A simple olfactometer for the investigation of sex pheromones and other olfactory attractants in fruit flies and moths

By B. I. KATSOYANNOS, E. F. BOLLER and U. REMUND

Abstract

A simple olfactometer is described for quantitatively measuring the response of Diptera and Lepidoptera to sex pheromones and other olfactory attractants. Selected data obtained with the fruit fly species Rhugoletis cerusi, Cerutitzs cupitata, Dacus oleue and the grape moth Eupoeczliu (Clysiu) urnbiguellu are presented to illustrate the practical applications of the instrument. This olfactometer has been adopted as one of the standard instruments for use in the international “RAPID Quality Control System” developed for the Mediterranean fruit fly, Cevutitis cupitutu.

1 Introduction

Since the earliest recognition that odors play an important role in the life of insects, many efforts have been made to investigate, qualitatively and quantita- tively, the various aspects of the insect-odor relationship. Olfactometers are devices often used for qualitative and/or quantitative assessment of insect response to odors; they consist basically of wind-tunnels where insects are exposed to odor treated airstreams. The behavioral response of the test animals to the given odors can be studied and measured in various ways that depend largely on the desi n of the olfactometer and of the experiments.

Olfactometers 8ave been very useful in the past for screening volatile chemicals and natural products as potential tools in pest management and population monitoring, as well as tools in the bioassays required for the isolation, identification and synthesis of sex pheromones or host odors. More recently, olfactometers have been applied in quality control programs for mass-reared fruit-flies (BOLLER and CHAMBERS 1977). It is beyond the scope and intention of this paper to discuss the pertinent theory behind olfac- tometry, or to enumerate the many designs of olfactometers that have been published in the past. We refer in this respect to the recent review of KENNEDY (1977).

The olfactometer described in this paper was primarily developed in order to conduct sex pheromone investi ations by the European cherry fruit fly,

simple in construcbion and operation; 2. It has a wide range of applications, such as the investigation of sex pheromones and other attractive odors in Diptera and Lepidoptera; 3. It produces data with small variability between replicates, and 4. It produces data that correlate well with similar observations made in the field. It has been used extensively with R. cerasi, the Mediterra- nean fruit fly, Ceratitis capitata Wied., and to a lesser extent with the olive fly, Dacus oleae Gmel. and the grape moth, Eupoecilia (Clysia) arnbiguella Hbn.

Rhagoletis cerasi L. It meets the f ollowing desirable requirements: 1. It is

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106 H. I . Krrtsoyannos, E . F. Holler, U. Reinund

and has produced highly satisfactory results. AS the presentation of all data obtained in the course of our investigations is not the primary objective of this report, we limit the experimental data to a few selected examples only, in order to supplement the more technical description of the device and illustrate its practical application.

2 Materials and methods

2.1 The test insects

The test insects used in our investigations had various rearing and handling histories. The specifics of those strains that were used in these experiments are outlined in Table 2.

The European cherry fruit flies (R. cerusz), were emerged from pupae collected from sweet cherries (Prunus avium L.), in the field. The Mediterranean fruit flies (C. cupirata), were reared from wild pupae collected in various European countries and shipped to our laboratory for testing within the framework of an international quality control program (BOLLER et al. in standard laboratory strain mentioned in this paper, was obtained from the IAEA lalz:>or;t Seibersdorf, Austria, and had been reared for three generations on our standard rearing system developed for R. cerasi(KATSOYANN0S et al. 1977). The olive flies (D. oleae), were sent to us by the Dernocritos Nuclear Research Center at Athens, Greece, as pupae either collected in the field from olives, or reared in the laboratory on an artificial diet for about 70 generations. All of these flies had been separated according to sex within 24 h after emergence, and held under standardized laboratory conditions (table 1) until they reached the appropriate age for the olfactometer tests.

The grape moth (€. ambiguella), were provided by our permanent laboratory colony that had reached approximately the 10th generation of laboratory propagation at the time of the test. Males and females were sexed in the late pupal stage, and the emerged moths were held separately in containers with a supply of 10 % sugar solution until tested in the olfactorneter.

2.2 Technical description of the olfactometer

The design of the olfactometer incorporates experience gained in earlier attempts with R. cerasi, certain aspects of other olfactometers developed for other fruit flies (e. g. NATION 1972; HANIOTAKIS 1974), and observations on the behavior of R. cerasi females in field-cage and orchard studies (KATSOYAN- NOS 1976, 1979). We used existing plexiglass cages and other suitable materials from the R. cerasi rearing program for the construction of the prototypes, and therefore did not carry out experiments with the aim of perfecting shape and dimensions. We describe here two types of olfactometers. The standard, extensively used one (Type A), and a modification of it (Type B).

2.2.1 The standard olfdctometer (Type A)

The olfactometers are usually used in series of 4 to 6 units that are operated simultaneously, but which function independently of each other. A schematic diagram of a Type A olfactometer unit is given in Figure 1. Each unit consists of three primary elements: test cage, tube system, and ventilator. The test cage (?) of plexiglass (40 X 30 X 30 cm) holds the test insects for response to the given odor. The bait cage (5), which is part of the test tube system, contains the source of attractant, either pheromone-producing insects or test chemicals applied to a dispenser such as a piece of cotton or other carriers. A fan (7) blows an air current through the tube system (2-6). The air picks up the odor in the bait cage (5) and carries it to the test cage (I) where it fills the entire cage volume, and is pushed through the wire-screened opening (23 cm diam) (8)

1

a - 1 - ; - 1 1 v--zi 2>31 \ I 4 ~ 5 6 c l l 8 /” - c I1

- 1 - 1 I1

4

Fig. 1. A: Schematic diagram (side view) of an olfactometer unit of Type A. 1 = test cage, 2 = wire-screen funnel leading into, 3 = catch chamber, 4 = intermediate tube, 5 = bait cage, 6 = end tube of tube system, 7 = ventilator, 8 = exit of airstream through ventilation window in test cage

ceiling. For details see text. B: Modified Type B olfactometer

into the experimental room. All individual units of the tube system are made of the same round, translucent, polystyrol boxes (14 cm long), which are slightly conical in shape (upper and lower diameter 11 .O and 11.5 cm, respectively), and can be stacked together. The narrow end of tubes 4, 5, and 6 are covered by wire-screening.

Fig. 2. Four olfactometer units of Type A in operation

108 B. I . Katsoyannos, E. F. Boller, I/. Remund

We have used commercially available fans (Mikrolufter R 90, Ventronic AG, Zurich) of 96 mm diameter, a capacity of 90 m 3/h, and a rotation of 2400 r.p.m. These small fans placed 5 cm in front of the tube system produce an airstream of ca. 1 m/s at the wide end of tube 6, as estimated with a commercially available hand-anemometer (Koch-Optik AG, Zurich). The experimental room itself should have ventilation adequate to remove odor- contaminated air and to provide the necessary amount of fresh air. Light banks installed ca. 1 m above the olfactometers produce the desirable light intensity in the test cages (usually 2500 lux).

Test insects in the test cage ( I ) responding to the odor stream orient themselves toward the odor source, land on the wire- screen cone (2) and are funneled into the catch chamber (3), where they are counted after termination of the test.

Because the entire tube system containing the odor source (units 5 and 6) is removed after each test, the test insects in the test-cage (1) can be exposed to different odor sources in sequence by inserting a different odor-cartridge.

Fig. 2 shows four units of the olfactometer in operation.

2.2.2 The modified olfactometer (Type B) The olfactometer of Type B (fig. 1) differs to the Type A in that the large test cage (1) is substituted by a cylindrical tube made of two units of the tube system (the screening of the inner container removed) and also that the air escapes through the screening of the test cage (8) at the same level as the fan.

Table 1. Experimental conditions for standardized sex pheromone bioassays of 3 fruit fly species and 1 lepidopteran species

Experimental parameter

Olfactometer type Tern erature ("C) Rel. [umidity ("0)

Photoperiod (h) Light intensity (lux)

Time of photoperiod for bioassay (h) Bioassay duration (min) Age of insects for phero- mone production and response (d) wild strains

lab strains Number of insects/cage Pheromone producing sex

Test insects Diptera Lepidoptera

Rhagoletis Ceratitir Dacur Eu oecilia cerast capitata oleae amLgue"a

A A B A B 25 25 25 25 25 60 60 60 60 60 18 14 14 12' 18

2500 2500 2500 2500 2500

9-13 1-8 9-12 second halP 1250

(dim light) of dark phase 45 15 5 15 15

7-20 14-20' 5-20 7-20 3-20 5-20 2-1 0 50 50 20 50 20

male male female female

' During the last 3 h of the photoperiod the light intensity was reduced by 50 %

the behavior

plant

During the dark-phase low light intensity (below 10 lux) can be used without interfering with

Sexual maturation of females was very slow and influenced by geographic origin and host

A simple olfactometer f o r the investigation of sex pheromones

2.3 Experimental procedures

109

Because all elements of the olfactometer can easily be dismantled, the test insects can be put into the olfactometer the day of the experiment. However, when experiments must start early in the morning we recommend introducing the test insects in the evening of the previous day to allow them to adapt to the new environment. Prior to the first test, the lid covering the cage entrance (2) is removed, and the units of the tube system 3 and 4 are fitted to the test cage and secured with tape. At the beginning of the bioassay, the bait cage (5) with connected end tube (6), is introduced into the unit 4. The entire system is then positioned at the proper distance (5 cm) from the fan, the airstream generated, and a time-clock started. Upon termination of the test (usually after 15 min) the airstream is interrupted, the odor-cartridge is removed and the olfactome- ter is turned around 180 degrees. Because the insects caught in the catch- chamber (3) are now orienting themselves towards light from behind the cage, the concentrate on the funnel-shaped screening (2), and can be easily counted

conducted with the same insects after about 1 hour. Usually half of the olfactometer units are used as control units without an odor source in the bait cage (5), or with solvents alone where solutions of chemicals are tested.

Whereas only virgin females can be used for tests that assess response to male- roduced sex pheromone, this is not an essential prerequisite for tests

schedule for tests which measure the responses to sex pheromones, it is important to take into account the intrinsic rhythmicity of pheromone pro- duction and response, as determined for each species in preliminary tests.

The experimental conditions for standardized sex pheromone bioassays for the investigated species are summarized in table 1.

an di put back into the same test cage. The next series of experiments can be

with f ood lures or host odors. However, when establishing the experimental

3 Results and discussion

Some selected examples of data obtained with the described two types of the olfactometer are given in table 2. The test insects used in these investigations show a strong response to the attractant whereas their response to control, odor-free bait cages is very low, and in the case of C. capitata even close to zero when the olfactometer of type A is used (table 2, expt. 3). In such situations we can reduce the number of untreated replicates to a minimum, especially when a simultaneous comparison of two or more strains makes the availability of additional olfactometer units highly desirable. Although we present only a limited amount of experimental data in order to demonstrate the information output, we would like to point out that with this olfactometer we were able to measure for the first time the response of R. cerasi to sex pheromones in the laboratory. Furthermore, we have increasing evidence that the data obtained with the laboratory olfactometer can be confirmed in field experiments. Such data have now been accumulated for R. cerasi with respect to daily periodicity, influence of mating history and pheromone concentration on the response of females to the male sex pheromone (KATSOYANNOS 1979), and for C. capitata with respect to daily periodicity of response, and the influence of strains and quality characteristics on the response to sex phero-

Tabl

e 2.

Rep

rese

ntat

ive

data

fro

m 2

dif

fere

nt t

ypes

of

olfa

ctom

eter

s und

er s

tand

ardi

zed

test

con

ditio

ns

% R

espo

ndin

g in

dwid

uals

T

est

spec

ies

Exp

t. T

ype

of a

ttra

ctan

ts a

nd o

f tr

eatm

ents

' N

O.

Rhag

olet

is ce

rasi

1 Se

x ph

erom

one

(A)

50 P

? 50

66

15

31.1

8.

7 (w

ild)

50 P

O no

flie

s 15

3.

6 3.

3

2 Fo

od lu

re (A

) 50

P?'

fo

od lu

re4

7 33

.3

5.8

NO

. ol

fact

omet

er (A

, B)

T

est

cage

B

ait

cage

re

ps.

X

50 9

9 fo

od l

ure

8 6.

0 5.

9

Cer

atiti

s cap

itata

3

Sex

pher

omon

e (A

) 50

??

50

8d

15

48.8

6.

3 (l

abor

ator

y st

rain

) 50

99

no f

lies

15

0.5

0.5

4 Se

x ph

erom

one

(B)

20 9

9 20

66

6 81

.7

9.8

20 P

O 20

dd,

no

6 31

.7

11.3

20 P

O

no fl

ies

6 15

.0

6.3

vent

ilato

r5

Dac

us o

leae

5

Sex

pher

omon

e (A

) 50

88

(lab

) 50

P?

(lab

) 11

40

.5

8.1

50 d

d (w

ild)

50 P

P (w

ild)

5 45

.6

12.2

50

66

(lab

) no

flie

s 6

14.3

5.

3 Eu

poec

ilia

6 Se

x ph

erom

one

(B)

20 88

20 P

P 8

57.6

8.

0 am

bigu

ella

(la

b)

20 d

d no

mot

hs

8 2.

5 3.

8

All

inse

cts

used

wer

e vi

r in

s ex

cept

in

expe

rim

ent n

o. 2

with

foo

d lu

res.

-

Flie

s w

ere

star

ved

for

15-2

2 h

befo

re th

e te

st. -

U

nsta

rved

flie

s. -

10 Y

o ye

ast

hydr

olis

ate.

-

Air

ffo

w t

hrou

gh o

lfac

tom

eter

cau

sed

by a

ir-c

ondi

tioni

ng.

A simple olfactometer for the investigation of sex pheromoncs 111

mone (BOLLER et al., in prep.). This relationship between laboratory data and observations made in the field indicates that these olfactometer experiments carried out under defined laboratory conditions produce valuable data with respect to the field behavior of a given species.

Many modifications of the described olfactometer could be made without significantly reducing the inherent quality of the device as long as three principles are maintained, namely (a) the air has to be blown through the system and not be drawn through by aspiration, (b) the experimental room needs a good ventilation system adequate to remove odor-contaminated air, and (c) a suitable funnel-shaped screen has to be used at the entrance to the catch chamber which will allow a wide airstream to pass through the test cage. The two types of the olfactometer described here are good examples of such modifications. Although both types performed very well, each has its advanta- ges and shortcomings. For example, C. capitata flies in the Type B olfactome- ter respond and enter into the catch chamber too rapidly with most of the flies entering even during the first minute of the test, thus rendering difficult quantitative comparisons between strains. Also, larger numbers of this fly species enter the catch chambers of untreated control units (table 2, Expt. 4). O n the other hand, we obtained better results in this olfactometer with E . ambiguella (table 2, expt. 6). This was clearly caused by different behavioral patterns of flies and moths.

In preliminary experiments with the standard olfactometer (Type A) we observed that most of the responding moths were concentrated on the opposite wall facing the tube system and were forming there a swarm of approximately the diameter of the tube system. Only a small number of individuals, but still significantly more than in untreated control units, were able to land on the funnel and pass into the catch chamber within the defined period of the experiment. O n the other hand, no swarming could be observed in the control ca es. It is possible that the pheromone concentration at the back wall was hig K er than in the main airstream coming out of the tube system and thus the moths concentrated in that area. It was this observation that led us to develop the olfactometer of Type B. We anticipated that this artificial pheromone concentration at the back wall could be eliminated by replacing the solid wall by a wire screen and that the moths would stop swarming and would follow the odor plume into the catch chamber. The results obtained with the olfactometer of Type B, which incorporates this characteristic (table 2, expt. 6), confirm this hypothesis and indicate also the need for direct observations of the insects’ behavior. The type B olfactometer could be an interesting tool for measuring the response of mass-reared fruit flies under simple experimental conditions where two or more strains could be compared. However, we prefer the standard Type A olfactometer for investigations requiring more precision. The Type A olfactometer has been recommended for application in the international “RAPID Quality Control S stem” for C. cupitatu now being implemented in the major medfly rearing Y acilities (BOLLER et al., in prep.).

Acknowledgements

We thank Drs. C . 0. CALKINS, L. GRINGORTEN (IAEA Seibersdorf Laboratory, Austria) and D. L. CHAMBERS, USDA, Gainesville/Fla., USA, for critically reading the manuscript and making valuable suggestions. The help of Michelle Flury in the preparation of the manuscript is gratefully acknowledged.

112 A 1. Katsoyannos, E . F. Boller, II. Rrmund

Zusammenfassung

E-171 einfaches Olfaktometer zur quantitativen Messung der Reuktion von Fruchtfliegen und Motten auf Sexuufpheromone und andere gerucbliche Lockstoffe

Ein einfaches Olfaktometer wird technisch beschrieben und eine Anzahl ausgewahlter Resultate vorgestellt, welche mit der Kirschenfliege Rhugoletis cerasi, der Mittelmeerfruchtfliege Ceratitis cupituta, der Olivenfliege Dacus ofeue, sowie dem einbindigen Traubenwickler Eupoecifiu (CIysia) ambiguellu erarbeitet wurden. Mit dieser Apparatur gelang es zum ersten Mal, bei der Kirschen- fliege das Sexualpheromon im Laboratorium nachzuweisen. Das beschriebene Olfaktometer ist inzwischen als integrierter Bestandteil in das internationale “RAPID” Qualitatspriifungspro- gramm fur Ceratitis cupituta aufgenommen worden.

References

BOLLER, E. F.; CHAMBERS, D. L., 1977: Quality Control - An Idea Book for Fruit Fly Workers. IOBC/WPRS Bulletin 1977/5. 162 pp.

HANIOTAKIS, G. E., 1974: Sexual attraction in the olive fruit fly, Ducus oleae (Gmelin). Environ. Entomol. 3, 82-86.

KATSOYANNOS, B. I., 1976: Female attraction to males in Rhugoletis cerusi. Environ. Entomol. 5, 474-4 76.

- 1979: Zum Reproduktions- und Wirtswahlverhalten der Kirschenfliege, Rhagoletis cerusi L. (Diptera: Tephritidae). Diss. ETH Zurich No 6409.

KATSOYANNOS, B. I.; BOLLER, E. F.; REMUND, U., 1977: Beitrag zur Entwicklung von Methoden fur die Massenzucht der Kirschenfliege, Rhagoletis cerasi L., auf kiinstlichen Substraten. Mitt. Schweiz. Ent. Ges. 50, 25-33.

KENNEDY, J. S., 1977: Behaviorally discriminating assays of attractants and repellents. In: H. H. SHOREY and J. J. MCKELVEY, Jr. (eds.), Chemical Control of Insect Behavior-Theory and Application, 215-229, New York: Wiley-Interscience.

NATION, J. L., 1972: Courtship behavior and evidence for a sex attractant in the male Caribbean fruit fly, Amstrepha suspensu. Ann. Entomol. SOC. Am. 65, 1364-1367.

Authors’ addresses: Dr. E. F. BOLLER; U. REMUND, Fruit Fly Laboratory, Swiss Federal Research Station for Arboriculture and Horticulture, CH-8820 Wadenswil; B. I. KATSOYANNOS, Faculty of Agriculture and Forestry, University of Thes- saloniki, Greece