage to peaches. Journal of Economic Entomology 83: 505-5 10
Wellington, W.G. 1950. Effects of radiation on the temperature of insects' habitats. ScientiJic Agriculture 30: 209-233
Weseloh, R.M. and T.G. Andreadis. 1982. Possible mechanism for synergism between Bacillus thuringiensis and the gypsy moth (Lepidoptera: Lymantriidae) parasitoid, Apanteles melanoscelus (Hymenoptera: Braconidae). Annals of the Entomological Society of America 75: 435-438
Winfield, A.L. 1964. The biology and control of the cherry-bark tortric moth. Plant Pathology l3:ll5-l2O
Figure 2- 1 Parasitoid community structure associated with the larval and pupal stages of Enarmonia formosana. Arrows connect host stages attacked and killed by each of the four parasitoid guilds: (1) larval, (2) larval - pupal, (3) late larval ecto, and (4) pupal. Arrow width indicates the relative abundance of each parasitoid species interacting with the host. Arrows drawn through the host stage bubble represent endoparasitism while those not passing through the bubble represent ectoparasitism.
Guild Member S ~ e c i e s
@ larval - pre-pupal Campoplex dubitator Horstmann (Ichneumonidae) endoparasitoid
larval - pupal Lissonofa sp. (Ichneumonidae) @ endoparasitoid
@ late larval Isadelphzrs inimicus (Gravenhorst) (Ichneumonidae) ectoparasitoid
@ pupal Tycherus vagus Bertoumieu; Pimpla spuria Gravenhorst; Pimpla contemplator endoparasitoid Miiller; Pimpla turionellae Linnaeus; Cyclogastrella simplex Walker; Gelis
longicauda (Thornson); Mastrus sp.; Phygadeuontini (all Ichneumonidae); and Dibrachys afinis Masi (Pteromalidae
Figure 2- 2 Association between Enarmonia formosana density and tree trunk circumference. (a) Number of total CBT (healthy and parasitised) per 1000 cm2 at the tree base (r2 = 0.001; P = 0.449). (b) Number of only parasitised CBT per 1000~ at the tree base (y = 0 . 0 0 6 ~ + 0.444; r2 =
0.03, P = 0.013).
40 60 80 100 120 140 160 180 200 220
Trunk circumference (cm)
Trunk circumference (cm)
Figure 2- 3 Comparison of within-tree distributions of Enarmoniaformosana frass tubes (*SE) on the trunks of cherry trees in central Europe and Canada. The European within-tree distribution of frass tubes differs significantly from that of Vancouver (chi-square test for heterogeneity: ~2 = 70.596; df = 5; P < 0.001).
North North North South South South Bottom Middle TOP Bottom Middle TOP
Trunk Section
Central Europe 0 Vancouver, Canada
Figure 2- 4 Spatial distribution of healthy and parasitised Enarmonia formosana on the trunks of cherry trees in central Europe. Error bars indicate 1 standard error. Significantly more healthy (P < 0.001) and parasitised (P < 0.001) E. formosana specimens were collected from the bottom section than the middle and top sections of trees.
North North North South South South Bottom Middle TOP Bottom Middle TOP
Tree section
Healthy CBT 0 Parasitised CBT
Figure 2- 5 Parasitism response of Campoplex dubitator to host density per tree, based on the collection and rearing of all host (late instar) Enarmonia formosana from selected trees, between 19 April to 14 June (y = -15.8Ln(x) + 67.89; r2 = 0.57, P < 0.001).
# hosts per tree
Figure 2- 6 Change in the relative abundance of Enarmonia formosana pupae (columns) and the mean head capsule width (points) of field-collected E. formosana larvae (* S E ) over the 2001 and 2002 field seasons.
Date
Figure 2- 7 Frequency distribution of measurements of 2 133 Enarmonia formosana larval head capsule widths collected during the summer of 2002 in central Europe. Columns with different fill patterns show the head capsule size ranges of seven instars. Horizontal lines above columns indicate the head capsule size ranges of the five instars described by Roediger (1956).
9L.1-1L.1
99'1-19.1
99'1-1S.L
9P.1-1b.1
9•’~1-1•’.1
9Z' 1-12'1
91-1-11.1
SO' 1- LO' 1
96'0-16'0
Table 2- 1 List of identified parasitoid species eclosing from pupal and larval Enarmonia formosana hosts collected in central Europe between 2000 and 2002. Regions of discovery and relative abundance are given for each species.
Collection Sites * # Specimens Parasitoid Community Parasitoid Species B BF SRV Collected Composition (%)
ICHNEUMONIDAE
Campoplex dubitator Horstmam
Tycherus vagus Bertoumieu
Pimpla spuria Gravenhorst
Theroscopus hemipteron (iiiche j * *
Isadelphus inimicus (Gravenhorst)
Pimpla contemplator Miiller
Pimpla turionellae Linnaeus
Cyclogastrella simplex Walker
Gelis longicauda (Thomson)
Lissonota sp.
Mastrus sp.
Phygadeuontini
PTEROMALIDAE
Dibrachys afinis Masi
294 100 * B = Baselland (Switzerland); BF = Black Forest (Germany);
SRV = southern Rhine Valley (France and Germany)
* * hyperparasitoid
Table 2- 2 Comparison of overall percent parasitism of Enarmonia formosana by region during 2001 and 2002. Parasitism rates are further divided into larval and pupal host groups.
Percent Parasitism . -. --... . ~
Region Surveyed 2001 2002
Black Forest larvae + pupae 16.6 12.2 (Germany) larvae 12.9 9.7
Pupae 20.0 i 5.4
Baselland larvae + pupae 15.7 11.8 (Switzerland) larvae 14.5 10.9
Pupae 41.7 18.2
Southern Rhine Valley larvae + pupae 16.2 --- (Germany) larvae 16.3 ---
pupae 20.0 ---
Alsace (France)
larvae + pupae 17.4 --- larvae 17.4 --- pupae 0 ---
Missing data = no larvae or pupae collected in that year
Table 2- 3 Association of host tree features with the probability and density of Enarmonia formosana infestations on cherry trees in central Europe (2001-2002).
Table 2- 4 Association of host tree features with the probability and density of parasitised Enarmonia formosana on cherry trees in central Europe (2001-2002).
Mea
n d
ensi
ty o
f
par
asiti
sed
Pro
bab
ility
of
E. f
orm
osa
na
Tre
e A
ttri
bu
te
par
asiti
sm *
n
F
df
P
(wh
en p
rese
nt)
**
n
F
df
P
Veg
etat
ion
1
0.47
14
0 1.
856
2 0.
191
den
sity
ran
k: **
* 2
0.36
17
4
3 0.
53
45
Mo
ss:
pres
ent
0.30
29
5 0.
351
1 0.
563
abse
nt
0.36
64
No
stat
istic
al d
iffer
ence
s ar
e sh
own
in th
is ta
ble.
* "P
roba
bilit
y of
par
asiti
sm"
refe
rs to
the
prop
ortio
n of
infe
sted
tree
s co
ntai
ning
at
leas
t one
par
asiti
sed E. fo
rmos
ona
spec
imen
.
" D
ensi
ty is
cal
cula
ted
as th
e m
ean
num
ber o
f par
asiti
sed
host
s pe
r tre
e, in
clud
ing
only
thos
e tr
ees
with
at
leas
t one
par
asiti
sed
host
'** V
eget
atio
n de
nsity
ran
k "1
" =
little
or
no v
eget
atio
n su
rrou
ndin
g tr
unk;
"2"
= s
ome
vege
tatio
n bu
t pat
chy;
"3"
veg
etat
ion
was
mos
t den
se.
CHAPTER 3 Biology and Rearing of Campoplex dubitator
Abstract
The successful implementation of classical biological control against the cherry bark tortrix
(CBT), Enarmonia formosana Scopoli (Lepidoptera: Tortricidae), in North America requires the
development of an efficient rearing method for the host and its European parasitoid, Campoplex
dubitator Horstmann (Hymenoptera: Ichneumonidae). Because this parasitoid has not been
investigated previously, a detailed study of its reproductive biology is a prerequisite to designing
a rearing system. The CBT larval hosts were reared on a pinto-bean diet prior to, and following,
parasitism by C. dubitator. Parasitoid females were provided with hosts of all instars and readily
attacked each instar on the condition that fresh host frass was present to serve as a tactile stimulus
for oviposition. First instar CBT larvae appeared to be unsuitable hosts because every specimen
died shortly after the parasitism event. While the development time of immature C. dubitator was
suspected to depend on the host stage attacked, no difference was detected between the instars
from which parasitoids successfully emerged. The male-biased sex ratio of C. dubitator did not
differ from that observed in natural communities. Females lived, on average, 26 days at 20' C
with honey and water while males survived only 20 days. This species is synovigenic. Based on
dissections, the number of mature eggs that females held in the lateral oviducts was observed to
increase over the first 48 hours after emergence. The ovariole number varied substantially
between individuals and this was correlated with variation in the number of mature eggs present.
Finally, no correlations were found between female adult body size and host instar at the time of
parasitism.
3.1 Introduction
Classical biological control requires the successful establishment of natural enemies in the
area of release. Proper establishment alone does not guarantee adequate pest suppression, but
without it, pest reduction can be short-lived (unstable) or may not occur at all. Natural enemy
release is one of the final major challenges of any classical biological control programme and
mistakes made during this phase have been blamed for the failure of certain past projects (Stiling,
1993). Some of the factors affecting establishment success rates are climate, host abundance,
alternative food sources, availability of overwintering sites, natural enemy genetic variation, and
the number of natural enemy species released at a given site (van Driesche and Hoddle, 2000).
Unfortunately, the exact impact of such factors can only rarely, if ever, be predicted prior to field
releases.
Beirne (1975 j and Stiiing (i993 j suggest that natural enemy estabiishment for ciassicai
biological control is often dependent, in part, on the number of agents released into a new area.
Low number introductions, for example, may lead to genetic constraints on the population
(Hopper et al, 1993) or reduced mating frequencies (Stiling, 1993). To avoid these problems, it is
suggested that biological control practitioners release higher numbers of individuals and conduct
repeated releases over several years (Beirne, 1975). However, mass-rearing systems to obtain
sufficient parasitoid numbers for establishment may not always be economically or logistically
feasible. Rearing programmes commonly face problems including high cost of required materials
(Brinton et al., 1969), low availability of host specimens (Rojas et al., 1995), long generation
time of the host or parasitoid (Rappaport and Page, 1985), or male-biased sex ratios (Shon and
Shea, 1975). Overcoming such obstacles is an important step in the successfid implementation of
any biological control initiative.
An extensive assessment of potential biological control agents is also important before final
decisions can be made regarding their release. Studies of attack rates and parasitoid behaviour can
provide valuable information, which may be critical for successful rearing and release
programmes. For instance, knowing which host stage is the most vulnerable to parasitoid attack is
elemental in determining the most effective release period. Similarly, understanding the
generation time and intrinsic rate of increase of a parasitoid species may be important for
establishing the release schedule.
The cherry bark tortrix (hereafter referred to as CBT), Enarmonia formosana Scopoli
(Lepidoptera: Tortricidae), is a bark pest on ornamental cherries and other rosaceous trees in
British Columbia, Washington State, and Oregon State. It is naturally suppressed in its native
range of Europe and Eurasia, and parasitoids are believed to play a key role in this. Campoplex cf.
dubitator Horstmann (Hymenoptera: Ichneumonidae) has been found to be a dominant mortality
factor within the central European parasitoid community that attacks the immature stages of the
CBT (Chapter 2). This koinobiotic larval endoparasitoid was reared from field-collected CBT
larvae and was subsequently maintained on the same host species in 2001 and 2002 for further
study of its biology and foraging behaviour. There is currently some confusion regarding the
identification of this species. Dr. Klaus Horstmann (University of Wuerzburg, Germany)
currently identifies it as Campoplex cf, dubitator Horstmann, but plans to closely review
specimens with the same identification reared from other host species.
In this chapter I describe the laboratory rearing of C. dubitator on its univoltine CBT host.
First, there is a discussion of the methods used to rear the host colony (see Chapter 2 for
additional information on CBT biology). Then an explanation is given for the approach used to
maintain the parasitoid culture. I report on features of C. dubitator that are of potential
importance for rearing. These include mating and parasitism behaviour (see also Chapter 5), egg
load, larval development time and adult longevity, host instar suitability, laboratory sex ratio, and
the test of whether adult body size varies as a function of development time and host instar at the
time of parasitism.
3.2 Methods
3.2.1 Host Rearing
To initiate a culture of parasitoid hosts, live specimens of the CBT were collected from the
bark of cherry trees in central Europe and reared at the CAB1 Bioscience Centre in DelCmont,
Switzerland. Upon eclosion, adults were transferred directly into clean 5 cm diameter, tight-
sealing Petri dishes containing a small piece of wet cotton. In most cases, each Petri dish held two
males and two females to increase the chances of at least one pair mating. Mating pairs were kept
at 23 A 2' C under a 16L:8D photoperiod. Most adults mated readily with subsequent oviposition
occurring at least three days following eclosion. Adults were kept in the Petri dish until the last
female had died. Eggs were laid on all surfaces of the Petri dish interior. Once the dead adults
were removed, moth scales and other debris were gently washed from the Petri dish to prevent
contamination of the eggs. Washed Petri dishes were left for several minutes to air-dry. CBT eggs
64
were kept at 23 * 2" C under a 16L:SD photoperiod with a small piece of wet cotton placed into
the Petri dish to prevent desiccation. Shortly before neonate hatching, identified by a darkening of
the sclerotised head capsule now visible inside the egg, a freshly cut piece of cherry bark (4 cm x
4 cm) was placed into the Petri dish. Following emergence, the larvae immediately began feeding
on the bark provided.
CBT larvae were allowed to feed on bark until they reached the second or third instars (using
the instar description given by Roediger, 1956; see Chapter 2), by which point they were more
likely to accept, and survive on, a meridic pinto bean-based diet, modified from Shorey and Hale
(1965). Before the larvae in a Petri dish were large enough to be transferred to diet, the bark
typically had to be changed at least once due to contamination by moulds or desiccation of the
sap tissues. When larvae reached the third or fourth instar they were transferred to individual 5
cm diameter P e t i dishes with meridic diet. Each CRT pupa and parasitoid cocoon was placed
individually into an empty plastic vial (7 cm height x 1.5 cm diameter) until adult emergence. All
hosts (larvae and pupae) were monitored daily for the calculation of developmental rates.
3.2.2 Biology and Rearing of Campoplex dubitator
Adult C. dubitator eclosing from cocoons were placed into plastic cylindrical cages (10 cm
height x 10 cm diameter) with solid honey and water in a cotton wick and kept at 23 & 2" C under
a 16L:SD photoperiod. Females were always kept individually while males, depending on
eclosion dates, were kept either singly or in groups of 2 to 5 wasps. Within three days of
emergence, each female was placed with a single male for 5-10 minutes to achieve mating. If,
after 10 minutes, no courtship behaviour was observed, the female was transferred to another
arena with a new male. To increase the males' ability to locate and follow females, mating trials
were run in small 5 cm Petri dishes. Males used in mating trials ranged in age from one to 21
days, although male wasps under four days of age were rarely responsive to the pesence of
females.
Parasitism of hosts was accomplished by placing a single female wasp into an arena with
hosts. Generally, the parasitoid was presented with only a single host each time. To ensure a
parasitoid would recognise its host, a fresh frass tube, constructed of faecal pellets and silk
produced by host larvae feeding on cherry bark, was placed on or near the larva. Frass tubes built
by meridic diet-feeding hosts were net recognised by C. dubitator. Following an oviposition
event, the host was removed and returned to its labelled Petri dish for the remainder of its (and
the parasitoid's) development. The time between oviposition and adult emergence was recorded
for each parasitoid to determine whether development time varied for wasps in different host
instars. Only those parasitoids that developed fully before the host went into diapause were
included in this comparative analysis.
As with development time, the general body size of female C. dubitator can vary notably
between individuals. In previous studies of correlations between body size and fecundity, body
size has typically been described by the relative width or length of the head, thorax, or hind tibia
(Jervis and Copland, 1996). The right hind tibia of each adult C. dubitator parasitoid was
measured and these lengths were then graphed as a function of host head capsule width. The
relationship between tibia length and development time was also examined to test for an effect of
the number of days between parasitism and adult emergence.
All statistical analyses were run using JMP 5.0.
3.3 Results and Discussion
The longevity of adults kept individually in cages was measured, showing that females lived,
on average, for 26.5 * 3.5 SE days (n = 20) following emergence while males lived only 20.3 * 1.5 SE days (n = 6 1). This difference was statistically significant (t-test: t = 1.986, df = 8 1, P =
0.05).
Most attempts to establish mating pairs under laboratory conditions failed. Fewer than 50%
of males selected gave the appropriate wing-fanning response apparently necessary to initiate
courtship. It is not known if this was due to lack of recognition of the female or whether the males
were physiologically impaired or unprepared. Even when male wasps did respond to the females,
they were often rejected. Rejected males eventually ceased their wing-fanning display after a few
minutes of continued rejection and trials were subsequently terminated. Each pair was separated
after several minutes and returned to their respective cages whether copulation had occurred or
not. Placing more than one male with a single female did not increase the chances of success
since more insects within a small space typically caused the female to become more distressed.
We therefore adopted a more time-consuming approach of transferring females from one mating
arena to the next until either mating occurred or all males had been rejected. All male-female
interactions were observed so that the mating status of each female could be recorded.
When mating was successful, a male mounted a female after a wing-fanning display that
lasted anywhere from 30 seconds to 20 minutes. The mean duration of copulation was 260 i 10.3
SE seconds (n = 33). Males sometimes attempted to force copulation. When a male was
successful in mounting a female, the female tried to dislodge the male with her hind legs. The
mean duration of forced copulations was 83 h 22.3 SE seconds (n = 5). It is not known if C.
dubitator females mate multiple times in the wild. For reasons of efficiency, laboratory wasps
were mated only once in their lifetime. Males, on the other hand, were given several opportunities
to inseminate females. On two separate occasions a single male fertilised two females within 15
minutes. In each case the second copulation lasted approximately 4 times longer than average.
Nonetheless, each of the second inseminated females produced female progeny, indicating that
mating was successful in those cases.
Under laboratory conditions, C. dubitator attacked CBT hosts of all larval instars Females
used their ovipositors to probe frass tubes built by any instar of host, so long as the faecal matter
was relatively fresh. Faeces was considered to be fresh if it had been deposited no more than
approximately 3 days earlier, although older females were typically less likely to reject older,
dryer frass (pers. obs.). The parasitoids took longer to locate younger and smaller larvae with
their ovipositors and in many cases these hosts escaped parasitism altogether. None of the first
instars stung during parasitism trials survived long enough to determine whether parasitism had
been successful. Much of this early mortality resulted from the trauma of the oviposition. In
general, parasitisation of the CBT in the laboratory was very successful, with 88% (n = 389) of all
host attacks resulting in the development of parasitoid offspring. This success was consistent
regardless of whether C. dubitator attacked second, third, fourth, or fifth instar hosts (Table 3-1).
Since a host represents a fixed resource for a developing parasitoid, parasitoid adult body size
or weight is often positively correlated with host size (Godfray, 1994) and this has been shown
for other ichneumonids like Pimpla turionellae, Coccygomimus turionellae, and Venturia
canescens (Arthur and Wylie, 1959; Sandlan, 1979; Harvey et al., 1994, respectively). For C.
dubitator, there was no effect of host instar on female parasitoid size, based on right hind tibia
measurements (Figure 3-1) (ANOVA: F = 0.122, r2 = 0.004, P = 0.730). It was also not possible
to detect any significant differences in parasitoid development rates for different host instars
parasitised (Table 3-2). Overall, parasitoid development from oviposition to adult emergence took
from 14 to 133 days (excluding any specimens that were overwintered prior to emergence).
Parasitoids never emerged from hosts parasitised in the second and third instars in fewer than 27
days. Those developing in hosts parasitised in the ultimate instar were occasionally able to
develop completely in fewer than 16 days, while others took up to 125 days. The reason for this
variance is not known. The failure to detect significant differences may have been due to an
insufficient sample size (j3 > 0.45).
Contrary to these findings, Harvey et al. (1994) showed a strong effect of host instar at
oviposition on development time and adult body size (also measured by hind tibia length) for the
koinobiotic ichneumonid V. canescens. They found the development of this parasitoid to be
delayed in the early host stages, with accelerated growth occurring in the final host instars. In
contrast, other parasitoids are known to develop at a constant rate regardless of the host instar
attacked (Shu-Sheng, 1985; Sequeira and Mackauer, 1992) with the benefit of avoiding
developmental delays but at the expense of having smaller adults emerging from smaller hosts.
The strategy used hy V canporeno is helieved to he adaptive because it ensures a large adult 47e
(Gauld, 1988; Harvey et al., 1994). In order for this reasoning to be complete, variation in body
size must, in turn, be positively correlated with fitness. This has indeed been shown for certain
fitness measurements such as ovariole number, egg load, lifetime fecundity, longevity, and
mating success in several parasitoid and predator species (Jervis and Copland, 1996; Ellers et al.,
1998; Sagarra et al., 200 1). More specifically, Harvey et a1 (1994) were able to demonstrate that
the egg load and longevity of V. canescens adults increases with increased adult body size.
Hence, these parasitoids may maximise their fitness by selecting larger hosts for oviposition.
In several instances, a host was stung at least twice within a minute by the same parasitoid. A
z-test reveals that multiple host probes with the ovipositor occurred more often in fourth and fifth
instar hosts than first to third instars (z = 2.580, df = 5 16, P = 0.010). It is not known why hosts
may be re-attacked so suddenly by the same wasp. Oviposition occurred rapidly (normally < 1 s)
and parasitised larvae were not dissected to determine whether a parasitoid oviposits during the
first penetration (see Chapter 5 for a further discussion on discrimination against self-parasitised
hosts). In 15% of the attacks on hosts, oviposition lasted > 1 s and was characterised by a
noticeable delay before the ovipositor was withdrawn from the host. These longer ovipositions
lasted from 2 to 15 s and were significantly more likely to occur with first, second, and third
instar hosts (ANOVA: F = 15.8 13, df = 4, P < 0.000 1). Table 3-1 gives the percent success and
sex ratios resulting from single or multiple, and rapid or delayed ovipositions. There is no evident
change in the likelihood of success or in the sex ratio associated with either of these behaviours
during parasitism. Thus, no explanation is currently available for either.
6 8
In the field, the fema1e:male sex ratio for C. dubitator is approximately 0.39 : 0.61 (n = 205)
(Chapter 2). In the laboratory, based on parasitism from mated females only, the equivalent ratio
was 0.38 : 0.62 (n = 170). Hence, laboratory rearing does not appear to be restricted by
unnaturally low female abundance. There were no seasonal differences in this sex ratio in the
laboratory. Although the mean head capsule width of host larvae selected for male progeny
(0.904 mm) was slightly larger than that of hosts selected for females (0.804 mm), this difference
was not statistically significant (t-test: t = 1.292, df = 42, P = 0.203). This indicates that foraging
parasitoids probably do not determine offspring sex based on the developmental stage or size of
the host. The sex ratio of F, parasitoids was very consistent when estimated separately for hosts
parasitised in each instar (Table 3-1). Although the sex ratio appears to be 0.50 : 0.50 for second
instar hosts, it must be noted that this was calculated from only six eclosing wasps. Finally, there
:;r~s 29 cl_iffer~nce h~+.veer! the ma!e srm! fema!e dew!opment rates from esrrh host instar
parasitised (Table 3-2).
Campoplex dubitator lies more toward the synovigenic end of the "continuum of ovigeny"
(Jervis et al., 2001). Based on ovary dissections from 28 unmated, naive females, C. dubitator
produces small (length = 0.3 1 mm) hydropic eggs. Each ovary consists of several ovarioles,
ranging in number from 11 to 28 (mean = 18). The ovariole number is rarely identical between
the two ovaries of an individual parasitoid, but is usually very similar (Table 3-3). On the day of
eclosion there is one nearly mature egg per ovariole, but no eggs are found in the lateral oviducts.
At 24 hours after eclosion, a female has, on average, 10.0 * 2.2 SE mature eggs (n = 8) in the
lateral oviducts. Females dissected after three days have approximately 40.0 * 8.6 SE mature
eggs (n = 6) held in the lateral oviducts (Table 3-3). Because the dissected wasps were given only
water, they appear to be capable of maturing eggs without further consumption of a nitrogen food
source. In support of this, females used for parasitising hosts have never been observed to host
feed.
In the field, most C. dubitator larvae developing after mid July diapause in their
overwintering larval hosts (Chapter 2). However, this parasitoid species was also able to complete
development without a diapause phase. Two complete parasitoid generations were reared in the
CAB1 Bioscience laboratory within a single summer, between May and September. As observed
in 200 1 and 2002, , by mid-September the majority of healthy and parasitised CBT larvae showed
signs of reduced feeding and development, suggesting the onset of diapause. The exceptions to
69
this were the early instars, which fed actively until reaching a later instar stage. A short phase (5
weeks in 2001 and 7 weeks in 2002) of cool temperatures (5-6' C) was adequate to overwinter the
colony. Nearly all of the CBT and C. dubitator adults had emerged within two months following
the diapause period in both years.
3.4 Conclusion
A colony of C. dubitator can be maintained in the laboratory. Modifications of the techniques
adopted in this study are required for an increased production of wasps, since current methods are
costly for time and material resources. One of the greatest impediments to rearing C. dubitator
has been low host availability. Collecting large numbers of the CBT from the field is an arduous
task (see Chapter 2). Subsequent rearing of the CBT exclusively on its proper food source (cherry
bark) in the laboratory is possible but costly. While the meridic diet is relatively cheap and can be
used efiicientiy, there is a high mortaiity of iarvae, particuiariy eariy instars, deveioping on it.
This mortality may be due to contamination of the food source or starvation resulting from
rejection of the diet.
The pinto bean-based diet seems suitable for the development of parasitoids and has not
caused any detectable abnormalities in the eclosing adults. For instance, the ability of C.
dubitator to recognise the frass of its hosts appears to be inherent and is not affected by rearing on
diet. Unfortunately, the CBT hosts must consume bark tissues in order for their faeces to serve as
tactile stimuli for parasitoid oviposition. Parasitoids will typically not recognise, much less attack,
hosts burrowed into the diet substrate. This means that preparing hosts for parasitism requires
either the laboratory-production or field-collection of the necessary frass material.
It had been assumed from early observations that the timing of parasitoid emergence was
constrained by the host's development rate. Therefore, parasitoids were given mainly late instar
hosts to accelerate development. However, according to the development times shown in Table 3-
2, host age may not be a significant factor (further testing is required to clarify this). Two
alternative ways in which the development rate of parasitoids might be accelerated in the future
include increasing rearing temperatures above 22' C and providing hosts with a more suitable diet
to stimulate immediate feeding. There is currently no known way to increase the fema1e:male sex
ratio, since host instar or size does not seem to affect the outcome. Nonetheless, a greater
proportion of females would benefit this programme in providing more specimens for further
assessment of this parasitoid as a potential classical biological control agent of the CBT.
70
3.5 References
Arthur, A.P. and H.G. Wylie. 1959. Effects of host size on sex ratio, development time and size of Pimpla turionellae. Entomophaga 4: 297-301
Beirne, B.P. 1975. Biological control attempts by introductions against pest insects in the field in Canada. The Canadian Entomologist 107: 225-236
Brinton, F.E., M.D. Proverbs, and B.E. Carty. 1969. Artificial diet for mass rearing production of the codling moth, Carpocapsapomonella (Lepidoptera: Olethreutidae). The Canadian Entomologist 101: 577-584
Ellers, J., J.J.M. van Alphen, and J.G. Sevenster. 1998. A field study of size-fitness relationships in the parasitoid Asobara tabida. Journal ofAnima1 Ecology 67: 3 18-324
Gauld, I.D. 1998. Evolutionary patterns of host utilization by ichneumonid parasitoid (Hymenoptera: Ichneumonidae and Braconidae). Biological Journal of the Linnean Society 35: 35 1-377
Godfray, H.C.J. 1994. Parasitoids: Behavioural and Evolutionary Ecology. Princeton University Press, Princeton 473 pp.
Harvey, J.A., I.F. Harvey, and D.J. Thompson. 1994. Flexible larval growth allows use of a range of host sizes by a parasitoid wasp. Ecology 75: 1420-1428
Hopper, K.P., R.T. Roush, and W. Powell. 1993. Management of genetics on biological control introductions. Annual Review of Entomology 38: 27-5 1
Jervis, M.A. and M.J.W. Copland. 1996. The life cycle. Pp. 63-160. In: Jervis, M.A. and N. Kidd (Eds.) Insect natural enemies: practical approaches to their study and evaluation. Chapman and Hall, London
Jervis, M.A., G.E. Heimpel, P.N. Ferns, J.A. Harvey, and N.A.C. Kidd. 2001. Life-history strategies in parasitoid wasps: a comparative analysis of 'ovigeny'. Journal of Animal Ecology 70: 442-458
Shu-Sheng, L. 1985. Development, adult size and fecundity of Aphidius sonchi reared in two instars of its aphid host, Hyperomyzus lactuacae. Entomologia Experimentalis et Applicata 37: 41-48
Rappaport, N. and M. Page. 1985. Rearing Glypta fumrferanae (Hym.: Ichneumonidae) on a multivoltine laboratory colony of the western spruce budworm (Choristoneura occidentalis) (Lep.: Tortricidae). Entomophaga 30: 347-352
Rojas, M.G., S.B. Vinson, and H.J. Williams. 1995. Supplemental feeding increases the utilization of a factitious host for rearing Bracon thurberiphagae Muesebeck (Hymenoptera: Braconidae) a parasitoid of Anthonomus grandis Boheman (Coleoptera: Curculionidae). Biological Control 5: 591-597
Roediger, H. 1956. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) Zeitschrift fur Angewandte Entomologie 38: 195-321
7 1
Sagarra, L.A., C. Vincent, and R.K. Stewart. 2001. Body size as an indicator of parasitoid quality in male and female Anagyrus kamali (Hymenoptera: Encyrtidae). Bulletin of Entomological Research 91: 363-367
Sandlan, K.P. 1979. Sex ratio regulation in Coccygomimus turionellae L. (Hymenoptera: Ichneumonidae) and its ecoiogical implications. Ecological Entomology 4: 365-378
Sequeira, R. and M. Mackauer. 1992. Nutritional ecology of an insect host-parasitoid association: the pea aphid-Aphidius ervi system. Ecology 73: 183-1 89
Shon, F.L. and P.J. Shea. 1975. Increased rearing efficiency of two hymenopterous parasites using a non-diapausing host species, Choristoneura occidentalis. Environmental Entomology 5: 277-278
Shorey, H.H. and R.L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58:522-524.
Stiling, P 1991, Why do natural enemies fail in classical biological control programs? American Entomologist spring: 3 1-3 7
van Driesche, R.G. and M S . Hoddle. 2000. Classical arthropod biological control: measuring success, step by step. Pp. 39-75. In: Gurr, G. and S. Wratten (Eds.) Biological Control: Measures of Success. Kluwer Academic Publishers, Dordrecht, Netherlands
Figure 3-1 Relationship between Enarmonia formosana host size at oviposition and hind right tibia length of eclosing adult Campoplex dubitator. (r2 = 0.004; P = 0.730)
0.8 1 1.2
Head capsule width (mm)
Table 3-1 Comparison of the results of parasitism on the five Enarmonia formosana instars and of the effect of single vs multiple and brief vs prolonged ovipositions.
Successful Female : Male Categories Compared N Parasitism (%) Sex Ratio
Host instar attacked I st instar --- --- --- 2nd instar 6 83.3 0.50 : 0.50 3rd instar 45 82.2 0.37 : 0.63 4th instar 246 88.2 0.37 : 0.63 5th instar 92 90.2 0.37 : 0.63
Number of ovipositor insertions into host
1 434 86.4 0.38 : 0.62 2 73 95.0 0.32 : 0.68
>2 11 85.7 ---
Oviposition duration < 1 second 439 86.9 0.39 : 0.61 > I second 79 88.5 0.27 : 0.73
No data are available for the first instar since none of these hosts survived long enough to determine whether parasitism was successful.
Table 3-2 Comparison of female and male parasitoid development time within three age classes of Enarmonia formosana hosts.
Mean Development Host lnstar Attacked Time (days) n S E F df P
I st instar 2nd instar 3rd instar 4th instar 5th instar
Sexes sep2:stecl: 2nd instar
female parasitoids male parasitoids
3rd instar female parasitoids
male parasitoids
4th instar female parasitoids
male parasitoids
5th instar female parasitoids
male parasitoids
No data are available for the first instar since none of these hosts survived long enough to determine whether parasitism was successful.
Table 3-3 Ovariole and mature egg counts from dissections of Campoplex dubitator ovaries.
has i to id Parasitoid Age at Ovariole k m b e r Number of Mature Specimen Dissection (days) Ovary 1 Ovary 2 Eggs in Oviducts
Parasitoid Parasitoid Age at Ovariole Number Number of Mature Specimen Dissection (days) Ovary 1 Ovary 2 Eggs in Oviducts
CHAPTER 4 Response of the parasitoid Campoplex dubitator
to host- and habitat-related odours in an olfactometer
Abstract
The ichneumonid parasitoid Campoplex dubitator attacks the bark-boring larvae of the cheny
bark tortrix (CBT), Enarmonia formosana Scopoli (Lepidoptera: Tortricidae). Because the larvae
feed in complete concealment beneath the bark, parasitoids must use indirect cues to locate and
make preliminary assessments of their hosts. The frass of a CBT larva accumulates at the
entrance of the feeding tunnel and was known to be a strong tactile stimulus for oviposition. In
this study, the frass was shown to release volatiles, which attracted C. dubitator females over
short distances. Similarly, uninfested cherry bark emitted chemical cues that directed parasitoid
movement inside the olfactometer. Bare larvae, however, could not be shown to attract C.
dubitator through volatile emission alone. In a subsequent comparison of the attractiveness of
uninfested cherry bark and host frass (digested bark), the parasitoids moved toward the frass
stimulus significantly more often than the bark.
4.1 Introduction
Insect parasitoids use an array of mechanisms to detect their host targets. Since the fitness of
parasitoids is directly linked with the discovery and parasitism of hosts, there is strong selection
pressure for parasitic wasps to be efficient foragers. A variety of host detection tactics exist
among parasitoids today, many of which are highly specialised for specific parasitoid-host
interactions. These different modes of search may result in a partial reduction of niche overlap,
which could facilitate coexistence of natural enemies foraging for the same host species (van
Dijken and van Alphen, 1998).
Parasitoids may either actively seek herbivore hosts or rely on more passive means to
successfully parasitise their hosts (Godfray, 1994). Passive strategies are less common among
parasitoids. Examples include phoresy, in which parasitoids hitch rides on reproductive adults of
the host species and wait untii those aduits oviposit or return to a nest (Ciausen, i976), and the
ingestion of parasitoid eggs by the herbivore hosts (Hagen, 1964). The more ubiquitous approach
to host finding by parasitoids is an involved search, whereby parasitoids, often relying on
extremely subtle cues, must travel to their hosts (Godfray, 1994). While these can include the use
of visual (Monteith, 1956; Rice, 1968), chemical (Mitchell and Mau, 1971; Harris and Todd,
1980), tactile (Ryan and Rudinsky, 1962; Glas and Vet, 1983), andlor auditory (Cade, 1975)
signals, chemical cues are perhaps the most universal and best understood. Stimuli involved in
parasitoid foraging may arise from the host habitat or food plant, the host itself ("direct"), or from
some interaction between the host and its food source ("indirect") (Mackauer et al., 1996). It is
argued that interactions between the first and second trophic levels, or between the second and
third trophic levels, cannot be understood fully without the inclusion of all three levels
simultaneously (Price et al., 1980; Vet and Dicke, 1992). This is reflected in the common usage
of, and dependency on, cues from both plants and hosts by foraging parasitoids.
The foraging process for parasitoids is generally divided into three (Hassell and Southwood,
1978; Waage, 1979) or four or five (Vinson, 1976) phases. The parasitoid first locates the host's
habitat and then searches within it to find the host itself. Following some sort of assessment, the
host is either accepted for oviposition or rejected. Additional steps sometimes added to the
process that results in successful oviposition include host suitability and host regulation (Vinson,
1976). These latter two phases, however, are not a part of the initial host selection process.
In some systems, this hierarchical search may be quite simple, while in others it might
incorporate the use of a number of different forms of stimuli (Fischer et al., 2001). When
herbivores exist in a structural refuge, parasitoids may be required to adapt by developing new
sensory "equipment" to deal with finding their hosts. The physical concealment of hosts within
plant tissues will likely affect the relative usefulness of various stimuli, compared to more
exposed hosts (Vet et al., 1991). For instance, if relying on vision, parasitoids would be forced to
use less reliable indirect cues from the host (perhaps signs of feeding) to locate their quarry.
The system described in this paper exemplifies a relationship in which the parasitoid depends
upon indirect cues in finding its bark-boring hosts. The larvae of the cherry bark tortrix (hereafter
referred to as CBT), Enarmonia formosana Scopoli, (Lepidoptera: Tortricidae), live and feed
between the cork and cambium of the bark of a variety of rosaceous plants, most notably cherry
trees (Roediger; 1956). These larvae remain within their mined tunnels until they emerge as
adults. Throughout their development, they deposit their faecal matter at the entrance of their
feeding galleries, forming a silk-lined, tube-like structure. While this faecal accumulation, or frass
tube, was known to serve as a tactile stimulus for oviposition behaviour by Campoplex cf.
dubitator Horstmann (Hymenoptera: Ichneumonidae), it was not clear by which mechanisms this
parasitoid was able to locate CBT larvae from distances greater than a few centimetres.
This paper describes an examination of materials associated with the herbivore and its
habitat, which are potentially important to C. dubitator in its search for hosts. The research
objectives were to: (1) determine whether semiochemicals play a role in host location by this
larval parasitoid (see Nordlund, 1981 for a review of infochemical use by parasitoids), (2)
identify the source(s) of such volatile cues, and (3) compare the attractiveness of kairomones
and/or synomones intercepted by C. dubitator.
4.2 Materials and Methods
4.2.1 Study Organisms
CBT specimens were obtained from field collections in central Europe throughout the
summer seasons (May - September) during which experiments were run. The larvae were reared
on a meridic pinto bean-based diet, modified from Shorey and Hale (1965), at the CAB1
Bioscience Centre in Delkmont, Switzerland. Larvae and pupae were kept solitary in labelled
plastic vials and monitored for parasitoid emergence. During 2001 all immature CBT were kept at
20" C under a 16L:8D photoperiod. In 2002 the larvae and pupae were reared at slightly warmer
temperatures (23 * 2" C) to increase the developmental rate.
All C. dubitator parasitoid adults eclosing from CBT hosts were placed into plastic
cylindrical cages (I 0 cm height x 10 cm diameter) with solid, unpasturised honey and water in
cotton wicks on the day of eclosion. Females were kept individually whereas males, depending on
eclosion dates, were kept either singly or in groups of two to five wasps. Within three days of
eclosion, females were placed with males for short periods to achieve mating. Following the
monitored mating trials, C. dubitator females were given host larvae for oviposition experience.
As with the mating trials, all oviposition events were observed and the oviposition history was
recorded. All females were provided with hosts for oviposition one day prior to the olfactometer
tests. This egg-laying opportunity was provided because experienced females are generally more
responsive to host-related c ~ s .
Only females reared from CBT hosts parasitised in the field were used in the static chamber
olfactometer assay in 2001. In contrast, the Y-tube olfactometer experiments conducted in 2002
used C. dubitator parasitoids reared from both field-parasitised and lab-parasitised host larvae.
4.2.2 Experiments 1-3: Parasitoid Response to Volatile Cues
4.2.2.1 Materials Tested
Three materials were tested as possible sources of volatiles used by parasitoids for orientation
during foraging. These include uninfested cherry bark, host larval frass, and host larvae (Table 4-
1). Cherry bark was selected for bioassay as it has been shown that some parasitoids will orient
and move toward the food sources of their hosts (Vinson, 1981). The frass of the CBT was known
to be a strong tactile stimulus, eliciting probing behaviour in C. dubitator (pers. obs.), however, it
was not clear if this faecal material produced volatiles that could be detected at greater distances
by the wasps. Finally, the CBT larvae themselves were tested to determine whether the foraging
process involved the response of C. dubitator to kairomones from its host.
4.2.2.2 Experiment I
Small sections (2 * 0.2 g) of cherty bark were collected from trees (Prunus avium) just prior
to olfactometer assays. The fragment of bark used in each experiment was composed of
approximately 50% outer rough tissue and 50% phloem tissue. While the bark selected was free
of insect feeding damage, it was inevitably physically damaged during removal from the tree.
84
Slices of cork (not a host of the CBT), washed with acetone, rinsed with distilled water, and
dried, were used as a control.
4.2.2.3 Experiment 2
Fresh host frass was collected directly from infested trees prior to experiments. Frass
consisted of larval faecal pellets, silk, and small quantities of chewed but undigested bark. Old-
appearing, and likely evacuated, frass tubes were avoided. A mass of 0.20 * 0.05 g of frass was
used in each replicate. This consisted of the fecal pellets (digested bark tissues) and mandibular
silk of several host larvae of variable instars. Frass tubes from late instar hosts, however, made up
more than 90% of the total mass of the fecal matter collected. As a control, fine-grained pebbles,
similar in size to the fecal pellets, were used. These pebbles were also washed with acetone,
rinsed with distilled water, and dried prior to running the bioassay.
4.2.2.4 Experiment 3
Host larvae were prepared for the experiment immediately before the trials began. In each
replicate, three healthy (not parasitised) larvae were used as test subjects, each larva representing
one of three instar stages (third, fourth, and fifth instars). Approximately two hours before the
bioassays, the larvae were removed from their feeding galleries and placed into large clean Petri
dishes (2 cm height x 10 cm diameter). Hosts were separated from their food source in this way to
avoid having parasitoid responsiveness (or repulsion) confounded by possible plant-related
volatiles carried by the larvae. During this non-feeding pre-trial period, the gut of each larva was
also emptied through forced defecation, which was achieved by pressing each larva between a
small section of soft plastic film and dental cotton and gently pressing the abdomen with a fine
paintbrush. This effectively prevented the larvae from excreting faeces during the experiment.
Small sections of clean filter paper, rolled and secured with copper wire, were used as the inert
control material. These filter paper rolls were prepared to match the sizes of the larvae selected
for the experiment.
4.2.2.5 Static Chamber Olfactometer Bioassay
Potential kairomone sources were tested for attractiveness to C. dubitator in a sealed
olfactometer box design without the flow of air through the system. The olfactometer consisted of
a plastic box (25 cm height x 24 cm length x 12 cm width) with two holes cut into the ceiling at
opposite ends through which test materials were inserted. A single hole was cut into the side to
serve as the entrance for parasitoids (see Figure 4-1). This design was selected to permit flight by
8 5
the parasitoids (as opposed to a Y-tube design), as it was suspected that flight was a typical, and
perhaps essential, behaviour in the mid-range search phase of foraging. Each olfactometer was
washed with Sparkleen (Fisherbrand, Pittsburgh), rinsed with distilled water, and dried bemeen
uses. In every trial, the treatment odour source was randomly assigned to either end of the test
arena, while the control material was placed at the opposite end. The treatment and control
substrates were hung from the ceiling of the static chamber by a length of thread attached to foam
plugs, which were inserted into the holes. To reduce the effect of visual or tactile cues on
parasitoid responsiveness, the test materials were placed into double-layer, white gauze bags (6
cm x 4 cm). These gauze sacs were sufficiently porous to allow the emission of odours, yet
effectively concealed the materials from view and prevented the parasitoids from contacting the
treatment and control substrates. External visual cues were standardised by enclosing the
olfactometer on three sides with white screening. Diffuse light was provided by a single 60 W
incandescent light bu!b (3 10 LUX), p ! a d 30 err, behind the h!!y tr~csparect test srena.
Trials were conducted from 11 July to 26 September in 2001 and from 9 May to 12 June in
2002, always between 1l:OO and 19:00, at a temperature of 22 h 2" C with ambient relative
humidity. Two minutes before each trial began, the treatment and control materials were inserted
into the box through the assigned holes at each end of the chamber. To start the trial, a single
female parasitoid was introduced to the arena through the entrance hole at the centre line of the
test area. For seven minutes (420 s), the behaviour and location (with respect to the centre line) of
the parasitoid were logged using event-recording software (Observer), with 0.1 s time resolution.
Of primary interest was the total time the parasitoid spent on the treatment and control halves of
the olfactometer.
Every parasitoid female (n = 20 in 200 1 ; n = 7 in 2002 ') was tested on four occasions, with
an average of 12 days between sessions, following eclosion. In each session of experiments, a
female was tested with each of the three suspected kairomone
sources. All females were ultimately used in 12 trials (3 trials per day, 4 days). The order
of testing of the three materials was randomised for each female in each session so
there would be no confounding effect of treatment order (ie. learning or experience).
By preventing the females from initiating oviposition behaviour (ie. blocking tactile cues),
experience effects were further minimised. In this way, the parasitoids did not experience either
good hosts or bad hosts, or hosts present versus hosts absent in association with the odours
presented to them.
The number of parasitoids responding to volatile stimuli was analysed with the X2 goodness-
of-fit test (a = 0.05) to determine whether the observed frequency deviated significantly from
expected frequencies, under the null hypothesis that C. dubitator did not show any preference for
either treatment or control odours. Each parasitoid was classified as having chosen the treatment
or control material based on the amount of time the wasp spent with each substrate. This time
value was a pooled total from each of the four experimental sessions (4 x 420 s = 1680 s per
tested odour source).
To check for any mating effect on response to semiochemicals, a z-test (Sigmastat 2.03) was
employed to test for differences in the time spent or! the treatmefit and co~tro! !m!ves ~f the arena
by mated and unmated female wasps. This analysis was run separately for the three substrates.
4.2.3 Experiment 4: Parasitoid Preference for Volatile Cues
4.2.3.1 Materials Tested
Based on the results of the preceding three experiments (see Results section), uninfested
cherry bark and host frass were used for further testing of parasitoid response to olfactory stimuli.
In this scenario, however, the two substrates were tested against one another to determine whether
C. dubitator is more attracted to the odours of either bark or frass.
The bark treatment was prepared by cutting 2.5 g of cherry bark from trees (Prunus avium)
immediately before the trials were run. This bark, as before, consisted of approximately 50%
coarse outer bark and 50% phloem. The bark was subsequently cut
into tiny fragments (5 x 3 mm) to increase the surface area of the material. Host faecal matter for
the frass treatment was gathered from sites of natural infestation on cherry trees (Prunus avium)
prior to the bioassay trials. A mass of 1.25 g of fresh frass was mixed with 1.25 g of cherry bark
(taken from the same sample used in preparing the bark treatment), also cut into small fragments.
Hence, a comparison was made between 2.5 g of uninfested cherry bark and a 2.5 g mixture of
uninfested cherry bark and host frass (digested cherry bark).
- - - -- --
I The seven mated females fiom 2002 were used only in trials testing the parasitoids' response to cherry
8 7
4.2.3.2 Y-tube Olfactometer Bioassay
A vertical glass Y-tube olfactometer (stem: 24 cm; side arms: 20 cm; diameter: 2.5 cm) was
used in this study. All replicates were conducted between 10:30 and 18:30 (1 1 June to 20
September, 2002) at 22 l o C with ambient relative humidity. The olfactometer was centered
beneath the only light source in the room: 3 tubes providing fluorescent "Cool White"
illumination (Osram L 36W/20,2200 LUX). Visual cues were standardised by having identical
structures associated with the treatment materials to the left and right of the apparatus and a white
wall opposite the observer. Air was pulled through the olfactometer at a rate of 2 Llmin (1 Llmin
for each arm) by a KNF Neuberger Miniport electric pump, connected to the base of the Y-tube.
For each arm, the air passed first through an acrylic air flow meter (Key Instruments) to gauge the
rate of air movement. It then traveled through a 50 mL Erlenmeyer flask housing the treatment
material to pick up volatiles, and finally entered the arm via a 30 cm section of 0.7 cm
polyethylene tubing.
The set-up was allowed to run for 2 minutes before a single, oviposition experienced, female
was introduced into the base of the stem of the Y-tube. Each parasitoid was given up to 10
minutes to adjust to the new environment and wind speed. The trial began once the parasitoid
crossed a start line drawn 10 cm from the base of the Y-tube stem. If the parasitoid did not climb
the stem far enough to cross this line within 10 minutes, the replicate was terminated. Once past
the start line, the parasitoid was given 10 additional minutes to reach the finish line, a line drawn
5 cm from the top of each arm. The experiment was terminated either when the parasitoid made a
choice by reaching the end of one arm or when the 10 minute time limit expired (no choice). All
non-responding parasitoids were excluded from statistical analyses.
A chi-square goodness-of-fit test (a = 0.05) was used on the response data to determine
whether the parasitoid females were discriminating between the odours provided for them. The
null hypothesis states that the frequency of parasitoids moving toward cherry bark will equal that
of parasitoids moving toward the frass-bark mixture.
A clean Y-tube (Sparkleen-washed and rinsed with distilled water) was employed for each
replicate and the test stimuli were randomly assigned to the side arms. Fifty-three females were
tested for a preference of either bark or frass odours and each female was used only once. Eleven
bark odours. No further testing of the fiass or host larvae treatments was conducted in 2002.
88
additional C. dubitator females were used in a control experiment (clean air flowing through both
arms) to test if the parasitoids were simply displaying either a negative geotactic or positive
phototactic response by moving upward in the apparatus. The proportion of parasitoids making a
choice in the control experiment was compared with that of parasitoids making a choice in the
treatment experiment, using a z-test (Sigmastat 2.03). 'bNon-choosers" were those individuals
remaining at the bottom of the Y-tube apparatus, while "Choosers" were those successfully
crossing one of the end lines in either arm of the olfactometer.
4.3 Results
4.3.1 Experiments 1-3: Parasitoid Response to Volatile Cues
The static chamber olfactometer design proved to be effective in testing the attraction or
arrestment response of C. dubitator to three odour sources (bark, frass, and larvae) related to its
hosts. The parasitoids often demonstrated ciear signs of orientation towards certain treatment
substrates, both while walking and during flight. This directional movement was observed at the
start and near the finish of trials, suggesting that a concentration gradient existed throughout the
course of the 420 s experiment.
Figure 4-2 displays the results from experiments 1-3. Foraging parasitoids displayed a strong
response to the bark treatment, with 8 1% of individuals spending more time on the treatment side
of the arena (x2 = 6.025; n = 27; df = 1; P = 0.014). From the trials using host frass material, 70%
of the test subjects chose the treatment over control, however, with a = 0.05, this was insufficient
to obtain a statistically significant difference (x2 = 1.667; n = 20; df = 1; P = 0.197). These results
must be interpreted with caution, due to small sample size. In experiment 3, the host larvae
treatment apparently did not elicit a directed response from the parasitoid females (X2 = 0.404; n =
20; df = 1; P = 0.525). Again, there is low power in this analysis due to a small sample group.
However, during trials with bare host larvae as the treatment materials, C. dubitator demonstrated
a much stronger phototaxic response, suggesting an attempt to disperse rather than search the
immediate environment. This behaviour was characterised by less time spent walking around the
entire chamber and more time spent on the back wall, which faced the sole light source.
Of the 20 parasitoids tested in 2001, only 9 had been successfully mated. Table 4-2 depicts
the output from the z-test comparing the responses of mated and unmated females. These results
show no difference in the responses to the tested odour sources, however caution must be taken
with the interpretation of this output, since the power of these tests was low.
89
4.3.2 Experiment 4: Parasitoid Preference for Volatile Cues
Experiment 4, a direct comparison of the attractiveness of uninfested cheny bark and CBT
host frass, provides compelling evidence for the attraction of C. dubitator to the frass of its hosts.
The results of this Y-tube bioassay (Figure 4-3) show a strong tendency for parasitoids to orient
towards odours associated with digested cherry bark and mandibular secretions (X2 = 5.580; n =
46; df = 1; P = 0.018). Seven of the 53 tested females were excluded from the analysis because
they did not make a choice before the 10-minute period expired. In the control experiment, only
36% of the tested parasitoids (compared to 87% in the treatment experiment) climbed upward
into the stem of the Y-tube (z = 3.705; P < 0.001). This low activity in the absence of any host-
related odours indicates that there was not a strong positive phototactic or negative geotactic
response in this experimental design. Movement in the olfactometer was, therefore, in response to
the cdours n r e c ~ n t ~ r l F a -V-A..--.
The parasitoids used were collected from CBT hosts parasitised under natural field conditions
and hosts parasitised in the laboratory. To ensure that the responsiveness to odours presented did
not differ between wasps of the two origins, the pooled response of parasitoids arising from field-
parasitised hosts (n = 18) was compared to that of the parasitoids reared from laboratory-
parasitised hosts (n = 28) using a z-test (Sigmastat 2.03). This analysis indicates no difference in
the choices (cherry bark versus host frass) made by the two sets of parasitoids (z = 1.209; P =
0.227).
4.4 Discussion
Volatile stimuli have been found to be an important part of the foraging process for a vast
number of predatory organisms. Parasitoids, certainly, are no exception. The use of odours by
insects to find food has been documented for decades and there is a wealth of such literature for
parasitoids. Reports of parasitoid attraction to volatile compounds produced by host plants, host
frass, and host individuals have appeared frequently over the last several years (Nordlund, 198 1;
Vet and Dicke, 1992; Godfray, 1994; Mackauer et al., 1996). Of the three materials tested on C.
dubitator in experiments 1-3, uninfested cherry bark and host frass were found to release alluring
scents while the host larvae apparently did not.
The results from this study indicate that females of C. dubitator can recognise and orient
toward certain materials associated with their host larvae, based on olfaction alone. These
90
chemical stimuli may play a vital role in the long- and short-range detection of hosts. The
conclusions described from this study do not rule out the possible use of other forms of stimuli,
such as visual, tactile, or auditory cues. They simply illustrate the olfactory capabilities of C.
dubitator. However, since the larvae and pupae of the CBT are so well concealed beneath the
bark of their host trees, it is conceivable that vision, for example, might not be as reliable a
mechanism as olfaction for host detection.
In much the same way that adult CBT perceive volatiles from their host trees (McNair et al.,
2000), C. dubitator parasitoids appear to be able to respond to the bark of cherry trees regardless
of the presence of host larvae. The attraction of parasitoids to the microhabitat or host plant in the
absence of prey is not uncommon and was first demonstrated over 60 years ago (Godfray, 1994).
It might be argued that such a stimulus could be disastrously misleading, for instance, in a
sitt~atinr! where mnst cherry trees were free nf CET ir.fest&inn. If 2 f ~ r ~ g e r C O E ~ ~ R U O ~ ~ S ! ~ ~ J *PS~OT,C!S & - r
to deceptive cues, it will inevitably lose search time and suffer a reduced fitness. Selection should
favour responsiveness to a cue only if that stimulus is consistently reliable. In other words,
responding to stimuli from the host's habitat could evolve in this CBT system if the probability of
finding hosts on an encountered tree is sufficiently high. Indeed, from a survey of mature cherry
trees in France, Germany, and Switzerland between 2001-2002, 72% of the trees investigated (n =
768) contained at least one immature CBT. While these infestations are normally not severe (low
host densities) in central Europe, having a directional response to the microhabitat of the host
might be an effective foraging strategy for these parasitoids.
The results from experiment 2, which failed to show an attraction of females to host frass,
were interpreted with skepticism for reasons discussed here. The chi-square calculations were
based on binomial data relating to choices of treatment versus control. If, however, one looks at
the actual time spent by parasitoids on the treatment side of the arena (on average, 60% with
treatment, 40% with control), the tendency for C. dubitator to move toward frass is more
apparent. In fact, the wasps spent the same proportion of time with the frass substrate as they did
with the bark treatment. Nonetheless, due to a smaller sample size in the frass experiment, no
difference was detected. A similar argument might be raised for the observed lack of response to
host larvae in experiment 3, however, in these trials, the parasitoids' movements were more
obviously phototactic. In contrast, in response to host frass and cherry bark, the wasps often
displayed unquesticnable signs of searching, including obvious antenna1 activity and walking in
tight circles when directly beneath the treatment bags. Hence, faecal material was used again in
experiment 4.
Theoretically, a parasitoid will be more efficient if it can quickly evaluate the quality of a
host plant as a foraging site upon arrival. The foraging pattern of parasitoids is generally
described as a step-by-step procedure in which the forager closes in on its host, using different
stimuli at each stage of the hunt (Hassell and Southwood, 1978; Waage, 1979). In this widely
accepted conceptualisation of the foraging process, parasitoids are thought to first find the
herbivore habitat, then to find the herbivore patch, and finally to locate the herbivore hosts within
that terminal patch. For C. dubitator, volatile cues emanating from the host faeces (digested
cherry bark) may be an example of a cue used by parasitoids to decide whether to remain at a
newly discovered tree or to move on to the next. In their description of chemical information in
tritmphic interactions, Vet and Dicke (1992) suggest that natnra! enemies h ~ ! d respond most
strongly to volatile cues that reliably indicate herbivore presence. In general, stimuli derived from
the herbivore itself are the most reliable information sources, while volatiles produced by the first
trophic level (food plant) are less likely to convey accurate information. Experiment 4 supports
this generalisation since parasitoids showed a stronger response to the host frass and bark mixture
than to bark alone. It must be stated, however, that the concentrations of volatiles emanating from
cherry bark and host frass in experiment 4 were not necessarily equal. Despite the use of equal
masses of the two substrates and fragmentation of the cherry bark, the frass may still have had a
greater surface area, thereby releasing higher concentrations of semiochemicals.
It has yet to be resolved whether C. dubitator actually relies on cherry tree odours when
foraging. If this is true, it may be that frass volatiles are not detectable beyond a certain distance
from the tree due to the miniscule size of the faecal deposits. The use of stimuli derived from the
second trophic level is commonly limited by low detectability, particularly at long distances.
Plants, on the other hand, which have a relatively larger biomass, usually release volatiles at more
easily detectable levels (Vet and Dicke, 1992). Hence, the ability to track odour plumes from the
microhabitat (cherry bark) may be useful for movement between patches of host plants,
particularly when the distance to travel is great and when any kairomones produced by the host
larvae occur in relatively low concentrations.
The fact that the parasitoids did not respond to host larvae in experiment 3 suggests that these
ichneumonids depend wholly on indirect cues to locate their hosts. This finding is supported by
the observation that C. dubitator wasps do not have an inherent ability to recognise their hosts
when they come into contact with the larvae. This is expected, however, considering the nature of
this host-parasitoid relationship. Only under rare circumstances are the CBT larvae actually
outside of their feeding tunnel. Under normal conditions, C. dubitator would never see or
antennate its hosts. The only contact between the parasitoid and host is with the very tip of the
parasitoid's ovipositor, which it uses to probe the host's feeding cavity. From an evolutionary
standpoint, there would be little or no selection for the ability to recognise exposed hosts.
Although no cue is more reliable than the host itself (Vet et al. 1991), the presence of frass is
likely a dependable indicator of hosts since the accumulation of faecal matter results directly from
larval feeding. Based on properties of the accumulation of frass, a parasitoid could potentially
acquire information on the availability, identity, density, and suitability of the host. Recently
deposited frass has been observed to be a stronger stimulus for oviposition behaviour than old
frass (pers. obs.). This implies that C. dubitator can also gauge the relative reliability of different
faecal tubes. For parasitoids of the CBT, this phenomenon must be a critical element for search
efficiency. In the field, there are often as many abandoned as there are occupied feeding galleries.
One key difference between old and new larval tunnels is that the frass tubes of the new larval
sites are less desiccated and likely releases more volatiles. More work must be done to determine
the limits of C. dubitator's ability to discriminate between new and old frass, or in other words,
occupied versus empty feeding tunnels. The lack of, or weakened, response of C. dubitator to
host larvae may be common among natural enemies of concealed hosts. The larval parasitoid
Olesicampe monticola Hedwig (Hymenoptera: Ichneumonidae), which attacks hidden sawfly
larvae, is known to show a stronger response to its hosts' faeces than to the hosts themselves.
Like C. dubitator, this wasp also exhibits an ability to distinguish between fresh and aged frass
(Longhurst and Baker, 198 1).
Host- and habitat-related odours likely play a critical role in C. dubitator's detection of hosts,
as they do for so many other parasitic wasps (Vinson, 198 1 ; Vet and Dicke, 1992; Godfray,
1994). Since C. dubitator is a potential candidate for classical biological control against the CBT
in North America, the identification of these foraging cues has at least four potential applications.
First, these known cues can be incorporated into experimental designs for studying host finding
and oviposition to ensure that parasitoids encounter natural stimuli and thus behave as they would
under field conditions. Second, recognising search cues may help in explaining patterns of
distribution of parasitism within a tree or an orchard. For instance, parasitoid activity might be
greatest on trees with severe structural damage, since cracked areas of the bark likely release
overwhelming concentrations volatiles. Third, information on foraging stimuli may be valuable
for the host range testing of this potential biological control agent. Ideally, one would test every
non-target arthropod species that would co-exist with such an agent in its new range. However,
the maintenance of a large culture of each non-target species would make this technically
impossible (Kuhlmann et al., 1998). Thus, it is necessary to select a sub-sample of representative
species. There are essentially two approaches to this selection process. The centrifugal
(phylogentic) approach, originally developed for weed biological control (Wapshere, 1974),
involves testing primarily closely related species, under the assumption that a particular agent has
evolved in a relatively strict association with the target host and is less prone to attack species that
may be unsuitable as food sources or for progeny development. This method has been deemed
equally appropriate for weed and arthropod biological control programmes (Sands, 1997; but see
Kuhlmann et al., 1998). The optional approach emphasises the importance of spatial overlap
hn+rlraan thn nvnt;r, n ;tn;rl o n r l nnn_tornnt cm~c;nc oqrl ;cr Q on onno* UuCvvuCIIL + ~ : ~ ~ I L v . u uIIu Cu16UL .,yVVIU., U1 ,., LL!aV wwua;dered tc be a suitabk
methodology for entomophagous biological control (Kuhlmann and Mason, in press). It
presupposes that species sharing the same niche or habitat as the target species are likely to be
encountered, even if accidentally, by the biological control agent (van Driesche and Hoddle,
1997). These non-target species should therefore be included in host range testing. If the latter
approach, or some combination of the two, is applied to the investigation of C. dubitator's host
range, knowing precisely which habitat and host features serve as foraging cues may assist in the
selection of non-target species for testing. For instance, as it is known that CBT frass is the
contact stimulus for oviposition behaviour, it may be of interest to select primarily non-target
species that similarly accumulate faecal matter at their feeding sites. Finally, this understanding
of parasitoid foraging may eventually enable practitioners of biological control to manipulate the
search efficiency of these parasitoids (Vet and Dicke, 1992). Modifying how these natural
enemies perceive their environment could lead to a desired level of pest control that might not
otherwise be attainable.
4.5 References
Cade, W. 1975. Acoustically orienting parasitoids: fly phonotaxis to cricket song. Science 190: 13 12-13 13
Clausen, C.P. 1976. Phoresy among entomophagous insects. Annual Review of Entomology 21: 343-368
Fischer, S., J. Samietz, F.L. Wackers, and S. Dorn. 2001. Interaction of vibrational and visual cues in parasitoid host location. Journal of Comparative Physiology: (A) Sensory Neural and Behavioral Physiology 187: 785-79 1
Glas, P.C.G. and L.E.M. Vet. 1983. Host-habitat location and host location by Diachasma aIloeumMuesebeck (Hym.: Braconidae), a parasitoid of Rhagoletispomonella Walsh (Dipt.: Tephritidae). Netherlands Journal of Zoology 33: 41-54
Godfray, H.C.J. 1994. Parasitoids: Behavioural and Evolutionary Ecology. Princeton University Press, Princeton 473 pp.
Hagen, K.S. 1964. Developmental stages of parasites. In: P. DeBach (Ed.) Biological Control of Insect Pests and Weeds. Reinhold, New York, Pp. 168-246
Harris, V.E. and J.W. Todd. 1980. Male-mediated aggregation of male, female, and instar tar southern green stink bugs and concomitant attraction of a tachinid parasite, Trichopodo pennipes. Entomologia Experimentalis et Applicata 27: 1 17- 129
Hassell, M.P. and T.R.E. Southwood. 1978. Foraging strategies of insects. Annual Review of Ecology and Systematics 9: 75-98
Kuhlmann, U. and P.G. Mason. In press. Use of field host range surveys for selecting candidate non-target species for physiological host specificity testing of entomophagous biological control agents. Proceedings of the First International Symposium on Biological Control of Arthropods USDA Forest Service, Honolulu, Hawaii
Kuhlmann, U., P.G. Mason, and D. Greathead. 1998. Assessment of potential risks for introducing European Peristenus species as biological control agents of Lygus species in North America: a co-operative approach. Biocontrol News and Information 19: 83N-90N
Longhurst, C. and R. Baker. 1981. Host location in Olesicampe monticola, a parasite of larvae of larch sawfly, Cephalcia Iariciphila. Journal of Chemical Ecology 7: 203-208
Mackauer, M., J.P. Michaud, and W. Volkl. 1996. Host choice by aphidiid parasitoids (Hymenoptera: Aphidiidae): host recognition, host quality, and host value. The Canadian Entomologist 128: 959-980.
McNair, C., G. Gries, and R. Gries. 2000. Cherry bark tortrix, Enarmonia formosana: olfactory recognition of and behavioural deterrence by nonhost angio- and gymnosperm volatiles. Journal of Chemical Ecology 26: 809-821
Mitchell, W.C. and R.F.L. Mau. 1971. Response of the female southern green stinkbug and its parasite, Trichopodapennipes, to male stink bug pheromones. Journal of Economic Entomology 64: 856-859
Monteith, L.G. 1956. Influence of host movement on selection of hosts by Drino bohemica Mesn. as determined in an olfactometer. Canadian Entomologist 88: 583-586
Nordlund, D.A. (Ed.) 198 1. Semiochernicals: their role in pest control. John Wiley and Sons, Inc., New York. 306 pp.
Rice, R.E. 1968. Observations on host selection by Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae). Contribution of the Boyce Thompson Institute 24: 53-56
Roediger, H. 1956. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) ZeitschrifttJitr Angewandte Entomologie 38: 195-32 1
Roitberg, B.D., J. Sircom, C.A. Roitberg, J.H.M. van Alphen, and M. Mangel. 1993. Life expectancy and reproduction. Nature 364: 108
Ryan, R.B. and J.A. Rudinsky. 1962. Biology and habits of the Douglas fir beetle parasite, Coeloides brunneri Viereck (Hymenoptera: Braconidae) in Western Oregon. Canadian Entomologist 94: 748-763
Sands, D.P.A. 1997. The "safety" of biological control agents: assessing their impact on beneficial and other non-target hosts. Memoirs of the Museum of Victoria 56: 6 1 1-6 15.
Shorey, H.H. and R.L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58: 522-524.
Tanigoshi, L.K., B.B. Bai, and T.A. Murray. 1998. Biology and control of the exotic cherry bark tortrix, Enarmonia formosana. Oregon Department of Agriculture Interim Project Report, 1998
van Dijken, M.J. and J.J.M. van Alphen. 1998. The ecological significance of differences in host detection behaviour in coexisting parasitoid species. Ecological Entomology 23: 265-270
van Driesche, R.G. and M. Hoddle. 1997. Should arthropod parasitoids and predators be subject to host range testing when used as biological control agents? Agriculture and Human Values 14: 21 1-226
Vet, L.E.M. and M. Dicke. 1992. Ecology of infochemical use by natural enemies in a tritrophic context. Annual Review of Entomology 37: 14 1- 172
Vet, L.E.M., F.L. Wackers, and M. Dicke. 1991. How to hunt for hiding hosts: the reliability- detectability problem in foraging parasitoids. Netherlands Journal of Zoology 41: 202-2 13
Vinson, S.B. 1981. Habitat location. Pg. 5 1-77. In: Nordlund, D.A. (Ed.), Semiochemicals: their role in pest control. John Wiley and Sons, Inc., New York.
Vinson, S.B. 1976. Host selection by insect parasitoids. Annual Review of Entomology 21:109- 133
Waage, J.K. 1979. Foraging for patchily-distributed hosts by the parasitoid, Nemeritis canescens. Journal of Animal Ecology 48: 3 53-37 1
Wapshere, A.J. 1974. A strategy for evaluating the safety of organisms for biological weed control. Annals of Applied Biology 77: 20 1-2 1 1
Figure 4-1 Illustration of the static chamber olfactometer used in identifying sources of attractive odours in experiment 1. Gauze bags were suspended >5 cm away from all walls of the arena to reduce the chance of accidental discovery of treatment and control substrates by the parasitoid.
Suspended gauze bags holding treatment/control materials
(randomly assigned)
Entrance hole for parasitoid
Mid-point dividing line
Figure 4-2 Response of Campoplex dubitator females to odours presented in a static chamber olfactometer. These figures show the response of parasitoids to cherry bark (2 * 0.2 g) (experiment 1: ~2 = 6.025; P = 0.014 ), host frass (0.20 h 0.05 g) (experiment 2: ~2 = 1.667; P = 0.197), and three naked Enarmonia formosana host larvae representing the third, fourth, and fifth instars (experiment 3: ~2 = 0.404; P = 0.525). A choice was determined based on a comparison of the total time spent on the treatment and control halves of the arena.
25
U) 20
B 0 .+1 15 U) m L x 10 y. 0
# 5
0 Bark Treatment Control
Odour source
Frass Treatment Control
Odour source
Larvae Control Treatment
Odour source
Figure 4-3 Response of Campoplex dubitator females to odours released from host frass (2.5 g) versus uninfested cherry bark (2.5 g) in a Y-tube olfactometer (experiment 4). A "choice" was made when parasitoids walked to within 5 cm of the distal end of either arm. Parasitoids demonstrated a strong preference for frass-related volatiles ( ~ 2 = 5.580; P = 0.01 8).
Frass Cherry Bark
Odour source
Table 4- 1 Summary of olfactory experiments investigating the response of Campoplex dubitator to volatiles associated with the host and host habitat.
Experiment Materials tested n Olfactometer Used
1 uninfested cherry bark (2.0 g) vs. 27 static chamber
control
host frass (0.02 g) vs. 20 static chamber
control
3 host larvae vs.
control
20 static chamber *
4 uninfested cherry bark (2.5 g) vs. 46 Y-tube **
cherry bark and host frass mixture (2.5 g)
Static chamber olfactometers involve a simple box design without airflow.
** Y-tube olfactometers use airflow to ensure that the parasitoids are exposed to odours
from the test materials.
Table 4-2 Effect of mating status on the response of foraging female Campoplex dubitator to stimuli in an olfactometer.
Mating Proportion
Treatment status n choosing treatment z P
Uninfested cherry bark mated 16 81.3
(2.0 g) unmated 11 82.0 0.0461 0.963
Host frass (0.2 g) mated 9 0.89
(digested cherry bark) unmated 11 0.55 1.655 0.098
E. formosana larvae mated 9 0.78
(third, fourth & fifth instar) unmated 11 0.55 1.087 0.277
CHAPTER 5 Oviposition behaviour and patch time allocation
of Campoplex dubitator
Abstract
Behavioural ecologists have described a number of patch leaving rules that parasitoids may
employ to determine the optimal residence time for a given patch. The current experiments
illustrate the flexibility of foraging behaviour of the ichneumonid wasp, Campoplex dubitator
Horstrnann. I tested the hypothesis that this parasitoid regulates the time it allocates to exploiting
a patch based on an initial assessment of host density and ovipositions into hosts. An ethogram
was constructed to portray C. dubitator behaviour prior to and immediately following oviposition.
The pre-oviposition behavioural sequence was highly structured. In contrast, behavioural
transitions became less predictable after oviposition, providing the parasitoids with an option to
abandon the patch or resume searching for new hosts. On average, C. dubitator females spent
more time on patches with higher host densities. Host encounters caused a significant reduction in
the leaving tendency. This effect was stronger when the encounters led to successful ovipositions.
The giving up time was only slightly affected by host encounters. Campoplex dubitator appears
to have only a marginal ability to distinguish between occupied and empty host tunnels, since
37% of all host frass tubes that were probed contained no larvae. Similarly, C. dubitator may not
effectively discriminate against already-parasitised hosts. Overall, 35% of all successful attacks
on hosts were cases of superparasitism.
5.1 Introduction
The hosts of most parasitoids occur in aggregations composed of variable numbers of
individuals (Godfray, 1994). A parasitoid, having encountered a host patch, must effectively
estimate the relative quality of that patch in order to maximise exploitation efficiency. The
parasitoid's perception of patch quality may be critical for determining how much time to spend
searching for hosts before leaving. Behavioural ecologists have proposed theoretical models to
predict the optimal residence time a parasitoid should allocate to a patch it encounters. The most
well known of these, for example, is the marginal value theorem (Charnov, 1976), which states
that a forager should abandon a patch when the rate of fitness gain in the patch falls below a
marginal value. This threshold should be equal to the mean rate of fitness gain in all the available
patches in the environment. A parasitic wasp should adjust its tendency to exit based on
information it collects while on the patch. But how does a single forager know when the quality
o f a patch has been reduced to a point where it is adaptive to seek new patches? The eariiest
models assumed parasitoids knew the distribution of all hosts and host patches (van Alphen et al.,
2003). Since this was unlikely, it was hypothesised that they must follow certain rules that will
provide the flexibility needed for optimal patch residence times in a heterogeneous environment.
More recently, studies have shown some of the specific mechanisms that allow parasitoids to
determine optimum patch-leaving strategies (van Alphen et al., 2003). A wasp's experiences
during foraging, such as encounters with healthy or already-parasitised hosts (Nelson and
Roitberg, 1995; Wajnberg et al., 2000), have been postulated to provide the information
necessary to evaluate patch quality.
Two distinct behavioural mechanisms that have been demonstrated by parasitoid species
include the incremental and decremental effects of an experience such as oviposition in or on a
healthy host. A mechanism is called incremental when the oviposition leads to an increase in
patch time (decrease in the leaving tendency). This phenomenon has been demonstrated in a
number of parasitic wasp species (Waage, 1979; van Alphen and Galis, 1983; Hemerik et al.,
1993; Nelson and Roitberg, 1995; Wajnberg et al., 2000). In contrast, ovipositions into healthy
hosts may also cause a decrease in patch time (increase in the leaving tendency) (Driessen et a[.,
1995; Wajnberg et al., 1999; Tenhumberg et al., 2001). This is a decremental mechanism.
The existence of opposite responses to the same experience (incremental versus decrernental
effects) suggests that features of a particular parasitoid-host interaction may govern which types
of decision rules are most appropriate. For instance, Iwasa et al. (198 1) proposed that patch
109
departure mechanisms should be determined by the distribution of hosts in the environment.
Parasitoids searching for hosts in an aggregated distribution should decrease the leaving tendency
with each oviposition while those foraging for evenly distributed hosts should be more likely to
leave a patch following each oviposition.
In theory, an incremental mechanism would be used in situations where the forager has only a
poor estimate of patch quality (Driessen and Bernstein, 1999). In this way, as long as the prey
encounter rate is high, a rich patch will not be left prematurely. This same mechanism will also
prevent the foraging animal from allocating too much time to a low quality patch. In contrast,
when the forager is well informed of patch value, prey captures indicate a loss in the future
quality of the patch, and a decremental mechanism would allegedly be most suitable (Driessen
and Bemstein, 1999). Some species may even switch from one decision rule to another within a
patch (O~.!treman et al. 2901~) or hetween patch visitsj based on bnst distribution patterns (see
Driessen et al., 1995).
The study of patch foraging by parasitoids and predators has been hindered by conceptual
hurdles from the outset. A universal problem has arisen in the process of identifying and
describing the patch itself (Addicott et al, 1987). Hassell and Southwood (1978) recognised three
fundamental hierarchical levels within a forager's environment: habitat, food patch, and food
item. These three scales of foraging may pertain to any predator seeking prey, or, as in the case
here, any parasitoid searching for hosts. While the boundaries of both the largest scale (habitat)
and finest scale (host) of this hierarchy can typically be identified without excessive argument,
those of the food/host patch are more obscure. Between the habitat and the hosts, there may be
one or more intermediate levels over which a parasitoid searches for its host targets. The
existence and shape of these transitional patches is determined by the forager's perception of its
surroundings (Hassell and Southwood, 1978), and not necessarily by the physical distribution of
host entities or the arbitrary lines drawn by the observer. The ability to detect aggregative
responses by parasitoids may be influenced by the scale at which interactions are observed
(Heads and Lawton, 1983; Ayal, 1987; Tenhumberg et al., 2001). To generate an adaptive
explanation for patch exploitation by a parasitoid, it is necessary to conduct the investigation of
foraging behaviour at the appropriate level.
Previously, considerable effort had been made to give a general definition to the appropriate
patch level, with each attempt contributing something new to the description (Wiens, 1976;
110
Hassell and Southwood, 1978; Waage, 1978; Waage, 1979; Ayal, 1987; Addicott et al, 1987). A
definition combining the suggestions of these authors was adopted for the current study. The
patch of interest was considered to be a basic unit of the environment containing a stimulus that
elicits characteristic arrestment behaviour in the searching parasitoids and within which a species
must demonstrate a patterned search programme shaped by natural selection. Finally, there are no
constraints on patch size or internal homogeneity. Tenhumberg et al. (2001) also highlight the
importance of properly identifying patch-leaving behaviour.
The aim of the current study was to examine patch use by the koinobiotic endoparasitoid
Campoplex cf. dubitator Horstmann (Hymenoptera: Ichneumonidae), using one proposed patch
definition. This parasitoid attacks the larvae of the cherry bark tortrix, Enarmonia formosana
Scopoli (Lepidoptera: Tortricidae). As the name suggests, the cherry bark tortrix (hereafter
referred to as CBT) larvae feed on the phloem tissues of cherry and other rosaceous t r e e
(Roediger, 1956), and are thus concealed beneath the protective outer bark. The CBT has a
relatively uniform distribution throughout a European orchard (treating each tree as a cell), but
within a tree, it tends to be highly aggregated within the bottom 40 cm of the trunk. By treating
the trunk base as a host patch (therefore, one patch per cell), we revealed that percent parasitism
by C. dubitator in the field can decrease with increasing host density (Chapter 2). This pattern of
inverse density dependence could result from a decremental mechanism or even a weak
incremental mechanism, each of which might lead to patch departure before the parasitoid has
thoroughly exploited the patch.
Elucidation of the causal mechanisms that determine patch time could therefore be useful in
understanding the inverse density dependent response observed under field conditions. The
experiments described in this paper also provide insight into the ability of C. dubitator to
distinguish between host galleries and frass tubes that contain a host and those that have been
abandoned by CBT larvae. In the field, at any one time, a high proportion of CBT frass tubes are,
in fact, empty. Parasitoids are expected to reject these because time spent investigating an empty
gallery is time wasted, which means a substantial decrease in foraging efficiency. Campoplex
dubitator's ability to discriminate against parasitised hosts and the implications this has on patch
foraging strategies is also discussed.
Before investigating foraging behaviour at the patch level, however, it was necessary to
identi@ parasitoid behaviours associated with parasitism of CBT hosts. Prior to this study of C.
11 1
dubitator foraging behaviour, nothing was known about this species' host acceptance or rejection
behaviour. Also, determining when an oviposition had taken place was complicated. Most
ovipositor insertions into hosts are extremely rapid and typically happen only once per host. This
is in contrast to the oviposition behaviour described for many other parastic wasps (Godfray,
1994), which often have lengthy host handling times and may repeatedly probe their hosts with
their ovipositors. An ethological approach was taken to describe the behavioural sequence
displayed by C. dubitator females during an oviposition event. The behaviour of C. dubitator
immediately before, during, and immediately after oviposition is described here. A catalogue of
behavioural categories was developed to depict typical successions of behaviours associated with
oviposition. An ethogram also was created to illustrate the sequence of behaviours during this
final phase of host search. The intent of indexing oviposition behaviour was to identify
behaviours that are linked with either successful or failed ovipositions, to assist in recognising
nvip~sitions i n the patch fnraging experiment;
5.2 Materials and Methods
5.2.1 Study Organisms
CBT specimens were obtained from field collections in the German Black Forest and
northwest Switzerland throughout the summer in 2002 while experiments were run. The larvae
were reared on a meridic pinto bean-based diet, modified from Shorey and Hale (1965), at the
CAB1 Bioscience Centre in Delemont, Switzerland. Larvae and pupae were labelled and kept
solitarily and monitored for parasitoid emergence. ,411 immature CBT were kept a: 23*2" C under
a 16L:SD photoperiod. Every larva used in these experiments was taken from the laboratory
culture to ensure that the hosts were not parasitised previous to the trials.
All C. dubitator parasitoid adults eclosing from the CBT hosts were placed into plastic
cylindrical cages (10 cm height x 10 cm diameter) with honey and water on the day of
emergence. Females were kept individually while males, depending on emergence dates, were
kept either singly or in groups of two to five. Within three days of eclosion, females were placed
with males for short periods to facilitate mating. Females that were not mated under observation
were placed into adult parasitoid cages with 2 or 3 males until needed for the experiment. Hence,
all female parasitoids were assumed to have mated before use in the experiment. Following
mating trials, C. dubitator females were given host larvae for oviposition experience. AS with the
mating trials, all oviposition events were observed and the oviporition history of each parasitoid
was recorded. All females were provided with hosts for oviposition one day prior to the
112
oviposition tests. This egg-laying opportunity was provided because experienced females were
known to be more responsive to hosts.
5.2.2 Experiment I: Description of Oviposition Behaviour
Host larvae were prepared by placing them into 5.5 cm Petri dishes with freshly cut sections
of cherry bark 2 to 3 days before the experiment. Each Petri dish held one fourth or fifth instar
larva on a single segment of bark (3 cm x 4 cm). During this pre-experimental period, the larvae
were permitted to feed on the bark. This length of time was sufficient for the larvae to produce
large frass tubes, yet was short enough to ensure that the larvae did not burrow too deeply into the
bark for parasitoids to reach.
A bark segment containing a single feeding host with an established frass tube was placed
into the centre of a 10 cm Petri dish with the host'< frasq tube and feeding gallery facing upward.
Fine sand was poured and packed firmly into the arena around the bark so that only the upper
surface of the cherry bark was exposed to the parasitoid. This reduced the surface area and was
assumed to increase the likelihood of successful parasitism. For each trial, a single experienced
parasitoid was placed into the arena on the underside of the lid of the Petri dish. The parasitoid
was then allowed to discover the bark and host larvae through its own search behaviour. Formal
observations began with the initiation of search by the parasitoid and ended when the wasp
attempted to fly away from the test area. Trials were always conducted between 11:OO and 19:00
(6 August to 16 September) at 22 * 2' C with ambient relative humidity.
Eight discrete behavioural categories were used to describe the foraging behaviour of C.
dubitator females leading up to, during, and following oviposition. A description of these
behaviours is given below.
1 . Walking: A rapid, primarily phototactic movement without orientation toward the host
target. Parasitoid exhibits a low turning frequency with antennae straight and held at a 25-45'
angle to the substrate.
2. Searching: Walking with frequent turning in response to volatile or tactile cues. This
typically occurs when the parasitoid first encounters cherry bark or is close to a host frass tube.
Antennae are often waved in circular motions presumably to sample the air, interrupted by brief
contacts with the substrate, which the parasitoid paipates with its antenna1 tips.
3. Probing: In association with continuous antenna1 palpation of host frass and strong
klinokinesis, the parasitoid arches its abdomen and rapidly probes the interior of the frass tube
with its unsheathed ovipositor. While probing, the parasitoid walks along the surface of the frass
tube. Once reaching the end of the frass substrate, the parasitoid typically turns 180" and
continues to probe the same frass tube.
4. Oviposition: Is always preceded by probing activity. Oviposition occurs extremely rapidly
and is not easily identified by observers. Under normal conditions where hosts are fully
concealed, they are neither paralysed nor handled by the wasp during parasitism.
5. Checking: A tactile investigation of the host's frass tube and surrounding area. The
parasitoid repeatedly contacts the frass and surrounding bark substrate with its mouthparts and
antennae. This behaviour presumably facilitates recognition of the frass tube to avoid self-
superparasirism in case o f a future encounter witin rhe same faecai pouch.
6. Grooming: Self-preening by the wasp. This behaviour includes cleaning of the antennae,
abdomen, wings, and legs.
7 . Resting: A sedentary phase, during which the parasitoid remains motionless. Antennae
tend to be held together, directly in front of the parasitoid's body, with no waving or trembling.
Slight abdominal movements are sometimes noticeable, suggesting the parasitoid may be
preparing itself physiologically for an oviposition.
8. Leaving: Rapid walking, running, or flying, often a phototactic response or otherwise
random movement. Antennae are straight and held at a 25-45' angle to the substrate. Parasitoid
may travel several centimetres before turning. This behaviour signals the termination of the
oviposition series; it suggests a loss of interest in the host and host frass by the parasitoid.
The interaction of the parasitoid and its host in the test arena was observed through a
binocular microscope and was recorded using event-recording software (The Observer 2.0) with
0.1 s time resolution. Neither intense nor natural light is required to motivate oviposition
behaviour (pers. obs.); therefore, a light level suitable for observation was used (650 LUX;
Philips projection lamp type 13 186, 14.5 V, 90 W, GX5.3). For each individual parasitoid, the
sequence and duration of behaviours was recorded from two consecutive parasitism events. The
stung host from the first trial was always replaced with a healthy larva on new bark prior to
commencing the second run. A total of 28 C. dubitator females were used to generate an
114
ethogam of oviposition behaviour. The frequency of transitions from one behaviour to any other
was averaged from the two observed ovipositicns with each female. The averages taken from
each parasitoid were then pooled and the frequencies associated with transitions from one
behaviour to another were organised into two contingency tables to create 1''- order transition
probability matrices as described by Fagen and Young (1978). The first matrix included
behaviours demonstrated only up until the moment of oviposition, while the second consisted of
transitions occurring after the egg laying event. To provide equal weighting to the individual
behaviours in each matrix, the modification of Charlton and Card6 (1990) was applied. Self-
transitions and impossible transitions were left as blanks. Also, transitions displayed by only a
single individual were excluded from the matrix. These probabilities were ultimately used to
construct an ethograrn of the interaction between C. dubitator and the CBT. A X2 analysis was
applied to determine whether the probability of certain behavioural transitions differed from
random, both before and after ovipositinn.
5.2.3 Experiment 11: Patch Residence Time
For C. dubitator, the elementary unit of foraging was considered to be the area of bark over
which a parasitoid would search for hosts while walking. This description was based on early
observations of foraging C. dubitator females. In the field, these parasitoids were observed to
hover around the bases of trunks, within 5-10 cm of the bark surface. Upon detection of host
frass, the parasitoids landed (arrestment) at the site of host infestation and resorted to a walking
search, which incorporated tactile cues. If a wasp walked approximately 10-1 5 cm away from the
last host without encountering more (patch boundary), it usually turned sharply and returned to
the previously discovered hosts before walking in a new direction. If no more hosts were
encountered with this search pattern, the parasitoid was likely to fly off the bark. Thus, the actual
size of a patch was determined by the distribution of larval hosts. If five CBT larvae were tightly
aggregated around a bark wound, the patch size would be considerably smaller than if those same
larvae were strung out in a line at 10 cm intervals. This idea of a patch was likened to the base of
a tree trunk, while the entire tree would be considered as a "super-patch". The surface area of the
patches used in experiment 2 was equivalent to, or greater than, the area typically covered by
these parasitoids during a patch visit (defined as the time spent on the bark between arrestment
and migration from the patch by flying).
Host patches containing mid to late instar larvae of the CBT were prepared using cherry logs
which were cut from either the bole or the lower limb of a wild-growing cherry tree (Prunus
115
miurn). All sections of the tree selected for use in this experiment were free of CBT damage prior
to manual infestation. To reduce desiccation of the wood, the sawed ends of each log were
painted with a tree-pruning paint (MioPlant, Migros-Genossenschafts-Bund, Zurich) known not
to deter either CBT larvae or foraging parasitoids (pers. obs.). The logs were also set upright on
one end in a shallow container holding approximately 2 cm of water. Predetermined numbers of
larvae were then transferred to the cherry logs. The upper cut surface was covered with a gauze
cap, secured with an elastic band, to block larval access to the saw wounds. The host larvae were,
therefore, allowed to distribute themselves over the log in a natural fashion. This resulted in a
tendency for the larvae to enter the bark through small lesions or around the nodes of stems.
While there appeared to be some clustering of feeding larvae (>1 frass tube within a 1-3 cm
radius), many larvae established feeding galleries more than 5 cm away from the nearest
neighbour. Larvae were left to develop in the cherry logs for several days before the logs were
fi~pd in plrasitoid feraging trials.
Four density categories were used to classify the number of CBT larvae on a given log. These
were: 0 larvae, 1-5 larvae, 6-10 larvae, and 11-15 larvae. At the start of each test, all the host
larvae were concealed within their feeding galleries. The actual larval density on the log was not
known until dissection of the cherry bark following the trial. Nor did the observer know the
occupancy status of the various frass tubes on the log before the larvae were extracted from the
feeding tunnels after the trial. Immediately following every foraging trial, the log used in the
experiment was dissected to collect all larvae from the bark and determine their exact
distribution. These hosts were subsequently reared out under laboratory conditions (see
description of rearing) to verify whether parasitism had occurred and to record the sex of the
resulting parasitoid progeny.
Immediately prior to testing, each C dubitator female was placed into a 5.5 cm Petri dish
with a small amount of frass. This was to test the responsiveness of the parasitoids to host-related
stimuli and to ensure that they were physically capable of detecting and parasitising their hosts
(Nelson and Roitberg, 1995). This process also served to standardise the parasitoids before they
entered the trial in that they were, in a sense, "activated" by the host-related cues in the frass.
Before entering the arena, the parasitoids had already done most of the grooming and cocking
actions necessary to prepare eggs for oviposition, which in some cases may take several minutes.
Parasitoids that did nct probe the frass tube during this preliminary test were not used in the
experiment.
116
Trials were run between 8:30 and 20:OO (29 May to 20 September) at 22 * 2" C with ambient
relative humidity. The test arena was centered beneath the only light source in the room: 3 tubes
providing fluorescent "Cool White" illumination (Osram L 36W120, 2200 LUX). For each
replicate in this experiment, a single prepared cherry log was placed into the centre of a
transparent plexi-glass arena (70 x 70 x 70 cm). Five minutes later, a C. dubitator female was
transported to the corner of the arena in a plastic vial (7 cm height x 1.5 cm diameter). Each
experimental parasitoid and cherry log was used only once. The experiment began once the
parasitoid landed on the log, at which point it began to walk over the surface of the cherry bark in
search of hosts. Every trial was continuously observed until the parasitoid flew away from the
log. The following parameters were measured while the parasitoid remained in the host patch:
- total time spent foraging on patch
- time between patch encounter (or last ovipo~itionj and departure from patch
(giving up time: GUT)
- frequency and duration of probing events at individual frass tubes
- number of successful and failed oviposition attempts
- initial and final rates of host encounters
- initial and final rates of oviposition
Successful ovipositions were identified by the behavioural pattern associated with oviposition, as
described in the results section for the preceding experiment. The collection of these data allowed
the calculation of search efficiency and density response, as well as an investigation of the
tendency to self-superparasitise hosts.
5.2.3.1 The Proportional Hazards Model
The patch leaving mechanisms employed by C. dubitator females were analysed using Cox's
proportional hazards model. The model is constructed in terms of the hazard rate, which is the
probability per unit time that a certain event (failure) occurs, given that it has not already
happened. For this experiment, the hazard rate refers to a parasitoid's tendency to leave a
foraging patch. Therefore, leaving the patch is defined as a failure.
The likelihood that a wasp will depart from a patch is assumed to be modified by certain
characteristics of the patch as well as by experiences the wasp has while on the patch. The effect
of these pre-defined factors (covariates) can be explained by the following equation:
(1)
where h(t) is the observed hazard rate, h,(t) is the baseline hazard (assumed to depend only on
time, and therefore corresponds to the scenario in which all covariates are equal to zero), t is the
time elapsed since the female entered the patch, and PI, ... P, are the regression coefficients giving
the relative contributions of the covariates. The strength of the effect of each covariate can be
determined by the exponential term (exp(P)), known as the hazard ratio. A hazard ratio greater
than one will indicate an increasing effect on the females' patch leaving tendencies, while a
hazard ratio lower than one would suggest parasitoids become less likely to leave. A more
thorough description of this model can be found in the literature dealing with survival analysis
(Kalbfleisch and Prentice, 1980).
Seven covariates were included in the proportional hazards model: host density, number of
frass tube encounters without oviposition, number of frass tube encounters with oviposition,
initial rate of encounter, final rate of encounter, initial rate of oviposition, and final rate of
oviposition. The significant effects of the covariates were tested with a likelihood ratio test. The
regression modelling was performed with SPSS 1 1.5 (Novell).
5.3 Results
5.3.1 Experiment I: Description of Oviposition Behaviour
Of the 28 females used in this experiment, all but three were successful in locating and
stinging their hosts. When parasitism was successful, the oviposition event lasted, on average, 0.8
seconds (0.3-2.3). The delivery of a parasitoid egg into the host must therefore happen nearly
instantaneously after the ovipositor pierces the larva's integument. At no point after ovipositor
insertion does the host larva suffer paralysis.
In the initial search phase leading up to oviposition into the host, the behavioural sequence of
C. dubitator appeared fixed, with a high predictability of the behavioural transitions. The most
typical behavioural sequence involved walking up to the infested bark substrate, locating the
host's frass tube, and probing for the host through the faecal material, which ultimately led to
oviposition. While the steps of this sequence did vary in some cases, such as the inclusion of
short resting periods between bouts of searching and probing, this variance was not substantial (as
indicated by the thickness of the arrows in Figure 5-1).
118
Following stinging of the host, the parasitoid action pattern lost its structure and
predictability. For this phase of the foraging process, it is not possible to define the most common
sequence of actions. Parasitoids almost always checked the substrate immediately after
oviposition, but following that, there was an approximately equal likelihood that they would
groom (29%), rest (27%), or resume probing the same frass tube (25%). This higher variability in
transitions is illustrated by the increased number and reduced thickness of the arrows in the post-
oviposition phase of Figure 5-1. Further probing of the host's frass tube following oviposition
occurred in 47% of the trials, but of these, only 23% resulted in a second successf~d oviposition
into the same host. On average, the females left the host site 74 seconds (5-289) following
oviposition (after the second oviposition, in instances with superparasitism).
. . The mean probabilities ~.rsed to generate the ethogram m F~gure - 1 are fm!nd in Tah!~ - 1
(pre-oviposition) and Table 5-2 (post-oviposition). The foraging process was divided into the pre-
and post-oviposition segments to illustrate the variation in transition probabilities before and after
parasitism of the host. Tables 5-1 and 5-2 also provide the chi-square statistics associated with the
pre- and post-oviposition behaviours, respectively.
5.3.2 Experiment 11: Patch Residence Time
As can be seen in Figure 5-2, C. dubitator females spent more time on patches with higher
numbers of hosts (ANOVA: F = 17.232, df = 3, P < 0.00 1). However, percent parasitism
remained constant at around 35% despite changing host densities (Figure 5-3) (r2 = 0.045, P =
0.222, n = 34). When hosts were present, the parasitoids always examined and probed at least one
of the frass tubes. On average, the parasitoid examined 57% * 4% SE of all the available frass
tubes on a patch. This proportion of frass tubes discovered was independent of the total number
of tubes in the patch (r2 = 0.02, P = 0.4661, n = 34). Parasitoids often re-encountered and re-
probed frass tubes that had been probed earlier in the foraging period; 5 1 % of all frass tube
encounters resulting in probing were with frass tubes that had already been probed at least once.
Correspondingly, of the total time allocated to probing, 46% was spent probing re-encountered
frass tubes. The frequency of re-probing events was significantly greater for trials with the
highest host densities (ANOVA: F = 5.634, df = 2, P = 0.009).
Ovipositions were observed in 26 of the 3 1 patch trials where at least one CBT larva was
present on the log. Parasitoids stung significantly more hosts when 5-10 or 11-15 larvae were
119
present than when only 1-5 were available in the patch (ANOVA: F = 8.9 109, df = 2, p = 0.0 10).
Of the five trials in which no ovipositions occurred, four were with host density class 2 (1-5
larvae) and the fifth was with host density class 3 (6-10 larvae). Although the number of hosts
parasitised per trial increased with host density, there is no evidence that the rate of parasitism
was dependent on host density (Figure 5-3). This result was consistent regardless of whether
parasitism was calculated based on oviposition events noted during the experiment (F = 1 S73, df
= 1, P = 0.222) or based on F, parasitoid emergence (as was done for field-collected specimens,
see Chapter 2) (F = 1.73 1, df = 1, P = 0.21 1). All parasitism rate data were normalised with
arcsine transformation.
Campoplex dubitator females may have a marginal ability to distinguish between occupied
versus abandoned frass tubes. Based on trials in which there was at least one empty and one
occ,ipied f ras tube ( n = M), 63% of the f r a s t u h e a wasp encountered contrined host ! z~ :2e ,
while on average, only 52% of all the frass tubes available actually held hosts. This difference,
however, is not statistically significant (z-test: z = 0.862, P = 0.389). From the 18 replicates in
which more than one oviposition occurred, 11 had cases of self-superparasitism, of which nine
were from trials with the highest host density. Overall, 35% of all successful host attacks were
instances of superparasitism during the same patch visit.
A proportional hazards model was fitted to the patch foraging data to determine the patch
leaving mechanisms used by these parasitoids. Table 5-3(a) gives the estimated effect of all the
covariates that had a significant impact on the time spent on the patch by each parasitoid. The
following terms were automatically removed from the model when found not to have an effect on
patch time: initial frass tube encounter rate, final frass tube encounter rate, initial oviposition rate,
and final oviposition rate. The number of encounters with frass tubes and number of ovipositions
significantly influenced the duration of patch visits while host density had a marginal impact.
Each of the covariates remaining in the model reduced the leaving tendency of the parasitoid
(negative p-value), therefore increasing total patch time.
Using 0 host density as the reference level (baseline hazard), the leaving tendency was 86%,
94%, and 99% less in trials with 1-5, 6-1 0, and 1 1-1 5 hosts per patch, respectively. Encounters
with frass tubes without subsequent oviposition reduced the parasitoids' tendency to leave by a
factor of 0.96, while frass tube encounters leading to successful oviposition resulted in a further
reduction of the leaving tendency by a factor of 0.8 1. This indicates that C. dubitator wasps use
an incremental mechanism in determining their patch residence time.
The Cox regression model was applied to the behavioural data a second time to assess the
effect of the same seven covariates on the GUT of the female wasps. Table 5-3(b) shows that only
a single covariate, the number of frass tube encounters without oviposition, affected the amount
of time between patch encounter (or last oviposition) and departure from the patch. The more
frass tubes that were discovered, the less likely a wasp was to leave the log in the final stage of its
patch visit, however, the effect was not strong (leaving tendency only 2% smaller after frass tube
encounter).
5.4 Discussion
5.4.1. Experiment I: Description of C)viposition Behaviour
The pre-oviposition and post-oviposition ethograms for C. dubitator behaviour clearly show a
higher predictability of transitions between actions before a host has been parasitised. In the pre-
oviposition sequence, certain actions were not expressed, including grooming, checking, and
leaving behaviour. It must be noted, however, that Figure 5-1 is based only on sequences in
which parasitoids were successful in parasitising their hosts. In situations where parasitoids
attempt, but fail, to sting their hosts, they may groom while resting, and at some point after giving
up, will inevitably leave. Such cases with attempted, but unsuccessful attacks on hosts were
observed repeatedly in experiment 2 and are likely quite common in the field where a large
proportion of CBT tunnels are abandoned or larvae are too deep inside the bark to be reached.
One parasitoid action that did have a relatively fixed position in the behavioural sequence
following oviposition was the host assessment (checking) behaviour. This unique behaviour never
occurred unless the wasp had struck the host with its ovipositor. It is not clear why parasitoids
perform the oral dabbing activity, called "checking". It may result in the application of a chemical
marker to the frass tube and surrounding bark, as is shown with other parasitoids attacking
concealed hosts (Potting et al., 1997; Hoffmeister, 2000; Hoffmeister and Roitberg 2002).
However, C. dubitator is not known to effectively discriminate between healthy and parasitised
hosts (see results for experiment 2). A second plausible explanation relates to the associative
learning process, in which a parasitoid correlates certain stimuli with ovipositions (Godfray,
1994). Touching antennae and mouthparts to a host's faecal tube may function in allowing
females to "memorise" particular cues. Regardless of the reason for it, the checking behaviour is
121
perhaps the only reliable indicator of successful parasitism. It is difficult to identify oviposition
events since no substantial host handling occurs and all other behaviours may be demonstrated
with or without oviposition.
The behavioural plasticity following oviposition may be an essential characteristic to achieve
optimal foraging since it is at this point that a parasitoid must decide what to do so that its fitness
is maximised. For instance, if the parasitoid is on a rich patch, it will benefit from remaining on
the patch rather than leaving directly after a successful attack. In contrast, if the parasitoid has
acquired sufficient patch information to know that the probability of finding another host is low,
its best option will be to abandon the patch without further search. As it is known that CBT patch
densities vary dramatically in the field (Chapter 2), the flexible post-oviposition behaviour
demonstrated by C. dubitator may be adaptive in allowing the parasitoids to respond optimally to
the cunent natrh n ~ i a l i t w . r-*-- -I-----J
5.4.2 Experiment 11: Patch Residence Time
Following Charnov's (1976) marginal value theorem, it is generally expected that a forager
will spend more time in patches that contain relatively more abundant resources. This study
examined the influence of host density and several intra-patch experiences on the patch leaving
behaviour of C. dubitator. These parasitoids remained longer in patches with greater host
densities. This was due to both the parasitoids' assessment of patch quality based on cues such as
levels of host-related kairomones (Waage, 1979) and experiences involving frass tube encounters
and ovipositions into hosts.
For C. dubitator, encounters with hosts and host frass tubes led to an increase in the patch
time (decrease in the leaving tendency). If C. dubitator females cannot effectively judge the
quality of a patch without actually sampling the hosts present, this sort of incremental mechanism
may serve them better in maximising their fitness within a patch. The timing of each successive
oviposition provides patch quality information and ultimately allows parasitoids to decide
whether they should remain on the patch (van Alphen et al., 2003). On cherry trees in central
Europe, the number of CBT larvae present on a tree may vary substantially. Approximately 30%
of those trees contain no larvae while 20% hold only one or two hosts and 16% have more than
10 hosts on the trunk (Chapter 2). When a wasp lands on a tree with no hosts, it will leave after a
fixed search time. This innate tendency to abandon a patch (baseline hazard) should be great
enough to ensure that no effort is wasted searching an empty patch, while allowing sufficient time
122
for the parasitoid to actually encounter a host when there is one present. It is therefore a delicate
balance between time squandered and attempting to parasitise every available host.
It has been suggested that "searching time" is a more appropriate variable to measure than
"total patch residence time" if trying to relate parasitoid behaviour to natural patterns of
parasitism (Waage, 1983; Morrison, 1986; Roermund et al., 1994; Nelson and Roitberg, 1995). In
the host-parasitoid system presented here, however, the non-search behaviours (resting,
grooming, and oviposition) were considered to have an insignificant impact on total time. That is,
"searching time", including olfactory, tactile, and visual search, did not differ greatly from "total
foraging time". Parasitoids spent nearly the entire patch time either probing discovered frass tubes
or searching the bark for new hosts. Whether this was an artifact of the experimental design is
unclear. But as the oviposition process is extremely rapid, host-handling time was virtually non-
existect. Simi!ar!y, bzuts cf restizg 2zd grzzmizg were very brief when they did sccur. 12
conclusion, "total foraging time" was selected as an accurate currency of foraging effort.
In contrast to some previous patch foraging behaviour studies relying on arbitrary definitions
of patch departure (Waage, 1979; van Alphen and Galis, 1983; Haccou et al., 199 1 ), determining
when the C. dubitator forager actually leaves the patch was simple. Abandonment of the patch,
due to habituation of the arrestment stimulus (Waage, 1979), was consistently associated with
obvious migratory flight behaviour. This patch-leaving flight differed greatly from the much
more brief (1-3 second) flights between points within the patch. The migratory (patch-leaving)
behaviour appeared to consist of an overriding phototactic response and drew the parasitoid to the
extreme edges of the test arena, well away from the infested log.
The number of frass tubes present on each experimental log was typically greater than the
number of CBT larvae. This was because, prior to the trials, the larval hosts occasionally
abandoned their initial feeding galleries, supposedly to find more profitable sites. This is
consistent with patterns observed in the field and results in an over-representative number of frass
tubes for the number of larvae present in the system. Nonetheless, the number of frass tubes per
log was linearly correlated with the actual host density per log (ANOVA: y = 4.1080 + 1.3999x,
r2 = 0.61, P < 0.001). This phenomenon was therefore not considered to be a problem while
assessing patch residence times.
A fundamental assumption underlying the marginal value theorem is that as a parasitic wasp
oviposits into the available hosts over time, the patch's productivity decreases (Charnov, 1976).
While declining patch quality is evident to predators through a reduced prey encounter rate,
parasitoids must deal with problems associated with re-encountering already parasitised hosts
because those hosts remain in the system. For instance, valuable time may be lost inspecting and
rejecting already parasitised hosts, or for species that cannot discriminate against such hosts, both
time and eggs may be wasted through superparasitism. Hence, the marginal value theorem
suggests that parasitoids should typically exit a patch before all hosts have been exploited.
Campoplex dubitator does not handle its hosts to assess them, but instead appears to rely on
recognition of the frass tubes of hosts it has already parasitised. This means that no time is lost in
handling hosts and additional time can be saved by not probing frass tubes containing already-
parasitised host. When host discrimination by C. dubitator does occur, it happens prior to probing
with the o v i p i t n r . A fe'~ma!e wil! &ten parasitise m!y cnce in a gix:: frzss +;be h~!ding o host.
If the parasitised larva is transferred to a new frass tube, it will likely be re-attacked. In contrast, a
healthy host may be rejected if it is placed into a frass tube that the wasp has already oviposited
into (pers. obs.). Superparasitism events were not included as a covariate in the proportional
hazards model since any attempts by a wasp to oviposit are interpreted to mean that the parasitoid
does not recognise that a host has already been parasitised. Based on the frequency of re-attacks
on already-parasitised hosts in this experiment, C. dubitator females appear to have a poor ability
to distinguish between frass tubes of healthy hosts and those of parasitised hosts. These
parasitoids thus risk wasting eggs through superparasitism. It is argued that parasitoids with an
imperfect ability to discriminate between healthy and already-attacked hosts may favour early
patch leaving to avoid self-superparasitism (Rosenheim and Mangel, 1994; Outreman et al.,
2001). Such behaviour could, in turn, create a pattern of inverse density dependence as was
observed from the earlier field surveys (Chapter 2).
The individual foraging strategy demonstrated in experiment 2 (Figure 5-3) cannot be scaled
up to explain the strong pattern of inverse density dependence in field parasitism by C. dubitator.
This might suggest that there are additional external factors that influence the final outcome of
parasitism in the field. There are at least four possible explanations for the inconsistency between
field and laboratory findings. First, the cherry logs prepared for experiment 2 had fundamental
differences from living cherry trees, which may have caused females to use a modified foraging
strategy. For instance, the patch surface area of the experimental logs was substantially smaller
than the actual tree bases surveyed in the field. A greater patch size may result in fewer host re-
124
encounters, thus changing the dynamics of the foraging process significantly. Perhaps more
importantly, a large cherry tree in the field might consist of a few separate aggregations of CBT
larvae, which a wasp would perceive as several smaller elementary units of foraging. Because the
density dependent response calculated from field data used the entire basal section of cherry trees,
several "patches" may have been considered at once. This approach may not give an accurate
indication of parasitoid effort (Heads and Lawton, 1983; Ayal, 1987), particularly if a wasp left a
tree before exploiting all the host patches present.
Second, it must also be noted that percent parasitism in experiment 2 was calculated from the
action of a single foraging parasitoid, which visited the host patch only once. In contrast, it cannot
be known from the available field data whether the parasitism on a particular tree was inflicted by
one or more females and during one or several patch visits. Therefore, despite having possibly
dmng patch !paving ru!q a d d i t i ~ n ~ ! er?virnnrt?enta! fzctors not tested experimect 2 =axr have --- --- J
contributed to the inverse density dependent pattern observed in the field.
Third, the method for calculating percent parasitism might have been a source of variation
when studying density dependence (Driesche, 1983). The inverse density dependent relationship
between parasitism rate and host density shown in Chapter 2 was calculated using data from field
collections. This approach is believed to underestimate the actual impact of parasitoids on the
host population since any early host death resulting from parasitism goes undetected. Also, this
procedure does not clearly indicate the foraging effort made by parasitoids that have visited host
patches on cherry trees. That is, a parasitoid may successfully attack half the hosts on a patch
before leaving, but due to factors such as failed parasitoid development, perhaps only a fraction
of these will lead to the development of parasitoid progeny. Conversely, the calculation of
parasitism rate using the observed number of ovipositions will often overestimate parasitoid
impact, since not all parasitised hosts will yield parasitoid offspring (Chapter 3). In experiment 2,
both of these approaches of computing percent parasitism were used to examine their accuracy.
Ultimately, there was no evidence of any difference between the two. Each method failed to
detect any form of density dependence by C. dubitator.
A final possible explanation for the discrepancy in determining density dependence between
field and laboratory studies is the relatively small sample size from experiment 2. A more
detectable pzttern in the density response of C. dubitator may emerge if a much larger set of
replicates is run.
Past studies have shown that information gained from previous patch visits may affect a
forager's leaving tendency in subsequent patch encounters. For instance, Tenhumberg et a/.
(2001), Outreman et al. (200 l), and Wajnberg et al. (1 999) demonstrated that patch leaving
tendency increases with successive patch visits. Rhagoletispomonella Walsh (Diptera:
Tephritidae), although not an entomophagous insect, forages for oviposition sites much like a
parasitoid. The fruit hosts of this parasite are analogous to the sedentary larval hosts of a wasp
like C. dubitator. During the exploitation of a host patch, R. pomonella also risks wasting time in
re-encountering already infested hosts and must decide on the optimum moment to leave the
patch. Roitberg and Prokopy (1982) showed that R. pomonella flies remained in patches longer
when neighbouring trees (patches) were farther away. These observations of inter-patch effects
suggest that studies of patch residence time should consider the possible effects of factors such as
previous patch quality or inter-patch d i t a ~ c ~ . The experiment~! desisq eof the current stcdl~ J l l c d ....-..
only a single patch and the pre-experimental oviposition experiences of the test subjects were
standardised. It is conceivable that incorporating additional patches into the system would have
changed the wasps' leaving tendencies and may have even altered the overall effects of the
covariates in the Cox regression model. Most patch time tests to date have not included inter-
patch effects in the experimental structure. It is perhaps assumed that a parasitoid's baseline
hazard is an adaptation for the interaction with a particular host species, whose distribution
remains relatively constant over multiple generations. Wajnberg et al. (2000) used naive female
Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) to demonstrate that
these parasitoids have innate patch leaving rules, which are, presumably, suited to the average
quality of patches they will encounter. Information gained from subsequent patch visits would
then allow the wasps to adjust their leaving tendencies accordingly.
Parasitoids must acquire information during patch visits to forage effectively in a
heterogeneous environment. To make adaptive decisions, they must compare current alternatives
with experiences they have had, and to be able to make such decisions, they must have flexibility
in their response to changing stimuli. This ability to modify search behaviour in response to
experiences within or even between patches should increase the overall efficiency of the
parasitoid species studied here. Campoplex dubitator is attracted to volatiles produced by even
uninfested cherry trees (Chapter 4) and may therefore search trees that are void of any suitable
hosts. The presence of host-rel~ted cues and host encounters, however, should ensure that females
spend more time on trees that do contain CBT larvae. It is now known that parasitoids obtain
126
information from a vast array of sources (van Alphen et al., 2003). For C. dubitator, as with other
species, foraging decisions may also be based on environmental conditions (Roitberg et al.,
1993), competition with other parasitoids (Bernstein and Driessen, 1996), or inter-patch
experiences (Tenhumberg et al., 200 1).
In conclusion, the patch definition applied to experiment 2 was adequate for identifying some
of the patch leaving rules used by C. dubitator. A higher number of hosts per patch led to a
greater search effort, particularly as a result of host encounters. However, under this model, it was
not possible to ascertain how host density affects percent parasitism. While the results show a
density independent response, it is not certain that this is the true response or whether the analysis
was too weak to detect either direct or inverse density dependence. One likely improvement to the
current patch concept would be the incorporation of information from previous patch visits. As
&scnssed elr!ier, it i f i ~ ~ e ~ ~ a p t ~ ) rnnrliirt the inj~pstioatiofi of fcranirln h~bawingr the J --"---- --' D b" b "- '-"" appropriate spatial level in order to formulate an adaptive explanation for a parasitoid's patch
exploitation strategy.
5.5 References
Addicott, J.F., J.M. Aho, MF. Antolin, D.K. Padilla, J.S. Richardson, and D.A. S&k. 1987. Ecological neighborhoods: scaling environmental patterns. Oikos 49: 340-346
Ayal, Y. 1987. The foraging strategy of Diaetetiella rapae: I. The concept of the elementary unit of foraging. Journal of Animal Ecology 56: 1057- 1068
Bernstein, C. and G. Driessen. 1996. Patch marking and optimal search patterns in the parasitoid Venturia canescens. Journal of Animal Ecology 65: 2 1 1-2 19
Charlton, R.E. and R.T. Carde. 1990. Behavioral Interactions in the courtship of Lymantria &spar (Lepidoptera: Lymantriidae). Annals of the Entomological Society of America 83: 89-96
Charnov, E.L. 1976. Optimal foraging: the marginal value theorem. Theoretical Population Biology 9: 129-136
Driessen, G. and C. Bernstein. 1999. Patch departure mechanisms and optimal host exploitation in an insect ~arasi toid .Joumnl of4nimnl Ecology 68. 445-459
Driessen, G., C. Bernstein, J.J.M. van Alphen, and A. Kacelnik. 1995. A count-down mechanism for host search in the parasitoid Venturia canescens. Journal of Animal Ecology 64: 1 17-125
Fagen, R.M. and D.Y. Young. 1978. Temporal patterns of behaviour: durations, intervals, latencies, and sequences. pg.79-114 In: Colgan, P.W. (Ed.) Quantitative Ethology. John Wiley & Sons, New York
Godfray, H.C.J. 1994. Parasitoids: Behavioural and Evolutionary Ecology. Princeton University Press, Princeton 473 pp.
Haccou, P., S.J. de Vlas, J.J.M. van Alphen, and M.E. Visser. 1991. Information processing by foragers: effects of intra-patch experience on the leaving tendency of Leptopilina heterotoma. Journal of Animal Ecology 60: 93-1 06
Hassell, M.P. and T.R.E. Southwood. 1978. Foraging strategies of insects. Annual Review of Ecology and Systematics 9: 75-98
Heads, P.A. and J.H. Lawton. 1983. Studies on the natural enemy complex of the holly leaf- miner: the effects of scale on the detection of aggregative responses and the implications for biological control. Oikos 40: 267-276
Hemerik, L., G. Driessen, and P. Haccou. 1993. Effects of intra-patch experiences on patch time, search time and searching efficiency of the parasitoid Leptopilina clavipes. Journal ofAnimal Ecology 62: 33-44
Hoffmeister, T.S. 2000. Marking decisions and host discrimination in a ~arasitoid adacking concealed hosts. Canadian Journal of Zoology 78: 1494-1499
Hoffmeister, T. and B. Roitberg. 2002. Evol~1tionar-y ecology of oviposition marking pheromones. Pg. 3 19-347 In: Hilker, M. and T. Meiner (Eds.) Chemoecology of Insect Eggs andEgg Deposition. Blackwell Press
128
Iwasa, Y., M. Higashi, and N. Yamamura. 198 1. Prey distribution as a factor determining the choice of optimal foraging strategy. The American Naturalist 117: 7 10-723
Kalbfleisch, J.D. and R.L. Prentice. 1980. The Statistical Analysis of Failure Time Data. Wiley, New York
Morrison, G. 1986. Stochastic aggregative responses and spatial patterns of parasitism in patchy host-parasitoid interactions. Oecologia 70: 402-4 10
Nelson, J.M. and B.D. Roitberg. 1995. Flexible patch time allocation by the leafminer parasitoid, Opius dimidiatus. Ecological Entomology 20: 245-2 52
Outreman, Y. A. Le Ralec, E. Wajnberg, J.S. Pierre. 2001. Can imperfect host discrimination explain partial patch exploitation in parasitoids? Ecological Entomology 26: 27 1-280
Potting, R.P.J, H.M. Snellen, and L.E.M. Vet. 1997. Fitness consequences of superparasitism and mechanism of host discrimination in the stemborer parasitoid Cotesia flavipes. Entomologia Experimentalis et Applicata 82: 342-348
Roediger, H. 1956. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) Zeitschrlftfir Angewandte Entomologie 38 : 1 95-3 2 1
Roermund, H.J.W., L. Hemerik, and J.C. van Lenteren. 1994. Influence of intrapatch experiences and temperature on the time allocation of the whitefly parasitoid Encarsia formosa (Hymenoptera: Aphelinidae). Journal of Insect Behaviour 7: 483-501
Roitberg, B.D. and R.J. Prokopy. 1982. Influence of intertree distance on foraging behaviour of Rhagoletis pomonella in the field. Ecological Entomology 7: 437-442
Roitberg, B.D., M. Mangel, R.G. Lalonde, C.A. Roitberg, J.J.M. van Alphen, and L. Vet. 1993. Seasonal dynamic shifts in patch exploitation by parasitic wasps. Behavioural Ecology 3: 156-165
Rosenheim, J.A. and M. Mangel. 1994. Patch-leaving rules for parasitoids with imperfect host discrimination. Ecological Entomology 19: 374-380
Shorey, H.H. and R.L. Hale. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58: 522-524.
Tenhumberg, B., M.A. Keller, H.P. Possingham, and A.J. Tyre. 2001. Optimal patch-leaving behaviour: a case study using the parasitoid Cotesia rubecula. Journal of Animal Ecology 70: 683-691
van Alphen, J.J.M., C. Bernstein, and G. Driessen. 2003. Infomation acquisition and patch time allocation in insect parasitoids. Trends in Ecology and Evolution 18: 8 1-87
van Alphen, J.J.M., and F. Galis. 1983. Patch time allocation and parasitization efficiency of Asohara tabida, a larval parasitoid of Drosophila, Journal of -4nimal Ecology 52: 937-952
van Driesche, R.G. 1983. Meaning of "percent parasitism" in studies of insect parasitoids. Environmental Entomology 12: 16 1 1-1 622
Waage, J.K. 1983. Aggregation in field parasitoid populations: foraging time allocation by a population of Diadegma (Hymenoptera: Ichneumonidae). Ecological Entomology 8: 447-453
Waage, J.K. 1979. Foraging for patchily-disrtibuted hosts by the parasitoid, Nemeritis canescens. Journal of Animal Ecology 48: 353-371
Waage, J.K. 1978. Arrestment responses of the parasitoid, Nemeritis canescens, to a contact chemical produced by its host, Plodia interpunctella. Physiological Entomology 3: 135- 146
Wajnberg, E., X. Fauvergue, and 0. Pons. 2000. Patch leaving decision rules and the marginal value theorem: an experimental analysis and a simulation model. Behavioural Ecology 11: 577-586
Wajnberg, E., M.C. Rosi, and S. Colazza. 1999. Genetic variation in patch time allocation in a parasitic wasp. Journal of Animal Ecology 68: 12 1-133
Wiens, J.A. 1976. Population responses to patchy environments. Annual Review of Ecology and Systematics 7: 8 1 - 120
Figure 5-1 Ethogram of Carnpoplex dubitator pre-oviposition and post-oviposition behaviour when attacking Enarmonia formosana hosts. Width of each solid line is associated with the transitional probability of the indicated behavioural event (n = 28). Following Check behaviour, the transitions between Rest, Probe, Search, Groom, and Walk become much less predictable. See Appendices 5-A and 5-B for actual values of transitional probabilities between behaviours. See text for definition of individual behaviours.
Figure 5-2 Mean (A SE) patch residence times of Campoplex dubitator on patches containing different densities of Enarmonia formosana hosts.
% 4000 V
3500 .- + $ 3000
2500 E 3 2000 L
5 1500 CI
8. 1000 - a
500 I-
0 0 1-5 6-10 11-15
Number of hosts
Figure 5-3 Proportion of Enarmonia formosana hosts parasitised by Campoplex dubitator versus host density. Percent parasitism is calculated based on the number of ovipositions observed during the patch visit (trials with zero ovipositions are kept in data set) ( r2 = 0.045; P = 0.222).
Number of hosts
Table 5-1. Average transitional probabilities between behaviours displayed by foraging Carnpoplex dubitator females from beginning of search to oviposition event.
Fol
low
in
Beh
avio
ur
IT
Lea
ding
B
ehav
iour
wal
k
sear
ch
prob
e
ovip
osit
chec
k
groo
m
rest
ovip
osit
ch
eck
1 groom
1 res
t 1 leav
e
Stat
isti
cs
Wal
k
---
anal
vsis
not
~o
ssib
le *
sear
ch
0.98
---
0.2
1
0.58
leav
e
prob
e
0.95
---
0.42
I I
wit
h an
equ
al fr
eque
ncy.
I
The
chi
-squ
are
anal
ysis
test
ed th
e nu
ll hy
poth
esis
that
par
asit
oids
wou
ld s
wit
ch f
rom
a p
artic
ular
beh
avio
ur to
any
oth
er
* Too
few
par
asit
oids
res
ted
prio
r to
ovi
posi
tion
to a
llow
a p
rope
r an
alys
is o
f th
e tr
ansi
tion
fre
quen
cies
in th
is
row
.
---
---
---
- 1 abie 5-2. Average transitional probabilities between behaviours displayed by foraging Campoplex dubitator females from oviposition to departure from host.
* Fai
lure
to r
ejec
t th
e nu
ll m
ust
be i
nter
pret
ed w
ith
caut
ion
as th
e po
wer
of t
he a
naly
sis
was
typi
cally
bel
ow t
he d
esir
ed l
evel
of
0.8
0.
Lea
ding
B
ehav
iour
wal
k
sear
ch
prob
e
ovip
osit
chec
k
groo
m
rest
1 le
ave
The
chi
-squ
are
anal
ysis
test
ed th
e nu
ll hy
poth
esis
that
par
asit
oids
wou
ld s
wit
ch f
rom
a p
artic
ular
beh
avio
ur to
any
oth
er w
ith
an e
qual
fre
quen
cy. wal
k
---
0.13
0.07
0.16
0.12
---
sear
ch
0.09
---
0.81
0.04
0.06
0.22
0.26
_
__
_
---
Fol
low
ing
Beh
avio
ur
Stat
isti
cs
prob
e
0.38
P
ovip
osit
---
0.25
---
chec
k
0.08
---
---
groo
m
0.1
1
0.14
0.78
---
0.3
0.3
---
rest
0.07
24.7
94
<0.
001
23.2
3 <
0.00
1
0.06
8.
727
0.19
0.
556
0.13
-
leav
e
0.63
x2
P
*
17.4
89
0.00
8
---
0.2
---
0.07
0.13
---
0.25
---
---
6.92
7 0.
328
0.45
4
7.96
3 0.
241
0.51
9
Table 5-3. (a) Estimated regression coefficients (P), standard errors (SE), and hazard ratio (exp(j3)) for only those covariates that had an effect on the patch leaving tendency of Campoplex dubitator females. (b) Estimated regression coefficient (P), standard error (SE), and hazard ratio (exp(j3)) for the single covariate that had an effect on the giving up time of C. dubitator females.
Covariate I3 SE exp(P) d f P 2
Patches with 0 hosts 0.00 --- 1 .OO Patches with 1-5 hosts -1.98 0.55 0.14 Patches with 6-1 0 hosts -2.88 0.62 0.06 Patches with 1 1-1 5 hosts -4.40 0.71 0.01 5.747 3 0.057
Frass tube encounters with -0.21 0.09 0.81 6.214 1 0.013 successful oviposition
Frass tube encounters without -0.04 0.01 0.96 9.721 1 0.002 successful oviposition
Covariate I3 SE exp(P) d f P 2
Frass tube encounters without -0.02 0.01 0.98 6.960 1 0.008 successful oviposition
CHAPTER 6 Conclusions and Final Remarks
6.1 Conclusion
The CBT, a recent invasive species in British Columbia, Washington, and Oregon, is
recognised as a serious potential threat to cherry and other rosaceous ornamental trees. As it
continues to slowly spread east and south across the Pacific coast, researchers in Canada and the
United States are taking action to reduce the number and severity of infestations. Traditional
approaches to combating the CBT in Europe and Asia have included the reduction of vegetation
from the bases of trees (Dickler and Zimmerman, 1972), mechanical removal of dead and peeling
bark, thinning of tree canopy, reduction of orchard density, and, most commonly, the application
of insecticides, creosote and tar oil (Roediger, 1956). In North America, apart from preliminary
studies of pyrethroid or organophosphate efficacy (Murray et al., 1998), insecticides were given
less attention due to an interest in an IPM approach. Other investigated methods of pest
management have included pheromone-based mating disruption (McNair et al., 1999),
entomopathogenic nematode application (McNair, pers. comm.), and biological control with the
indigenous egg parasitoid, Trichogramma cacoeciae Marchal (Hymenoptera:
Trichogrammatidae) (Tanigoshi, 2002).
Following repeated observations of lower levels of CBT infestations and a more robust
natural enemy community in Europe (Tanigoshi et al., 1998), the decision was made to
investigate the feasibility of a classical biological control programme against the CBT in North
America. The studies presented in Chapters 2 through 5 were conducted as part of the early phase
of this programme. With monitoring of the CBT distribution in North America ongoing, I studied
several aspects of the pest and its parasitoids in central Europe.
A large suite of hymenopteran parasitoids attacks the larvae and pupae of the CBT in Europe.
The majority of these species parasitise the host pupae, but none of the pupal parasitoids were
collected with any consistency. The most abundant of the sixteen species identified was the larval
endoparasitoid, Campoplex dubitator Horstmann. This species was recovered from CBT hosts
collected in all regions of the survey area, suggesting a strong host-parasitoid relationship with no
noticeable geographic or climactic restrictions.
The CBT occurs naturally on ornamental and orchard cherry trees in the Alsace of France, the
southern Rhine Valley and Black Forest of Germany, and the Jura Mountains of Switzerland.
While its distribution between trees and between habitats is patchy, its within-tree distribution is
almost always skewed toward the base of the tree. It is suspected that the microclimate near the
ground is more favourable for egg and larval development (Roediger, 1956), although it was not
possible to find supporting evidence for this assumption. It is interesting to note as well that the
CBT is more evenly distributed, if not showing the reverse within-tree distribution, on ornamental
cherry trees sampled in Vancouver, British Columbia. Reasons for this difference and its potential
significance can only be speculated.
The CBT has only a single generation per year, overwintering in the feeding gallery as a mid
to late larval instar. Although the stages of the CBT's life history are not strictly synchronised,
measurements of larval head capsules show a clear peak in adult flight tha t occurs hetween mid
June and early July. While this information is useful for pesticide application or parasitoid release
programmes, it is necessary to consider the proportion of the population that emerges either
earlier or later. In terms of biological control with a parasitoid agent, it may be necessary to
perform releases following a strict schedule, depending upon the oviposition needs of the
parasitoid species. Consider the egg parasitoid, I: cacoeciae, for example. Since neonate host
larvae can emerge within only 8 days following oviposition (Roediger, 1956), the window of
opportunity for successful parasitism is brief, Despite the relatively prolonged flight season of the
CBT, an inundative release of these wasps would be successful only when there were sufficient
numbers of host eggs available, and this would only occur during the peak in CBT flight. In
contrast, the larval parasitoid C. dubitator can parasitise any CBT instar but the first. Since this
species actively parasitised hosts throughout the entire summer in Europe and larvae are present
at all times, releases into North America could likely be made either early or later in the summer
with little change in the likelihood of establishment.
Following initial surveys of field parasitism, C. dubitator was selected as candidate classical
biological control agent, based on the following observations: (1) it made a significant
contribution to host mortality (approximately 85% of combined larval and pupal parasitism), (2)
it potentially had a high degree of host specificity, and (3) because it could be collected in
moderate numbers from CBT hosts, it was by far the most feasible to rear in culture for biological
studies. Despite rigorous field sampling, the remaining parasitoid species were collected in
insufficient numbers for the establishment of laboratory colonies and therefore could not be
studied.
Based on field data, the response of C. dubitator to host density per tree appeared to be
inversely density dependent. Over the last quarter-century, there have been many attempts to
identify foraging responses of parasitoids to host density that lead to population stability with low
host equilibrium (Hassell, 1982; Hassell et al., 1985; Lessells, 1985), features of paramount
importance for biological control. The conclusion generally agreed upon is that the density
dependence of a host-parasitoid relationship depends upon various characteristics of the system
(Hassell et al., 1985; Lessells, 1985). Nonetheless, with a basic understanding of a parasitoid's
density response under varying conditions and the critical parameters of a target host population,
one might predict the outcome of the host-parasitoid interaction. In Chapter 5, an experiment was
designed to further assess the response of C. dubitator to different CBT densities. This test found
that, although females spent more time on patches with higher host densities, particularly when
hosts were encountered, the rate of parasitism was not dependent on host density. The failure to
detect density dependence in these trials, in contrast from the field survey, may have stemmed
from either an inadequate sample size or the use of different scales in making the comparison
(Walde and Murdoch, 1988). While unable to explain the inverse density dependence observed in
the field, this experiment illustrated the flexible patch foraging behaviour and specific responses
of C. dubitator to contacts with and ovipositions into hosts.
Due to difficulties with rearing the CBT larvae on a meridic diet, much attention was given to
the maintenance of host and parasitoid cultures. I amassed information on the development rates
of hosts and parasitoids, the suitable instars for C. dubitator development, and the effect of the
host instar attacked on parasitoid size. In addition, the oviposition behaviour of C. dubitator was
studied in detail, providing insight into this wasp's ability to locate and subdue host larvae and to
discriminate against previously parasitised hosts to avoid wasteful superparasitism. An evaluation
of the response of C. dubitator to odours from the host and host habitat showed that foraging
females rely heavily on indirect cues associated with the host. The next major step in this
classical biological control programme is to conduct host range tests for C. dubitator. The
knowledge of this parasitoid's foraging process will be especially valuable for the selection of
non-target species for such host range testing. If volatiles emitted from trees attacked by the CBT
are an important cue for foraging C. dubitator, then it may be possible to assume that the risk to
non-target species closely related to the CBT, but in different habitats, will be minimal (van
Driesche and Hoddle, 1997; Kuhlmann and Mason, in press).
The work presented in this thesis, while broad in scope, contributed significantly to the
assessment of the feasibility of a classical biological control programme against the CBT.
Research on the CBT, from these and other studies, has formed an extensive source of
information that will valuable for future work with this species. Also, these first studies of the
biology and behaviour of C. dubitator have contributed to the general investigation of parasitoid
natural history, chemical ecology, behavioural ecology, and biological control. Ironically, the
labour-intensive rearing of these two species remains to be one of the largest hurdles yet to
overcome. To mass-rear the CBT or C. dubitator in the future, it will be necessary to develop a
rearing technique that will reduce the effort required to sustain laboratory cultures. A significant
improvement could be made by producing a meridic diet that is suitable for the establishment of
neonate larvae. Further studies of the parasitoid would also benefit from reduced development
times and a method of increasing the fema1e:male sex ratio.
6.2 References
Dickler, E, and H. Zimmerman. 1972. [Investigations on the control of the bark Tortricid Enarmonia formosana Scop. (Lepid., Tortr.)] Mitteilungen aus der Biologischen fur Land- und Forstwirtschaft, Berlin-Dahlem, 144: 143- 150
Hassell, M.P. 1982. Patterns of parasitism by insect parasitoids in patchy environments. Ecological Entomology 7: 3 65-3 77
Hassell, M.P., C.M Lessells, and G.C. McGavin. 1985. Inverse density dependent parasitism in a patchy environment: a laboratory system. Ecological Entomology 10: 393-402
Kuhlmann, U. and P.G. Mason. In press. Use of field host range surveys for selecting candidate non-target species for physiological host specificity testing of entomophagous biological control agents. Proceedings of the First International Symposium on Biological Control of Arthropods USDA Forest Service, Honolulu, Hawaii
Lessells, C.M. 1985. Parasitoid foraging: should parasitism be density dependent? Journal of Animal Ecology 54: 27-4 1
McNair, C., G. Gries, and M. Sidney. 1999. Toward pheromone-based mating disruption of Enarmonia formosana (Lepidoptera: Tortricidae) on ornamental cherry trees. The Canadian Entomologist 131: 97-1 05
Murray, T.A., L.K. Tanigoshi, B. Bai, and E. LaGasa. 1998. Cherry bark tortrix, Enarmonia formosana (Scopoli), bionomics, natural enemy survey and control research project, 1997-98. Washington State University Report
Roediger, H. 1956. Untersuchungen iiber den Rindenwickler Enarmonia woeberiana Schiff. (Lepid. Tortr.) Zeitschrijit fur Angewandte Entomologie 38: 195-32 1
Tanigoshi, L.K. 2002. Conservation and classical biological control of the cherry bark tortrix in the pacific northwest. Final Project Report 2002, Department of Entomology, WSU Vancouver Research and Extension Unit, Vancouver, Washington
Tanigoshi, L.K., B.B. Bai, and T.A. Murray. 1998. Biology and Control of the Exotic Cherry Bark Tortrix, Enarmonia formosana. Oregon Department of Agriculture Interim Project Report, 1998
van Driesche, R.G. and M. Hoddle. 1997. Should arthropod parasitoids and predators be subject to host range testing when used as biological control agents? Agriculture and Human Values 14: 21 1-226
Walde, S.J. and W.W. Murdoch. 1988. Spatial density dependence in parasitoids. Annual Review of E n t o m o l o ~ ~ 33: 44 1-466