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MANIPULATION, REARING AND STORAGE OFTAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI

(HEMIPTERA: PSYLLIDAE)

By

XULIN CHEN

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2013

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© 2013 Xulin Chen

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To my parents

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ACKNOWLEDGMENTS

Funding for this research was provided by Dr. Phil Stansly from Citrus Research

and Development Foundation.

I thank my major professor Dr. Phil Stansly for his patient guidance, support and

pertinent suggestions. I also thank my other two committee members, Dr. Howard Frank

and Dr. Eric Rohrig, for their continuous support and advice. I thank Dr. Jawwad

Qureshi, for providing suggestions in my research.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 LITERATURE REVIEW .......................................................................................... 12

Huanglongbing ........................................................................................................ 12 Distribution ....................................................................................................... 12

Symptoms ........................................................................................................ 12 Pathogen .......................................................................................................... 13

Diaphorina Citri ....................................................................................................... 13

Distribution ....................................................................................................... 14 Transmission Mechanism between Pathogen and Vector ................................ 14

Host Plants ....................................................................................................... 15 Biology of D. Citri .............................................................................................. 16

Life cycle .................................................................................................... 16

Seasonal history ........................................................................................ 18 Dispersion of D. citri ................................................................................... 19

Damage by D.citri ...................................................................................... 19

Influence of temperature and humidity ....................................................... 20

Influence of light ......................................................................................... 21 Natural Enemies ............................................................................................... 21

Predators ................................................................................................... 21

Parasitoids ................................................................................................. 22 Tamarixia Radiata ................................................................................................... 22

Distribution ....................................................................................................... 22 Taxonomy and Identification ............................................................................. 23 Host Specificity ................................................................................................. 23 Biology of T. Radiata ........................................................................................ 24

Life cycle .................................................................................................... 24 Mating ........................................................................................................ 25

Oviposition ................................................................................................. 25

Host preference ......................................................................................... 26 Host-feeding ............................................................................................... 26 Sex ratio ..................................................................................................... 27 Reproduction .............................................................................................. 27 Superparasitism ......................................................................................... 28

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Host density effects .................................................................................... 28

Influence of food ........................................................................................ 29 Development time ...................................................................................... 29

Temperature and humidity effects .............................................................. 30

2 CARBON DIOXIDE ANESTHESIA OF TAMARIXIA RADIATA (WATERSTON) (HYMENOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................................................................... 33

Introduction ............................................................................................................. 33

Materials and Methods............................................................................................ 34 Colonies ........................................................................................................... 34 Gas Chamber ................................................................................................... 34 Recovery Time ................................................................................................. 35

Survival Rate .................................................................................................... 35 Percent Parasitism ........................................................................................... 36

Results .................................................................................................................... 36 Discussion .............................................................................................................. 37

3 FUNCTIONAL RESPONSE OF TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE) TO DENSITIES OF ITS HOST, DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................................................................... 40

Introduction ............................................................................................................. 40 Materials and Methods............................................................................................ 42

Colonies ........................................................................................................... 42 Arenas .............................................................................................................. 43

Statistical Analysis .................................................................................................. 43 Results .................................................................................................................... 44

Fecundity, Percent Parasitism and Percent Superparasitism ........................... 44

Functional Response ........................................................................................ 44 Discussion .............................................................................................................. 45

4 THE INFLUENCE OF DIET ON EGG FORMATION IN TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE), A PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................................................................... 50

Introduction ............................................................................................................. 50 Materials and Methods............................................................................................ 51

Colonies ........................................................................................................... 51

Diets ................................................................................................................. 52

Egg Load .......................................................................................................... 53 Statistical Analysis .................................................................................................. 53 Results .................................................................................................................... 54 Discussion .............................................................................................................. 54

5 DISCUSSION AND CONCLUSIONS ...................................................................... 61

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LIST OF REFERENCES ............................................................................................... 64

BIOGRAPHICAL SKETCH ............................................................................................ 73

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LIST OF TABLES

Table page 1-1 Demographic parameters T (generation time) per day, R0 (net reproductive

rate) nymphs per female, and r (intrinsic rate of increase) per day. .................... 32

3-1 Number of parasitized hosts (Mean ± SEM) at different host densities .............. 47

3-2 Percent parasitism (Mean ± SEM) at different host densities ............................. 47

3-3 Percent superparasitism (Mean ± SEM) in different host densities, mean number ............................................................................................................... 47

4-1 Mean ± SEM number of eggs by treatment from dissections after 5, 10, 15,

and 20 days at 17 C. .......................................................................................... 56

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LIST OF FIGURES

Figure page 2-1 Carbon dioxide gas chamber .............................................................................. 38

2-2 Tamarixia radiata female and male recovery time frequency (%) distribution .... 38

2-3 Mean (SEM) survivorship of CO2 treated and untreated Tamarixia radiata adults. ................................................................................................................. 39

3-1 Number of parasitized host at different host densities, error bar stands for SEM .................................................................................................................... 48

3-2 Percent parasitism at different host densities, error bar stands for SEM ............ 48

3-3 Percent superparasitism in different host densities ............................................ 49

3-4 Number of D. citri parasitized by T. radiata and the functional response curve (Type II) .............................................................................................................. 49

4-1 Newly emerged (unfed) T. radiata female digestive system ............................... 57

4-2 Newly emerged (unfed) T. radiata female .......................................................... 57

4-3 Paired T. radiata ovaries after feeding on honey for 20 days (egg resorption) ... 58

4-4 T. radiata ovary after feeding on Nu-Lure+honey for 10 days ............................ 58

4-5 Paired T. radiata ovaries after feeding on nymphs for 5 days ............................ 59

4-6 Paired T. radiata ovaries after feeding on honey +nymphs for 10 days .............. 59

4-7 Paired T. radiata ovaries after feeding on Nu-Lure+ nymphs for 10 days........... 60

4-8 Paired T. radiata ovaries after feeding on Nu-Lure+honey+nymphs for 5 days .. 60

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the

Requirements for the Master of Science

MANIPULATION, REARING AND STORAGE OF TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI

(HEMIPTERA: PSYLLIDAE)

By

Xulin Chen

May 2013

Chair: Phil. Stansly Major: Entomology and Nematology

Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous

ectoparasite of the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera:

Psyllidae), vector of citrus greening disease or huanglongbing (HLB). Tamarixia radiata

is being tested as an augmentive biological control agent, since the number present in

the field is low following winter when few hosts are available. Any program aimed at

augmentation of a natural enemy would require an efficient system of mass production.

Key components would include methods of manipulating, rearing and storing large

populations of insects. This research investigated the use of carbon dioxide (CO2) to

anesthetize T. radiata, determined optimal host densities for rearing the parasitoid, and

evaluated diets to maintain fecundity during storage periods.

Tamarixia radiata adults held in an atmosphere of 100% CO2 for 5 min were

immobilized for about 4 min. However, survivorship and fecundity were reduced

significantly, although sex ratio of progeny from treated adults was not affected.

Consequently, lighter doses of CO2 or other methods of anesthesia are needed.

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One pair of three-day-old T. radiata was released in a centrifuge tube for five

days with different 4th instar D. citri nymphs densities: 10, 20, 30, 40, 50, and 60. At a

density 40, 4th instar nymphs per female fecundity, incidence of parasitism and

superparasitism were all optimal. The pattern of parasitism for the first five days

conformed to a Type II functional response. Estimated searching efficiency was 0.442 ±

0.036 per day and estimated handing time was 0.045 ± 0.008 days. Therefore, 40 hosts

per female T. radiata should be a target for mass rearing.

Females feeding on eight different diets were dissected every 5, 10, 15 and 20

days to check the number of eggs in ovaries. The result showed that T. radiata formed

more eggs feeding on mixed diets (Nu-Lure+ honey+ nymphs or Nu-Lure+ nymphs)

compared to nymphs alone. Thus, it is recommended that protein and carbohydrate

supplements along with host nymphs be provided to T. radiata before release in the

field.

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CHAPTER 1 LITERATURE REVIEW

This chapter reviews (1) distribution, symptoms and pathogen of Huanglongbing,

(2) biology, distribution of Diaphorina citri Kuyawama, (Hemiptera: Psyllidae), vector of

Huanglongbing, the transmission mechanism between pathogen and vector, and natural

enemies of D. citri, (3), biology of Tamarixia radiata (Waterston) (Hymenoptera:

Eulophidae), a major biological control agent for D. citri.

Huanglongbing

Distribution

Huanglongbing (HLB) is one of the most destructive diseases of citrus in the

world (Halbert and Manjunath 2004, Teixeira et al. 2005, Bové 2006, Wang et al. 2006,

Batool et al. 2007, Manjunath et al. 2008). It has been reported from Asia: eastern

Japan, southern China, Indian subcontinent and Pakistan, and also in the Arabian

peninsula for some time. An African form is found in eastern, central, and southern

Africa (Gottwald 2007). HLB was first discovered in the western hemisphere in Brazil in

July 2004 (Teixeira et al. 2005). The first discovery of HLB in North America occurred in

south Florida in August 2005 (Halbert 2005). By December 2007, it had spread through

30 counties which were all located south of Marion County (Hall 2008a).

Symptoms

Huanglongbing means "yellow dragon disease" or “yellow shoot disease” in

Chinese because of the yellow shoots which are early symptoms of the disease. In

addition to yellow shoots, typical symptoms also include blotchy (asymmetric) mottling,

chlorosis resembling zinc deficiency which is followed by leaf drop and twig dieback

(Halbert 2004). Symptomatic fruit are small, lopsided with small, dark aborted seed and

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discolored vascular bundles in the fruit axis. The stylar end of symptomatic fruit remains

green even as they mature (Gottwald 2007). Roistacher (1996) reported that HLB

damaged at least 10-15% of trees each year in Thailand, which could lead many citrus

areas to go out of business. Infected citrus trees do not die immediately but decline

within 8-9 years (Roistacher 1996).

Pathogen

The causative agent is a vector-borne α-proteobacterium (Jagoueix et al.1994)

which has not yet been isolated in pure culture (Garnier and Bové 1993). Three etiologic

agents of HLB have been implicated based on their 16S rRNA sequence: Candidatus

Liberibacter asiaticus (Asia, North America, and Brazil), Ca. Liberibacter americanus

(Brazil), and Ca. Liberibacter africanus (Africa) (Garnier et al. 1984, Jagoueix et al.

1996, Sagaram et al. 2009). Of the three agents, only Candidatus Liberibacter asiaticus

(Las) was discovered in Florida in August 2005 (Halbert 2005), which led to an

estimated 1.6% of orange trees in Florida being infected by 2008 (Morris et al. 2009a,b).

Candidatus Liberibacter asiaticus is transmitted by psyllid vectors, and can also be

transmitted by grafting, dodder, and possibly by seed, but not by contamination of

personnel and tools or by wind and rain (Halbert 2010).

Diaphorina Citri

To date, two vectors have been reported for HLB disease in the world, Asian

citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Sternorrhyncha: Psyllidae), and

African psyllid, Trioza erytreae (del Guercio) (Hemiptera: Sternorrhyncha: Triozidae). D.

citri is responsible for transmitting Ca. L asiaticus in North America, Brazil and Asia, and

Ca. L americanus in Brazil, while T. erytreae transmits Ca. L africanus in the Middle

East, Reunion and Africa (Halbert and Manjunath 2004). Of the two species, D. citri is

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more heat tolerant, while T. erytreae is sensitive to high temperatures (Massonie et al.

1976). D. citri was first detected in Florida in 1998 (Halbert 1998) and later become

established throughout the state (Halbert 2010) and in other southeastern states.

Distribution

Diaphorina citri is thought to be indigenous to tropical and subtropical Asia and

has been reported in the following geographical areas: China, India, Burma, Taiwan,

Philippine Islands, Malaysia, Indonesia, Ceylon, Pakistan, Thailand, Nepal, Sikkim,

Hong Kong, Ryukyu Islands, Afghanistan, Saudi Arabia, Reunion and Mauritius (Mead

1977, Halbert and Manjunath 2004). D. citri was also known to occur in South America

in Brazil (Lima 1942, Mead 1977). During the 1990s, D. citri invaded the West Indies

(Guadeloupe), Abaco Island, Grand Bahama Island, and Cayman Islands (Halbert and

Núñez 2004). In June 1998, D. citri was detected on the east coast of Florida, from

Broward to St. Lucie counties (Halbert 1998), by September 2000, this pest had spread

to 31 counties in Florida (Halbert et al. 2001). During 2001, it was found in the

Dominican Republic, Cuba (Halbert and Núñez 2004), Puerto Rico (Pluke et al. 2008)

and Texas (French et al. 2001).

Transmission Mechanism between Pathogen and Vector

Information about interactions between D. citri and the pathogen Las is quite

limited. Nymphal stages of D. citri have the ability to acquire HLB pathogen during the

later instar development when growing on the pathogen-infected trees. Adults emerging

from these infected nymphs can transmit the pathogen immediately after emergence

(Capoor et al. 1974, Xu et al. 1988). Uninfected adults feeding on infected trees can

also acquire the pathogen (Xu et al. 1988). However, compared with the adults, later

instar nymph stages have a higher efficiency in acquiring the pathogen in nature (Xu et

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al. 1988). In more recent studies, nymphs reared on Las-infected trees were also more

likely to acquire the pathogen than adults (Pelz-Stelinski et al. 2010). Acquisition access

period (AAP), latent period, and inoculation access period (IAP) all varied a lot in the

past studies. An AAP of 15min to 24h was reported for both D. citri and T. erytreae by

Capoor et al. (1974), and Buitendag and von Broembsen (1993), whereas Xu et al.

(1988) reported an AAP of between 30min to 5 h for D. citri.

The transmission mechanism between D. citri and the pathogen Las is persistent

and presumably propagative transmission. The pathogen apparently multiplies in the

vector, and the adult D. citri generally remain infective during their whole lifespan (Xu et

al. 1988, Hung et al. 2004); but Pelz-Stelinski et al. (2010) found that the ratio of Las

infected adults declined over time when reared on healthy trees, even though the

pathogen appears to multiply in the vector body.

Following acquisition, adults of T. erytreae may require 21 days of latent period

before they can transmit the pathogen (Moll and Van Vuuren 1977). Latent period for D.

citri was also reported to be 8 to 12 days by Cappor et al. (1974). IAP was reported from

15min to 7h for both psyllid species (Capoor et al. 1974, Buitendag and von Broembsen

1993), but a study with D. citri concluded that the disease could be transmitted after

adults had fed on healthy trees for 5hr to 7hr (Xu et al. 1988). Transovarial transmission

was not reported to occur between D. citri parents and offspring (Xu et al. 1988, Capoor

et al. 1974, and Hung et al. 2004). However, Pelz-Stelinski et al. (2010) reported that

transovarial transmission does occur at a rate from 2% to 6%.

Host Plants

Diaphorina citri has a restricted range of host plants including citrus, orange

jasmine (Murraya paniculata), orange boxwood (Severinia buxifolia), and some other

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related species of Rutaceae (Halbert and Manjanath 2004). Murraya paniculata was

reported to be more tolerant than citrus of direct feeding damage from D. citri (Skelley

and Hoy 2004). Murraya paniculata has been widely used to rear D. citri, and it is also

an ornamental plant and a host for both psyllids and bacteria. However, Walter et al.

(2012) suggested that, M. paniculata is serving as a minor source of Candidatus

Liberibacter asiaticus inoculum, because they found less than 1% of psyllids and 1.8%

of plants were positive, using sensitive quantitative polymerase chain reaction (qPCR)

targeting at two prophage genes of Candidatus Liberibacter asiaticus. The

development, longevity and fecundity of D. citri varied on different host plants (Fung and

Chen 2006, Nava et al. 2007). Tsai and Liu (2000) reported that D. citri has a higher

rate of development on grapefruit compared with rough lemon, sour orange and orange

jasmine. Nymphal viability tested on Sunki mandarin was lower than that on ‘Rangpur’

lime and M. paniculata (Nava et al. 2007). Fecundity was higher on M. paniculata.

Diaphorina citri developed better on Rangpur lime and orange jasmine compared to

Sunki mandarin (Nava et al. 2007).

Biology of D. Citri

Life cycle

The life cycle of D. citri is incomplete metamorphosis, so there are only three life

stages: egg, nymph and adult.

The egg has been described as oval or almond-shaped, elongated, slightly

curved with ends in a blunt point (Husain and Nath 1927). A slender stalk-like process

appears from its rounded base which links the egg with the plant tissue (Husain and

Nath 1927, Tsai and Liu 2000). Egg length was reported to be 0.3 mm on average

without the stalk, or about 0.038 mm with the stalk. The greatest diameter was around

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0.13 mm (Husain and Nath 1927). The color of the eggs is light yellow when newly

deposited, gradually turning bright orange with two distinct red spots, the eyes of the

nymph, just before hatching (Husain and Nath 1927, Hall 2008a).

D. citri development includes five nymphal instar stages. The general color of the

nymph is light yellow or dull orange (Husain and Nath 1927, Hall 2008a). As nymphs

mature, the abdominal color of some turn green while some others turn pale orange

(Tsai and Liu 2000). The first nymphal instar is 0.3mm long and 0.17 mm wide with two

visible red eyes (Tsai and Liu 2000). Wing-pads are not yet visible. Some setae arise

from the surface of the dorsum and two golf-club-shaped tarsal setae arise from of the

middle and hind legs with a single seta on each of the fore legs (Husain and Nath

1927). All three features differentiate the first instar from the other stages (Husain and

Nath 1927). The second instar is 0.45mm long and 0.25 wide (Tsai and Liu 2000), with

small triangular wing-pads distinct on the dorsum of the thorax (Tsai and Liu 2000,

Husain and Nath 1927). The third instar nymphs are 0.74mm long and 0.43 mm wide on

average, the wing pads are well developed and the antennal segmentation is visible

(Tsai and Liu 2000). There is a single lanceolate seta on each antenna (Husain and

Nath 1927). The fourth instar nymphs have two setae on each of the antennae (Husain

and Nath 1927). The fifth instar nymphs averaged 1.6mm long and 1.02mm wide (Tsai

and Liu 2000), with a distinctly marked thorax and well developed wing-pads (Husain

and Nath 1927). There is only one seta on each tarsus on the leg, and three setae on

each antenna (Husain and Nath 1927). Paiva and Parra (2012) found that, the egg to

adult survivorship was really low with only 7 adults emerging from 406 eggs under field

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conditions. Of all the generations, egg to first nymphal instar showed the lowest survival

of 38.8% in field

D.citri adults are 2.7 to 3.3 mm in length, with mottled brown wings (Hall 2008a).

When feeding or resting, they always align their heads against the surface of the leaves,

such that the axis of the body is at 45°angle with the leaf surface (Hall 2008a). Adults

can copulate soon after emergence, and eggs are laid if young shoots are available

(Husain and Nath 1927). Both oviposition and development of immature D. citri are

confined to young, new growth (Hall and Albrigo 2007), and all the activities are

restricted to the daylight (Wenninger and Hall 2007). Sex ratio of adult D. citri was

reported around 1:1 by Aubert and Quilici (1988). New adults become physically mature

in 2 to 3 days, and female adults begin to lay eggs 1 or 2 days after mating (Wenninger

and Hall 2007). Females can lay eggs continuously throughout their lifetime if new and

young shoots are available (Hall 2008a). Adult females live approximately 40 to 48

days, and lay 500 to 800 eggs on average during lifetime, and 1900 at most (Husain

and Nath 1927, Nava et al. 2007, Tsai and Liu 2000). Adults usually lay eggs in the

folds of partially opened leaves, push them between the bud and stem or petioles of

leaves and axillary buds, and other similar situations (Husain and Nath 1927). Eggs are

attached to plant tissue by means of their stalks, which can protect them from bad

climatic conditions, such as washing off by heavy rain (Husain and Nath 1927).

Seasonal history

All three stages of D. citri are found on citrus plants throughout the year, and no

hibernation of a specific stage has been discovered (Husain and Nath 1927). Adults

have been observed throughout winter, but usually lay no eggs until spring because of

the lack of new growth. Large numbers of eggs and nymphs were found during March

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and April, but in the northern Punjab, the most frequent stage found in the dry May and

June period is the adult stage (Husain and Nath 1927). If rains start in July, there will be

a second peak of the nymphal population coinciding with the burst of new growth.

Populations begin to decline in the middle of October, and always remain low until

January the next year (Husain and Nath 1927).

Dispersion of D. citri

Adults are very active; they can move a short distance through jumping and flying

(Husain and Nath 1927, Hall 2008a). Aubert and Hua (1990) reported that adults fly to

disperse all day long, but are most apt to move during warm, sunny afternoons between

4 to 6 pm. Hall (2008a) speculated that flying psyllids could be carried by wind over a

0.5 to 1.0 km distance according to wind speed and the duration of flight. Adults

congregate on young and fresh leaves, and move quickly when searching for a place to

oviposit (Husain and Nath 1927). Husain and Nath (1927) stated that adult D. citri are

positively phototropic and negatively geotropic (Husain and Nath 1927). Nymphs

concentrate on the young leaves close to the emergence site. Nymphs do not move

much, but will crawl down the stem to larger leaves when the population density is high

(Husain and Nath 1927).

Damage by D.citri

Besides being the vector of the HLB pathogen, D. citri can also cause damage

through feeding on plants. D. citri is a sucking insect that inserts its mouthparts into the

plant tissue to suck the sap (Husain and Nath 1927, Hall 2008). High adult populations

cause defoliation of shoots attacked and dieback of branches (Husain and Nath 1927).

Nymphs suck the cell sap and exclude thick sugary honeydew from the anus encased in

a waxy secretion secreted by the circumanal glands (Husain and Nath 1927). Nymphs

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move their abdomen to dislodge the wax-covered honeydew, and some fungi will also

grow on it, which makes the leaves look to be covered by a black sooty deposit (Husain

and Nath 1927).

Influence of temperature and humidity

Tsai and Liu (2000) recorded that eggs hatch in 4 days at 25 C. At this

temperature, the nymphal stage lasts over 13 d, leading to a total 17 days from egg to

adult. The life cycle varies significantly with different temperatures, 24 to 28 C being the

optimal temperature range. Diaphorina citri totally fails to develop at temperatures

above 33 C or below 10 C (Liu and Tsai 2000). Nava et al. (2007) reported that, with

temperature varying from 18 to 32℃, the duration of the egg and nymphal stages is 2.6

to 7.7 d and 9.4 to 35.8 d, respectively. Lower temperature development threshold (TT)

and thermal constant (K) for egg, nymph and whole life cycle were 12.0 C - 52.6

Degree-Day (DD), 13.9 C - 156.9 DD and 13.5 C - 210.9 DD respectively (Nava et al.

2007). Longevity differs between females and males even at the same temperature;

adult males live 21 to 25 d, while females live 31 to 32 d on average (Nava et al. 2007).

Different temperatures also influence adult longevity which ranges from 117 days at 15

C to 51 days at 30 C (Liu and Tsai 2000). Skelley and Hoy (2004) showed that D. citri

stopped laying eggs at 34 ℃ or above. However, fecundity returned if temperature was

later reduced. Fecundity is reduced at RH below 40% (Hall 2008a).

The survival of D. citri may increase with increasing relative humidity (McFarland

and Hoy 2001). Yang (1989) reported that nymphal mortality is low from RH 43% to

75% but increases at RH 85% to 92%. The combined influence of temperature and

humidity can reinforce the effect. Nymphal mortality was high at high temperature (34

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℃) and a high humidity (82-92%) and low at moderate temperature (20-30 ℃) and a low

humidity level (43-75%) (Yang 1989). However, in general, temperature plays a more

important role in influencing nymphal survival than humidity (Yang 1989).

Influence of light

Yang (1989) found that the number of eggs laid per female increased with

increasing light intensity and duration under constant conditions below 11000lux and

light duration less than 18 h per day. Both light duration and light intensity influenced

preoviposition period, fecundity, and mortality of females. However, light duration had a

slightly greater effect (Yang 1989).

Natural Enemies

Predators

Diaphorina citri is commonly attacked by ladybeetles (Coleoptera: Coccinellidae),

syrphid flies (Diptera: Syrphidae), lacewings (Neuroptera: Chrysopidae, Hemerobiidae),

and spiders (Araneae) (Michaud 2002, Michaud 2004, Gonzalez et al.2003, Qureshi

and Stansly 2009). Adults are not very vulnerable to these natural enemies (Husain and

Nath 1927). Coccinellid predators, such as Harmonia axyridis Pallas and Olla V-nigrum

Mulsant have been considered the most important predators in Florida (Michaud 2002,

2004). Exochomus childreni Mulsant, Cycloneda sanguinea L. (Michaud 2004), and

Curinus coeruleus Mulsant (Michaud and Olsen 2004) have also been observed to

attack D.citri in Florida. Two lacewings, Ceraeochrysa sp. and Chrysoperla rufilabris

Burmeister, were reported to contribute to psyllid mortality in Florida (Michaud 2004).

The spider Hibana velox (Becker) was reported to be of great importance in Florida

(Michaud, 2004). Ants can just prey on immature D. citri in Florida (Michaud 2002).

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(Juan-Blasco et al. 2012) found that Amblyseius swirskii (Acari: Phytoseiidae) showed

significant predation on D. citri eggs, and also suck body fluids of first instar nymphs.

Parasitoids

Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus

aligarhensis (Shafee, Alam and Agarwal) (Hymenoptera: Encyrtidae) are two well know

parasitoids of D. citri (Hall 2008b, Grafton-Cardwell et al.2013). The ectoparasitoid T.

radiata and endoparasitoid D. aligarhensis were recorded as primary parasites (Tang

1990). Marietta leopardina was found attacking immature T. radiata and D. aligarhensis

(Hoddle 2012). D. aligarhensis was released in Florida but has not yet been reported to

have established (Rohrig et al. 2011).

Tamarixia Radiata

Distribution

Tamarixia radiata, the arrhenotokous ectoparasite of D. citri, was first recorded In

Pakistan (Waterston 1922). In 1978, T. radiata was introduced to Reunion Island from

the Punjab of India and was credited with achieving control of D. citri (Etienne and

Aubert 1980). It was also reported to have been successfully introduced in Mauritius in

1984 (Aubert 1987). It is well known to occur in Jiangxi and Fujian provinces in China

(Tang 1998), but was not released there on purpose (Yang et al. 2006). It was

introduced to Taiwan in 1983 for control of D. citri, and is very effective there (Chien and

Chu 1996). Distributions of T. radiata in Asia include India (Husain and Nath 1927),

Japan (Kohon et al. 2002), Nepal (Lama et al. 1988), Thailand (Markote and Nanta

1995), and Vietnam (Hoy et al. 1999). The parasitoid was imported from Taiwan and

Vietnam and released in Florida in 1999-2001 (Hoy and Nguyen 2001). Tamarixia

radiata has been detected in Texas (French et al. 2001), Brazil (Gomez et al. 2006),

23

and Puerto Rico (Pluke at al.2008) although no releases were ever recorded for those

locations. Tamarixia radiata is established in Florida now, although the parasitism rate is

relatively low except for late in the growing season (Tsai at al. 2002, Qureshi et al.

2009).

Taxonomy and Identification

Tamarixia radiata was originally described as Tetrastichus radiatus by Waterston

(1922) from specimens collected at Lyallpur, India (now Faisalabad, Pakistan). The

genus Tamarixia was split off as a separate genus from Tetrastichus by Mercet (1924).

All are parasitoids of Psylloidea (LaSalle 1994). Tang and Aubert (1990) described

some distinguishing characteristics of T. radiata: about 1 mm long including head; eyes

red in fresh specimens; head and body blackish and shining but without a metallic

sheen; underside of gaster pale with a large whitish basal patch on the 5th tergite of

dorsum; legs totally pale-white and wings transparent. Male and female are the same in

color and body structure, except for antennae. The female antenna has eight segments,

both funicle and club with three segments covered with fine, short setae. The funicle is

slender with the 1st segment longer than the 2nd and the 2nd segment longer than the 3rd.

The length of the 3rd segment is almost equal to the width. The male antenna is more

slender, and nine-segmented. The four-segmented funicle is covered with long, slightly

curved hairs, and the ventral scapal sensorium is near to the base of the scape (Tang

and Aubert 1990).

Host Specificity

T. radiata is not known to attack any psyllid other than D. citri (Aubert and Quilici

1984). (Mann et al. 2010) reported that T. radiata can be attracted by volatiles

emanating from D. citri nymphs.

24

Biology of T. Radiata

Life cycle

Structure of the 4 life stages of T. radiata was described by Chen et al. (1991a).

The egg is translucent, ivory, and reniform, with one end adhesive to the host. There

are four larval instars, each distinguished by head length (Chen et al. 1991a).

Development of the immature stages was reported by Xu and Tang (1993) and Chien et

al. (1991a). The newly eclosed larva sucks fluids externally from the site where it is

closely attached to the nymph’s integument (Husain and Nath 1923, Tang and Huang

1991, Hall 2008b). The third instar crawls to the ventral side of the host thorax to feed

(Chien et al. 1991a). The parasitized nymph continues to live and secrete honeydew for

some time (Husain and Nath 1923). By the time the parasitoid molts to the fourth instar,

all contents of the nymph have been consumed and the nymph turns to a dark-brown

mummy (Husain and Nath 1923, Chien et al. 1991a). The mature larva ceases to feed

as it progresses to the prepupal stage which secures the mummy to the plant surface by

means of silken threads (Chien et al. 1991a). After expelling the meconium, it molts to

the pupal stage which turns yellow, with red ommatidia and ocelli (Chen et al. 1991a).

Emergence

As soon as the adult hardens, it makes its way out of the mummy by chewing a

round hole of approximately 0.5 mm diameter in the region of thorax (Husain and Nath

1923, Chien et al. 1991a, Aubert 1987). Over 80% of adults emerge between 5 a.m. to

10 a.m., with the peak between 7 a.m. to 8 a.m. Male emergence occurs 1.5 hours

earlier than the female on average (Chien et al. 1991a).

25

Mating

Males use their antennae to locate receptive females. Once found, the male

crawls onto the dorsum of the female to make contact for an average of 68±7 seconds

before mating which lasts an average 33±3 seconds (Chien et al. 1991a). About 93% of

females mate once and only once during the first day of emergence. The remainder

mate twice during the first two days following emergence. Fecundity and longevity of

females were not affected by mating frequency. Males are capable of multiple matings

over a lifetime (Chien et al. 1991a).

Oviposition

Eggs can be laid immediately after emergence by mated or unmated females

(Chien et al. 1991a). From 5 am to 10 am is the most active time of day for oviposition

(Chu and Chien 1991). Host volatiles mediate host location (Mann et al. 2010). The

female moves actively among D. citri nymphs using her antennae to search for a

suitable host (Husain and Nath 1923). After an acceptable host is found, she deposits

an egg or occasionally two on the underside of the nymph, usually next to the mid or

posterior coxae (Husain and Nath 1923, Aubert 1987, Chien et al. 1991a, Hall 2008b,

Tang and Huang 1991). Oviposition took 3 to 4 min according to Husain and Nath

(1923), but only 61±8 s according to Chien et al. (1991a). Chien et al. (1991a) also

reported that the female T. radiata injects venom into the host nymph through the

ovipositor, immobilizing it for 4 to 8 min. If the egg was removed, the host nymph could

not molt, and died 8 days later at a temperature of 25 C. A first, second, or third stage

parasitoid larva placed on an unparasitized 5th instar nymph could not attach, and

dropped off when the nymph began crawling (Chien et al. 1991a).

26

Host preference

Studies on host selection of T. radiata females have given varying results. Chien

et al. (1991a) and Chu and Chien (1991) reported that 5th instar nymphs were preferred

for oviposition. Survival rate was 85%, compared with 33% and 71% from 3rd and 4th

instars, respectively (Chien et al. 1991a). Body length was also greater among offspring

from 5th instar hosts compared to 4th instar hosts: 1.12 mm compared with 0.91 mm

(female), 1.03 mm compared with 0.86 mm (male). The pattern was repeated with

fecundity: 215 compared to 120 eggs per female, and longevity: 18.0 d compared to

14.4 d (females), or 11.6 d compared to 7.2 d (males) (Chien et al. 1991). However, 4th

instar nymphs are parasitized significantly more than 3rd or 5th instars according to Tang

and Huang (1991).

Host-feeding

Both sexes feed on honeydew from D. citri nymphs, and females can suck

hemolymph after puncturing the nymph with the ovipositor (Chien et al.1991a, Skelley

and Hoy 2004). The T. radiata population oviposited and host-fed almost simultaneously

(Chu and Chien 1991). Host-feeding occurs in daytime and takes an average of 21±2 s

(Chien et al. 1991a). The host will die once fed upon and females avoid laying eggs and

feeding on the same host (Chien et al. 1991a, Tang and Huang 1991). Ratio of host-

feeding to oviposition correlates with host density and parasitoid age (Chien et al.

1991a). One egg was laid for an average 0.18 hosts fed upon by females between 4 to

18 d old. Younger or older females laid one egg for an average 0.29-0.38 hosts fed

upon respectively (Chien et al. 1991a). An average of 80% mortality resulted from

parasitism with an additional 20% from host-feeding (Chien et al. 1991a, 1994b). In this

way, a single female T. radiata could kill up to 500 nymphs during her lifetime (Chu and

27

Chien 1991). However, Chien et al. (1993) estimated host-killing capacity at 16, 25, 245,

196 nymphs per female at 15 C, 20 C, 25 C and 30 C respectively.

Sex ratio

T. radiata is arrhenotokous, meaning virgin females deposit eggs which produce

males, while eggs deposited by mated females can develop into either sex. The

average number of eggs deposited by virgin and mated females in one study was 209.2

and 215.4 respectively (Chu and Chien 1991). Female progeny ratio is highly correlated

with parasitoid age (Tang and Huang 1991, Chu and Chien 1991). The proportion of

female progeny increased as the mother aged, from 0.5 from a 1 d old female to 0.77

from a 22 d old female (Chu and Chien 1991). Sex ratio is also correlated with host

stage, although published results differ. Tang and Huang (1991) reported female ratios

of 0.88 from 5th instar hosts, 0.75 from 4th instars and 0.41 from 3rd instars. However,

Chu and Chien (1991) found female ratios of 0.67 from 5th instar nymphs, compared

with 0.16 from 4th instars. Skelley and Hoy (2004) reported female ratios of 0.64 and

0.67 with 4th instar nymphs for their Taiwanese and Vietnamese colonies of T. radiata.

Reproduction

Tamarixia radiata can be characterized as synovigenic autogenous, meaning that

some eggs become mature in the newly emerged wasp without feeding, but that host-

feeding is later required to mature more eggs. Most synovigenic parasitoids, including T.

radiata, can resorb eggs when hosts are absent or scarce (Chien et al. 1994a), thereby

maintaining reproductive resources and synchrony with the host population (Jervis et al.

2001). Egg resorption is thus a mechanism that aids adaption to host biology and

ecology. It can occur in T. radiata at either 15 C or 25 C when only honey is provided

and is positively related to host deprivation time (Chien et al. 1991a, 1994a).

28

Once suitable hosts are fed upon, new mature eggs can be produced in the

ovary and oviposition can proceed normally (Chien et al. 1994a). Little or no effect on

total fecundity was observed after host deprivation for 10 days at 25 C with honey

provided as food (204 eggs), but there was a difference after host deprivation for 20 d

(156 eggs) (Chien et al. 1994a). Wasps thus stored for 10 to 20 d, wasps at 25 C laid

significantly more eggs (156 eggs) than wasps stored at 15 C (98 eggs). Fecundity

decreased greatly following host deprivation for 30 to 40 d at 15 C (25~59 eggs).

Superparasitism

Female T. radiata can discriminate between parasitized and unparasitized hosts

to avoid superparasitism (Chien et al. 1991a). Husain and Nath (1923) observed

superparasitism during December and January when hosts were scarce, but not when

hosts were abundant. Chien et al. (1991a, 1991b) observed superparasitism rates of up

to 5.6% when the host density was low and active space was limited.

Host density effects

Longevity, fecundity, sex ratio, and ratio of host-feeding to oviposition all

correlate with host density (Chu and Chien 1991, Chien et al. 1991a, 1995). The

relationship between host density and wasp longevity (both males and females) has

been described as following a domed parabolic response, meaning that female

longevity and fecundity ascend with host density to a peak and then decrease as host

density increases (Chien et al. 1995). Average female longevity increased from 15.9 to

18.6 to 20.3 d when host densities of 10, 20 and 30 were provided daily. However,

female longevity decreased from 23.6 to 17.2 to 11.2 over a range of 40, 60, 80 hosts

per day, respectively (Chien et al. 1995). Chu and Chien (1991) reported that females

lived an average 23.6 days and males 14.8 d when 40, 5th instars were provided at 25

29

C, 14:10 (L: D) photoperiod and 100% RH. Both daily and lifetime fecundity showed

similar parabolic responses to host density, with the peak at 40 hosts per day (Chien et

al. 1995).

Influence of food

Adults deprived of food or water survived 1.0 to 1.7 d (Chien et al. 1994b). These

authors found no difference in longevity among female adults deprived of hosts and fed

either honey alone (22.5 d), honey and pollen (23.0 d), or honey and yeast extract (23.4

d). However, all these food supplements increased fecundity and progeny survivorship

compared with adults held without food or water. Adults fed on a diet of honey and

yeast extract significantly decreased host-feeding while maintaining or improving

intrinsic rate of increase (0.2976 to 0.3014 per day) and the net reproductive rate (140

to 187 female eggs per female), respectively (Chien et al. 1994b).

Development time

Chien et al. (1991a) found the duration of one generation for T. radiata on orange

jasmine (egg to adult emergence) to be around 11.4 d at 25 C, 14:10 (L: D)

photoperiod, and 100% RH. This would include 45 h for the egg, 24, 24, 22, 26 h for the

1st through 4th instars, respectively, 14.4 h for the prepupal, and a 117.6 h pupal stage.

Xu and Tang (1993) reported a 12.6 d generation time at 25 ± 1 ℃, 14:10 (L:D)

photoperiod, and 75% to 85% RH: 40 h for the egg, 119 h = 25, 28, 32 and 34 h for 1st

to 4th larval instar respectively, 24 h for the prepupa and 120 h for the pupa. These

results could indicate that humidity may play an important role in development rate

especially in the prepupal and pupal stages.

30

Temperature and humidity effects

Tamarixia radiata completes development at 15 C to 32 C with an optimum

temperature of 25 ℃ (Chien et al. 1993). Gomez-Torres et al. (2012) found parasitism

rates to be highest at 25 and 30 °C (85.5 and 72.8%, respectively) compared to 23.1

and 40.2% at 15 and 35 °C respectively. They also found emergence rates to be

highest, 86.7 and 88.3%, at 25 and 30 °C respectively, compared to about 50% in the

15 to 20 °C range. At 70 ± 10% RH, and L: D= 14:10 they estimated maximum

parasitism rates of 77.2% at 26.3 ℃, whereas emergence was greatest (89.9%) at 30.8

℃. Pre-imaginal development was longer for females, varying from 489.6 h at 15° C to

247.2 h at 35 °C compared to males at 343.2 to 146.4 h over this same range.

Longevity with access to pure honey negatively correlated with temperature

between 8 C to 30 C (Quilici and Fauvergue 1990). These authors found that adult

longevity decreased from 34 d at 20 C to 22 d at 22 C, 10 d at 30 C and 8 d at 35 C.

Chien et al. (1993) found longevity to increase from 45.5 to 59.5 between 8 and 15 C

but then decrease to 22.5 and 9.6 days at 25 and 30 C respectively. Ten percent of T.

radiata adults survived for 50 d when stored at 25 C with access to honey and yeast

extract (Skelley and Hoy 2004).

McFarland and Hoy (2001) reported that T. radiata adults from Vietnam survived

longer without food and water compared to wasps from Taiwan over a range of RH from

7% to 97% at 25 °C and especially 30 °C. They attributed this difference to greater

moisture requirements of wasps from Taiwan.

Chien et al. (1993) estimated host-killing capacity at 16, 25, 245, 196 nymphs per

female at 15 C, 20 C, 25 C and 30 C. Estimates of intrinsic rate of increase (r), net

31

reproductive rate (R0), and mean generation time (T) for pairs of T. radiata provided 20

or 30, 5th instar nymphal hosts by Chien et al. (1993) and Gomez-Torres (2012)

respectively varied considerably, especially at low temperatures (Table 1-1). Different

results may have been due to different conditions during these two studies. Chien et al.

(1993) did theirs at host density 20 and 100% RH for five replications whereas Gomez-

Torres et al. (2012) conducted their study at density 30, 70 ± 10% RH for ten

replications, which may have led to the different results. Differences could also be

inherent in the races of T. radiata tested from Taiwan and Brazil respectively.

Skelley and Hoy (2004) showed that T. radiata stored for up to 35 d at 17 C with

honey and yeast suffered less than 5% mortality. Chien et al. (1994a) reported that

females stored for 20 d at 25 C were able to lay a total of 156 eggs, compared with 98

eggs when stored at 15 C. We may conclude that less mortality was experienced at low

temperature but that production suffered. Therefore, ideal storage temperature should

be determined according to specific objectives (establishment or augmentation) and

conditions (host availability).

32

Table 1-1. Demographic parameters T (generation time) per day, R0 (net reproductive rate) nymphs per female, and r (intrinsic rate of increase) per day as estimated by Chien et al. (1993) and Gomez-Torres et al. (2012).

Parameter°C Chien et al. (1993) Gomez-Torres et al. (2012)

T15 39.9 20.3 T20 22.8 18.8 T25 16.1 15.5 T30 12.3 11.8 T35 NA 10.4 R15 2 9.9 R20 6 23.6 R25 140 126.8 R30 90 58.6 R35 NA 21.3 r15 .0011 .18 r20 .0081 .25 r25 .31 .37 r30 .37 .34 r35 NA .25

33

CHAPTER 2 CARBON DIOXIDE ANESTHESIA OF TAMARIXIA RADIATA (WATERSTON)

(HYMENOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE)

Introduction

Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous

ectoparasite of the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera:

Psyllidae), vector of citrus greening disease or huanglongbing (HLB). The parasitoid is

reported to have controlled ACP populations to low levels on the islands of Réunion,

Guadeloupe and Puerto Rico (Aubert & Quilici 1984; Etienne et al. 2001, Pluke et al.

2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998

and released in 1999-2001 (Hoy & Nguyen 2001). A survey conducted in 2006-07

determined that T. radiata was well distributed in citrus orchards throughout the state

(Qureshi et al. 2009). However, incidence of parasitism was generally low, especially

early in the growing season, suggesting a need for augmenting parasitoid populations at

that critical time as component of an integrated management program (Qureshi et al.

2007, 2009). Studies of T. radiata biology and current efforts at mass rearing and

release of this species might benefit from an ability to inactivate adults by CO2

anesthetization, including separation of emergent wasps and psyllids.

Carbon dioxide (CO2) is widely used to anesthetize insects, but may also cause

deleterious side effects on biology and behavior. (Brooks 1957) found that development

rate of German cockroach, Blattella germanica L. (Blattodea: Blattellidae), nymphs

decreased 53% when exposed weekly to high CO2 concentrations for 3 min. (Crystal

1967) reported significantly decreased survival rates and fertility of New World

screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), exposed to

34

100% CO2 for 30 min. Sherman (1953) reported that CO2 anesthesia of Mediterranean

fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), led to increased mortality.

This study was undertaken as a first step toward possible use of anesthesia for

mass rearing by evaluating the response of T. radiata to CO2 exposure. The objective

was to test the effectiveness of CO2 anesthesia of T. radiata and to determine the

incidence and severity of side effects of CO2 anesthesia on longevity, parasitism rate

and sex ratio.

Materials and Methods

Colonies

A T. radiata colony was maintained at FDACS-DPI in Gainesville on ACP

nymphs using orange jasmine, Murraya paniculata (l.) Jack (Sapindales: Rutaceae), as

plant host. Six newly trimmed plants with new growth were held in an acrylic 62 cm

cubic cage and 600 D. citri adults were released for 72 h for oviposition in a greenhouse

under natural sunlight 25 ± 5 C and 50%~ 70% RH. Adults were removed and plants

held for 10 d until 4th instar nymphs were available. Plants were moved into another

clean cage of the same type for 20 days into which 100 T. radiata were released. Adult

progeny were later collected daily until no more emerged.

Gas Chamber

A gas chamber was constructed consisting of a vial, 6.50 cm in diam and 12 cm

in height (Fisher Scientific, Pittsburgh, Pennsylvania), provided with two 0.50 cm diam

holes in the lid into each of which was fitted a 0.5 cm plastic tube inserted either 1 cm or

11 cm into the chamber for a gas outlet and inlet respectively (Fig 2-1). Plasticine

modeling clay (Flair Leisure Products, Cheam, Surrey, England) was molded around the

openings of the lid to prevent leakage.

35

Flow time of gas at 3.8 kpa (2 psi) needed to displace all air in the chamber was

assessed by filling the vial with water and then replacing with CO2 through the inlet. All

the water was displaced in 10 s. A CO2 sensor (K-33 ICB 30% CO2 Sensor, CO2Meter

Inc., Ormond Beach, Florida, USA) was used to determine that 3 s of flow time were

necessary to attain a 30% CO2 concentration confirming the earlier result. The CO2

sensor was also used to test for leaks by confirming that a given concentration

remained constant over several min.

Recovery Time

Five wasps having emerged within 24 h or less were placed in the chamber. The

lower ¾ of the vial was covered with black cloth to induce the wasps to walk to the top

and thus avoid injury from inrushing gas. Gas was introduced through an inlet from a

CO2 tank at 3.8 kpa for 15 s to exchange all the air, and then the 2 tubes closed with

metal clamps. Wasps were removed after a 5 min exposure and observed with the

naked eye using a stop watch to record recovery time (normal movement). Males and

females were treated separately, each with eight replications so that a total of 80 wasps

was used.

Survival Rate

To evaluate survival, 5 anesthetized wasps were collected into each of 6 small

glass vials (1.5 cm in diam, 5.3 cm high) and provided pure honey on a tissue paper

strip. On the same day control wasps not anesthetized were placed in 6 other vials.

Vials with wasps were held in a growth chamber at 25 C, 14:10 h L: D and 60 ± 5% RH,

and checked daily, noting sex of all cadavers until all had died.

36

Percent Parasitism

Six newly trimmed plants were held in a ventilated 62 cm acrylic cubic cage until

there were at least 3 new shoots 3 cm in length upon which to evaluate parasitism.

Plants were infested by releasing 600 ACP adults for a 24-h oviposition period. ACP

adults were removed and the plants were held for 9 days in a rearing room at 25 ℃ and

60 ± 5% RH. A small brush was used to remove nymphs until exactly 120 fourth instars

remained on each plant. Each plant was then placed individually into a clear acrylic

cylinder (12.5 cm diam, 43 cm high) into which 3 T. radiata females and 2 males were

released. Cages were randomly selected to receive either anesthetized or untreated

wasps (N = 8). Newly emerged T. radiata offspring were collected daily from day-7 until

day-19 after which no more new progeny were found. Progeny were counted and sexed

and parasitism rate calculated based on 120 original hosts.

Results

Seventy percent of T. radiata females recovered from anesthesia with CO2 within

4 min, males recovered about as quickly. Indeed, there was no significant difference

between male and female recovery time (2 = 13.04, df = 7, P = 0.071, Fig 1). It was

noted that a wasp often would recover immediately after being crawled over by another

recovering individual.

Survival rate for the treated wasps was consistently lower than the control over

the entire study period (Fig. 2). Insect-days, the area under the curve of insect numbers

by time (Ruppel 1983), was significantly less for the CO2 treatment (3445.3 ± 348.6)

than the control (5610.5 ± 836.6) (t = 2.39, df = 7, P ˂ 0.05).

A mean of 59.0 ± 3.8 wasps emerged over 12 days from 120 fourth instar hosts

exposed to 3 female and 2 male T. radiata treated with CO2 compared to a mean of

37

85.2 ± 4.5 in the control. This corresponded to a parasitism rate of 49.2 ± 3.2% for

treated wasps compared to 71.0 ± 3.7% for untreated wasps (t = 4.46, df = 14, P =

0.00054). There was no significant difference in progeny sex ratio (t = 1.03, df = 14, P =

0.32) between the treated and the control.

Discussion

Carbon dioxide anesthesia is a convenient tool for manipulating insects, but can

cause deleterious side effects. In this case, a 5 min exposure of Tamarixia radiata

adults to 100% CO2 concentration caused a knockdown of about 4 min, significantly

reduced survivorship and fecundity, but did not affect the sex ratio of progeny from

treated adults. Future research will focus on using less concentrated doses or shorter

exposure times to inactivate the wasps in order to improve survival and fecundity.

38

Figure 2-1. Carbon dioxide gas chamber (Photo courtesy of Xulin Chen)

Figure 2-2. Tamarixia radiata female and male recovery time frequency (%) distribution

39

Figure 2-3. Mean (SEM) survivorship of CO2 treated and untreated Tamarixia radiata adults.

40

CHAPTER 3 FUNCTIONAL RESPONSE OF TAMARIXIA RADIATA (HYMENOPTERA:

EULOPHIDAE) TO DENSITIES OF ITS HOST, DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE)

Introduction

Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous

ectoparasite of the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera:

Psyllidae), vector of citrus greening disease or huanglongbing (HLB). The parasitoid is

reported to have controlled ACP populations to low levels on the islands of Réunion,

Guadeloupe and Puerto Rico (Aubert & Quilici 1984, Etienne et al. 2001, Pluke et al.

2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998

and released in 1999-2001 (Hoy & Nguyen 2001). A survey conducted in 2006-07

determined that T. radiata was well distributed in citrus orchards throughout the state.

However, incidence of parasitism was generally low, especially early in the growing

season, suggesting a need for augmenting parasitoid populations at that critical time as

component of an integrated management program (Qureshi et al. 2007, 2009). The

success of such a program would depend, in part, on the efficiency of mass-rearing the

parasitoid. Thus optimization of the host: parasitoid ratio is of critical importance.

Optimal host: parasitoid ratio may also be an important consideration for guiding field

release strategies.

It has been reported that the fecundity of T. radiata is highly correlated with host

density (Chu and Chien 1991, Chien et al. 1991a 1995). The relationship between both

daily and lifetime fecundity with host density showed similar parabolic responses,

meaning that female fecundity ascends with host density to a peak of 40 hosts, and

then decreases as host density increases (Chien et al. 1995). The decline in fecundity

41

at high host density could be due to host defense or excessive honey dew. In fact, he

experiments were conducted at 100% RH which would increase honeydew and

consequent sooty mold that would interfere with the T. radiata female’s search for hosts.

Another issue may be that late 5th instar nymphs are not suitable hosts especially when

they are about to emerge as adults. Similar behaviors have been observed in

Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae), parasitoid of whitefly

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). The female E. mundus never

attacked late 4th instar white fly nymphs once adult development was initiated (Gelman

et al. 2005). Therefore, it is necessary to repeat these experiments under more

favorable conditions of relative humidity and host availability.

The relationship between number of hosts parasitized per parasitoid and host

density is known as functional response. It has been used to illustrate the complex

interactions between parasitoids and their hosts, and is an important character

influencing biocontrol success (Jones et al. 2003). Functional response has been

described as Holling (1959) Type I, II or III, characterized by linear, decelerating and

sigmoidal responses of parasitism rate to increasing host density. Most laboratory

studies show that parasitoids exhibit Type II functional response, Type III functional

responses are also common (Holling 1959). The shape of the functional response curve

varies with parasitoid species, and also parasitoid age (Jones et al. 2003).

My objective was to evaluate the influence of host density on percent parasitism

and the number of hosts parasitized by T. radiata (functional response), to help optimize

rearing conditions in the laboratory as well as release conditions in the field.

42

Materials and Methods

Colonies

Murraya paniculata (L.) Jack (Rutaceae) contained in 3.92 L pots with 40: 60 mix

of Canadian sphagnum peat (Fafard 4P mix Professional Growing Mix soil, Conrad

Fafard Inc.) was used to rear D. citri. Each plant selected had 2-5 shoots of new growth,

with each new shoot about 4 cm in length. Plants were maintained using M-pede soap

(Dow AgroSciences LLC) as a contact chemical to control unwanted psyllids and other

pests, soap sprayed plants cannot be used within 3 d. All plants used in this experiment

were grown in an unheated screen houses (hoop style trussed greenhouse with insect

screen mesh) with natural ventilation and sunlight.

A wood framed cubic cage was used (62 cm in each dimension) 3 sides of which

were covered with fine mesh for ventilation with the other 3 sides enclosed with clear

acrylic. Six flushing M. paniculata plants were placed in the cage and 600 D. citri

released and held there for 24 h for oviposition. Plants were then moved to a similar

clean cage for about seven days until the eggs hatched and nymphs developed to the

4th instar. All cages were held in an air-conditioned rearing room at 25-30 C, 60-80%

RH (Extech RH10 humidity and temperature datalogger, Grainger, Inc) and L: D=14:10.

Murraya paniculata with 4th nymphal instar psyllids were transferred into a clear

acrylic cylinder (12.5 cm diam, 43 cm high) with mesh on top in a growth chamber

(Percival model I36LLC8, Perry, Iowa) at 24 ± 4 C, RH 60-80%, L: D=14:10 (Extech

RH10 humidity and temperature datalogger, Grainger, Inc). Five pairs of newly emerged

T. radiata were released into this cylinder for three days to allow the parasitoids to

mature.

43

Arenas

Arenas were prepared using centrifuge tubes (11 cm long and 3 cm diam). Into

each arena was placed a 7 cm young shoot of M. paniculata infested with 10, 20, 30,

40, 50, or 60, 4th instar psyllid nymphs; the number of nymphs was controlled using a

small paintbrush. Optimal young shoots were chosen as those with more than the

number of hosts needed. A small paintbrush was used to remove extra nymphs to

achieve the desired density. Placing additional nymphs onto the young growth was

avoided, because the newly placed nymphs always crawled off the new shoot. One

randomly-chosen 3-d old parasitoid pair was released into each centrifuge tube and

sealed with Parafilm provided with small holes made by a No.1 insect pin for ventilation.

Arenas were placed in the growth chamber and shoots replaced every 24 h. Exposed

nymphs were inverted under a stereoscopic microscope to check for parasitoid eggs.

Each pair of T. radiata was held in the arena for 5-d, and six replications were

completed for each host density.

Statistical Analysis

Data on number and proportion of parasitized and superparasitized hosts in each

arena among six host densities over the 5-d period were transformed using square root

for normality fitness and then subjected to Analysis of Repeated Measures using JMP

software (SAS Institute, 2012) with mean separation by Fisher’s LSD (P < 0.05). A

polynomial regression of proportion of hosts consumed versus density was used to

distinguish between Type II and Type III functional response (Juliano 2001). A

significant positive linear term coefficient indicates Type III whereas a significant

negative linear term coefficient implies a Type II functional response. Data were then

fitted to the corresponding functional response equation using nonlinear regression in

44

JMP and the functional response parameters: searching efficiency (a) and handing time

(TH) were reported from the regression.

Results

Fecundity, Percent Parasitism and Percent Superparasitism

Inspection of the box plot revealed no outliers among the data, and no significant

differences were observed among replications (F = 0.6461, df = 5, P = 0.6648).

Fecundity was significantly different among the six host densities (F = 111.2432, df = 5,

P < 0.0001), and it increased with host density to a maximum of 11 to 12 eggs per day

with no significant differences among host densities of 40, 50, and 60 (Table 3-1 and

Fig 3-1).

Percent parasitism was significantly different among six host densities (F =

49.6352, df = 5, P < 0.0001): highest at 43% with 10 hosts and least at 18.7% with 60

hosts (Table 2, Fig. 2). Percent superparasitism (superparasitized hosts number over

parasitized hosts) was also significantly different among the six host densities (F =

140.7006, df = 5, P < 0.0001); highest (37.9%) at the lowest host density level of 10

hosts per female, followed by 15.4% at 20 hosts per female. Superparasitism was

negligible at densities over the range of 30 to 60 hosts per female (Table 3)

Functional Response

The estimated linear term coefficient for the polynomial regression was -0.0099 ±

0.0015 and significant (t = 4.01, df = 177, P < 0.001), indicating a type II functional

response (Juliano 2001). When the data were fitted to the disc equation (Holling 1959),

the estimated searching efficiency (a) was 0.442 ± 0.036 per day (95% confidence

interval was 0.378~0.520) and estimated handing time (TH) was 0.045 ± 0.008 d (95%

confidence interval was 0.032 ~ 0.063), which were all significant (Fig 4).

45

Discussion

Percent parasitism and percent superparasitism changed with host density. The

number of hosts parasitized increased from density 10 to 40 per female, after which it

remained constant. Host availablility was limited at low host densities, indicating that

fecundity was constrained by lack of hosts. The number of eggs laid did not increase

beyond 40 hosts per female, indicating that the upper limit had been reached.

Parasitism rate was highest at density 10, but 37.9% of the parasitized hosts were

superparasitized, which meant the hosts were not used effectively as only one

parasitoid adult can emerge and supernumerary parasitoids are thus wasted (Chien et

al. 1991a). Therefore a host density of 10 per female would not be efficient for

parasitoid mass-rearing.

A maximum number of hosts were parasitized a host density of 40, and percent

parasitism was highest at densities of 20 and 40 hosts per female. However, 15.4%

superparasitism was observed at 20 hosts per female whereas superparasitism was

negligible at a host density of 40. Therefore a density of 40 host per female optimal

because it maximized number of percentage of hosts parasitized with minimal

superparasitism. When host density exceeded 40, the number of hosts parasitized did

not increase but the percent parasitism declined. However, no decrease in number of

hosts parasitized was observed at host densities above 40 per female as reported by

Chien et al. (1995). In conclusion, a host density of 40 was the optimal choice maximal

progeny and the best for usage of D. citri.

Superparasitism was highest at a host density of 10, and was not significantly

different when the density exceeded 30. Chien et al. (1991) reported that female T.

radiata were able to discriminate between parasitized and non-parasitized hosts. But

46

gravid females still laid eggs even though there were not enough suitable hosts

available resulting in superparasitism. Superparasitism is wasteful because only one

egg can mature to the adult and any others will die.(Chien et al. 1991a) To avoid

superparasitism, host number should be controlled in excess of 20 per female.

The pattern of hosts parasitized by T. radiata over the first five days conformed to

a functional response Type II, which means the searching efficiency and the handing

time were all constant in different host densities. These two parameters will be useful to

compare T. radiata with other parasitoids, and they may help to make a better study of

the behavior of T. radiata in the future.

47

Table 3-1. Number of parasitized hosts (Mean ± SEM) at different host densities

Host Density (4th Instars/arena/day)

Parasitized Hosts (Mean ± SEM)

10 4.30 ± 0.16 a 20 5.60 ± 0.3 b 30 7.23 ± 0.42 c 40 11.24 ± 0.24 d 50 11.50 ± 0.40 d 60 11.22 ± 0.30 d

Table 3-2. Percent parasitism (Mean ± SEM) at different host densities

Host Density (4th Instars/arena/day)

Percent parasitism (Mean ± SEM)

10 43.0 ± 0.016 a 20 28.0 ± 0.015 b 30 24.1 ± 0.006 c 40 28.1 ± 0.006 b 50 23.0 ± 0.008 c 60 18.7 ± 0.005 d

Table 3-3. Percent superparasitism (Mean ± SEM) in different host densities, mean

number

Host Density (4th Instars/arena/day)

Percent superparasitism (%) (Mean ± SEM)

10 37.9± 0.03a 20 15.36± 0.03 b 30 1.24± 0.004c 40 0.35± 0.007c 50 0.43± 0.004 c 60 0.27± 0.002c

48

Figure 3-1. Number of parasitized host at different host densities, error bar stands for

SEM

Figure 3-2. Percent parasitism at different host densities, error bar stands for SEM

49

Figure 3-3. Percent superparasitism in different host densities

Figure 3-4. Number of D. citri parasitized by T. radiata and the functional response curve (Type II)

50

CHAPTER 4 THE INFLUENCE OF DIET ON EGG FORMATION IN TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE), A PARASITOID OF DIAPHORINA CITRI

(HEMIPTERA: PSYLLIDAE)

Introduction

Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous

ectoparasite of the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama) (Hemiptera:

Psyllidae), vector of citrus greening disease or huanglongbing (HLB). The parasitoid is

reported to have controlled ACP populations to low levels on the islands of Réunion,

Guadeloupe, and Puerto Rico (Aubert & Quilici 1984, Etienne et al. 2001, Pluke et al.

2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998

and released in 1999-2001 (Hoy & Nguyen 2001). A survey conducted in 2006-07

determined that T. radiata was well distributed in citrus orchards throughout the state.

However, incidence of parasitism was generally low, especially early in the growing

season, suggesting a need for augmenting parasitoid populations at that critical time as

component of an integrated management program (Qureshi et al. 2009). It would thus

be necessary to mass-rear parasitoids to obtain adequate numbers for release,

requiring temporary storage. During the holding period, food provided to females may

affect the number of eggs formed in ovaries, which may influence their efficiency as a

biocontrol agent upon release.

Oogenesis is a nutrition-limited process, and nutrition is obtained during the

larval or adult stage; insufficient nutrients always affect egg production (Wheeler 1996).

Carbohydrate is the major energy source for most insects; but it is not the main nutrition

that triggers egg formation in female insects. Varley and Edwards (1957) reported that

female Nasonia vitripennis (Wlkr.) (Pteromalidae) fed on carbohydrates only survived,

51

but resorbed eggs resulting in gradual decreaseof eggs in the ovaries. (Bownes and

Blair 1986) found that when Drosophila were feeding on sugar diets, the number of

eggs laid and the number of vitellogenic oocytes in ovaries were reduced significantly.

This was due to reduced transcription of yolk protein in the fat bodies resulting in

reduced availability of yolk protein for oogenesis.

Host hemolymph is rich in protein and an important source of nutrients to many

hymenopterous female parasites for increasing fecundity (Howard 1910, Rockwood

1917). Varley and Edwards (1957) observed egg development by female N.

vitripennis,was initiated and/or accelerated shortly after host hemolymph was. (Leius

1961) reported that female Itoplectis conquisitor (Say) (Hymenoptera: Ichneumonidae)

which fed on host body fluid laid more eggs than those fed on carbohydrates alone.

Additionally, Leius (1961) found that pollen in diets significantly increased female I.

conquisitor fecundity, and females fed on a mixed diet (host body fluid and

carbohydrates) deposited even more eggs.

The objective of this experiment was to investigate which foods promote egg

production during storage, presumable improving oviposition rate upon release.

Materials and Methods

Colonies

Murraya paniculata (L.) Jack (Rutaceae) was used as a host plant to maintain a

D. citri colony. Plants were grown in 3.9 L pots using 40: 60 mix of Canadian sphagnum

peat potting soil (Fafard 4P Professional Growing Mix soil, Conrad Fafard Inc.) in a

screen house (hoop style trussed, with insect screen mesh) under natural sunlight and

passive ventillation. Plants sprayed with 35% M-pede soap (Dow AgroSciences LLC) as

needed to control psyllids insects and mites.

52

The D. citri colony was maintained in an air-conditioned rearing room at 28± 2 C,

60-80% RH (Extech RH10 humidity and temperature datalogger, Grainger, Inc), and L:

D= 14: 10. Six M. paniculata plants, each with five to eight, 4 cm long new sprouts were

placed a wood frame cage (60 cm 60 cm 120 cm) covered all three sides with fine

mesh for ventillation. Six hundred D. citri adults were released in each cage for 24 h for

oviposition. All adult psyllids were then collected and removed. Plants were held for

approximately 10 d (depending on temperature and humidity until all nymphs reached

4th instar.

Six M. paniculata plants with 4th instar nymphs were transferred into a clear

acrylic cage (60 cm 80 cm 90 cm) with 3 sides of fine mesh in an air conditioned

greenhouse maintained at 28± 2 C, 75± 5% RH. Fifty T. radiata females and 30 males

were released in the cage for 48 hours to parasitize hosts, then collected and removed.

Plants were held for another 6 d until the parasitized hosts d mummified. Then, the

young shoots with nymphs were excised and nymphs were examined under a

microscope. When a nymph was found parasitized with its parasitoid wasp developed

almost to the pupal stage, that part of the shoot (about 2 cm) was excised and placed in

a glass tube (75 mm long, 12 mm in diameter, Fisher Scientific, Pyrex 9820) in an air-

conditioned rearing room at 28±2 ℃, 60-80% RH (Extech RH10 humidity and

temperature datalogger, Grainger, Inc). Tubes were checked frequently, and once the

wasp removed immediately after emerge.

Diets

Each pair of newly emerged T. radiata was placed in a 50 ml centrifuge tube

(Kendall Labware, Mansfield, MA), sealed with Parafilm (Pechiney Plastic Packaging

53

Company, Chicago, Illinois) and stored in a growth chamber (Percival Scientific, model

I36LLC8,Perry Iowa) at 17 C, 75~ 85% RH , and L: D = 14:10. Wasp were provided

with 8 different diet treatments: water alone, honey, Nu-Lure (Miller Chemical &

Fertilizer Corporation, Hanover, Pennsylvania), host nymphs, honey+ Nu-Lure, honey+

host nymphs, Nu-Lure+ host nymphs, and honey+ Nu-Lure + host nymphs (water stripe

was provided in every treatment). Nu-Lure (Miller Chemical and Fertilizer, Hanover, Pa.)

is a proteinaceous dark brown liquid made from 44% hydrolyzed corn gluten meal, and

56% inert ingredients. Water, honey, and N-Lure were all provided on 8 cm long, 1.5 cm

wide cellulose strips (Wypall-L30, Kimberly-Clark Professional). Host nymphs were

provided on a fresh cut shout, on which 2nd, 3rd, 4th instar nymphs were mixed together.

Water strips were changed every 24 h, and honey, Nu-Lure strips, and host nymphs

were changed every 48 hours to make sure they did not dry out and continued providing

efficient food sources. Wasps in each treatment were held separately for 5, 10, 15, and

20 d. There were 10 replications of every treatment day combination.

Egg Load

T. radiata females under each different treatment were dissected after 5, 10, 15,

and 20 d as well as newly emerged T. radiata under a dissecting microscope (Olympus

SZX 16, Olympus Corporation). Pictures were taken using an Olympus DP 21, and the

number of eggs was recorded.

Statistical Analysis

Data from the eight treatments at each 5 day dissection interval were compared

using Tukey’s Student’s t-test (JMP, SAS Institute Inc. 2012).

54

Results

Newly emerged T. radiata females averaged 4.6 eggs each. Egg loads were

significantly different among the eight treatments at 5 days (F = 11.1702, df = 55, P <

0.0001), 10 days (F = 12.72, df = 40, P < 0.0001), 15 days (F = 7.8989, df = 35, P <

0.0001), and 20 days (F = 10.28, df = 34, P < 0.0001). Wasps fed on water and Nu-Lure

all died within 10 days. At each of the five-day checks, females fed on honey had

significantly fewer eggs compared with the other treatments except for water and Nu-

Lure. During the first five days, females fed on nymphs formed the same number of

eggs as honey + Nu-Lure, but with time , females fed on honey+ Nu-Lure formed fewer

and fewer eggs until the 20th day, when the difference was significant compared to

those fed on nymphs. Females fed on Nu-Lure + nymphs, and honey+ Nu-Lure+

nymphs formed significantly more eggs than those provided with nymphs alone, and a

little more than those on honey + nymphs, although the difference was not significant

(Table 4-1). Wasps fed on honey alone formed fewer and fewer eggs after 10 days;

wasps fed on honey +Nu-Lure also formed gradually fewer eggs

Discussion

An average of 4.6 eggs were observed in ovaries of newly emerged female T.

radiata, despite not feeding on any diets (Fig 4-1, 4-2), so they may use reserved

nutrition from larval stages to form the first clutch of eggs. Mating was not necessary for

egg formation.

This study showed that honey alone was enough to keep wasp females alive, but

egg resorption took place within 5 days after emergence (Fig 4-3). This may mean that

T. radiata females lack the ability to transfer carbohydrates into amino acids to form

55

eggs, or oogenesis requires essential amino acids which cannot be transferred from

carbohydrates.

Nu-Lure alone was not sufficient to keep the females alive for more than 5 days,

which indicated that carbohydrate is a necessary energy resource needed for survival,

and they may lack the capability of transforming amino acids into carbohydrate.

The combination of honey + Nu-Lure resulted in female survivorship similar to a

diet of host nymphs, but egg formation was still less than if provided with nymphs (Fig 4-

4). Females with access to host nymphs (Fig 4-5) matured significantly more eggs than

those fed on honey, Nu-Lure, or honey+ Nu-Lure, indicating that host body fluid

contained essential nutrients not available in the food supplements. However, the

combinations of honey+ Nu-Lure+ nymphs (Fig 4-8) and Nu-Lure+ nymphs (Fig 4-7)

resulted in significantly higher fecundity than a diet of nymphs alone, honey + nymphs

(Fig 4-6) was a little better, but not significant (Table 4-1). I concluded that host nymphs’

body fluid is an irreplaceable nutrition source for females’ egg formation. However, the

extra source of carbohydrates and amino acids provided by the supplements had a

positive effect both in the absence and presence of nymphs. In future studies, it will be

interesting to investigate which amino acids or other constituents of hemolymph are

important for egg formation.

56

Table 4-1. Mean ± SEM number of eggs by treatment from dissections after 5, 10, 15,

and 20 days at 17 C. Means in the same column followed by the same letter are not significantly different (Tukey’s Student’s t-test HSD, P<0.05).

5 days 10 days 15 days 20 days

Water 1.83±0.6 D ______ ______ ______ Nu-Lure 2.14±0.7 D ______ ______ ______ Honey 3.50±0.4 C 3.67±0.6 C 2.40±0.4 D 1.00±0.4 D Nymphs 5.50±0.6 B 6.30±0.8 AB 6.88± 0.7 BC 6.50±0.8 B Honey+ Nu-Lure 5.38±0.7 B 5.30±1.0 B 5.00± 0.7 C 4.60±0.8 C Honey+ Nymphs 6.00±0.5 AB 7.00±0.8 AB 7.70± 0.6 AB 8.00±1.5 A Nu-Lure+ Nymphs 7.56±0.6 A 8.00±0.9 A 8.00± 0.9 A 8.30±0.7 A Honey+ Nu-Lure+ Nymphs 7.20±0.6 A 7.80±0.6 A 8.00± 0.8 A 8.20±0.8 A

57

Figure 4-1. Newly emerged (unfed) T. radiata female digestive system (Photo courtesy of Xulin Chen)

Figure 4-2. Newly emerged (unfed) T. radiata female (Photo courtesy of Xulin Chen)

58

Figure 4-3. Paired T. radiata ovaries after feeding on honey for 20 days (egg resorption) (Photo courtesy of Xulin Chen)

Figure 4-4. T. radiata ovary after feeding on Nu-Lure+honey for 10 days (Photo courtesy of Xulin Chen)

59

Figure 4-5. Paired T. radiata ovaries after feeding on nymphs for 5 days (Photo courtesy of Xulin Chen)

Figure 4-6. Paired T. radiata ovaries after feeding on honey +nymphs for 10 days (Photo courtesy of Xulin Chen)

60

Figure 4-7. Paired T. radiata ovaries after feeding on Nu-Lure+ nymphs for 10 days (Photo courtesy of Xulin Chen)

Figure 4-8. Paired T. radiata ovaries after feeding on Nu-Lure+honey+nymphs for 5 days (Photo courtesy of Xulin Chen)

61

CHAPTER 5 DISCUSSION AND CONCLUSIONS

Tamarixia radiata, is an arrhenotokous ectoparasite of the Asian citrus psyllid

(ACP) Diaphorina citri, vector of citrus greening disease or huanglongbing (HLB).

Tamarixia radiata is being tried as an augmentive biological control agent, since the

number present in the field reduced in winter and by insecticide applications. Any

program aimed at augmentation of a natural enemy would require an efficient system of

mass production. Key components would include methods of manipulating, rearing and

storing large populations of insects.

To make counting easily and separating D. citri and T. radiata after collection,

carbon dioxide was used as anesthesia. Carbon dioxide anesthesia can be a

convenient tool for manipulating insects, but can also cause deleterious side effects. In

this study, a 5 min exposure of Tamarixia radiata adults to 100% CO2 concentration

caused a knockdown of about 4 min, and significantly reduced survivorship and

fecundity, but did not affect the sex ratio of progeny from treated adults.

The harmful effects of CO2 proscribe its routine under the tested exposure

conditions for rearing T. radiate. Nevertheless the wasp showed surprising tolerance to

high CO2 concentration: the wasps acted normally when sealed in a gas chamber with

30% CO2. Perhaps lower concentrations and/or exposure times would provide more

acceptable alternatives. Also, the physiological mechanism of CO2 catabolism could be

an interesting research aspect. The effects of chilling to immobilize the insects should

also be tested.

Host density is an important factor influencing mass rearing efficiency. Fourth

and early 5th instars are the preferred stage for oviposition by T. radiata. My study

62

showed that a density 40, 4th instar nymphs per female optimized fecundity, maximized

incidence of parasitism and minimized incidence of superparasitism. This result could

be used to determine the idea number of parasitoids to be released into a rearing cage.

The number of flushes in the rearing cage can be counted, and the number of proper

stage nymphs on one shoot can be estimated to calculate the number of nymphs in the

whole rearing cage. Then, knowing the sex ratio and the ideal 4th instar host to female

ratio of 1:40, the best number of T. radiata females to release will be clear.

The pattern of parasitism observed over the first five days conformed to a Type II

functional response, estimated searching efficiency was 0.442 ± 0.036 per day and

estimated handing time was 0.045 ± 0.008 days. These two parameters are important in

comparing a T. radiata female’s parasitism efficiency with other parasitoids, to evaluate

its parasitic capacity.

When T. radiata were stored for release, certain nutritional food is necessary to

maintain fecundity. My results showed that, T. radiata formed more eggs feeding on

mixed diets (Nu-Lure+ honey+ nymphs or Nu-Lure+ nymphs) compared to nymphs

alone. Until now, no diet has been found to completely substitute nymphal hemolymph.

The present practice is to hold wasps in centrifuge tubes provided with honey on

paper strips preparatory to release. My research would indicate that wasps should also

be provided with a supplementary protein source such as Nu-Lure, and also with hosts

in order to maintain the fecundity.

Tamaraxia radiata is an efficient biological control agent of D. citri under optimal

temperature, humidity, host densities and nutritional supplement in lab conditions. More

63

work need to be done to apply these lab results in field to get a more optimal parasitism

capacity of D. citri.

64

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BIOGRAPHICAL SKETCH

Xulin Chen was born in Shandong Province, China. She began her

undergraduate study in Shandong Agriculture University majoring in Plant Quarantine in

2007. After she graduated in July, 2011, she started her graduate study in University of

Florida, Entomology and Nematology Department under the supervision of Dr. Phil

Stansly.