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Life History, Host Choice, and Behavioral Plasticity of Trichopria nigra (Hymenoptera

Permanent Link: http://ufdc.ufl.edu/UFE0021395/00001

Material Information

Title: Life History, Host Choice, and Behavioral Plasticity of Trichopria nigra (Hymenoptera Diapriidae), a Parasitoid of Higher Diptera
Physical Description: 1 online resource (118 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: conditioning, diapriidae, diapriinae, olfactometer, parasitoid, trichopria
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Trichopria nigra (Nees) (Hymenoptera: Diapriidae) is a gregarious pupal endoparasitoid of several common fly species. Furthermore, it is a parasitoid of two of the most common pest fly species in North America: the stable fly Stomoxys calcitrans (L.), and the house fly Musca domestica L. T. nigra was first established in a North American laboratory from specimens that emerged from stable fly pupae collected in Russia and Kazakhstan in 1999. Little is known about the life history and definitive host range of this insect. No records of this insect in North America have been made. Its ability to successfully parasitize multiple common pest fly species, however, as well as its small size and inexpensive, simple rearing methods make it a potentially valuable biological control agent against stable flies and house flies. The first photographs of T. nigra are presented with a detailed analysis of adult external morphology and sexual dimorphism. The rearing methods used in maintaining colonies of this parasitoid are provided. Dissections of T. nigra ovarioles were made to determine mean number of ova. Weights of, and head capsule widths from, parasitoids reared on two different hosts were made to determine whether variance in body size exists with regard to host size, and it was determined that a larger host does produce, on average, larger parasitoids of this species. A longevity experiment was conducted to determine the mean lifespan of male and female parasitoids when provided with honey, water and host pupae. It was determined that providing honey lengthened adult lifespan, an effect that was increased when host pupae were resulted in conjunction with honey. Providing hosts in the absence of honey and water led to the shortest lifespan for male and female parasitoids. Two variations of a choice test were conducted, one in which adult parasitoids were conditioned for 48 h on the pupae of one of three host species, and another in which wasps were reared on two host species. Conditioning parasitoids for 48 h significantly increased the proportion of female wasps that chose that host species to which they were conditioned in an open-arena assay for house fly-conditioned parasitoids only. Rearing parasitoids on a particular host species led to a significant difference in host choice in an open-arena assay, with parasitoids strongly preferring to oviposit in the host species in which they had developed. Y-tube olfactometer experiments were conducted to corroborate findings from the choice test experiments as well as to determine whether response of adult female parasitoids changed significantly with age and previous exposure to a host insect. Conditioning parasitoids increased the likelihood, compared to unconditioned controls, of females choosing their conditioning host when presented with two choices in a Y-tube olfactometer. Rearing parasitoids on a host species greatly increased the likelihood of females choosing their natal host when presented with two choices. Lastly, it was determined that the strength and speed of female response to host odors does not significantly change in the first five days of adult emergence, although the most number of females responded, and demonstrated the fastest response, between two and three days post-emergence.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Geden, Chris J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021395:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021395/00001

Material Information

Title: Life History, Host Choice, and Behavioral Plasticity of Trichopria nigra (Hymenoptera Diapriidae), a Parasitoid of Higher Diptera
Physical Description: 1 online resource (118 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: conditioning, diapriidae, diapriinae, olfactometer, parasitoid, trichopria
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Trichopria nigra (Nees) (Hymenoptera: Diapriidae) is a gregarious pupal endoparasitoid of several common fly species. Furthermore, it is a parasitoid of two of the most common pest fly species in North America: the stable fly Stomoxys calcitrans (L.), and the house fly Musca domestica L. T. nigra was first established in a North American laboratory from specimens that emerged from stable fly pupae collected in Russia and Kazakhstan in 1999. Little is known about the life history and definitive host range of this insect. No records of this insect in North America have been made. Its ability to successfully parasitize multiple common pest fly species, however, as well as its small size and inexpensive, simple rearing methods make it a potentially valuable biological control agent against stable flies and house flies. The first photographs of T. nigra are presented with a detailed analysis of adult external morphology and sexual dimorphism. The rearing methods used in maintaining colonies of this parasitoid are provided. Dissections of T. nigra ovarioles were made to determine mean number of ova. Weights of, and head capsule widths from, parasitoids reared on two different hosts were made to determine whether variance in body size exists with regard to host size, and it was determined that a larger host does produce, on average, larger parasitoids of this species. A longevity experiment was conducted to determine the mean lifespan of male and female parasitoids when provided with honey, water and host pupae. It was determined that providing honey lengthened adult lifespan, an effect that was increased when host pupae were resulted in conjunction with honey. Providing hosts in the absence of honey and water led to the shortest lifespan for male and female parasitoids. Two variations of a choice test were conducted, one in which adult parasitoids were conditioned for 48 h on the pupae of one of three host species, and another in which wasps were reared on two host species. Conditioning parasitoids for 48 h significantly increased the proportion of female wasps that chose that host species to which they were conditioned in an open-arena assay for house fly-conditioned parasitoids only. Rearing parasitoids on a particular host species led to a significant difference in host choice in an open-arena assay, with parasitoids strongly preferring to oviposit in the host species in which they had developed. Y-tube olfactometer experiments were conducted to corroborate findings from the choice test experiments as well as to determine whether response of adult female parasitoids changed significantly with age and previous exposure to a host insect. Conditioning parasitoids increased the likelihood, compared to unconditioned controls, of females choosing their conditioning host when presented with two choices in a Y-tube olfactometer. Rearing parasitoids on a host species greatly increased the likelihood of females choosing their natal host when presented with two choices. Lastly, it was determined that the strength and speed of female response to host odors does not significantly change in the first five days of adult emergence, although the most number of females responded, and demonstrated the fastest response, between two and three days post-emergence.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Geden, Chris J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021395:00001


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LIFE HISTORY, HOST CHOICE AND BEHAVIORAL PLASTICITY OF Trichopria nigra,
(HYMENOPTERA: DIAPRIIDAE), A PARASITOID OF HIGHER DIPTERA






















By

KIMBERLY MARIE FERRERO


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA INT PARTIAL FULFILLMENT
OF THE REQUIREMENT S FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008


































O 2008 Kimberly Marie Ferrero



































To my father, the first and greatest scientist in my life.









ACKNOWLEDGMENT S

The list of people who have contributed to my reaching this point in my education is both

long and impressive. I am indebted to so many men and women whose advice, encouragement

and criticism have made this body of scientific work what it is. I can only hope that everyone

mentioned within these pages knows that they are as much a part of thi s writing as I am.

First and foremost, the deepest of thanks go to the scientists who served on my Master' s

committee: Dr. Chris Geden and Dr. Jerry Hogsette. These two men have devoted time, patience,

countless hours both in the field and in the laboratory, endless reams of paper and red ink, and an

embarrassment of intellectual riches to my education. From my committee I have known nothing

but genuine caring and encouragement tempered with the ability to let me know when my

research (or my work ethic) needed tweaking, and the ability to point out my strengths and

weaknesses as a graduate student. I am fortunate to have been mentored by true scientists, and

hope that I will do their tutelage justice.

I would like to thank the scientists and staff in my laboratory: Hank McKeithen and Ashley

Campbell. Hank had passed onto me both the importance of rigorous and proper scientific

method as well as memories of many hours of field work and laboratory assays that would have

otherwise been much more tedious. His friendship, his guidance and advice both on professional

and personal matters has left me feeling that I have always, and will always, have a friend at my

lab bench. Ashley kept the laboratory running flawlessly through many of my experiments, kept

me sane during many a data analysis and writing flurry, and kept me intrigued and humored with

her knowledge of biology and the scientific world. I would not be as patient a scientist if Ashley

had not taught me the limits of human endurance in the face of scientific tedium.

During my assistantship in Dr. Geden' s laboratory, many scientists, researchers and

employees at the United States Department of Agriculture Center for Medical, Agricultural and









Veterinary Entomology became friends and mentors to me. In no particular order (except

perhaps as I ran into them every morning) I would like to thank with all warmth and caring Dr.

Gary Clark, Dr. Matt Aubuchon, Dr. Brian Quinn, Dr. Sandra Allan, Dr. Ulrich Bernier, Dr. Dan

Kline, Natasha Elejalde, Greg Allen, Kathleen Smitherman, Mike Brooks, and Lindsay Clark

among others. Writing my thesis, papers and grants would not have been possible without their

input, nor would working at the USDA have been so enj oyable, without their friendship,

professional candor and willingness to help.

While it has been nearly a decade since I was a student in his classroom, I would like to

thank Mr. Mark Schiffer, my biology teacher of four wonderful years and the reason I am so

detail-obsessed and well-versed in molecular genetics and the virtues of caffeination. He has

inspired more young scientists than he will ever know.

I would like to extend a personal note of gratitude to the fellow students who I came to

know as friends while at the University of Florida. The first is Christine Bertrand, who has been

a best friend and comrade in academia since our first day of undergraduate classes seven years

ago. Christine has been my anchor through years of classes, graduation and graduate school,

writing our theses concurrently, and the many trials and tribulations of growing up. She is as

much family to me as are my sisters and brothers. I would also like to thank Roxanne Burrus, a

fellow entomologist who came from the same laboratory as I did, and quickly became a woman I

admired and leaned on for help and advice. She is a model for young female scientists to

emulate; I have learned so much from her in a very short time.

The faculty and staff at the University of Florida' s Entomology and Nematology

department will always command my respect and gratitude for the time and effort they devote to

each of the graduate students who pass through their laboratories and lecture halls. I am grateful









to Dr. Phil Kaufman for inspiring a love of medical and veterinary entomology in me, and for

making my induction into the field of entomology as a new graduate student an enj oyable and

comfortable one. Every student of his is a fortunate student. I would also like to thank Dr. Carl

Barfield for the opportunity to teach his students, as well as for his practical and honest advice on

the professional aspects of being a scientist. I am also indebted to Debbie Hall for several years

of reminders, deadlines, crisis-solving skills and an innate ability to keep an entire department of

students on track. A final thanks is extended to Dr. Richard Patterson, his wife, daughter and

granddaughter: a family of entomologists who have treated me like family as well, and instilled

in me a sense of academic diplomacy, hard work and honesty.

The most personal of thanks I leave to the end, not for lack of importance but because

wording such gratitude is difficult. My family has always been an ongoing source of love,

support and encouragement. I would like to thank my parents, Patti and Frank, as well as my

siblings Ashley, Francesca, Christopher and Shawn, for always being a light in the window, an

answer to a phone call, a willing eye to read my papers before a deadline, and a group of open

arms that never cease to remind me how important I am to them. My father, a heart surgeon and

professor of medicine, was my role model and the inspiration for my becoming a scientist. He

passed away in winter of 2006, a tragedy that forever changed my life. While I will never be the

scientist he was, I can only be grateful that I was part of the life of a man who saved so many

others. Last, but far from least, I would like to thank Matthew Cmar, who has been an

understanding and supportive partner through all these semesters of research, class, experiments

and writing. He made me laugh many times, prompted me to cry a few, cheered me on to stay up

late to meet deadlines, and made me feel like a good scientist even when I doubted myself. His

love and encouragement will never be taken for granted.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ................. ................. 4...............


LI ST OF T AB LE S............... ............... 9


LI ST OF FIGURE S ................. ................. 11......... ....


AB STRACT ................. ...............12.................


CHAPTER


1 INTRODUCTION.............................. 14


2 LITERATURE REVIEW ............... .................... 16


Evolution of the Parasitic Hymenoptera .................. .. .. ........... .... ............... 16...
Biological Control of, and Economic Damages Caused by, Pest Flies ................. ................ 18
Family Diaprii dae ................. ................. 2......... 1....
B iology ............... ............... 21...
D distribution ............... ............... 21...

Trichopria nigra ................. ................ 22......_ ....

Origin and Distribution .............._. ................ 22......_ ...
Biology .............._. ................ 23......_ ....
Food and Hosts .............._. ...............23....._.......


3 NOTES ON SPECIES RECOGNITION AND REaRING METHODS .............. .............25


Introduction .........._.... .. .... _._ .............. 25...
Positive Identification of Species ............ ...... __ ...............25.
A dult Insect .............. ........... .... ............ ..._ ............. 2
Female Reproductive Potential with Respect to Body Size............_ .. ......._ ........27
Rearing Methods ............ ...... __ .............. 29...
C ontai ner s ............ ..... ._ ............... 3 0..
D iet ............... ...............30...
Host Provisioning ............... ...............30....


4 LONGEVITY AND FECUNDITY EXPERIMENTS ......___ ...... .. __ .........._......42


Introduction ............ .... __ .............. 42...
Material s and Method s............ ...... __ .............. 46..

Study Site ............ .... __ .............. 46...
Parasitoid s ............._ ......... ..............._ 46....

Longevity Arena ............._ ......... ..............._ 47....
Experimental Design ............._ ......... ..............._ 47....
Longevity determination ............._ ......... ..............._ 47....












Fecundity estimation ................. ...............48.................
Stati stical Analy si s ................. ................. 49.............
R e sul ts ................... .......... ................. 5 0....

Longevity Determination ................. ................. 50..............
Fecundity Estimation ................. ...............51.................
Di scu ssi on........._...... ......___. ................ 52..

Longevity Determination ........._...... ................ 52..._... ....
Fecundity Estimation ................. ................. 54..............


5 ARENA-CHOICE EXPERIMENTS ................. ...............69................


Introduction ................. ...............69.......... ......
Material s and Method s............... .................72

Study Site ................. ...............72.......... ......
H osts ................ ...............72.......... ......
Parasitoid Strains ................... .. ......... ...............72......

Conditioning parasitoids for short-term assay............... ..................72
Arena for short-term assay............... ..................73
Parasitoids for rearing assay ................. ...............74......... ....
Arena for rearing assay ................. ...............74......... ....
Statistical Analysis ................. ...............75........_ .....
Results ........ ................. ...............76 .....
Di scu ssi on ........._...... ...............77..__._. .....


6 Y-TUBE OLFACTOMETER EXPERIMENT S ................ ...............87........... ...


Introduction ................. ................. 87..............
Material s and Method s............... ................ 90

Study Site ................. ................. 90..............
Parasitoids ............._.. ........ ...._.. .... .. .._ ..... ...... ........9

Experiment 6-1: Effect of Three Days of Conditioning on Host Choice.............._.._. ......91
Experiment 6-2: Host Choice Response After 1 d, 3 d, and 5 d of Conditioning on
S. bullata Pupae................ ... .. .. ......... .. ..........92

Experiment 6-3: Host Choice by Parasitoids Reared on Different Hosts ................... .....93
Experiment 6-4: Response of Unconditioned Parasitoids to Hosts during Five Days
Post-Emergence................ .............94
Statistical Analy si s ..........._...__........ ..............._ 94....
Results ..........._...__........ ...............96.....
Di scu ssi on ................. ...............97................


LIST OF REFERENCES ................. ...............107................


BIOGRAPHICAL SKETCH ................. ...............117......... ......










LIST OF TABLES


Table page

Table 3-1. Head capsule widths and egg numbers of Trichopria nigra females and males
(50/50 sex ratio) reared on pupae of Salrcophaga bullata.....__.___ ........___ .............32

Table 3-2. Head capsule widths and weights for female and male Trichopria nigra (50/50
sex ratio) reared on pupae of Salrcophaga bullata and Stomoxys calcitrans. ................... .. 33

Table 4-1. Mean lifespans (+SE) (d) of Trichopria nigra adults under different feeding
treatments at 25 TC ................. ................. 57.............

Table 4-2. Female 7 nigra, two-way ANOVA results for influence of presence/absence of
honey and host pupae on survival time ................. ...............58........... ..

Table 4-3. Male 7 nigra, two-way ANOVA results for influence of presence/absence of
honey and host pupae on survival time ................. ...............59........... ..

Table 4-4. Effect of diet on mean number (+SE) of pupae killed by female T. nigra, pupae
producing wasps, having dead wasps, and total number of parasitized pupae. ........._.......60

Table 4-5. Cumulative (+SE) numbers of male and female progeny, respectively, produced
by parasitoids given only water and pupae, or water, honey and pupae..............._...__.........61

Table 5 -1. Proportion of female 7 nigra responding to pupae of 3 host species after prior
conditioning for 48 h on pupae of a single host species. Means in rows............................ 81

Table 5 -2. Proportion of male 7 nigra responding to pupae of 3 host species after prior
conditioning for 48 h on pupae of a single host species ......... ................. ............... 82

Table 5-3. Mean proportions of female parasitoids reared on either S. calcitrans or S. bullata
pupae ................. ...............83.................

Table 5-4. Mean proportions of male parasitoids reared on either S. calcitrans or S. bullata
pupae ................ ................. 8......... 4.....

Table 6-1. First choices made by female parasitoids conditioned for 3 d on either S.
calcitrans or S. bullata pupae ................. ...............100........... ...

Table 6-2. First choices made by female parasitoids conditioned on S. calcitrans pupae and
tested at 1 d, 3 d and 5 d ............... ....................101

Table 6-3. First-turn choices made by 3 -d old female parasitoids reared on either S.
calcitrans or S. bullata pupae ................. ...............102........... ...

Table 6-4. First-turn choices made by female parasitoids run in a Y-tube olfactometer at 24-
h interval s after emer gence. ................. ................. 103........ ...











Table 6-5. Results of G tests for the four olfactometer experiments ................. ......................104










LIST OF FIGURES


Fiu~re page




Figure 3-1. Adult female Trichopria nigra, lateral view. .............. .....................34

Figure 3 -2. Adult male Trichoprianigra, lateral view. ................. ...............35........... .

Figure 3 -3. Probing female Trichopria nigra............................. ........36

Figure 3 -4: Dissection of 7: nigra abdomen ................. ...............37........... .

Figure 3 -5. A) Empty puparium of Salrcophaga bullata; and B)Dissected host remnants. ...........38

Figure 3 -6. Microscopy image (400x) of 3 -day-old Trichopria nigra ovarioles. ................... ....... 39

Figure 3 -7. Regression analysis for body size and egg load of female Trichopria nigra ..............40

Figure 3-8. Rearing containers used for colonies of Trichopria nigra ............. ....................41

Figure 4-1. Rearing containers for longevity determination ................. ............... ......... ...62

Figure 4 -2. A) 60-ml cups containing indivi dually-encap sulated S. calcitrans pupae; and B)
Example of successfu~lly-parasitized pupa ................. ...............63................

Figure 4-3. Mean adult A) female and B) male Trichopria nigra mortality for all treatments.....64

Figure 4-4. Total number of S calcitrans pupae parasitized by female parasitoids for each of
two treatments ..........._...__........ ...............65.....

Figure 4-5. Mean number of adult parasitoids that emerged daily for each of two treatments .....66

Figure 4-6. The average number of parasitoids that emerged per pupa per day for A) water
and hosts only.; and B) food, water and hosts ................. ...............67.............

Figure 4-7. Cumulative mean number of adult parasitoids that emerged from S. calcitrans
pupae for each of two treatments ........._. .......___ ...............68..

Figure 5-1. A) Containers for parasitoids conditioned for 48 h on different host pupal types;
and B) containers with tops secured during experimentation ................. ............. .......85

Figure 5-2. Containers for parasitoids that emerged from different host pupal types .........._.._......86

Figure 6-1. A) Y-tube olfactometer used in experimentation; and B) choices presented in
screened chambers. .............. .....................105

Figure 6-2. Y-tube olfactometer arms and entry capsule. ....._.__._ .... ... .__. ......._._.......10









Abstract of Thesi s Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

LIFE HISTORY, HOST CHOICE AND BEHAVIORAL PLASTICITY OF Trichopria nigra,
(HYMENOPTERA: DIAPRIIDAE), A PARASITOID OF HIGHER DIPTERA

By

Kimberly Marie Ferrero

December 2007

Chair: Christopher J. Geden
Major: Entomology and Nematology

Trichopria nigra (Nees) (Hymenoptera: Diapriidae) is a gregarious pupal endoparasitoid of

several common fly species. Furthermore, it is a parasitoid of two of the most common pest fly

species in North America: the stable fly Stomoxys calcitrans (L.), and the house fly M~usca

domestic L. I nigra was first established in a North American laboratory from specimens that

emerged from stable fly pupae collected in Russia and Kazakhstan in 1999. Little is known about

the life hi story and definitive host range of this insect. No records of thi s insect in North America

have been made. Its ability to successfully parasitize multiple common pest fly species, however,

as well as its small size and inexpensive, simple rearing methods make it a potentially valuable

biological control agent against stable flies and house flies.

The first photographs of 7 nigra are presented with a detailed analysis of adult external

morphology and sexual dimorphism. The rearing methods used in maintaining colonies of this

parasitoid are provided. Dissections of 7 nigra ovarioles were made to determine mean number

of ova. Weights of, and head capsule widths from, parasitoids reared on two different hosts were

made to determine whether variance in body size exists with regard to host size, and it was

determined that a larger host does produce, on average, larger parasitoids of this species.









A longevity experiment was conducted to determine the mean lifespan of male and female

parasitoids when provided with honey, water and host pupae. It was determined that providing

honey lengthened adult lifespan, an effect that was increased when host pupae were resulted in

conjunction with honey. Providing hosts in the absence of honey and water led to the shortest

lifespan for male and female parasitoids.

Two variations of a choice test were conducted, one in which adult parasitoids were

conditioned for 48 h on the pupae of one of three host species, and another in which wasps were

reared on two host species. Conditioning parasitoids for 48 h significantly increased the

proportion of female wasps that chose that host species to which they were conditioned in an

open-arena assay for house fly-conditioned parasitoids only. Rearing parasitoids on a particular

host species led to a significant difference in host choice in an open-arena assay, with parasitoids

strongly preferring to oviposit in the host species in which they had developed.

Y-tube olfactometer experiments were conducted to corroborate findings from the choice

test experiments as well as to determine whether response of adult female parasitoids changed

significantly with age and previous exposure to a host insect. Conditioning parasitoids increased

the likelihood, compared to unconditioned controls, of females choosing their conditioning host

when presented with two choices in a Y-tube olfactometer. Rearing parasitoids on a host species

greatly increased the likelihood of females choosing their natal host when presented with two

choices. Lastly, it was determined that the strength and speed of female response to host odors

does not significantly change in the first five days of adult emergence, although the most number

of females responded, and demonstrated the fastest response, between two and three days post-

emergence .









CHAPTER 1
INTRODUCTION

Because the hosts of many parasitic wasps are flies commonly considered pests of humans

and livestock, the significance of parasitoids as biological control organisms is considerable

(Rueda and Axtell 1985, Anderson and Leppla 1992). Two of the most important pest flies with

regard to human health, well-being and economics in North America are often considered M~usca

domestic L., the house fly, and Stomoxys calcitrans L., the stable fly. Other flies which are

considered pests of humans and commercially-reared animals include the face fly M~usca

autumnalis De Geer, the horn fly Haematobia irritans (L.), and a variety of blow flies and flesh

flies in the families Calliphoridae and Sarcophagidae.

One of the primary motives for fostering research in the field of biological control is the

growing resistance of pest insects to chemicals utilized in controlling them. House flies were

among the first insects to demonstrate documented resi stance to in secticides, as DDT became

commercially available for house fly control in 1944. By 1947, DDT had failed to control fly

populations in Europe (Decker and Bruce 1952). House fly resistance to pyrethroids emerged

within ten years of introduction of these chemicals as commercial products for house fly control

(Hogsette 1998). Resistance of insects has long been known to develop swiftly in insect

populations subj ected to frequent, and high-dosage, pesticide applications (Mallis et al.. 2004).

An additional dimension to the problem of pest fly resistance to insecticides is the apparent

genetic basis for pesticide resistance exists in the house fly, which is allelic in nature and subject

to both genetic mutation and the migration of pest fly populations with novel alleles (Rinkevich

et al. 2007). In the future, agriculturists may find that relying solely on pesticidal methods to

prevent insect damage to livestock and crops will have become wholly ineffective. Currently it

proves impossible (Hogsette 1998) to maintain livestock pests such as horn flies at or below their









damage thresholds with registered pesticides. Treating agricultural pest problems with biocontrol

organisms, however, does not incur resistance to that treatment method as do pesticide

applications.

In light of the reduced efficacy of traditional chemical control and the effects of pesticides

on the environment, there exists an increasing need for novel and effective biological control

organi sms which can be used to reduce the number of pest insects and their effects on humans

and agricultural products. The aims of this research were to better understand the biology and

behavior of a species of endoparasitic wasp, Trichopria nigra (Nees) (Hymenoptera: Diapriidae),

which parasitizes numerous species of flies commonly considered pests of humans and livestock.

Because the facility in which this research was conducted is the first known location of in-

laboratory rearing of 7: nigra, a detailed identification of this species, along with this

researcher' s rearing methodology, is included (Chapter 3). A longevity experiment was

conducted to determine the longevity of this species when presented with a food source, water,

and hosts in several combinations, as well as to compile a greater understanding of the life

history of this species (Chapter 4). Additionally, two experiments were performed to determine

(1) the preference of this species when provided with multiple choices for a host, and whether

that choice can be influenced (Chapter 5); and (2) whether olfaction is an important method this

species uses to locate a host, and if the strength of an olfaction-based host choice changes with

the age of the adult insect (Chapter 6).









CHAPTER 2
LITERATURE REVIEW


Evolution of the Parasitic Hymenoptera

Within the order Insecta, parasitic life history strategies account for the second-most

popular life hi story strategy in insects after phytophagy (Althoff 2003). Within Hymenoptera,

parasitism of other insects is so prevalent across the families of waisted wasps (Hymenoptera:

Apocrita) that an early evolutionary origin of parasitic Hymenoptera is likely (Whitfield 1992).

Furthermore, the prevalence of parasitic lifestyles with the maj ority of families of Apocrita

possessing genera that are considered parasitoids of plants or other insectst indicates that such

life history strategies are highly successful.

Sy stematic coevolution of insects and the plants and animals which provide them with

food, shelter and reproductive niches is well-documented across taxa. A common entomological

example is louse-host specificity (Bush and Clayton 2006). Parasitoid-host coevolution is

similarly apparent at least among plant parasitoids. Sidhu (1984) observes that genetic

selection in many plants important to humans (and to insects as hosts) is the result of breeding

and crop selection. The evolution of host animals, in contrast, is often driven more by ecology

and behavior, making closely-related coevolutionary changes more difficult to observe.

However, diversity of parasitoids seems, generally, to be under the control of host diversity

(Sidhu 1984, Hufbauer 2001).

According to Quicke (1997), the fossil record contains preserved hymenopteran fossils

dating as early as the mid-Triassic period. There are currently two extant suborders of

Hymenoptera (Johnson and Triplehorn 2004): the Symphata (including sawflies and wood

wasps) and the Apocrita (all other hymenopterans including all known parasitoids), although the

outset of the Jurassic period saw diversif ication of Hymenoptera to include the extinct suborders









Karatavitidae and Ephialtitidae (Quicke 1997). The appearance of morphological features that

would allow for parasitism of a host, such as a lengthened ovipositor, hint at the emergence of

the first parasitoid hymenopterans during the Jurassic period.

Within Hymenoptera, the suborder Apocrita is primarily defined (Johnson and Triplehorn

2004) by the fusion of the first abdominal segment to the thorax and the constriction or "waist"

between the first and second abdominal segments. Other defining characteristics, such as the

number of basal hindwing cells (Quicke 1997), further separate Apocrita from the evolutionarily

primitive sawflies. Within Apocrita, ten of the twelve superfamilies largely include parasites of

either plants (gall wasps, fig wasps) or animals (overwhelmingly other members oflnsecta)

(Johnson and Triplehorn 2004). Superfamilies within Hymenoptera which are known to contain

parasitoid members include Ichneumonidea, Chalcidoidea, Evanioidea, Proctotrupoidae,

Bethyloidea, Scolioidea and Vespoidea (Whitfield 1998, Johnson and Triplehorn 2004).

The question as to why parasitism evolved across multiple insect orders has been under

speculation for decades (Quicke 1997). Parasitism most likely evolved only once in basal

Hymenoptera (Eggleton and Belshaw 1992) as, driven by competition for food sources, early

mycophagic wasps began to kill larvae already feeding on fungi and superimpose their own

larvae on the food source. The most primitive (from a cladistic viewpoint) parasitic superfamilies

- the Ichnauemonidea and Evanioidea are often found in close relation to detritus and fu~ngal-

heavy environments (Eggleton and Belshaw 1992, 1993); the more advanced parasitoid

hymenopterans practice life history strategies ranging from parasitizing developing larvae to take

advantage of a longer developmental time (Gelman et al.. 2005b), to drilling into host puparia

and laying eggs outside the host. Further discussion on the advantages and disadvantages of a

parasitic lifestyle in Hymenoptera follows below.









From an evolutionary viewpoint, it might be assumed that if parasitism evolved once in a

clade and was not only retained as a life history strategy but became the overwhelming life

history strategy of an entire suborder (Borror et al. 1974, Johnson and Triplehorn 2004), that life

history strategy must offer distinct and significant benefits to the insects practicing it. One

advantage that parasitoids encounter is that they do not have to devote time as immature insects

to foraging for, locating, and processing food (Quicke 1997). Parasitoid wasps live within their

source of food, water, and (often) endocrine compounds (Gelman et al.. 2005a). Their hosts

confer protection from changes in temperature and humidity, and unless the host insect is

predated upon, the parasitoids are safeguarded from predation themselves.

Obvious risks are associated with a parasitic life history for parasitoid hymenopterans.

The quality of the host often determines the quality of the next generation of para sitoids (Ellers

et al. 1998, Consoli and Vinson 2004), as the available nutrients for the developing parasitoids

are limited by the size and carrying capacity of the host insect. Another drawback to parasitism is

the risk of choosing a host species that is poorly suited to parasitism, especially in non-specific

solitary parasitoid wasps (Quicke 1997, Ferrero 2006 from unpublished research).

Biological Control of, and Economic Damages Caused by, Pest Flies

Because the hosts of many parasitoid wasps are fli es commonly considered pests of

humans and livestock, their significance to human populations is considerable (Rueda and Axtell

1985, Anderson and Leppla 1992). The most important pest flies with regard to human health,

well-being and economics in North America are often considered M~usca domestic L., the house

fly, and Stomoxys calcitrans (L.), the stable fly (Geden and Hogsette 1994). Other flies which are

sometimes considered pests of humans and commercially -reared animals include the face fly,

M~usca autumnalis, the horn fly, Haematobia irritans, the flesh fly, Salrcophaga spp., and the

black garbage fly, Ophyra spp.









According to the USDA, confined and range cattle contributed the largest share of

agricultural profit in the United States with 37.9 billion dollars in income (Geden and Hogsette

1994). Of this amount, 17.6 billion dollars in damage come from confined cattle facilities; the

highest infestations of pest flies which are hosts of numerous parasitoid species occur in confined

cattle facilities. While traditionally considered pests of confined cattle only (Smith and Rutz

1991), stable flies have been known to disturb cattle in open fields in geographic locations that

are subject to periodical rainfall (Geden and Hogsette 1994). Directly, stable flies impact cattle

by blood-feeding, which reduces overall health and feeding behavior of the animal and,

ultimately, quality of product (Campbell and Berry 1989).

Poultry contribute a large portion of income to the agricultural industry in the United

States, and with an increase in poultry rearing in the past five decades from small agribusinesses

to large-scale poultry rearing facilities, pest flies have become an increasingly problematic issue.

The primary pest of poultry agribusinesses, house flies, present a sanitation and public health

problem (Mann et al. 1990). Transmission of avian pathogens as well as bacteria known to cause

human illness (Geden and Hogsette 1994) create a potentially hazardous working environment in

poultry houses. The house fly has been implicated as a potential vector of pathogens that pose

serious risks to human health (Eldridge and Edman 2003) such as poliomyelitis, cholera, typhoid,

tuberculosis and dysentery. Additionally, house flies swarming in confined spaces such as

rearing facilities causes an annoyance such that poultry farms may incur a monetary loss in

employee turnover, reduced efficiency of work, and time spent implementing control methods

for pest flies in response to complaints from public health agencies.

Insect resistance to chemical control is another justification for the importance of

parasitoids in improving agricultural and domestic aspects of human life. Insect physiological









resistance to chemical control has, since the implementation of en masse pesticide application in

the forties, become a growing problem for agriculturalists worldwide (Learmount et al.. 2002).

Hogsette (1998) documents that house fly resistance to pyrethroids emerged within ten years of

implementation; resistance of pest filth flies to avermectins such as abamectin (Clark et al..

1995) developed swiftly in insect populations and established readily in the gene pool. In the

future, agriculturists may find that relying solely on pesticidal methods to prevent insect damage

to livestock and crops will have become wholly ineffective, even as currently it proves

problematic (Hogsette et al. 1991) to maintain livestock pests such as horn flies at or below their

damage thresholds with registered pesticides. Treating agricultural pest problems with biocontrol

organisms, however, does not incur resistance to that treatment method as do pesticide

applications.

Most commercially-utilized biocontrol organisms are wasps which are pupal parasitoids

parasitoid in the family Pteromalidae (Hymenoptera: Chalcidoidea). Biological control of pest

flies involves releasing either adult parasitoids of the pest fly, or fly pupae with developing

parasitoids within, en masse at an infested location (Geden and Hogsette 2006). Adult parasitoids

increase the mortality of the host insect at a specific point in development by killing the host,

usually prior to adult eclosion or adult emergence from the puparia (Rueda and Axtell 1985,

Quicke 1997). Little is known about the efficacy of other parasitoid families in controlling pest

flies. Trichopria nigra (Hymenoptera: Diapriidae) is a gregarious endoparasitoid of pest fly

pupae that has been shown to significantly reduce population levels of house fly and stable fly

pupae in the research conducted within this body of work. It is this researcher' s goal to

demonstrate that 7 nigra, a novel and little-known parasitoid wasp is an efficient biocontrol









organism for the stable fly S. calcitrans, and potentially of other pest fly species within its host

repertoire via selective conditioning.

Family Diapriidae

Biology

The family Diapriidae (superfamily Proctotrupoidea) consists of minute wasps, all of

which are parasitoids of other insect taxa (Nixon 1980). Diapriid wasps are known

endoparasitoids of members of Diptera, Coleoptera (Masner 1993), and in the unique case of

genus Ismarus, other Hymenoptera (Loiacono 1987). Diapriidae is divided into four subfamilies:

Ismarinae, Ambositrinae, Belytinae and Diapriinae. Some members of Diapriidae are solitary;

many, such as Trichopria nigra, are gregarious endoparasitoids (Nixon 1980). Solitary

parasitoids are defined as those insects which lay one egg per host; gregarious parasitoids lay

multiple eggs per host (Johnson and Triplehorn 2004). Subfamily Diapriinae, to which T. nigra

belongs, shares the fewest traits and is often considered by insect taxonomists as the subfamily to

which parasitoids not fitting the other three subfamilies are assigned (Buss, personal

correspondence).

Medvedev (1988) provides a thorough catalog of characteristics shared among members

of Diapriidae. Among these features include a dark and glossy exoskeleton, a small and rounded

head with long antennae, and a markedly narrow first abdominal segment (petiole). Despite most

members of Diapriidae having large wings relative to absolute body size, they are poor fliers

compared to many other Hymenoptera (Medvedev 1988).

Distribution

While genera of Diapriidae are distributed worldwide, (Masner 1976, Masner 1993), the

exact distribution of most of the approximately four thousand species placed within Diapriidae is

largely unknown (Masner and Garcia 2002).









Trichopria nigra


Origin and Distribution

Roger Moon (University of Minnesota) collected pupae of Stomoxys calcitrans on dairy

farms near the cities of Almaty, Kazakhstan, and Kraznodar, Russia, in 1999 (C.J. Geden,

USDA/ARS, personal correspondence). The purpose of such collections was to discover novel

parasitoids of the stable fly and house fly. Pupae collected by Dr. Moon were transported to the

United States and the first documented emergence and colony establishment of 7 nigra occurred

in 2000 at the USDA/ARS CMAVE facility in Gainesville, Florida. This is the first documented

laboratory colony of 7 nigra.

The distribution of 7 nigra is not completely known; its discovery in Eastern European

and Eurasian sites by Dr. Moon suggests that distribution is at least partially Palearctic;

Medvedev (1988) cites the distribution as Romania (Moldavia). The most recent record of 7

nigra in the wild is a 2002 collection of Trichopria spp. taken from sites in Kalambaka, Greece

(Petrov 2002); the collector lists the known distribution of 7 nigra as Germany north to Sweden

and east to Moldova. Given the proximity of Moldova and Sweden to Russia, it is likely that the

distribution of 7 nigra extends from central Europe into northern Asia.

Related species in the genus Trichopria are distributed worldwide; collections have been

made in the eastern (Bradley et al. 1984) and western United States(Krombein et al. 1979a;

Krombein et al 1979b), as well as European countries such as Hungary (Hogsette et al. 1994). In

Africa, Trichopria spp. have been observed in countries such as Zimbabwe (Huggert and

Morgan 1993; Morgan et al. 1990) and Ethiopia (Huggert 1977).

Given that many discoveries of Trichopria species are made in and near poultry houses on

all continents the genus has been discovered (Hogsette et al. 1994; Morgan et al. 1990), and that

7: nigra emerged from S. calcitrans pupae collected by Dr. Moon on dairy farms, it is likely that










T nigra may live and reproduce at least in the proximity of livestock- and poultry-rearing

facilities.

Biology

The first entomological description of Trichopria nigra is attributed to Nees (1 83 4); thi s

species was included in Kieffer' s 1916 taxonomic treatise on Diapriidae (Kieffer 1916), and

more recently in Masner and Garcia' s (2002) description of Diapriidae. Few taxonomic keys

provide an adequately detailed identification of the genus. Medvedev (1988) offers the following

couplet for identification of members of Trichopria:

...Prothorax with collar of white or silvery hairs; collar broader
than 1st antennal segment, and these hairs discumbent. Temples
with white or silvery hairs. Longitudinal axis of eye usually not
shorter than length of middle coxa. Head roundish or rectangular
on dorsal surface. Females: Antennae at least with dark clava.
Antennal segments, commencing from the 3rd, round...

In addition to the above couplet, 7 nigra adults possess features that distinguish the

species from sister species; the 2nd and 3rd antennal segments are approximately equal in length

and the legs, in contrast to the glossy black body color, are a dark yellow color and nearly

translucent (Medvedev 1988). Males and females are dimorphic with respect to antennal shape;

females possess a clubbed terminus at the distal end of each antenna whereas males possess

comparatively more uniform antennae. The sexes are not dimorphic with respect to size,

although a larger host (e.g., S. bullata is larger than S. calcitrans) will usually produce larger

parasitoid offspring of both sexes (see Chapter 3: Effect of Host Size on Parasitoid Size).

Food and Hosts

7 nigra has not, to the author' s knowledge, been observed feeding in the wild. A related

species, T. stomoxydis, which is a gregarious endoparasitoid of S. calcitrans, shares a similar life

history to 7 nigra. It was determined that T. stomoxydis does not host feed prior to, or following,









host location and oviposition; without a food source such as plant nectar, longevity was

calculated to be approximately three days (Morgan et al. 1990). Other shared life history features

include the endoparasitic nature of larval development; both T. nigra and T. stomoxydis

immatures feed and develop within the body of the pupating host. Unlike T. nigra, however, 7

stomoxydis is host-specific to S. calcitrans (Nash 2005).

In colony at the USDA/ARS CMAVE facility, adult 7 nigra are provisioned with honey

on an ad libitum basis. Despite being a koinobiont species, adult female 7 nigra do not host-feed

and thusly do not require a meal to complete ovariole maturation (Chan and Godfray 2005).

Water i s also provided ad libitum. Although it i s likely that thi s insect procures moi sture from

nectar food sources in the wild; the moisture content of most commercial honeys, at 20-40% is

considerably lower than that of plant nectars due to the concentration of sugars (Bijlsma et al.

2006). For a detailed description of rearing methodology, see Chapter 3.









CHAPTER 3
NOTES ON SPECIES RECOGNITION AND REARING METHODS


Introduction

Very few references to Trichopria nigra (Nees) exist in the scientific literature. A 1984

collection of seaweed fly pupae from two species collected from Kieler Forde, Germany

(Heitland 1988), Coelopa frigida (F.) and Fucellia tergina (Zett.), yielded specimens identified

as 7 nigra. Petrov (2002) mentions 7 nigra in a catalogue of Diapriid parasitoids found in

Greece, although first identification of this species is attributed to Nees in 1834 (noted in the

Hymenoptera collection of the Zoological Museum, University of Copenhagen). A field survey

by Hogsette et al. (1994) in central Hungary lists two species of Trichopria which were found in

proximity to pupae of2~usca domestic L. and Stomoxys calcitrans (L.); given the discovery of

7 nigra in Russia and Kazakhstan by Moon (personal communication) as well as in China by

Yujie et al (1997), it is possible that this species has a broadly Palearctic range.

The lack ofa definitive and readily available species description, in addition to the lack of

known photographs and images of this insect, indicate the need for a detailed analysis of T.

nigra's morphology and differentiation from related species. Furthermore, rearing methodology

of this insect is included for the benefit of scientists who wish to rear 7 nigra in a laboratory

setting. To this researcher' s knowledge, both strains of 7 nigra (the Russian-collected

population and the Kazakhstan-collected population) are morphologically and behaviorally

similar, and are reared identically.

Positive Identification of Species

Adult Insect

7 nigra adults share many of the general traits of family Diapriidae, being described as

small (1-6 mm), black parasitoid wasps with large eyes and geniculate antennae possessing 11-









15 segments, and an ovipositor that is not visibly protruding (Masner and Garcia 2002). The

insect is readily identified as a wasp, with three well-defined body segments, a narrow waist, and

two pairs of wings. The sexes are not sexually dimorphic with regard to size, as both male and

female adults grow to a maximum mean length of 2-3 mm from head capsule to terminal

abdominal segment.

It has been documented (Ueno 2004) that the size of many pupal parasitoids, as well as the

sex ratio of progeny produced, varies in direct relation to the size of their hosts. The sizes of 7

nigra offspring reared on two host fly species' pupae of different sizes were analyzed to

determine if size plasticity with regard to host size is correlated in this parasitoid species. Adult

7 nigra reared on pupae of Stomoxys calcitransrt~r~rt~rtrt~rt (L.) were on average one-third lighter (mean

mass 0.069 & 0.001mg.) than individuals reared on pupae of Sarcophaha bullata Parker (mean

mass 0.106 & 0.001mg.), with a head capsule diameter that is~-80% that of individuals reared on

S. bullata (Table 3 -2).

The head is rounded and possessing of large, black eyes that are the most prominent

feature of that segment. Antennae attach superiorly and medially to the eyes, and sexing of the

adult insect is most easily performed by examining the shape of the antennae. In female insects

the antennal segments enlarge distally into a distinct club (Figure 3 -2). Male 7 nigra adults,

however, possess antennae that are nearly uniformly filamentous (Figure 3-3). Both male and

female insects possess, as do all Diapriine wasps (Masner 1976), thirteen antennal segments.

Second and third antennal segments are equal in length; this feature along with the unique

coloration of the legs is considered the defining trait for 7 nigra, separating it from 7 socia in a

dichotomous key (Medvedev 1988).









The exoskeleton is uniformly black in color and highly reflective, almost lustrous; the legs

are of dark amber to golden yellow color, with femoral and coal segments distinctly darker than

the tarsal segments. The entire insect is sparsely covered with slender bristles. The cephalic point

of the first thoracic segment, as well as thoracic segments 2, 3 and the thoracic-abdominal

juncture, are covered in dense collars of short, golden hairs.

I: nigra ovipositor size is small in relation to body length, and markedly short in

comparison to ovipositor length in taxa such as Ichneumonidae. The length of the ovipositor is

retracted except during probing and oviposition (Figure 3-4). This feature is shared among

Diapriidae (Medvedev 1988). Females are gregarious in oviposition behavior, and adult 7 nigra

chew multiple exit holes in the host puparia (Figure 3-5A). Developing parasitoids require most

of the room inside the developing fly host, and dissected puparia reveal an empty "mummy" or

cuticle which protected the parasitoid larvae inside (Figure 3-5B).

Female Reproductive Potential with Respect to Body Size

Female reproductive anatomy of 7 nigra consists of two bi-lobed ovarioles (Figure 3 -6)

of the meroistic type, germ cells differentiating into both oocytes as well as nurse cells. Like

most koinobionts, female 7 nigra emerge with competent autogenouss) oocytes (Waage and

Greathead 1986) and do not need to feed in order to oviposit, and oocytes are likely not

replenished as they are used. As demonstrated in Figure.3-1, 3-2, and 3 -3, adult body size can

vary greatly in this species, to the extent that it is questioned whether the size of adult female 7

nigra is indicative of their reproductive potential.

To determine whether a correlation exists between adult female size and fecundity (egg

load), dissections of female T. nigra reared on two hosts were conducted. Twenty 2-5 day old

female 7 nigra reared on Stomoxys calcitrans and twenty 2-5 day old female 7 nigra reared on

S. bullata were ovariectomized. The number of eggs for each female was counted (Figure 3 -6),









and the width of the head capsule measured. The mean (+SE) head widths and number of eggs

are listed in Table 3-1. A regressional analysis was conducted for each treatment as well as both

treatments combined, with head capsule width and number of eggs as variables (Figure3 -7).

Furthermore, PROC GLM was conducted (SAS 2000) to determine whether rearing host has an

effect on egg load.

For females reared on S. calcitrans pupae, the average head capsule width was 0.320 mm,

approximately 84% of the head capsule width of females reared on S. bullata pupae (0.3 80 mm)

(Table 3-1). This difference was statistically significant (SAS, 2000) and was consistent with

hypotheses about the 7 nigra size/fitness relationship, and previous parasitoid research (see

Quicke 1997; Cohen et al. 2005). Because head capsule width is an indicator of overall body size

and often, of fecundity and adult fitness (Liu 1985, Visser 1994, respectively), it was expected

that larger females (i.e., females reared on S. bullata, the larger host) would possess more eggs.

This was found to be the case as well, with 7 nigra reared on S. calcitrans possessing on

average approximately 76% of the egg load their S. bullata cohorts developed.

The observed differences in body size and fecundity of 7 nigra when reared on two hosts

of differing host sizes agrees with literature on parasitoid fitness with respect to host fitness. In

general, parasitoids whose hosts are larger have more food available and therefore can acquire

more nutrition for growth and development (e.g., of eggs). Some literature suggest that for

koinobiont parasitoids, who develop internally and allow their hosts to continue physiological

development, the correlation between host size and parasitoid fitness is not necessarily valid (see

Jenner and Kuhlmann 2005). For T. nigra, however, which is an unusual koinobiont in that it

parasitizes its hosts during their pupal stage, host growth in a spatial context has mostly ceased

and in fact is constrained by the puparium containing the host. It may be assumed that a clearly-









lineated relationship between body size and size of the host is a characteristic more commonly

associated with idiobionts and those koinobionts which parasitize later stages of their hosts.

Rearing Methods

At 25' C, development requires ~26 d from oviposition for adult emergence (personal

observation). Males and females emerge from puparia at the same time. It is not known whether

adults are competent to oviposit immediately following adult emergence. It is also not known

whether any mating occurs inside host puparia although such mating would prove unlikely given

the spatial constraints of adult parasitoids inside the puparia. A related species, Trichopria

stomoxydis, mates shortly after emergence of adults from host puparia, with male insects

emerging approximately 24 h prior to female insects (Morgan et al. 1990). It is likely that T.

nigra follows a similar mating behavior. For a detailed life history of 7 nigra, see Chapter 4.

In this researcher' s laboratory, 7 nigra has successfully parasitized pupae of Stomoxys

calcitrans (L.), Ophyra aenescens (Wiedemann), Haemotobia irritans (L.), and Salrcophaga

bullata Parker. Colonies of 7 nigra have been successfully maintained utilizing S. bullata and

S. calcitransrt~r~rt~rtrt~rt as hosts. Additionally, 7 nigra females readily examine and probe with their

ovipositors the pupae of2~usca domestic, and have been observed ovipositing inside M~

domestic puparia; regardless of a high pupal mortality in the host, no adult parasitoids have

emerged in numerous laboratory rearing attempts (personal observation). Other instances of 7

nigra parasitizing a dipteran host include the cheese skipper, Piophila ca~sei L. (Teodorescu and

Ursu 1979). All of the above hosts are flies in the infraorder Muscamorpha. Additionally, 7

nigra has been reared on the small pest fly Sturmia bella (Meigen) (Diptera: Tachinidae) (Nash

2005). The majority of demonstrated T. nigra hosts are commonly considered pest flies in close

association with human habitats; as data on this species is highly incomplete a detailed list of all

hosts is not available.









Containers

Clear plastic storage containers (ca. 25 x 14 x 12 cm) were utilized as rearing containers.

Container tops were discarded and the containers sealed across their opening with a white cotton

tube and two rubber bands to prevent wasp escape and to allow for periodic access to the

container (Figure 3-8). Rearing containers were placed on one side to that access via cotton tube

was from the side rather than the top of the container. Water was provided ad libitum on cotton

balls placed in one-ounce plastic cups (Sweetheart Cup co., Owings Mills, MD); one or two

water-soaked cotton balls in cups were placed in each rearing container. When access to the

interior of the container was not required, the end of each cotton tube was tied securely into a

single knot.

Diet

Commercially-purchased (human consumption grade) honey was provided to adult wasps

on cotton balls placed in two-ounce plastic food service containers (Sweetheart Cup co., Owings

Mills, MD). Additionally, a square piece of muslin cloth (ca. 10 x 6 cm) was covered in a thin

layer of honey and placed on the inside superior wall of the rearing container. This was done to

prevent drowning of parasitoids in the honey due to their small size.

Since 7 nigra does not host feed, no pupae as a protein food source were required for

colony maintenance. See Chapter 4 for a discussion on whether honey is required for colony

maintenance. This researcher has also successfully reared 7 nigra on a 50% v/v solution of

honey and water provided on cotton balls in one-ounce plastic food service containers

(Sweetheart Cup co., Owings Mills, MD).

Host Provisioning

Both strains of 7 nigra (Russian and Kazakhi,) were reared on ~2-day old pupae of

Sarcophaga bullata in ~3 00 cc paper containers covered loosely with a piece of muslin cloth










and a rubber band. Paper containers of stung pupae were covered to prevent accidental

hyperparasitism from other species reared in the laboratory, and kept in an incubator at 25" C

until adult emergence was first noted. Time required for development, from stinging of pupae to

emergence of adults, was approximately 26 days at a temperature of 25' C. At first sign of

emergence from puparia, adults were transferred to rearing containers (see above) by placing the

paper food service containers containing pupae and new adults, uncovered, in the rearing

containers. Adult wasps, beginning at 5 -6 d post-emergence, were provisioned with~-250 cc

fresh unstung 2- to 3 -day old S. bullata pupae for 5-6 days on a weekly basis, to establish the

subsequent generation. For the purpose of experimentation requiring T. nigra reared on hosts

other than S. bullata, host provisioning methods were identical except that ~250 cc of pupae

from S. calcitransrt~r~rt~rtrt~rt or M domestic were utilized.

An interesting aspect of host provisioning for T. nigra is that, while adult females will

readily attempt to parasitize M. domestic pupae, and very few host pupae survive to adult

emergence, no parasitoids emerged from puparia. The mechanism for this phenomenon is

unknown, although it is hypothesized that rapid melanization of wounds caused by probing kill

parasitoid eggs, if any are laid (personal observation). Dissections of house fly puparia stung by

7: nigra at 2, 5 and 10 days post-sting have not yielded any parasitoid larvae.









Table 3-1. Head capsule widths and egg numbers of Trichopria nigra females and males (50/50
sex ratio) reared on pupae of Sarcophaga bullata and Stomoxys calcitrans. For S.
bullata reared, F=8.99; df= 1 P<0.05. For S. calcitrans reared, F=8.60; df= 1 P<0.05.
Rearing Host Head Capsule Width (mm) Egg Load n

S. bullata emerged 0. 382 (.007)a 114.3 (6.4)a 20
S. calcitransrt~r~rt~rtrt~rt emerged 0.320 (.010)b 87.2 (4.5)b 20
Means in the same column followed by the same letter are not significantly different (SAS
2000).















S. bullata emerged 0.384 (.005) 0.106 (.001)
S. calcitransrt~r~rt~rtrt~rt emerged 0.322 (.005) 0.069 (.001)


Table 3-2. Head capsule widths and weights for female and male Trichopria nigra (50/50 sex
ratio) reared on pupae of Salrcophaga bullata and Stomoxys calcitrans.


Rearing Host


Head Capsule Width (mm)


Weight (mg)
























































Figure 3-1. Adult female Trichopria nigra, lateral view.







34


























































Figure 3-2. Adult male Trichopria nigra, lateral view.






35


























































Figure 3 -3. Probing female Trichopria nigra. The ovipositor is extended and inserted into the
puparium of Stomoxys calcitrans.
























































Figure 3-4: Dissection of 7 nigra abdomen reveals the retracted ovipositor in close relation to
the ovarioles.









37










A)



















B)



















Figure 3 -5. A) Empty puparium of Salrcophaga bullata. Multiple exit holes created by
Trichopria nigra are visible. B) Dissected puparium of S. bullata. B) Dissected host
remnants. During parasitoid development, the interior of the host fly is consumed.
























































Figure 3 -6. Microscopy image (400x) of 3 -day-old Trichopria nigra ovarioles. Small dark circles
at the base of the ovarioles (arrow) are the spermathecae.






39



















180

160

e 140 mm R2 =0.4612

E 120



u, 80 -1 m-

S60 -1 a m observed
O -- predicted
40
-Linear (observed)
20


0.21 0.26 0.31 0.36 0.41 0.46

head width (mm)

Figure 3 -7. Regression analysis for body size of female Trichopria nigra (directly correlated to
head capsule width in mm) and egg load size (in eggs/female).




















































Figure 3-8. Rearing containers used for colonies of Trichopria nigra at the USDA/ARS Center
for Medical, Agricultural and Veterinary Entomology in Gainesville, FL.










41









CHAPTER 4
LONGEVITY AND FECUNDITY EXPERIMENTS

Introduction

Reproduction is nutritionally costly in all animals; insects prove no exception. Female

parasitoids that oviposit must have competent eggs, and must be competent themselves to lay

eggs. Many parasitoids, before laying eggs inside or on a host, will utilize a host as a food source

(Godfray 1994). This feeding strategy of utilizing a potential host to feed both an adult parasitoid

and its offspring is widespread among idiobionts. Females of idiobiont parasitoid species emerge

without competent eggs and must host-feed to acquire protein for egg maturation. Additionally,

odopbionts parasitize their hosts in later host life stages (Quicke 1997). In contrast, koinobionts -

parasitoids which parasitize their hosts early in development and allow hosts to continue

development rarely host-feed.

The life history of Trichopria nigra (Nees) is largely unknown. It has been observed that 7

nigra does parasitize multiple species of higher flies, and that it is not observed to feed on

potential hosts before or during oviposition. From an evolutionary perspective, these behaviors

are unusual. 7 nigra is a koinobiont parasitoid. Its hosts continue to develop post-oviposition

(see chapter 3 for experiments and observations on this topic). Many of the characteristics of 7

nigra's reproductive behavior and physiology however, reflect those ofidiobiont parasitoids

(Hawkins et al. 1990); for example, idiobionts tend to have a relatively wide host range that

encompasses multiple host species. Preliminary data indicate that 7 nigra readily parasitizes

several closely-related host species. Additionally, laboratory rearing of this insect has

demonstrated a low female fecundity, despite gregarious immature development within the host

(Geden, personal observation). Lastly, 7 nigra parasitizes its hosts later in their development, a









trait normally ascribed to idiobiont species (Quicke 1997), yet is an endoparasitoid with larval

wasps demonstrating a koinobiont trait by feeding and developing inside the host viscera.

When a parasitoid develops inside a host that is growing (such as a larva), nutritional

resources are continually acquired by the host. Less need exists for a well-yolked, larger egg,

because the host's continual growth provides a renewed food source (Giron et al. 2002). Because

of this, koinobionts tend to possess smaller eggs and are proovigenic (i.e., eggs are chorionated

and the female insect does not require a protein meal to produce yolk). In general, it is assumed

that the main nutritional components gleaned from feeding on host hemolymph are sugars such

as trehalose and amino acids (Giron et al. 2002, Quicke 1997), although results from longevity

experiments (Ferracini et al. 2006, Jervis and Kidd 1991) offer inconclusive evidence that host-

feeding imparts any benefits such as longevity to the maternal parasitoid.

Because the purpose of host-feeding is solely to develop the ovaries and produce

competent eggs (Quicke 1997), it would not be expecteded that host-feeding incurs any lifespan-

lengthening effects to female parasitoids. Therefore, 7: nigra, a parasitoid which has never been

documented host-feeding, should not benefit from the presence of host pupae with regard to

lifespan because it would not be expected that such hosts would be utilized as a food source.

However, it is not known whether T. nigra males and females, when provided with a

carbohydrate food source such as honey or plant nectar, live longer or produce more progeny

than individuals maintained solely with a source of water. Additionally, it is not known whether

the presence of host pupae for the purpose of oviposition appreciably lengthens or shortens the

lifespan of male and female 7: nigra.

The effects of providing food and hosts to parasitoid wasps have been studied in many

non-diapriid parasitoids. In an early experiment conducted on Nasonia vitripennis (Walker)










(Hymenoptera: Pteromalidae) it was determined that the longevity of unfed parasitoids was

approximately 7 days for females and less than two days for males (Nagel and Pimintel 1963). It

is generally assumed that for many insects the addition of an energy source (i.e., food) extends

the lifespan of individuals. Various parasitoid species in the families Pteromalidae and

Diapriidae which are reared at CMAVE, for example, live as long as four or five weeks in a

colony situation, where rearing containers include a food source in the form of honey (Ferrero,

personal observation).

Irvin et al.. (2007) determined that when three species of egg parasitoids in the genus

Gonatocerus were given a honey-water solution, the longevity of male and female parasitoids

increased between 1400% and 1800% in all three species, compared to parasitoids given only

water. Furthermore, studies which involved feeding parasitoids a variety of potential

carbohydrate food sources in the form of insect-derived sugars (such as trehalose) or plant-

derived sugars (such as fructose), indicate that plant-derived sugars increase the longevity of

parasitoids more appreciably (Jacob and Evans 2000, 2004). The Diapriid parasitoid, Trichopria

stomoxydis Huggert, provides compelling evidence that feeding on a carbohydrate source such as

a sugar to parasitoids in a laboratory setting changes the lifespan of the adult insect. Morgan et

al.. (1990) claimed that I: stomoxydis presented little potential as a biological control agent in

part because of its short adult lifespan. In a laboratory setting, provided water but no food source,

the maximum longevity for all parasitoids was I 3 days.

Similar results were obtained in rearing of the Encyrtid parasitoid Tachinaephagus

zealandicus Ashmead (De Almeida et al. 2002a), where at temperatures from 16-22.C both male

and female parasitoids given food and water lived up to two times longer than those with access

to water alone. Interestingly, this effect diminished with increasing temperatures at which adults









were held. 7: zealan2dicus was later observed (De Almeida et al.. 2002) to attack significantly

more pupae when females had access to both food (provided as honey) and water than when

given only water; females given neither food nor water had the lowest attack rates. Additionally,

no correlation could be made between feeding treatment and fecundity, except in the instance of

females given neither food nor water. Since sugar metabolism is not required for maturation of

parasitoid eggs (Quicke 1997), it is logical to assume that providing most parasitoids with a

sugar-based food source would not substantially affect the number, i f not the quality, of progeny

produced.

The number of offspring produced by many parasitoids is dependent on adult body size

and food availability. It has been demonstrated (Ueno and Ueno 2007) that providing the

Ichneumonid parasitoid Itoplectis naranyae Ashmead with hosts increases their fecundity; while

L. naranyae females could mature (chorionate) eggs without host-feeding, feeding produced an

increase in future egg clutch size. Egg clutch size appears to be at least partially connected to the

geographical regions in which strains develop, as is documented in the case of Muscidi~irax

raptor Girault and Sanders strains (Legner 1969; Legner 1979). M~ raptor strains from Peru are

almost completely solitary in laying of egg clutches, whereas the Chilean strain of this same

insect demonstrates a higher instance of gregariousness. If tendency toward solitary oviposition

is favored with smaller hosts, and gregarious oviposition more common in larger hosts (Godfray

1994), then it could be hypothesized that T. nigra is more likely a gregarious parasitoid.

The obj ectives of these experiments were twofold. The first obj ective, with regard to adult

parasitoids, was to establish how long male and female 7: nigra parasitoids live, respectively,

when provisioned solely with water, with water and a carbohydrate food source, with water and a

potential host, and with water, a carbohydrate food source plus potential hosts. The second










objective, with regard to the Fl generation produced by adults in Obj ective (1), was to examine

how the above four treatments affect female fecundity with emphasis on parasitism rate, number

of progeny produced by female parasitoids, and the sex ratio of progeny produced in the Fl

generati on.

Materials and Methods

Study Site

Male and female 7: nigra specimens for the bioassay were combined from two strains

reared at the USDA/ARS Center for Medical, Agricultural and Veterinary Entomology

(CMAVE), Gainesville, FL. Rearing of parasitoids occurred at the CMAVE's Quarantine

facility. All host flies, including Sarcophaga bullata Parker and Stomoxys calcitransrt~r~rt~rtrt~rt (L.), were

reared in the CMAVE' s colony facility. The following experiment was conducted at the

CMAVE' s Quarantine facility.

Parasitoids

To provide adequate numbers of insects for experimentation, a Kazakh strain (KzTn) and

a Russian strain (RuTn) of T. nigra were combined. Both strains were reared on ~2-day old

pupae ofSarcophaga bullata. Newly emerged T. nigra adults were transferred to clear plastic

containers (25W x 14L x 12H cm). Honey was applied to muslin strips (approx. 15 x 8 cm) and

placed on the upper interior wall of the containers, and to cotton balls placed inside ~30ml

plastic cups (Sweetheart Cup co., Owings Mills, MD). Water was provided ad libitum on cotton

balls in 30ml plastic cups. Adult wasps were provisioned with~-250 cc fresh, unstung 2- to 3 -day

old S. bullata pupae. Parasitoids were exposed to the pupae for 5-6 days on a weekly basis until

the death of adult wasps, to establish an Fl generation, which was used in experimentation.










Longevity Arena

Arenas consisted of translucent 60-ml plastic containers (Sweetheart Cup co., Owings

Mills, MD) measuring 5.5 cm in height and 7 cm in diameter with matching snap-lock plastic

lids. To provide air flow and exposure to honey and water, and to prevent parasitoids from

escaping, a 5 -cm dia. circular piece was removed from the center of each lid with a scalpel and

replaced with circles of fine copper mesh (#50 size) fastened over the holes with a hot glue gun.

Twenty arenas were constructed to test four treatments with five repetitions per treatment.

Food and water were provided to wasps by placing a cotton ball saturated with either a

honey-water solution or water placed atop the mesh cover, allowing a portion of the mesh to

remain uncovered for air flow (Figure 4-1).

Experimental Design

Longevity determination

The experiment was designed to assess the survivorship of male and female adult 7

nigra when provided solely with water, with water and a carbohydrate food source (honey), with

water and a host, and with honey and water as well as a host. Food consisted of a 50% w/w

solution of clover honey and water kept refrigerated at 12-13oC to prevent spoilage. Water was

obtained from the tap. Hosts consisted of 2-day old pupae of S. calcitrans (L.). Prior to exposure

to 7 nigra in arenas, S. calcitrans pupae were washed, dried and sorted into groups of 200 for

ease of replacement on a daily basis.

Host pupae were presented to the pupae-and-water and pupae-food-and-water treatments

for a period of 24 h by gently pouring 200 pupae into the rearing container. Exposed pupae were

removed from the arenas at the end of each 24 h period. Adult parasitoids were manually

separated from the pupae by gently shaking them through a metal sieve (#12 mesh size). Dead

wasps were removed, counted and sexed. The remaining, live wasps were returned to the










appropriate arenas with 200 fresh S. calcitrans pupae. Ten sets of 200 pupae (five repetitions of

two treatments) were recovered from the arenas after each 24 h period.

Fecundity estimation

Fecundity of parasitoids with respect to treatment was determined by holding exposed S.

calcitrans pupae from each day of the longevity experiment. The number of pupae which did not

produce adult flies and the number of pupae which produced adult parasitoi ds were counted.

Pupae collected daily from the longevity experiment were placed in ~60 ml translucent plastic

cups (Sweetheart Cup co., Owings Mills, MD) with plastic lids and retained in an incubation

chamber at 25 C until S. calcitrans adults emerged (emergence time was between two and four

days post-wasp exposure). Adult flies were not given food or water, and died within 48 h.

Dead flies and empty puparia were removed by hand. Pupae which did not produce adult

flies were placed individually into size "O" gelatin capsules, which were then retained in ~60 ml

translucent plastic cups (Figure 4-2), sorted by treatment (i.e., food, water and hosts or water and

hosts). Cups of encapsulated pupae were returned to the 250 C (+0.40C, 70% RH) incubator until

adult wasps emerged. The numbers of wasps that emerged per pupa per day were counted for

each treatment, and total numbers of male and female progeny counted to observe the sex ratio

of the Fl generation.

Pupae which produced neither flies or parasitoids were considered "dud" pupae (Petersen

and Meyer 1985). Ten of these pupae were dissected per treatment daily to determine whether

they had been parasitized by T. nigra but the developing parasitoids did not survive to adult

eclosion ("mummy" pupae), or if no observed parasitism occurred. The total number of mummy

parasitoids for each day was estimated with the equation (Ferrero)

Thl= (M/10) (TN -PP)

where










Thl= total number of pupae which did not produce adult flies or parasitoids,

M = total number of pupae from ten dissections which contained dead immature parasitoids,

TN = total number of pupae which did not produce adult flies, and

PP = total number of pupae which did not produce flies but did produce adult parasitoids.

The total number of parasitized S. calcitrans pupae for each day and treatment was

calculated as

TP = PP + Thl

where

Thl= total number of parasitized pupae which did not produce adult flies or parasitoids, and

PP = total number of pupae which did not produce flies but did produce adult parasitoids.

Statistical Analysis

The adult longevity data for each treatment and for each sex were analyzed via two-way

analysis of variance (Proc GLM; SAS Institute 2000) using honey, pupae, and honey pupae as

model effects. Treatment means were separated using Tukey' s means separation analysis. Unless

otherwise stated, P values of less than 0.05 were considered statistically significant.

For fecundity data (i.e., fly pupae that were parasitized, and the resultant parasitoid

progeny) statistical tests factored in sex of 7: nigra progeny when noted; for all other progeny

data analyzed, male and female numbers were combined. One-way analysis of variance

(ANOVA) was performed (SAS Institute 2000) with the total number of parasitized pupae, the

total number of mummy pupae, the cumulative number of male progeny, the cumulative number

of female progeny, the percentage of female progeny, and the total number of parasitoid

progeny, respectively, as dependent variables. This was done to determine if the water treatment

only versus the water and honey treatment produced significant differences in any of the above










parameters. Data analyzed in the one-way ANOVA were subjected to arcsine transformation and

analyzed as proportions.

Regression analyses were performed on the numbers of T. nigra progeny that emerged

per pupa for both of the pupae-included treatments to determine whether a relationship exi sted

between the presence or absence of parental feeding and resultant progeny. Regression analyses

were also performed to determine whether a relationship existed between parental feeding and

number of progeny (and whether 7: nigra fecundity could be influenced by parental feeding).

Results

Longevity Determination

The longest lifespan for both sexes was ob served in the treatment in which both honey

and pupae were available to parasitoids, and the shortest lifespan for both sexes was observed in

the treatment providing pupae and water but no honey. Mean longevity for females was between

11 and 13 days for the water-only treatment, the water-and-honey treatment, and for the water,

honey and pupae treatment, respectively (Table 4-1). For females given only water, the lifespan

was less (under ten days). Mean longevity for males (in days) was between five and ten days for

all treatments (Table 4-1). Mean mortalities for all four treatments are plotted for males (Figure

4-3A) and females (Figure 4-3B)..

The provi sioning of parasitoid females with honey resulted in a significant increase in

longevity (F = 11.73, df= 1, 367; P = 0.007) (Table 4-2). There was, however, a significant

pupae-honey interaction (F = 5.91; df = 1, 367; P < 0.05), which indicated that parasitoids given

pupae and honey lived significantly longer than parasitoids given pupae alone (Table 4-1).

Female parasitoids given pupae and water did not express a significant increase in lifespan (F =

1.83; df = 1, 367; P > 0.05) over females given only water. Additionally, females with access to

pupae but not honey died sooner than females which were provided only water (Table 4-1).









Results with male parasitoids were similar to those with females. Provisioning male T. nigra

with honey significantly lengthened lifespan (F = 8 1.96; df = 1, 386; P <0.000 1); providing

males with pupae did not lengthen lifespan compared to the water-only males, however (F =

0.05; df = 1, 386; P > 0.05). Overall, the effect of providing parasitoids with either honey, or

honey and pupae, was stronger in males than in females, with the mean lifespan of males

provided with honey and pupae nearly double that of the control males which received neither

(Table 4-1). The interaction of honey and pupae on males was statistically significant, with males

provided both living the longest of the four treatments (Tables 4-3).

Fecundity Estimation

Because only two of the four experimental treatments in the Longevity Estimation

involved pupae and therefore produced 7 nigra progeny, only two treatments (water and pupae;

water, pupae and honey) are discussed below. The mean number of pupae that were parasitized

(killed) by 7 nigra adults provisioned with water did not differ significantly from the mean

number of pupae parasitized by adults provisioned with honey and water (Table 4 -4). The

number of mummy pupae (labeled "Pupae with dead immature wasps" in Table 4-4) produced

did not differ significantly between treatments. The cumulative number of male and female

progeny, respectively, did not differ between treatments, although the percentage of female

parasitoids that emerged from successfu~lly-parasitized pupae did differ significantly (F = 6.2; df

= 1, 8; P < 0.05), with slightly more female progeny emerging from pupae which were stung by

females provisioned with honey as well as water (Table 4-5).

The cumulative number of pupae parasitized over time is presented in Figure 4-4. Females

began parasitizing hosts immediately, and within 2 days had parasitized over half of the number

of hosts that they would parasitize during their lives. By day 5, females in both treatment groups

had parasitized over 90% of the total hosts that they would parasitize. When progeny production









is viewed in terms of successful production of adult progeny per day, a similar pattern is evident

(Figure 4-6), with a maximum progeny production occurring in the first 2 -3 days of adulthood.

The number of adult progeny produced per parasitized host in the honey -fed group showed a

noticeable decline over time, with pupae stung by young females producing about twice as many

parasitoids (ca. 6 per host) as older females (Figure 4-6B) (R2=0.5876). In contrast, pupae stung

by female parasitoids given only water showed no significant trend in parasitoid progeny

produced over their reproductive lifetime (Figure 4-6A) (R2=0.0175).

Discussion

Longevity Determination

Because 7 nigra is proovigenic, its eggs are fully chorionated upon emergence of adult

female parasitoids from host puparia (Quicke 1997) and, as such, female parasitoids do not need

to feed on their hosts in order to obtain amino acids for yolking (Heimpel et al. 1996; Heimpel

and Rosenheim 1998). It was not expected that the presence of S. calcitrans pupae, in the

absence of food, would significantly increase the lifespan of either female or male parasitoids.

Likewise, because from a physiological perspective little incentive remains for female

parasitoids to stay alive once they have utilized all eggs, it was predicted that giving 7 nigra

females access to host pupae would decrease their life expectancies compared to withholding

pupae. In this study, female T. nigra lived approximately two days longer in the absence of

pupae than when given free access to fresh pupae on a daily basis (Table 4-1), but this difference

was not statistically significant.

In invertebrate models, the correlation between bearing young and life expectancy in

females has been studied extensively, with data demonstrating that producing offspring has a

negative impact on female longevity in the post-reproductive phase (Chapman et al. 1998).

Mukhopadhyay and Tissenbaum (2006) suggest that the energy required to produ ce gametes and









develop offspring may be taken from energy that would otherwise be utilized to maintain

somatic cells (those which will not produce gametic cells); if a tradeoff occurs when an animal

attempts reproduction, a shortening of life expectancy would be, and indeed is, observed. This

phenomenon may explain why male 7 nigra, despite not producing or laying eggs, experienced

a shortened lifespan when provided with pupae but not honey. If production of gametes is, for

both sexes, energetically costly, then males that mated with females who had potential hosts

available (as unstung pupae) would have utilized their gametes and no longer have any

requirements to remain alive.

The increase in longevity for 7 nigra females and males which were provisioned with both

honey and pupae is unusual. It was hypothesized that the presence of a food source would

increase the mean lifespan of all parasitoids because of an input of energy for physiological

maintenance as well as providing additional energy to locate mates (male and female parasitoids)

or hosts (female parasitoids only) (Wackers 1994; Wackers 1998). However, while the presence

of honey does significantly affect male parasitoid longevity for T. nigra, the presence of pupae

should not because male parasitoids neither feed nor oviposit inside pupae. The analysis

indicated that the effect of honey on longevity was stronger when pupae were present than when

they were absent. This was likely due to the continued presence of pupae allowing females to

oviposit fewer eggs each day over a longer period of time, and therefore be more selective in

their choice of host pupae.

When provided with pupae and water but no honey, both male and female parasitoids did

not demonstrate a significant difference in longevity compared to conspecifics that were only

given water (Table 4-1). It was not expected that providing hosts to parasitoids in the absence of

a food source would extend lifespan for males. Because most female parasitoids control the









timing of fertilization of eggs and oviposition (Quicke 1997), there is little pressure on males to

mate with females as quickly as possible. It was predicted that providing pupae to female T.

nigra adults in the absence of a carbohydrate food source such as honey would decrease their

lifespan due to a greater pressure on females to oviposit before depleting the energy allocated for

host seeking and oviposition; however, the greater mean lifespan of females provided with water

alone was not significantly different from the mean lifespan of those females provided with

water and pupae (Table 4-1). Indeed, this indicates that, as with males, the life-lengthening

effects of honey were greater when host pupae were present than when they were absent. Thi s

may be because the energetic demands required for drilling and oviposition by female T. nigra

were lessened with the availability of energy via honey.

Fecundity Estimation

Of the parameters analyzed via two-way ANOVA (Table 4-4), only the cumulative

numbers of killed pupae differed significantly between treatments (pupae only, versus pupae and

honey). The increase in number of pupae killed by female parasitoids given both honey and

pupae (856.8+3 0.3) compared to females provided pupae and no honey is because a greater

number of pupae were killed between d 4 and 9 (Figure 4-4). Both treatments demonstrated

greatly reduced killing of host pupae by d 1 1, with the maj ority (>90%) of cumulative pupae

killed by d 10. Since many parasitoids have demonstrated a higher fecundity rate and produce

longer-lived progeny when provided with older hosts (de Almeida et al. 2002; Bellows, Jr.

1985), the influence of host age on data collected from parasitoid progeny in this experiment was

controlled for by utilizing host pupae of a strictly defined age.

On average, each female of T. nigra produced about 23 adult progeny over her lifetime.

This is substantially higher than the 10.5 and 3.6 progeny produced per female of 7 stomoxydis

(Morgan et al.. 1990) and T. painter (Huggert and Morgan 1993), respectively. It is similar to









the lifelong fecundity figure of 2 1.4 progeny per female of the encyrtid larval parasitoid

Tachinaephagus zealandicus Ashmead when 7 zealan2dicus is supplied with house fly pupae

(Geden et al.. 2003). Most of the research on filth fly parasitoids has concentrated on pupal

ectoparasitoids in the family Pteromalidae. Spalangia endius Walker, which has been used

successfully as a fly biological control agent, produces only about half as many progeny per

female as 7 nigra (Morgan et al.. 1976). Spalangia cameroni Perkins, another commonly used

fly parasitoid, has a longer lifespan than S. endius and produces about 30-40 progeny per female

(Legner and Gerling 1967, Moon et al. 1982).

Perhaps the most widely used parasitoid for fly control are M~uscidifurax raptor and

related species in the same genus. Lifelong fecundity estimates for M~ raptor vary from 26-185

progeny per female over her lifetime depending on the strain and health of the colonies under

consideration (Morgan et al.. 1979, Zchori-Fein et al. 1992). Fecundity of T. nigra therefore is

squarely within the range of several other economically important species of muscoid fly

parasitoids. The geographic distribution of T. nigra (Geden and Moon, unpublished data) across

eastern Europe is also within the distribution of other common filth fly parasitoids, such as S.

endius Walker, S. camneroni Perkins, and M. raptor (Hogsette et al. 1994, 2000). Having identical

host milieus and overlapping geographical distributions indicate that in the wild, these species

likely inhabit complementary niches. It would be interesting to determine in the future whether

introduction of 7 nigra in conjunction with other commercially-important parasitoids of filth

flies (e.g., S. endius, S. cameroni. and M. raptor) increases control of flies that both species are

known to parasitize, such as M~ domestic and S. calcitrans.~rt~t~rtrt~r~rt

Future experimentation involving the life history of Trichopria nigra will utilize a larger

sample size for longevity studies. It would also be prudent to feed adult T. nigra, particularly









females, on a variety of sugar-based food sources (such as plant nectar, honey, glucose solution),

to more accurately determine whether an artificial (laboratory) diet significantly extends or

shortens the lifespan of this parasitoid, compared to the lifespan when fed on a diet more

accurately mimicking their natural diet. Additionally, it is not known at this time whether feeding

females before allowing them access to host pupae, rather than providing both at the same time,

affects the number of progeny that are produced successfully. In this experiment, 7: nigra in the

water, honey and pupae treatment were given access to all three factors from day 0; perhaps

withholding pupae and feeding parasitoids first would incur a benefit of some sort.











Treatment n Mean (SE) lifespan (d) $ n $ Mean (SE) lifespan (d)
Water only 90 11.94 (10.51)ab 102 6.71 (10.33)b
Water + honey 102 12.53 (10.52)a 97 8.97 (10.35)a
Water + pupae 80 9.85 (10.52)b 94 5.46 (10.30)b
Water + honey + pupae 99 12.99 (10.52)a 97 10.05 (10.49)a
Means in the same column followed by the same letter are not significantly different according to Tukey' s means separation analysis
(SAS 2000).
For females, F=6.49; df=3, 367; P<0.001. For males, F=30.54; df=3, 386; P<0.001.


Table 4-1. Mean lifespans (+SE) (d) of Trichopria nigra adults under different feeding treatments at 25 TC.










Table 4-2. Female 7: nigra, two-way ANOVA results for influence of presence/absence of
honey and host pupae on survival time. Numbers marked with an asterisk are
considered significant (P < 0.05). Water alone was considered the control, therefore
water is not included as a model effect.

Model effect df Sum of Squares Mean Square F; P
Honey 1 297.5 297.5 11.73 0.0007*
Hosts 1 46.4 46.4 1.83 0.1769
Honey x Hosts 1 150.0 150.0 5.91 0.0155*
Error 367 9309.3 25.4










Table 4-3. Two-way ANOVA results for influence of presence/absence of honey and host pupae
on survival time of male 7: nigra. Water alone was considered the control, therefore
water is not included as a model effect.

Model effect df Sum of Squares Mean Square F P
Honey 1 1129.2 1129.2 81.96 <0.0001*
Hosts 1 0.8 0.8 0.05 0.8151
Honey x Hosts 1 132.3 132.0 9.60 0.0021*
Error 386 5318.2 13.8
Numbers marked with an asterisk are considered significant (P<0.05).









Table 4-4. Effect of diet on mean number (+SE) of pupae killed by female T. nigra, pupae producing wasps, having dead wasps, and
total number of parasitized pupae.
Treatment Numb er of Pupae Number of Pupae Number of Pupae with Cumulative Number of
Killed Producing Wasps dead immature wasps Pupae Parasitized

Pupae Only 715.4 (148.1) 47.0 (16.0) 163.6 (19.9) 210.6 (15.5)
Honey and Pupae 856.8 (130.3) 46.4 (17.5) 172.1 (+21.4) 218.5 (127.7)
ANOVA F 6.2* <0.1ns 0.09ns
df = 1, 8; P< 0.05









Table 4-5. Cumulative (+SE) numbers of male and female progeny, respectively, produced by parasitoids given only water and pupae,
or water, honey and pupae.

Treatment Cumulative Number of Cumulative Number of Cumulative Number Percent
Male Progeny Female Progeny Progeny Produced Female

Hosts Only 141.8 (142.9) 83.3 (122.1) 225.0 (127.2) 37.0 (11.9)
Honey and Pupae 229.4 (141.3) 107.8 (143.3) 237.2 (136.7) 45.4 (12.6)

























Cotton ball saturated with
either water or 50% V/V
water and honey solution


Figure 4-1. Rearing containers for longevity determination. Air flow and access to water and/or
food was provided via mesh screen on container lids. Five repetitions were provided
with water, five with water and 200 S. calcitrans pupae, five with a 50% V/V water
and honey solution, and five with a 50% V/V water and honey solution and 200 S.
calcitrans pupae.






























B)







Figure 4-. 0-lcus otaiigidvdal-nasltdS.clirn ua.A olwn
eme3C~rgec ffisfo naaiie ua, hs ua hc i o rdc le
we~~re eahpae noagltncpues httenme fwsseegdfo
eahscesfllyprstzdppecudb bevd )Scesul-aaiie




pupa.












A) z



i~20

,s Water
a 15
SHoney
a5
L..~ Water and
X "O Pupae
5 CHoney and
r: Pupae
S5




1 2 3 4 5 6 7 8 9 1011 12 1314 15 161718 1920 2122
Days



B)
25



i~20


.4 Water

SHoney

L. Water and


a: Pupae





1 2 3 4 5 6 7 8 9 1011 12 1314 15 161371.81920 2122
Days




Figure 4-3. Mean adult A) female and B) male Trichopria nigra mortality for all treatments.















| 250

200


-150

100

-50



1 3 5 7 9 11 13 15

Days

-A Hosts Only -* Honey and Hosts


Figure 4-4. Total number of S. calcitrans pupae parasitized by female parasitoids for each of two
treatments (water and hosts, food and hosts, respectively). The total number of
parasitized pupae was defined as the sum of pupae which successfully produced adult
parasitoids and pupae which contained immature parasitoids that never emerged.
After days 9-10, no adult parasitoids emerged from pupae.






































__


12 34 56 7 8 9 1 -1
Days


-A- Hosts Only

-*- Honey and Hosts


Figure 4-5. Mean number of adult parasitoids that emerged daily for each of two treatments
(water and hosts, food and hosts, respectively). After days 6-7, very few parasitoids
emerged. The highest numbers of adult emergence were observed from day 1 through
day 3.

























S5-
**A
4-
o






0 2 4 6 8 10

Days


B 12

S10-
*
a- *

u, 6- +* +
o a *

2 -( *
ca *



0 2 4 6 8 10 12

Days

Figure 4-6. The average number of parasitoids that emerged per pupa per day. A) Water and
hosts only. B) Food, water and hosts. The average number of parasitoids per pupa
was calculated for each replicate, and averages plotted. The R2 ValUe for the
regression line in A) = 0.5876. The R2 Value for the regression line in B) = 0.0175.
























250


200


S150
E ~- -Hosts Only
ur -4 Hloney and Hosts
-C 100



50


1 2 3 4 5 6; 7 8 9 10 11 12 13 14 15

Day

Figure 4-7. Cumulative mean number of adult parasitoids that emerged from S. calcitrans pupae
for each of two treatments (water and hosts, food and hosts, respectively). After days
6-7, very few adult parasitoids emerged.










CHAPTER 5
ARENA-CHOICE EXPERIMENTS

Introduction

Host specificity is a common feature found in many parasitoid genera. In general,

koinobionts (insects which parasitize a host in the early stages of host development, and in which

the host continues to develop) demonstrate a higher level of host specificity than do idiobionts

(Althoff 2003); thi s aspect of parasitoid evolution i s most likely due to the necessity of a

koinobiont parasitoid being able to develop and mature in confluence with a host with a

continuously changing endocrine milieu. Idiobionts, by contrast, parasitize their hosts in later

stages of development and, because the host insect may go through few if any endocrinological

changes such as change of instar that influence parasitoid development, the host is often

"arrested" in development and does not survive long (Quicke 1997). Because the nutritional and

physiological dependence of koinobiont parasitoids on their hosts is greater compared to

extemnally-living idiobiont parasitoids, host ranges of koinobionts tend to be narrower than those

of idiobionts (Hochberg and Ives 2000; Godfray 1994).

Parasitoid species possess specific and innate ranges of hosts they can successfully

parasitize. Askew and Shaw (1986) hypothesized that koinobiont parasitoids would be more

host-specific than idobionts because of the internal development and nutritional and

endocrinological dependencies that koinobionts have on their hosts. Other Trichopria species

have been found to parasitize Stomoxys calcitrans (L); 7 stomoxydis Huggert and 7 painter

Huggert and Morgan are known only to parasitize that species (Morgan et al. 1990; Huggert and

Morgan 1993). Another closely related species, 7 anastrephae Lima, was found to parasitize

pupae ofAnastrepha species in western Brazil (Garcia and Corseuil 2004).










The question of whether parasitoids are innately competent to locate hosts, or whether

prior exposure to a host (or a host' s surroundings) improves chances of locating a host and laying

eggs, is an important one. Little is known about the effects of conditioning on fly pupal

parasitoids, and even less about conditioning on parasitoid species in the family Diapriidae, such

as 7 nigra (Nees). More generally, it is known that for many parasitoids, associative learning is

an important aspect of host location and selection. Hopkins' Host-Selection Principle has been

observed in many insects (Craighead 1921; Smith and Cornell 1979; Davis and Stamps 2004).

According to this principle, first observed in herbivorous insects (Craighead 1921), the host an

insect develops within provides the first visual and olfactory conditioning, so that adult insects

can later locate the same host they developed within, for laying their eggs.

The odors and visual presence associated with pupae often serve as stimuli (see Jandt and

Jeanne 2004 for example) that parasitoids associate with a reward (i.e., oviposition, or feeding

prior to oviposition). Odor stimuli can be either compounds from either potential host pupae or

their surrounding environment (Sullivan et al. 2000; de Jong and Kaiser 1992). In some

instances, host location stimuli are visual rather than olfactory, as is the case for the phorid fly

parasitoid of fire ants, Apocephalus paraponera Borgmeier (Morehead and Feener 2000).

Regardless of the type of stimulus, learning i s an adaptive behavior. Once conditioned to seek

out olfactory or visual cues, the foraging time for future oviposition events is often shortened,

maximizing the number of hosts parasitized and eggs laid by the parasitoid (Stireman III 2002).

Diadromus pulchellus Wesmael (Hymenoptera: Ichneumonidae), a parasitoid wasp which

attacks larvae of the moth Acrolepiopsis assectella Zeller, appears to utilize volatile compounds

emitted from A. a~ssectella larvae and their frass to locate possible hosts for oviposition

(Lecomte and Thibout 1993). When adult female parasitoids are exposed to either frass or larval









hosts prior to placement in an olfactometer, the number of turns made before choosing the

host/frass odor over a control was fewer than those made by females never exposed to hosts or

frass post-emergence (Lecomte and Thibout 1993). However, when native D. pulchellus females

were exposed to volatiles from A. a~ssectella larvae and a control in an olfactometer, they did not

show any significant differences in behavior when compared to female D. pulchellus reared on

an atypical host, Plodia interpunctella (Hubner). These findings indicate that, for some species,

post-developmental exposure to a host is a critical step for females to learn host selection.

In the laboratory, Trichopria nigra (Nees) successfully parasitizes pupae of a number of

nuisance flies, including Stomoxys calcitransrt~r~rt~rtrt~rt L., Ophyra aenescens (Wiedemann), Haemotobia

irritans (L.), and Sarcophaga bullata (Parker) in the laboratory (Geden and Ferrero, personal

observation). Additionally, colonies of T. nigra have been successfully maintained utilizing these

same fly species as hosts. I nigra readily probes pupae of2~usca domestic and appears to

oviposit; although pupal mortality in the host is high, no adult parasitoids have emerged in

numerous laboratory rearing attempts (Geden, unpublished data). Other dipteran hosts

parasitized by 7 nigra include Piophila casei (L.) (Teodorescu and Ursu 1979) and tachinid flies

(Nash 2005).

I: nigra demonstrates an unusual life history, with respect to the interplay of physiology

and behavior of parasitic wasps. Unlike many other endoparasitoids, it develops inside a pupal

host rather than a larval one. Furthermore, the ability of T. nigra to develop successfully in a

number of hosts is atypical of endoparasitoids. Conditioning studies involving T. nigra cannot be

found in the literature and the effects of conditioning on this parasitoid are unknown. Therefore,

the first obj ective of these experiments was to determine if conditioning 7 nigra to a particular

host via exposure would bias 7 nigra females to choose their "conditioning type" host when










presented with multiple host choices. The second obj ective was to determine if rearing T. nigra

on two different host species affects their likelihood of choosing the same species, when newly-

emerged Fl parasitoids are presented with a choice of multiple hosts.

Materials and Methods

Study Site

Parasitoids and host flies were reared at the USDA/ARS Center for Medical, Agricultural

and Veterinary Entomology (CMAVE), Gainesville, FL. The following experiment was

conducted at the CMAVE' s Quarantine facility.

Hosts

The host fly species Stomoxys calcitrans (L.), M~usca domestic L. and Salrcophaga

bullata Parker were taken from USDA colonies. Pupae utilized for both experiments were

collected from rearing containers 2-3 days following pupation and either used immediately, or

stored in chambers at 140C (70% RH) for up to four days to retard physiological development

and emergence of adult flies. It has been demonstrated that holding pupae of these species at

similar temperatures (see Moribayashi et al. 1999, Leopold et al. 1997) does not significantly

affect quality or survivorship of the insect.

Parasitoid strains

The 7 nigra adults used for the bioassay were combined from two strains reared separately

and were later pooled to provide adequate numbers of parasitoids. A Kazakhi strain and a

Russian strain, both maintained on S. bullata pupae and assumed to be identical in physiology

and behavior, were used for experimentation. For details on rearing of 7 nigra, see Chapter 3.

Conditioning parasitoids for short-term assay

Three days after emergence from S. bullata pupae [to provide adult wasps with adequate

time for wing and exoskeleton hardening, maturation of ovarioles, and opportunities. to mate]









male and female 7 nigra were removed from their rearing containers with an aspirator. The

wasps were combined then divided into fourths and placed into 250 ml paper cups containing

140 cm3 Of either S. bullata, S. calcitrans, or M. domestic pupae or no pupae (control) for the

purpose of conditioning wasps to pupae of one species.

The 250 ml paper cups containing wasps and treatments were secured across their

openings with cotton cloth to allow air flow and prevent wasp escape. Atop each of the four

cotton cloth covers was placed a cotton ball saturated with distilled water, and a streak of honey

was applied to the cotton to provision wasps with food. Cups were then placed in an incubator

(25 C +0.4' C, 70% RH) for 48 h, after which the wasps were separated from the pupae by

placing both in a #12 mesh screen sieve and shaking gently so T. nigra adults would fall onto a

clean piece of white paper for collection. Wasps were then immediately introduced to arenas for

experimentati on.

Arena for short-term assay

Arenas consisted of clear plastic containers measuring ca. 24 x 24 x 10 cm (L x W x H)

with plastic airtight snap-closure tops (Glad Products company, Oakland CA). Into each arena

were placed four ~5 cm-dia. plastic Petri dish bottoms each containing 2.0 g. S. bullata pupae,

2.0 g. S. calcitrans pupae, 2.0 g. M~ domestic pupae, or an empty (blank) Petri plate serving as a

control (Figure 5-1). Petri plates were arranged in a randomized block design between

repetitions. Five repetitions were conducted for each of four treatments, and the assay was

repeated twice for a total of ten repetitions per treatment. Arrangement of Petri plates was to

provide maximum distance between choices (approx. 8 cm between plates).

The wasps which had been conditioned to different fly species were introduced to arenas

by aspirating them following sieving procedure, and then gently blowing a pea-sized ball of

parasitoids into the center of each arena. Plastic lids were immediately secured tightly on









containers to prevent wasp escape during the assay. Containers were left for 2 hours to provide

wasps with adequate time to choose a host species and initiate probing and/or oviposition. At 2 h,

plastic lids were removed from the arenas, covers were placed on Petri plates to contain wasps,

and the Petri plates were frozen for 12h to kill wasps. Dead male and female wasps in each of the

Petri plates were counted.

Parasitoids for rearing assay

Adult (5-day old) 7 nigra from the combined strains were divided into two plastic

rearing containers (25 cm L x 14 cm W x 12 cm H, see Chapter 3 for further details on rearing

methods). One container of wasps was provisioned with 150 cm3 Of S. bullata pupae, and the

other with 150 cm3 Of 2-3-d old S. calcitrans pupae, in 250 ml paper cups. The layer of pupae

was approximately 3 -5 cm deep. The cups of pupae were held inside their respective 7 nigra

rearing containers for 6 d to allow time for females to oviposit in the puparia.

After 6 d, the paper food cups of pupae were removed from the 7 nigra rearing

containers, adult parasitoids shaken off with a #12 mesh sieve and discarded, and cloth covers

secured across the opening of the containers (now containing only potentially-stung pupae). The

containers were placed in an incubator (25'+~0.4"C, 70% RH) and held until the emergence of the

next generation of parasitoids. Upon emergence of parasitoids, a cotton ball saturated with

distilled water and a streak of honey was placed atop the cloth cover to provide parasitoids with

moisture and a food source. Wasps were introduced to the experimental arena following a 3 -d

maturation period.

Arena for rearing assay

Arenas consisted of clear plastic containers measuring 24 x 24 x 10 cm (L x W x H) with

plastic airtight snap-closure tops (Glad Products company, Oakland CA), of similar design to

those in the short-term host exposure assay. Into each arena were placed three ~5-cm dia. plastic









Petri dish bottoms each containing 2.0 g. of S bullata pupae, 2.0 g. S. calcitrans pupae, or an

empty (blank) Petri plate serving as a control (Figure 5 -2). Petri plates were placed in a

randomized block design between repetitions. Five repetitions were conducted for each of two

treatments. Petri plates were arranged to provide maximum distance between choices (approx. 10

cm between plates).

The wasps reared on either S. bullata pupae or S. calcitrans pupae were introduced in

pea-sized balls to arenas via a manual aspirator, one group for each arena. Plastic lids were

immediately secured tightly on containers to prevent wasp escape during the assay. Containers

were left for 2 h as in the previous test. At 2 h, plastic lids were removed from the arenas, covers

were placed on Petri plates to contain wasps, and all arenas were frozen for 12 h. Dead male and

female wasps in each of the Petri plates were counted.

Statistical Analysis

For the short-term host exposure assay, numbers of wasps in each Petri plate were

separated and analyzed by sex. The numbers for the five repetitions were averaged for each

treatment and the mean raw numbers of male and female wasps exposed to each treatment

compared using Tukey's HSD test (P=0.05; SAS Institute 2000). Conditioned wasps were

compared with unconditioned parasitoids for host preference via one-way analysis of variance

(SAS Institute 2000). Additionally, an arcsin transformation was performed on mean numbers of

wasps to correct for significant variation in total numbers of wasps among repetitions, and the

proportions analyzed using Tukey' s HSD test (P=0.05; SAS Institute 2000).

For the rearing assay, numbers of wasps in each Petri plate were separated and analyzed by

sex. Following sexing of parasitoids in each of the Petri plates, numbers for repetitions were

averaged for each treatment and the mean numbers of male and female wasps exposed to each

treatment were compared using Tukey' s HSD test (P=0.05; SAS Institute 2000). Additionally, an









arcsin transformation was performed on mean numbers of wasps to correct for significant

variation in total numbers of wasps among repetitions, and the proportions analyzed using

Tukey's HSD test (P=0.05; SAS Institute 2000).

Results

No significant differences were observed between the proportions of female parasitoids

conditioned on S. calcitrans pupae which were counted in (e.g., assumed to have oviposited in)

Petri plates containing different pupal types (Table 5-1). The Petri plate of M domestic pupae

lured a significantly greater proportion of female parasitoids (df=1; P<0.0001) than the pupae

of the other two species (Table 5-1). Although numerically more female parasitoids which were

conditioned with S. bullata pupae went to that species' pupae than did female parasitoids

conditioned to other hosts, the difference was not significant (0.051). Male parasitoids

conditioned to S. bullata pupae demonstrated no significant preference to any of the Petri plates

of pupae, regardless of conditioning (Table 5 -2). Unconditioned parasitoids of either sex did not

demonstrate any significant preference for a particular host fly species' pupae.

The host in which the female parasitoids were reared had a significant impact on their

behavior in the experiment (df=1; P<0.0001), with approximately twice as many females

choosing their natal host rather than a non-natal host (Table 5-3). This preference for natal host

was not observed in males (df=1; P=0.57) reared on either S. calcitrans or S. bullata pupae

(Table 5-4).

For the host-exposure experiment, post-experiment counts (total number of wasps were

counted for five of the twenty containers) determined that the groups introduced to the arenas

averaged 589 + 16 wasps of mixed sex per arena, equating to a "pea-sized ball". For the rearing

experiment, post-experiment counts (total number of wasps were counted for five of the twenty









containers) determined that the groups introduced to the arenas averaged 432 + 21 wasps of

mixed sex per arena.

Discussion

The large proportion of female 7 nigra responding to the visual and olfactory cues

provided in the experimental arena, as well as females observed and counted while attempting to

oviposit on all fly species' pupae presented in the experimental arenas suggest that in the wild T.

nigra may attempt to oviposit inside any of these species and use them as hosts. The results of

these experiments indicate that the host range of 7 nigra is fairly broad, especially for a

koinobiont (Whitfield 1998; Quicke 1997). Additionally, other species of Trichopria have been

shown to have wide host ranges (Morgan et al. 1990; Garcia and Coseuil 2004). Such an

assumption must be made with caution, however, as it has been demonstrated that

hymenopterous insects can be readily conditioned to artificial stimuli (Jong and Kaiser 1991;

Jandt and Jeanne 2004).

Another reason for caution is the role of host habitats in directing searching behavior in

7: nigra. Parasitoids typically follow a series of steps that begin with host habitat location

(Vinson 1976). If T. nigra is conditioned to search for carrion rather than dung, then carrion-

inhabiting flies, e.g., calliphorids and sarcophagids, should be parasitized by 7 nigra more often

than stable flies. Field research in the home range of this species is needed to determine whether

parasitism of stable flies is common, or the result of chance encounters. The experiment

conducted utilizing parasitoids which had successfully emerged from both S. bullata and S.

calcitrans pupae, indicates that this species utilizes both host species where they both occur in

the wild. Moreover, laboratory studies can sometimes suggest stronger host preferences than are

evident in the field, where parasitoids are subject to more complex sets of stimuli (Mandeville

and Mullens 1990a, b).









Learning among parasitoid hymenoptera is well-documented, and follows the general

pattern of insect reward-driven association. Females of the fruit fly larval parasitoid Leptopilina

boulardi Barbotin et al. (Hymenoptera: Eucoilidae), for example, were exposed to an artificial

odor (i.e., one not naturally produced by the host or plants fed on by the host) in conjunction

with an oviposition experience in a laboratory (de Jong and Kaiser 1992). The exposed females

would demonstrate a much stronger preference for the scent they were exposed to during

stimulus/reward conditioning compared to naive female parasitoids. Conditioned females also

demonstrated a much stronger preference than did their control counterparts, which were not

classically conditioned to any stimulus.

Selection of a suitable host at close range is essential, and the availability of visual or

olfactory cues may greatly increase a parasitoid' s likelihood of encountering a potential host if

that parasitoid has learned that certain cues signal host availability (Hochberg and Ives 2000).

Most parasitoids whose host range is greater than one host species will often choose the most

abundant host for the greatest number of oviposition events (Hastings and Godfray 1999); while

possibly the result of a female parasitoid accidentally encountering the most abundant host

species at a greater statistical rate, may also be explained by a greater number of visual or

olfactory cues presented by the most abundant host. The number of viable host pupae available is

often an indicator of host fecundity (Hochberg and Ives 2000), and is perhaps, as well, an

indicator of host attractiveness to parasitoids. To this end, an abundance of all potential host

species was presented in the experiments, to prevent one potential host being more available than

the others.

The stronger response of female T. nigra tested in the rearing assay compared to those

conditioned for 48 h indicate that rearing of this wasp on a particular host increases the fidelity









of adult females to forage for and oviposit in the same host as their natal species. Hopkins' Host

Selection principle has often been cited as a strong motivator for insects to search for and utilize

hosts which resemble their own natal hosts (Michaud and Grant 2005), although the life stage at

which this induction occurs remains somewhat controversial (Barron 2001; Davis and Stamps

2004). It would seem logical that, for parasitoids with more than one potential host, initial

exposure to host kairomones predisposes female parasitoids to forage for that particular host.

This preference for natal host appears to be established when newly-emerged parasitoids exit

their host puparia and encounter the odors associated with that species and location (Vet 1983;

Vet and Groenewald 1990). Such a preference would be particularly adaptive in relatively

constant habitats that support sequential generations of hosts (Vet 1983; Turlings et al.. 1992).

Although the literature on insect learning is voluminous, little is known about

conditioning and host selection in parasitoids of muscoid flies, and even less is known about host

selection behavior in the family Diapriidae. Nasonia vitripennis (Walker) shows a moderate

preference for the host species on which is reared, but it is not clear whether this preference

represents pre-imaginal conditioning or an induction event in which newly emerged adults

experience host remains and empty puparia at the time of eclosion (Oghushi 1960, Smith and

Cornell 1979). In contrast, there is little evidence for rearing-host effects on host preference in

parasitoids in the genera M~uscidufurax, Dirhinus, or Spalangia (Mandeville and Mullens 1990b,

Oghushi 1960). The only known report of adult conditioning of fly parasitoids is that of

Mandeville and Mullens (1990b). In this study, the strong innate preference of Muscidi~irax

zaraptor for house fly over Faznni cannicularis (L.) pupae was shifted in favor of the latter

species by 2 days of experience ovipositing on that host. This shift occurred in spite of the fact

that M~ zaraptor is substantially more successful on M~ domestic than on F. canicularis hosts










(Mandeville and Mullens 1990b). The observation that 7 nigra can be conditioned to favor a

host on which it can not develop (house fly [Geden and Moon 2008]) is a curious parallel to the

host shift seen in M~ zaraptor.

Due to the inability to rear 7 nigra on pupae ofM~ domestic, choices for the rearing assay

were limited to S. bullata and S. calcitrans pupae (see Chapter 2). The host-selection behavior of

7 nigra when presented with multiple potential host insects, and the shift in that behavior (in

most instances) to preferentially seek pupae of the host previously conditioned to is consistent

with the current literature on associative learning in other parasitic Hymenoptera. While

Hopkin' s host selection principle may not be strictly valid in explaining the stronger response of

parasitoids reared on multiple host species (as opposed to a brief post-emergent exposure to a

particular host), certainly initial exposure of female parasitoids to host kairomones presents a

case for de facto conditioning to a parasitoid' s natal host that, in the absence of later associative

learning that exposes parasitoids to different host species, establishes a default mechanism for

seeking out hosts that are native to a certain geographical area. Further research should be

performed to examine the means by which 7 nigra might learn to seek out a particular host, and

establish the strength of conditioning' s effect on host-seeking behavior. Further research

conducted involving host choice with 7 nigra should utilize a much higher sample size,

especially with regards to the rearing assay.









Table 5 -1. Proportion of female 7: nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single
host species. Means in rows followed by different letters are significantly different according to Tukey' s HSD-test (P=0.05;
SAS Institute 2000).

Mean (SE) proportion of parasitoids recovered on host
S. calcitrans M~ domestic S. bullata ANOVA P
Conditioning Host pupae pupae pupae F
S. calcitrans 0.34(10. 10)a 0.36(10. 12)a 0.30(10.05)a 0.81 0.50
M~ domestic 0.27(10.07)a 0.43(10. 10)b 0.30(10.10)a 9.81 0.0002
S. bullata 0.29(10.07)a 0.31(10.09)a 0.40(10. 10)a 2.95 0.0512
No conditioning 0.32(10.07)a 0.35(10.13)a 0.34(10.07)a 0.80 0.50









Table 5 -2. Proportion of male 7: nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single host
species. Means in rows followed by different letters are significantly different according to Tukey' s HSD-test (P=0.05;
SAS Institute 2000).

Mean (SE) proportion of parasitoids recovered on host
S. calcitrans M~ domestic S. bullata ANOVA P
Conditioning Host pupae pupae pupae F
S. calcitrans 0.45(10.05)a 0.23(10.04)b 0.34(10.04)ab 6.36 0.0056
M~ domestic 0.40(10.03)a 0.31(10.02)a 0.29(10.03) a 2.83 0.0771
S. bullata 0.38(10.04)a 0.31(10.05)a 0.30(10.03)a 0.78 0.4697
No conditioning 0.37(10.04)a 0.35(10.05)a 0.29(10.02)a 1.07 0.3593





Table 5-3. Mean proportions of female parasitoids reared on either S. calcitrans or S. bullata
pupae, who selected either S. calcitrans or S. bullata pupae as a first host choice.
Means followed by the same letter are not statistically significant by PROC GLM
(P-0.051.


Emerged from
S. calcitr ans ~rt~t~rtrt~r~rt
S. bullata
Df=1, 8; F= 33.25; P< 0.0001


On S. calcitrans pupae
0.70a
0.36b


On S. bullata pupae
0.30b
0.64a










Table 5-4. Mean proportions of male parasitoids reared on either S. calcitrans or S. bullata
pupae, who selected either S. calcitrans or S. bullata pupae as a first host choice.
Means followed by the same letter are not statistically significant by PROC GLM
(P-0.05).
Emerged from On S. calcitrans pupae On S. bullata pupae
S. calcitr ansrt~r~rt~rtrt~rt 0.45a 0.55a
S. bullata 0.60a 0.40a
Df=1, 8; F=1.72; P-0.202




























































Figure 5-1. Containers for parasitoids conditioned for 48 h on different host pupal types, with
Petri plates containing three choices of pupal host as well as a blank control Petri
plate. A) Containers were arranged in a randomized block pattern to control for
variations in light intensity, temperature and surroundings. B) During the experiment,
plastic tops were securely fastened onto containers to prevent escape of insects.


~. ~
i...






i,


" I) ~


... ;.t
ii i.,
iiii;ii;










































Figure 5-2. Containers for parasitoids that emerged from different host pupal types, with Petri
plates containing two choices of pupal host as well as a blank control Petri plate.
Containers were arranged in a randomized block pattern to control for variations in
light intensity, temperature and surroundings











CHAPTER 6
Y-TUBE OLFACTOMETER EXPERIMENT S

Introduction

For most insects, olfaction serves as a vital form of communication. The necessity of

locating and identifying odors is important for insects that cannot utilize visual or auditory cues

to locate mates, food, hosts and water (Hallem et al. 2006). Female insects, especially those

whose mates must fly great distances for mating opportunities, may give off pheromone

compounds to signal their location to males, e.g., the tobacco hornworm moth Manduca sexta L.

(Daly et al. 2001) and silkworm moths (Lepidoptera: Saturniidae) (Hansson 1995). Olfactory

cues are provided by different insect species, vertebrates and plants as well as an insect' s own

species. The use of kairomone compounds is well-documented in entomology (Johnson and

Triplehorn 2004), mainly to attract insects to a source. The gall wasp, Antistrophus rujis Gillette,

for example, has demonstrated a sex-mediated development schedule in which adult males

emerge from galls prior to female emergence to increase their chances of mating with a virgin

female (Tooker et al. 2002). Males ofA. rufits locate unemerged females by following olfactory

cues produced by stems of their host plants (genus Silphium) rather than from olfactory cues

provided by the females of A. rufus. Honey bees (Hymenoptera: Apidae) provide another classic

example of olfaction-based resource location (Reinhard et al. 2004), in which a scent (e.g., from

food) is associated with a location. Bees exposed to a scent specific to a location will return

again to that location.

Research on the relationship between associative learning and olfaction in insects has

yielded interesting examples of how strongly an initial exposure to a scent that is tied to a reward

(e.g., food or host source) can affect the future behavior of that insect. Foraging members of the

yellow acket species Vespula germanica (Fab.) will, when simultaneously exposed in their nest









to a food source and a scent, will preferentially visit a source outside the nest with that particular

scent profile, compared to a control (Jandt and Jeanne 2004). While much entomological data

suggest that associative learning is an important component of behavior for many insects, those

species with a life history strategy dependent on a host (e.g., parasitoids) present the question of

whether prior exposure to a host will affect the later behavior of that insect. Specifically, it is

unknown for many parasitoids whether close contact with a host during the larval stages will

affect the adult behavior of that insect with regards to locating and ovipositing in that same host.

Many species of parasitoid wasps have demonstrated measurable changes in behavior

following exposure to chemicals associated with hosts or host-fostering habitats. In many cases,

the surroundings of the host may provide the olfactory stimuli, rather than the host itself. Such

odors may be produced by plants that a host feeds on, decaying plant or animal matter that host

larvae develop in, or host frass. Both of the parasitoid wasps, Dibrachys cavus (Walker) and

Roptrocerus xylophagorum (Ratzeburg) (Hymenoptera: Pteromalidae), for example,

demonstrated a greater preference for the frass of their respective host insects than for the hosts

(Chuche et al. 2006; Sullivan et al. 2000, respectively).

The host insect itself may be the source of volatile chemicals that attract parasitoids, rather

than the surroundings of the host. The Diapriid parasitoid Trichopria drosophilae Perkins was

found to be attracted to kairomones emitted by the anterior spiracles of its host, Drosophila

melanogaster L. (Romani et al. 2002). Host recognition by M~uscidiferax raptor Girault and

Sanders involves chemicals emitted by its host M~usca domestic L. rather than from the feeding

and rearing medium of the host (in this case, manure) (McKay and Broce 2003). In an

olfactometer, M~ raptor preferred the odor of host pupae alone over the odor of manure. When









both manure and pupae were presented in one arm of the olfactometer, M~ raptor was not

attracted to the odors from that arm (McKay and Broce 2003).

Utilizing an olfactometer to document insect choice-making behavior is a common

technique in associative learning research with parasitoids. de Jong and Kaiser (1991), for

example, demonstrated that fruit fly larval parasitoid Leptopilina boulardi Barbotin et al.

(Hymenoptera: Eucoilidae) females exposed to an artificial odor in conjunction with an

oviposition experience would demonstrate a much stronger choice in an olfactometer for the

scent they were exposed to during stimulus/reward conditioning compared to native female

parasitoids, as well as to controls which were not classically conditioned. In this study, artificial

odors were considered ones not naturally produced by the host or plants fed on by the host. Later

studies with L. boulardi indicated that when females were exposed to multiple odors at different

times, they demonstrated a strong preference for all odors compared to native parasitoids, with

the strongest preference for the most recently-learned host (de Jong and Kaiser 1992). Learning

by parasitoids to prefentially follow an odor has been documented in Cotesia marginiventris

Cresson (Turlings et al. 1989), Leptopilina heterotoma (Thomson) (Papaj and Vet 1990) and

M~icroplitis croceipes (Cresson) (Lewis and Tumlinson 1988). In the case of M croceipes, which

is a parasitoid of Helilnthi\ zea (Boddie), female wasps could be successfully conditioned to

locate odors which are not naturally associated with their host, and in fact are unattractive, such

as vanilla (Lewis and Tumlinson 1988).

Because Trichopria nigra (Nees) has demonstrated a host range consisting of multiple fly

species (chapter 3) as well as significant changes in behavior following conditioning to a host

pupal type (chapter 5), the first obj ective was to determine whether conditioning females of T.

nigra to a host species' pupae creates a stronger preference to odors from that host they were










exposed to, compared to native female parasitoids (e.g., females not previously exposed to pupal

odors). The second obj ective was to determine whether the length of time females of 7 nigra are

exposed to a potential host influences their host-seeking behavior. Lastly, it was unknown

whether rearing 7 nigra on different host species affects female host preference, and whether the

speed and strength of native female 7 nigra response to host odors change with age.

Materials and Methods

Study Site

All experiments were conducted in a glass Y-tube olfactometer (Agricultural Research

Systems, Gainesville FL) (Figure 6-1) approximately 24 cm in length, with a tube diameter of

approximately 5 cm. Parasitoids were prevented from escaping during experiments by closing

the main arm of the Y-tube with a glass entry capsule containing a mesh opening (for air escape).

Flow rate was 0.3 L per minute (LPM) for all experiments. A clean air source was provided by

the air system at the CMAVE facility, which was supplied via a nalgene tube and filtered by

bubbling through H20 before flowing through the arms of the olfactometer. To negate the

possible effect of phototaxis, lighting was diffuse, being provided by four 32 W fluorescent

lights positioned centrally approximately 1.2 m above the olfactometer. Room temperature was

22-240C.

Parasitoids

For all experiments except 6-3 (rearing effect on host choice), female 7 nigra adults

from both the Russian and Kazakh strains were pooled immediately following emergence. Only

female parasitoids were utilized in experiments since Chapter 5 data suggest that male

parasitoids do not react significantly in a closed arena to volatiles from host pupae. Strains were

pooled by placing rearing containers (see chapter 3) in a refrigerator at~-13oC for five minutes,

then aspirating females from the bottom of both containers with a small handheld aspirator. All










aspirated females were gently blown into a Petri plate bottom to pool both strains, and gently

dropped into gelatin capsules for holding prior to experiments. For each repetition, five females

were counted and placed into an empty gelatin capsule (size "O") and were introduced to the

olfactometer by gently tapping the gelatin capsule against a hard surface to stun the parasitoids

and prevent them from flying away before placement in the olfactometer, and twisting it open

with the parasitoid-containing half shaken into the terminal end of the olfactometer. The entry

capsule was then placed into the terminal end of the olfactometer, preventing escape.

Controls were run prior to each experiment, consi sting of five replicates of five naive

female parasitoids introduced to the olfactometer with an air flow of 0.3 LPM. Both arms were

empty, and parasitoids were monitored for bias toward either arm of the olfactometer. The Y-

tube apparatus and associated stimulus capsules (Figure 6-2) were not washed between

replicates, as one arm of the Y-tube was designated for one of two stimuli, and attraction to that

arm did not change between replicates. Between experiments, all glass parts of the Y-tube

apparatus were washed with detergent and rinsed with tap and deionized water, respectively. All

pieces were allowed to air-dry, to avoid accidental marking of glass pieces with volatiles from

drying materials and fabrics.

For all experiments, strength of response was determined as being the proportion of females

which made a choice by traveling at least halfway down one arm of the olfactometer.

Experiment 6-1: Effect of Three Days of Conditioning on Host Choice

7: nigra adults (both strains) were aspirated from rearing containers within 12 h of adult

emergence and divided into three groups. A surplus (<1000) of parasitoids was collected to

ensure an adequate number of surviving females. Approximately 150 cc of 2-d-old Sarcophaga

bullata Parker pupae and 150 cc of Stomoxys calcitrans (L.) pupae were each placed into ca. 500

ml paper cups. One group of parasitoids introduced to each container for conditioning. The third










group was placed into an empty paper cup as a control. Containers were covered with muslin and

secured with rubber bands. A streak of honey and a water-dampened cotton ball placed atop the

muslin cover provided food and water. The paper containers were placed in a 250C incubator (+

0.40C, 70% RH) and 7 nigra females were allowed 3 d to associate host pupal odors with

oviposition (i.e., conditioning).

After the 3-d conditioning interval, parasitoids were knocked down by refrigerating the

paper cups for several minutes (~13oC) and then aspirating females from the inside of the cups.

Four replicates of five females each (i.e., five females at a time), conditioned to S. bullata pupae,

were introduced for 3 min into the olfactometer with ca. 1.0 g of either S. bullata or S. calcitrans

pupae (ca. 2 d old) in the arms, respectively. Pupae had, immediately prior to experimentation,

been collected from colonies at the USDA' s CMAVE facility, washed, dried gently, and placed

into the attractant chambers at the ends of the arms. Four replicates of five females each (e.g.,

five at a time), conditioned to S. calcitrans pupae, were introduced for 3 min into the

olfactometer with ca. 1.0 g of S. bullata and S. calcitrans pupae (ca. 2 d old) in the arms,

respectively. Additionally, four replicates of five unconditioned females each were introduced to

the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca. 2 d old) in the

arms. First choice was noted for each female within a replicate, as well as time required for each

female to make a first choice.

Females which did not choose either arm of the olfactometer within 3 min were

considered non-responders and not included in data analysis.

Experiment 6-2: Host Choice Response after 1 d, 3 d, and 5 d of Conditioning on S. bullata
Pupae

7 nigra adults (both strains) were aspirated from rearing containers within 12 h of adult

emergence and divided into two groups. A surplus (<1000) of parasitoids was collected to ensure









an adequate number of surviving females. Approximately 150 cc of 2-d-old S. calcitransrt~r~rt~rtrt~rt pupae

were placed into a ca. 500 ml paper cup. One group of parasitoids was placed in the container

with S. calcitrans pupae, and the other was placed in an empty paper cup as a control. Containers

were covered with muslin and secured with rubber bands. A streak of honey and a water-

dampened cotton ball placed atop the muslin cover provided food and water. The paper

containers were placed in a 250C incubator (A 0.40C, 70% RH). After 1 d, females were

aspirated from each container in groups of five (eight groups total) with a manual aspirator and

placed in gelatin capsules for experimentation, and the remaining parasitoids returned to the

incubator.

Eight replicates of five females each, conditioned to S. calcitrans pupae, were introduced

for 3 min into the olfactometer with ca. 1.0 g of S. calcitrans pupae in one arm and a control arm

not containing a stimulus. First choice was noted for each female within a replicate, as well as

time required for each female to make a first choice. Females which did not choose either arm of

the olfactometer within 3 min were considered non-responders and not included in data analysis.

Eight more sets of five females were collected at 3 d and 5 d after parasitoids were exposed to

pupae, and the above experimental procedure repeated.

Experiment 6-3: Host Choice by Parasitoids Reared on Different Hosts

7: nigra adults were reared on pupae of S. bullata and S. calcitrans, respectively (for

detailed rearing methodology, see Chapter 3). Within 12 h of parasitoid emergence, the ~500-ml

paper cups of pupae and newly-emerged parasitoids were provided with food and water via a

streak of honey and a water-dampened cotton ball placed atop the secured muslin covers. The

paper cups were placed in a 250C incubator (A 0.40C, 70% RH) for 48 h to allow for cuticle

hardening and sexual maturity of parasitoids.









Six replicates of five females each, reared on S. bullata pupae, were introduced for 3 min

into the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca. 2 d old) in the

arms, respectively. Six replicates of five females each, reared on S. calcitrans pupae, were

introduced for 3 min into the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans

pupae (ca. 2 d old) in the arms. Additionally, four replicates of five unconditioned females each

were introduced to the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca.

2 d old) in the arms. First choice was noted for each female within a replicate, as well as time

required for each female to make a first choice.

Experiment 6-4: Response of Unconditioned Parasitoids to Hosts During Five Days Post-
Emergence

7: nigra adults were aspirated from rearing containers within 12 h of adult emergence and

placed into a ca. 500 ml paper cup which was then covered with muslin, secured with rubber

bands, and food and water provided via a streak of honey and a water-dampened cotton ball

placed atop the muslin cover. A surplus (<1000) of parasitoids was collected to ensure an

adequate number of surviving females. The paper cup was placed in a 250C incubator (+ 0.40C,

70% RH) for 1 d to allow for cuticle hardening and sexual maturity of parasitoids. After 1 d, six

replicates of five females were aspirated from the paper container and introduced for 3 min into

the olfactometer with ca. 1.0 g of S. calcitrans pupae (ca. 2 d old) in one arm and a control

(empty) arm. First choice was noted for each female within a replicate, as well as time required

for each female to make a first choice. The above procedure was repeated on days two, three,

four and five after emergence.

Statistical Analysis

For Experiment 6-1, G tests were conducted to determine if the sampling distribution of

frequency of choices female parasitoids made in the olfactometer was random, or if there were










significant differences between the distributions of the three treatments. Parasitoids conditioned

to S. calcitransrt~r~rt~rtrt~rt were compared to parasitoids conditioned to S. bullata. Additionally, parasitoids

conditioned to S. calcitrans as well as parasitoids conditioned to S. bullata were individually

compared with the controls. G test statistics were rounded to two significant figures, and an

alpha level ofP<0.05 was considered significant.

For Experiment 6-2, two sets of G tests were conducted to test for variance in the

frequency of choices made by unconditioned and S. calcitrans conditioned parasitoids with

respect to time and treatment. S. calcitrans conditioned parasitoids were compared to

unconditioned controls at one day, three days and five days. Individually, both S. calcitrans

conditioned parasitoids and unconditioned controls were subj ected to two G tests: one comparing

day one choices for that treatment with day three choices, and another comparing day one

choices with day five choices. G test statistics were rounded to two significant figures, and an

alpha level ofP<0.05 was considered significant.

For Experiment 6-3, a single G test was conducted to test for variance in the frequency of

choices made by S. calcitrans conditioned parasitoids versus S. bullata conditioned parasitoids.

Additionally, for parasitoids that did respond to a stimulus in the olfactometer, a two-way

ANOVA was conducted on the mean response times of each treatment to the two choices, to

determine if any significant differences in response time emerged as a result of rearing host (SAS

Institute 2000).

For Experiment 6-4, G tests were conducted to test for differences in response strength

(e.g., number of parasitoids that chose pupae or the control arm) over time. Four comparisons

were made, examining cumulative choices made by female parasitoids on days one and two,

days one and three, days one and four, and days one and five. G test statistics were rounded to









two significant figures, and an alpha level ofP<0.05 was considered significant. A two-way

ANOVA was conducted to test for any significant differences in mean response time and mean

response strength for the five days the experiment was conducted. Response strength was

determined as the proportion of five females that made a decision within 3 min for each

repetiti on.

Results

Unconditioned female parasitoids preferred S. calcitrans pupae over S. bullata pupae

(Table 6-1). Host conditioning made a significant difference in the number of 7 nigra females

which responded to their conditioning host versus an unfamiliar host (Table 6-5). Comparing S.

calcitrans conditioned parasitoids to unconditioned controls (G=0.02; df=1) did not demonstrate

any significant difference in strength of response, nor did S. bullata conditioned parasitoids

compared to the unconditioned ones (G=2.16; df=1).

Comparing S. calcitrans conditioned parasitoids with their unconditioned counterparts did

not yield any significant differences on day one, day three or day five (Table 6-2). Comparing

the cumulative numbers of S. calcitrans conditioned parasitoids responding to pupal odors on

days one and three yielded a significantly greater response on day three (G=4.70; df=1), although

comparing day one and day five responses ofS. calcitrans conditioned parasitoids did not

(G=3.42; df=1). Additionally, no significant changes were observed between responses of

unconditioned parasitoids when days one and three and one and five were compared (G=0.75;

0.27 respectively; df=1). The greatest increase in response rate was seen in S. calcitrans

conditioned parasitoids between day one and day three (Table 6-2).

For parasitoids reared on two different hosts, there was no significant correlation between

host rearing type and pupal choice in the olfactometer (Table 6-5). While a greater number of S.









calcitrans conditioned parasitoids chose their natal pupal type over an unfamiliar stimulus (Table

6-3), S. bullata-conditioned parasitoids did not choose their natal pupal type.

The number of naive parasitoids responding to odors from S. calcitrans pupae increased

from day one to day three, then decreased slightly to day five (Table 6 -4). While response

strength did not change with respect to time in Experiment 6-4, the mean response time did

change significantly (P=0.04; df=4) over the five days. Comparing the strength ofunconditioned

7 nigra female response during the first five days after adult emergence yielded no significant

differences in G tests (Table 6-5). The fastest mean response time for female parasitoids to S.

calcitrans pupae was on day three.

Discussion

The results of Experiment 6-1 agree with the first arena experiment conducted in Chapter

4; i.e., that short-term conditioning of 7 nigra females to volatiles produced by host pupae does

indeed influence their host-seeking behavior to preferentially search for hosts of that exposure

type. Interestingly enough, while 48 h of female T. nigra exposure to one of two pupal types

created a preference for conditioning host, experiment 6-3 does not corroborate with the second

arena experiment conducted in Chapter 4. While in Chapter 4 we found that rearing does

influence host selection in an enclosed arena, the female parasitoids who were reared on S.

calcitrans and S. bullata pupae did not demonstrate a significant preference for their rearing host

when presented with those pupal types as choices in a Y-tube olfactometer. de Jong and Kaiser

(1992) suggest that the most recent exposure to positive stimulus in association with an odor has

the greatest influence on insect behavior, compared to earlier conditioning experiences. From an

ecological perspective, this assumption makes sense because, in the wild, a female parasitoid

who encounters multiple positive stimuli (e.g., an odor in association with an oviposition event)

is most likely to receive a reward if she seeks out the most recent location or type of stimulus.









Several explanations exist for the lack of differences in the behavior of unconditioned

parasitoids compared to those conditioned in S. calcitransrt~r~rt~rtrt~rt pupae. One possibility is that utilizing

clean, dried pupae does not provide a strong enough attractant for parasitoids, who are instead

seeking out odors of the environment associated with that host, such as odors associated with

larvae, their frass, or their larval rearing substrate (Sullivan et al. 2000). Additional research is

needed to determine whether 7 nigra utilizes a hierarchy of stimuli which includes host habitat

and proximity of host larvae in addition to cues that direct them to host pupae.

For many parasitoids, a combination of cues is necessary to successfully locate hosts.

This may be a combination of olfactory cues, of which some are attractive at a distance and

others at close range (Morehead and Feener 2000), or a combination of visual and olfactory cues.

Diacha;smimorpha juglandis for example, a parasitoi d of Rhagole tis fli es, l ocate s it s ho st vi a the

Rhagoletis food source, walnut fruit husks (Henneman et al. 2004). In an olfactometer, D.

juglan2dis can discern between intact walnuts and those which have been damaged by Rhagoletis.

However, when presented with visual rather than olfactory cues, such as when both mechanically

damaged and host-damaged walnut husks were presented, D. juglandis had little success

discerning between husks with hosts and husks without (Henneman et al. 2004). Clearly, visual

clues are often important for host location at a distance, luring parasitoids close enough to locate

olfactory cues.

For pupal parasitoids, it is possible that a combination of odors contributes to host-

seeking behavior. However, for parasitoids with a broad host range, such as 7 nigra, the

question arises of whether parasitoids naturally seek out multiple, and possibly quite different,

odors. McKay and Broce (2003) found that, for M~uscidifurax zaraptor Kogan and Legner, the

odors emitted from house fly puparia were much more attractive than the manure house fly









larvae naturally occur in. Furthermore, female parasitoids could not differentiate manure with a

combination of pupae and manure as a stimulus, when presented with both choices in a y -tube

olfactometer. I nigra, with a host range which encompasses flies that develop in a variety of

substrates and media (e.g.,, manure and carrion), the smaller and more closely-related milieu of

odors emitted from related host species may be the primary attractant, rather than a broad

spectrum of complicated odors.

The increase in response time to S. calcitrans pupae by unconditioned parasitoids in a Y-

tube olfactometer through day three, followed by a decrease in both response rate and response

time, indicates that the optimal oviposition time for this species is likely around three days post-

emergence. If 7 nigra does not mate inside the host (as is suggested due to spatial constraints),

the delay in optimal host location behavior may be explained by 7 nigra requiring this amount

of time to mate and complete hardening of the cuticle. Clearly, further research is required to

examine whether conditioning to host pupae does indeed override preference for natal host type,

as well as whether post-emergent conditioning to the same pupal type as the natal host type

strengthens the response of female 7 nigra searching for a host to oviposit in. In addition, it

remains to be determined whether olfactory cues associated with a host, such as the media in

which S. calcitrans and S. bullata develop, are stronger attractants for female 7 nigra compared

to the odors associated with pupae alone.










Table 6-1. First choices made by female parasitoids conditioned for 3 d on either S. calcitrans or
S. bullata pupae in a Y-tube olfactometer. The two choices presented in the
olfactometer arms were S. calcitrans and S. bullata pupae, respectively.

Conditioning Cumulative number of parasitoids attracted to
host S. calcitrans S. bullata
No conditioning 30 17
S. calcitrans 45 16
S. bullata 26 28










Table 6-2. First choices made by female parasitoids conditioned on S. calcitrans pupae and
tested at 1 d, 3 d and 5 d against unconditioned female parasitoids of the same age.
The two choices presented in the olfactometer arms were S. calcitrans pupae and a
blank arm, respectively.

Conditioning Day after Cumulative number of parasitoids attracted to
host emergence S. calcitrans blank
No conditioning 1 19 15
No conditioning 3 28 7
No conditioning 5 24 7
S. calcitr ansrt~r~rt~rtrt~rt 1 14 6
S. calcitr ansrt~r~rt~rtrt~rt 3 25 6
S. calcitr ansrt~r~rt~rtrt~rt 5 26 8










Table 6-3. First-turn choices made by 3-d old female parasitoids reared on either S. calcitrans or
S. bullata pupae and run in a Y-tube olfactometer. The two choices presented in the
olfactometer arms were S. calcitrans and S. bullata pupae, respectively.

Rearing Cumulative number of parasitoids attracted to
host S. calcitrans S. bullata
S. calcitr ansrt~r~rt~rtrt~rt 13 6
S. bullata 12 11









Table 6-4. First-turn choices made by female parasitoids run in a Y-tube olfactometer at 24-h
intervals after emergence. Day 1 was defined as 24 h post-emergence. ANOVA F
values are listed below means. Numbers marked with an asterisk are considered
significant (P<0.05).
Day Cumulative number of parasitoids attracted to Response time to
S. calcitrans blank S. calcitrans (s)
1 19 9 44.0 (4.9)
2 23 6 52.6 (5.6)
3 25 6 35.7 (3.9)
4 32 7 36.5 (3.7)
5 23 10 49.2 (6.1)

ANOVA F 2.54*
df=4,1; P<0.05










Table 6-5. Results of G tests for the four olfactometer experiments, comparing likelihood ratios
between variables. Numbers marked with one asterisk are considered significant at
P<0.05, while numbers marked with two asterisks are considered significant at
P<0.01.


At P <0.05. G >3.8. and at P <0.01.G >6.6.


Experiment Variables

6-1 Host conditioning on
S. calcitrans or S. bullata

6-2 Host conditioning on
S. calcitrans

Host conditioning on
S. calcitrans, with time


6-3 Rearing on S. calcitrans
or S. bullata pupae
6-4 Unconditioned, per day
post-emergence


Comparison: conditioning, time

S. calcitrans v. S. bullata
S. calcitrans v. no conditioning
S. bullata v. no conditioning
Day 1: S. calcitrans v. no conditioning
Day 3: S. calcitrans v. no conditioning
Day 5: S. calcitrans v. no conditioning
Unconditioned, day 1 v. day 3
Unconditioned, day 1 v. day 5
S. calcitrans, day 1 v. day 3
S. calcitrans, day 1 v. day 5
S. calcitrans v. S. bullata

Unconditioned, day 1 v. day 2
Unconditioned, day 1 v. day 3
Unconditioned, day 1 v. day 4
Unconditioned, day 1 v. day 5


G

8.04**
1.23ns
2.52ns
1.07ns
<0.01ns
<0.01ns
4.69*
3.42ns
0.75ns
0.27ns
1.15ns

0.97ns
1.27ns
1.79ns
0.02ns



































B)














Figure 6-1. Y-tube olfactometer used in experimentation. A) Air flow and humidity were
controlled for by a regulator bubbling air through distilled water. B) Up to two
choices were presented to parasitoids in screened chambers that allowed air flow of
volatiles. Choice was counted as the first arm a parasitoid moved halfway down,
regardless of future turns or time spent in that arm.


























































:*
1
ll1


Figure 6-2. Y-tube olfactometer arms and entry capsule. Between experiments, all glass pieces

were washed, rinsed twice and allowed to air-dry.










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BIOGRAPHICAL SKETCH

Kimberly Marie Ferrero was born on March 18, 1983, the first child of Patti A. Ferrero and

Dr. Frank A. Ferrero. Her father delivered her at Miami Mercy Hospital, where he was a heart

surgeon. Kimberly is the oldest of five children, and has two younger sisters, Ashley and

Francesca, as well as two younger brothers, Christopher and Shawn. A first generation Italian-

American, she grew up in Miami, Florida and attended high school at Lake Mary High in

Orlando, Florida. As a young child, her love of science blossomed under the guidance of many

wonderful teachers, so that at the age of seventeen she became one of only two students to teach

honors biology at her high school.

Following high school, she began attendance at the University of Florida, and in 2001

completed her Bachelor' s degrees in Anthropology and Zoology, with a focus on the

evolutionary biology of primates. Her years at the University of Florida allowed her to take part

in research travel to places such as Indiana University in the United States, St. Andrews

University in Scotland, and Suriname in South America. A summer internship in the final year of

her undergraduate education, at the University of Florida' s Entomology and Nematology

department, convinced Kimberly to remain at the university to pursue a Master' s degree in

medical and veterinary entomology, with an emphasis on control of medically important Diptera

(flies and mosquitoes).

Kimberly subsequently spent the next two years working at the United States Department

of Agriculture' s on-campus Center for Medical, Agricultural and Veterinary Entomology, and

her love of public health issues was rewarded when she was permitted to conduct her Master' s

research on a little-known parasitoid from Eastern Europe that attacks and kills filth flies that are

common worldwide. She has given numerous talks and lectures on the importance of control of

arthropod disease vectors, and has taught Introductory Entomology at the University of Florida









as a Graduate Teaching Assistant. She spends much of her free time collecting insects in the

wilderness of north-central Florida, and enjoys swimming, kayaking, sailing, rock climbing and

gourmet cooking on a graduate student budget.

As a final note, Kimberly' s inspiration for pursuing a career in the sciences is attributed

to her father, Dr. Ferrero. A gifted doctor and scientist with degrees in biology and physics, his

unwavering support of her education and insistence that women can be influential figures in the

sciences has allowed her to complete this thesis. Although Dr. Ferrero passed away one year

before seeing his daughter obtain the first of her graduate degrees, his passion for helping people

through a greater understanding of the natural world lives on in his daughter.





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1 LIFE HISTORY, HOST CHOICE AND BEHAVIORAL PLASTICITY OF Trichopria nigra ( HYMENOPTERA: DIAPRIIDAE), A PARASITOID OF HIGHER DIPTERA By KIMBERLY MARIE FERRERO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLO RIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Kimberly Marie Ferrero

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3 To my father, the first and greatest scientist in my life.

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4 ACKNOWLEDGMENTS The list of peop le who have contributed to my reaching this point in my education is both long and impressive. I am indebted to so many men and women whose advice, encouragement and criticism have made this body of scientific work what it is. I can only hope that everyone mentioned within these pages knows that they are as much a part of this writing as I am. committee: Dr. Chris Geden and Dr. Jerry Hogsette. These two men have devoted time, patience, countless hours both in the field and in the laboratory, endless reams of paper and red ink, and an embarrassment of intellectual riches to my education. From my committee I have known nothing but genuine caring and encouragement tempered with the ability to let me know when my research (or my work ethic) needed tweaking, and the ability to point out my strengths and weaknesses as a graduate student. I am fortunate to have been mentored by true scientists, and hope that I will do their tute lage justice. I would like to thank the scientists and staff in my laboratory: Hank McKeithen and Ashley Campbell. Hank had passed onto me both the importance of rigorous and proper scientific method as well as memories of many hours of field work and labo ratory assays that would have otherwise been much more tedious. His friendship, his guidance and advice both on professional and personal matters has left me feeling that I have always, and will always, have a friend at my lab bench. Ashley kept the labora tory running flawlessly through many of my experiments, kept me sane during many a data analysis and writing flurry, and kept me intrigued and humored with her knowledge of biology and the scientific world. I would not be as patient a scientist if Ashley h ad not taught me the limits of human endurance in the face of scientific tedium. employees at the United States Department of Agriculture Center for Medical, Agricultural a nd

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5 Veterinary Entomology became friends and mentors to me. In no particular order (except perhaps as I ran into them every morning) I would like to thank with all warmth and caring Dr. Gary Clark, Dr. Matt Aubuchon, Dr. Brian Quinn, Dr. Sandra Allan, Dr. U lrich Bernier, Dr. Dan Kline, Natasha Elejalde, Greg Allen, Kathleen Smitherman, Mike Brooks, and Lindsay Clark among others. Writing my thesis, papers and grants would not have been possible without their input, nor would working at the USDA have been so enjoyable, without their friendship, professional candor and willingness to help. While it has been nearly a decade since I was a student in his classroom, I would like to thank Mr. Mark Schiffer, my biology teacher of four wonderful years and the reason I am so detail obsessed and well versed in molecular genetics and the virtues of caffeination. He has inspired more young scientists than he will ever know. I would like to extend a personal note of gratitude to the fellow students who I came to know as fri ends while at the University of Florida. The first is Christine Bertrand, who has been a best friend and comrade in academia since our first day of undergraduate classes seven years ago. Christine has been my anchor through years of classes, graduation and graduate school, writing our theses concurrently, and the many trials and tribul ations of growing up. She is as much family to me as are my sisters and brothers I would also like to thank Roxanne Burrus, a fellow entomologist who came from the same labor atory as I did, and quickly became a woman I admired and leaned on for help and advice. She is a model for young female scientists to emulate; I have learned so much from her in a very short time logy and Nematology department will always command my respect and gratitude for the time and effort they devote to each of the graduate students who pass through their laboratories and lecture halls. I am grateful

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6 to Dr. Phil Kaufman for inspiring a love o f medical and veterinary entomology in me, and for making my induction into the field of entomology as a new graduate student an enjoyable and comfortable one. Every student of his is a fortunate student I would also like to thank Dr. Carl Barfield for th e opportunity to teach his students, as well as for h is practical and honest advice o n the professional aspect s of being a scientist. I am also indebted to Debbie Hall for several years of reminders, deadlines, crisis solving skills and an innate ability t o keep an entire department of students on track. A final thanks is extended to Dr. Richard Patterson his wife, daughter and granddaughter: a family of entomologists who have treated me like family as well, and instilled in me a sense of academic diplomac y, hard work and honesty. The most personal of thanks I leave to the end, not for lack of importance but because wording such gratitude is difficult. My family has always been an ongoing source of lov e, support and encouragement I would l ike to thank my parents, Patti and Frank, as well as my siblings Ashley, Francesca, Christopher and Shawn, for always being a light in the window an answer to a phone call, a willing eye to read my papers before a deadline, and a group of open arms that never cease to re mind me how important I am to them My father, a heart surgeon and professor of medicine, was my role model and the inspiration for my becoming a scientist. He passed away in winter of 2006, a tragedy that forever changed my life. While I will never be the scientist he was, I can only be grateful that I was part of the life of a man who saved so many others. Last, but far from least, I would like to thank Matthew Cmar, who has been an understanding and supportive partner through all these semesters of resea rch, class, experiments and writing. He made me laugh many times, prompted me to cry a few, cheered me on to stay up late to meet deadlines, and made me feel like a good scientist even when I doubted myself. His love and encouragement will never be taken f or granted.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .................... 4 LIST OF TABLES ................................ ................................ ................................ ................................ 9 LIST OF FIGURES ................................ ................................ ................................ ............................ 11 ABSTRACT ................................ ................................ ................................ ................................ ........ 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ....................... 14 2 LITERATURE REVIEW ................................ ................................ ................................ ........... 16 Evolution of the Parasitic Hymenoptera ................................ ................................ ................... 16 Biological Control of, and Economic Damages Caused by, Pest F lies ................................ ... 18 Family Diapriidae ................................ ................................ ................................ ........................ 21 Biology ................................ ................................ ................................ ................................ 21 Distribution ................................ ................................ ................................ .......................... 21 Trichopria nigra ................................ ................................ ................................ .......................... 22 Origin and Distribution ................................ ................................ ................................ ....... 22 Biology ................................ ................................ ................................ ................................ 23 Food and Hosts ................................ ................................ ................................ .................... 23 3 NOTES ON SPECIES RECOGNITION AND REaRING METHODS ................................ 25 Introduction ................................ ................................ ................................ ................................ 25 Positive Identification of Species ................................ ................................ ............................... 25 Adult Insect ................................ ................................ ................................ .......................... 25 Female Reproductive Potential w ith Respect to Body Size ................................ .............. 27 Rearing Methods ................................ ................................ ................................ ......................... 29 Containers ................................ ................................ ................................ ............................. 30 Die t ................................ ................................ ................................ ................................ ....... 30 Host Provisioning ................................ ................................ ................................ ................ 30 4 LONGEVITY AND FECUNDITY EXPERIMENTS ................................ .............................. 42 Introduction ................................ ................................ ................................ ................................ 42 Materials and Methods ................................ ................................ ................................ ................ 46 Study Site ................................ ................................ ................................ ............................. 46 Parasi toids ................................ ................................ ................................ ............................ 46 Longevity Arena ................................ ................................ ................................ .................. 47 Experimental Design ................................ ................................ ................................ ........... 47 Longevity det ermination ................................ ................................ .............................. 47

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8 Fecundity estimation ................................ ................................ ................................ .... 48 Statistical Analysis ................................ ................................ ................................ ............... 49 Re sults ................................ ................................ ................................ ................................ .......... 50 Longevity Determination ................................ ................................ ................................ .... 50 Fecundity Estimation ................................ ................................ ................................ ........... 51 Disc ussion ................................ ................................ ................................ ................................ .... 52 Longevity Determination ................................ ................................ ................................ .... 52 Fecundity Estimation ................................ ................................ ................................ ........... 54 5 A RENA CHOICE EXPERIMENTS ................................ ................................ ......................... 69 Introduction ................................ ................................ ................................ ................................ 69 Materials and Methods ................................ ................................ ................................ ................ 72 Study Site ................................ ................................ ................................ ............................. 72 Hosts ................................ ................................ ................................ ................................ ..... 72 Parasitoid S trains ................................ ................................ ................................ ................. 72 Conditioning pa rasitoids for short term assay ................................ ............................ 72 Arena for short term assay ................................ ................................ ........................... 73 Parasitoids for rearing assay ................................ ................................ ........................ 74 Arena for rearing assay ................................ ................................ ................................ 74 Statistical Analysis ................................ ................................ ................................ ...................... 75 Results ................................ ................................ ................................ ................................ .......... 76 Discussion ................................ ................................ ................................ ................................ .... 77 6 Y T UBE OLFACTOMETER EXPERIMENTS ................................ ................................ ....... 87 Introduction ................................ ................................ ................................ ................................ 87 Materials and Methods ................................ ................................ ................................ ................ 90 Study Site ................................ ................................ ................................ ............................. 90 Parasitoids ................................ ................................ ................................ ............................ 90 Experiment 6 1: Effect of Three Days of Conditioning on Host C hoice ......................... 91 Experiment 6 2: Host Choice R espo nse A fter 1 d, 3 d, and 5 d of C onditioning on S. bullata P upae ................................ ................................ ................................ ................ 92 Experiment 6 3: Host Choice by Parasitoids Reared on Different H osts ........................ 93 Experiment 6 4: Response of Unconditioned P arasit oids to Hosts during Five Days Post E mergence ................................ ................................ ................................ ................ 94 Statistical Analysis ................................ ................................ ................................ ...................... 94 Results ................................ ................................ ................................ ................................ .......... 96 Discussion ................................ ................................ ................................ ................................ .... 97 LIST OF REFERENCES ................................ ................................ ................................ ................. 107 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ........... 117

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9 LIST OF TABLES Table page Table 3 1. H ead capsule widths and egg numbers of Trichopria nigra females and males (50/50 sex ratio) reared on pupae of Sarcophaga bullata ................................ ................... 32 Table 3 2. H ead capsule widths and weights for female and male Trichopria nigra (50/50 sex ratio) reared on pupae of Sarcophaga bullata and Stomoxys calcitrans ..................... 33 Table 4 1. Mean lifespans (SE) (d) of Trichopria nigra adults under different feeding treatments at 25 C. ................................ ................................ ................................ .................. 57 Table 4 2. Female T. nigra t wo way ANOVA results for influence of presence/absence of honey and host pupae on survival time ................................ ................................ ................. 58 Table 4 3. Male T. nigra t wo way ANOVA results for influence of presence/absence of honey and host pupae on survival time ................................ ................................ ................. 59 Table 4 4. Effect of diet on mean number (SE) of pupae killed by female T. nigra pupae producing wasps, having dead wasps, and total number of parasitized pupae. ................. 60 Table 4 5. Cumulative (SE) numbers of male and female progeny, respectively, pro duced by parasitoids given only water and pupae, or water, honey and pupae. ............................ 61 Table 5 1. P roportion of female T. nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single host species. Means in rows ............................ 81 Table 5 2. P roportion of male T. nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single host spe cies. ................................ ..................... 82 Table 5 3. Mean proportions of female parasitoids reared on either S. calcitrans or S. bullata pupae ................................ ................................ ................................ ................................ ....... 83 Table 5 4. Mean proportions of male parasitoids reared on either S. calcitrans or S. bullata pupae ................................ ................................ ................................ ................................ ....... 84 Table 6 1. First choices made by female parasitoids conditioned for 3 d on either S. calcitrans or S. bul lata pupae ................................ ................................ .............................. 100 Table 6 2. First choices made by female parasitoids conditioned on S. calcitrans pupae and tested at 1 d, 3 d and 5 d ................................ ................................ ................................ ...... 101 Table 6 3. First turn choices made by 3 d old female parasitoids reared on either S. calcitrans or S. bullata pupae ................................ ................................ .............................. 102 Table 6 4. First turn choices made by female parasitoids run in a Y tube olfactometer at 24 h intervals after emergence. ................................ ................................ ................................ 103

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10 Table 6 5. Results of G tests for the four olfactometer experiments ................................ ............. 104

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11 LIST OF FIGURES Figure page Figure 3 1. Adult female Trichopria nigra lateral view. ................................ ................................ 34 Figure 3 2. Adult male Trichopria nigra, lateral vi ew. ................................ ................................ .... 35 Figure 3 3. Probing female Trichopria nigra ................................ ................................ .................. 36 Figure 3 4: Dissection of T. nigra abdomen ................................ ................................ ..................... 37 Figure 3 5. A) Empty puparium of Sarcophaga bullata ; and B)Dissected host remnants ........... 38 Figure 3 6. Microscopy image (400x) of 3 day old Trichopria nigra ov arioles. .......................... 39 Figure 3 7. Regression analysis for body size and egg load of female Trichopria nigra .............. 40 Figure 3 8. Rearing contain ers used for colonies of Trichopria nigra ................................ ......... 41 Figure 4 1. Rearing contain ers for longevity determination ................................ ............................ 62 Figure 4 2. A) 60 m l cups containing individually encapsulated S. calcitrans pupae ; and B) Example of successfully parasitized pupa ................................ ................................ ........... 63 Figure 4 3. Mean adult A) female and B) male Trichopria nigra mortality for all treatments. .... 64 Figure 4 4. Total number of S. calcitrans pupae parasitized by female parasi toids for each of two treatments ................................ ................................ ................................ ........................ 65 Figure 4 5. Mean number of adult parasitoids that emerged daily for each of two treatments ..... 66 Figure 4 6. The average number of parasitoid s that emerged per pupa per day for A) w ater and h osts only. ; and B) food, water and hosts ................................ ................................ ...... 67 Figure 4 7. Cumulative mean number of adult parasitoids that emerged from S. calcitrans pupae for each of two treatments ................................ ................................ .......................... 68 Figure 5 1. A) Containers for parasitoids conditioned for 48 h on different host pupal types; and B) containers with tops secured during experimentation ................................ ............. 85 Figure 5 2. Containers for parasitoids that emerged from different host pupal types .................... 86 Figure 6 1. A) Y tube olfactometer used in experimentation ; and B) choices presented in screened cham bers ................................ ................................ ................................ .............. 105 Figure 6 2. Y tube olfactometer arms and entry capsule. ................................ .............................. 106

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science LIFE HISTORY, HOST CHOICE AND BEHAVIORAL PLASTICITY OF Trichopria nigra (HYMENOPTERA: DIAPRIIDAE), A PARASITOID OF HIGHER DIPTERA By Kimberly Marie Ferrero December 2007 Chair: Christopher J. Geden Major: Entomology and Nematology Trichopria nigra (Nees ) (Hymenoptera: Diapriidae) is a gregarious pupal endoparasitoid of several common fly species. Furthermore, it is a parasitoid of two of the most common pest fly s pecies in North America: the stable fly Stomoxys calcitrans (L.), and the house fly Musca domestica L T. nigra was first established in a North American laboratory from specimens that emerged from stable fly pupae collected in Russia and Kazakhstan in 199 9. Little is known about the life history and definitive host range of this insect. No records of this insect in North America have been made. Its ability to successfully parasitize multiple common pest fly species, however, as well as its small size and i nexpensive, simple rearing methods make it a potentially valuable biological control agent against stable flies and house flies. The first photographs of T. nigra are presented with a detailed analysis of adult external morphology and sexual dimorphism. Th e rearing method s used in maintaining colonies of this parasitoid are provided. Dissections of T. nigra ovarioles were made to determine mean number of ova. Weights of, and head capsule widths from parasitoids reared on two different hosts were made to determine whether variance in body size exists with regard to host size and it was determined that a larger host does produce, on average, larger parasitoids of this species.

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13 A longevity experiment was conducted to determine the mean lifespan of male and female parasitoids when provided with honey water and host pupae. I t was determined that providing honey lengthened adul t lifespan, an effect that was increas ed when host pupae were resulte d in conjunction with honey Providing hosts in the absence of hon ey and water led to the shortest lifespan for male and female parasitoids. Two variations of a choice test were conducted, one in which adult parasitoids were conditioned for 48 h on t he pupae of one of three host species, and another in which wasps w ere reared on two host species. Conditioning parasitoids for 48 h significantly increased the proportion of female wasps that chose that host species to which they were conditioned in an open arena assay for house fly conditioned parasitoids only Rearing para sitoids on a particular host species led to a significant difference in host choice in an open arena assay, with parasitoids strongly preferring to oviposit in the host species in which they had developed. Y tube o lfactometer experiments were conducted to corroborate findings from the choice test experimen ts as well as to determine whether response of adult female parasitoids changed significantly with age and previous exposure to a host insect. Conditioning parasitoids increased the likelihood, compared to unconditioned controls, of females choosing their conditioning host when presented with two choices in a Y tube olfactometer. R earing parasitoids on a host species greatly increased the likelihood of females choosing their natal host when presented with t wo choices. Lastly, it was determined that the strength and speed of female response to host odors does not significantly change in the first five days of adult emergence, although the most number of females responded, and demonstrated the fastest response between two and three days post emergence.

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14 CHAPTER 1 INTRODUCTION Because the hosts of many parasitic wasps are flies commonly considered pests of humans and livestock, the significance of parasitoids as biological control organisms is considerable (Rue da and Axtell 1985, Anderson and Leppla 1992). Two of the most important pest flies with regard to human health, well being and economics in North America are often considered Musca domestica L. the house fly, and Stomoxys calcitrans L., the stable fly. O ther flies which are considered pests of humans and commercially reared animals include the face fly Musca autumnalis De Geer, the horn fly Haematobia irritans (L.) and a variety of blow flies and flesh flies in the families Calliphoridae and Sarcophagida e. One of the primary motives for fostering research in the field of biological control is the growing resistance of pest insects to chemicals utilized in controlling them. House flies were among the first insects to demonstrate documented resistance to in secticides, as DDT became commercially available for house fly control in 1944 By 1947, DDT had failed to control fly populations in Europe (Decker and Bruce 1952). House fly resistance to pyrethroids emerged within ten years of introduction of these chem icals as commercial products for house fly control (Hogsette 1998). Resistance of insects has long been known to develop swiftly in insect populations subjected to frequent, and high dosage, pesticide applications (Mallis et al. 2004). An additional dimen sion to the problem of pest fly resistance to insecticides is the apparent genetic basis for pesticide resistance exists in the house fly, which is allelic in nature and subject to both genetic mutation and the migration of pest fly populations with novel alleles (Rinkevich et al. 2007). In the future, agriculturists may find that relying solely on pesticidal methods to prevent insect damage to livestock and crops will have become wholly ineffective. C urrently it proves impossible (Hogsette 1998) to maintai n livestock pests such as horn flies at or below their

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15 damage thresholds with registered pesticides. Treating agricultural pest problems with biocontrol organisms, however, does not incur resistance to that treatment method as do pesticide applications. I n light of the reduced efficacy of traditional chemical control and the effects of pesticides on the environment, there exists an increasing need for novel and effective biological control organisms which can be used to reduce the number of pest insects an d their effects on humans and agricultural products. The aims of this research were to better understand the biology and behavior of a species of endoparasitic wasp, Trichopria nigra (Nees) (Hymenoptera: Diapriidae), which parasitizes numerous species of f lies commonly considered pests of humans and livestock. Because the facility in which this research was conducted is the first known location of in laboratory rearing of T. nigra a detailed identification of this species, along with this ing methodology, is included (Chapter 3). A longevity experiment was conducted to determine the longevity of this species when presented with a food source, water, and hosts in several combinations, as well as to compile a greater understanding of the life history of this species (Chapter 4). Additionally, two experiments were performed to determine (1) the preference of this species when provided with multiple choices for a host, and whether that choice can be influenced (Chapter 5); and (2) whether olfact ion is an important method this species uses to locate a host, and if the strength of an olfaction based host choice changes with the age of the adult insect (Chapter 6)

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16 CHAPTER 2 LITERATURE REVIEW Evolution of the Parasitic Hymenoptera Within the order Insecta, parasitic life history strategies account for the second most popular life history strategy in insects after phytophagy (Althoff 2003). Within Hymenoptera, parasitism of other insects is so prevalent across the families of waisted wasps (Hymenopt era: Apocrita) that an early evolutionary origin of parasitic Hymenoptera is likely (Whitfield 1992). Furthermore, the prevalence of parasitic lifestyles with the majority of families of Apocrita possessing genera that are considered parasitoids of plant s or other insectst indicates that such life history strategies are highly successful. Systematic coevolution of insects and the plants and animals which provide them with food, shelter and reproductive niches is well documented across taxa. A common en tomological example is louse host specificity (Bush and Clayton 2006). Parasitoid host coevolution is similarly apparent at least among plant parasitoids. Sidhu (1984) observes that genetic selection in many plants important to humans (and to insects a s hosts) is the result of breeding and crop selection. T he evolution of host animals in contrast, is often driven more by ecology and behavior, making closely related coevolutionary cha nges more difficult to observe. H owever, diversity of parasitoids seem s, generally, to be under the control of host dive rsity (Sidhu 1984, Hufbauer 2001 ). According to Quicke (1997), the fossil record contains preserved h ymenopteran fossils dating as early as the mid Triassic period. There are currently two extant suborders of Hymenoptera (Johnson and Triplehorn 2004): the Symphata (including sawflies and wood wasps) and the Apocrita (all other hymenopterans including all known parasitoids), although the outset of the Jurassic period saw diversification of Hymenoptera to inc lude the extinct suborders

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17 Karatavitidae and Ephialtitidae (Quicke 1997). The appearance of morphological features that would allow for parasitism of a host, such as a lengthened ovipositor, hint at the emergence of the first parasitoid hymenopterans durin g the Jurassic period. Within Hymenoptera, the suborder Apocrita is primarily defined (Johnson and Triplehorn between the first and second abdominal segments. Other defining characteristics, such as the number of basal hindwing cells (Quicke 1997), further separate Apocrita from the evolutionarily primitive sawflies. Within Apocrita, ten of the twelve superfamilies largely include parasites of either plants (ga ll wasps, fig wasps) or animals (overwhelmingly other members of Insecta) (Johnson and Triplehorn 2004). Superfamilies within Hymenoptera which are known to contain parasitoid members include Ichneumonidea, Chalcidoidea, Evanioidea, Proctotrupoidae, Bethyl oidea, Scolioidea and Vespoidea (Whitfield 1998, Johnson and Triplehorn 2004). The question as to why parasitism evolved across multiple insect orders has been under spec ulation for decades (Quicke 1997 ). Parasitism most likely evolved only once in basal H ymenoptera (Eggleton and Belshaw 1992) as, driven by competition for food sources, early mycophagic wasps began to kill larvae already feeding on fungi and superimpose their own larvae on the food source. The most primitive (from a cladistic viewpoint) par asitic superfamilies the Ichnauemonidea and Evanioidea are often found in close relation to detritus and fungal heavy environments (Eggleton and Belshaw 1992, 1993); the more advanced parasitoid hymenopterans practice life history strategies ranging fr om parasitizing developing larvae to take advantage of a longer developmental time (Gelman et al. 2005 b ), to drilling into host puparia and laying eggs outside the host. Further discussion on the advantages and disadvantages of a parasitic lifestyle in Hy menoptera follows below.

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18 From an evolutionary viewpoint, it might be assumed that if parasitism evolved once in a clade and was not only retained as a life history strategy but became the overwhelming life history strategy of an entire suborder (Borror et al. 1974, Johnson and Triplehorn 2004), that life history strategy must offer distinct and significant benefits to the insects practicing it. One advantage that parasitoids encounter is that they do not have to devote time as immature insects to foraging f or, locating, and processing food (Quicke 1997). Parasitoid wasps live within their source of food, water, and (often) endocrine compounds (Gelman et al. 2005 a ). Their hosts confer protection from changes in temperature and humidity, and unless the host i nsect is predated upon, the parasitoids are safeguarded from predation themselves. Obvious risks are associated with a parasitic life history for parasitoid hymenopterans. The quality of the host often determines the quality of the next generation of para sitoids (Ellers et al. 1998, Consoli and Vinson 2004), as the available nutrients for the developing parasitoids are limited by the size and carrying capacity of the host insect. Another drawback to parasitism is the risk of choosing a host species that is poorly suited to parasitism, especially in non specific solitary parasitoid wasps (Quicke 1997, Ferrero 2006 from unpublished research). Bi ological Control of, and Economic Damages Caused by, P est F lies Because the hosts of many parasitoid wasps are fli es commonly considered pests of humans and livestock, their significance to human populations is considerable (Rueda and Axtell 1985, Anderson and Leppla 1992). The most important pest flies with regard to human health, well being and economics in North Am erica are often considered Musca domestica L. the house fly, and Stomoxys calcitrans (L.) the stable fly ( Geden and Hogsette 1994 ). Other flies which are sometimes considered pests of humans and commercially reared animals include the face fly Musca aut umnalis the horn fly Haematobia irritans the flesh fly Sarcophaga spp ., and the black garbage fly Ophyra spp

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19 According to the USDA confined and range cattle contributed the largest share of agricultural profit in the United States with 37.9 billion dollars in income (Geden and Hogsette 1994) Of this amount, 17.6 billion dollars in damage come from confined cattle facilities; the highest infestations of pest flies which are hosts of numerous parasitoid species occur in confined cattle facilities. Wh ile traditionally considered pests of confined cattle only (Smith and Rutz 1991), stable flies have been known to disturb cattle in open fields in geographic locations that are subject to periodical rainfall (Geden and Hogsette 1994). Directly, stable flie s impact cattle by blood feeding, which reduces overall health and feeding behavior of the animal and, ultimately quality of product (Campbell and Berry 1989). Poultry contribute a large portion of income to the agricultural industry in the United States and with an increase in poultry rearing in the past five decades from small agribusinesses to large sc ale poultry rearing facilities pest flies have become an increasingly problematic issue. The primary pest of poultry agribusinesses, house flies, prese nt a sanitation and public health problem (Mann et al. 1990). Transmission of avian pathogens as well as bacteria known to cause human illness (Geden and Hogsette 1994) create a potentially hazardous working environment in poultry houses. The house fly has been implicated as a potential vector of pathogens that pose serious risks to human health (Eldridge and Edman 2003) such as poliomyelitis, cholera, typhoid, tuberculosis and dysentery. Additionally, house flies swarming in confined spaces such as rearin g facilities causes an annoyance such that poultry farms may incur a monetary loss in employee turnover, reduced efficiency of work, and time spent implementing control methods for pest flies in response to complaints from public health agencies. Insect re sistance to chemical control is another justification for the importance of parasitoids in improving agricultural and domestic aspects of human life. Insect physiological

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20 resistance to chemical control has, since the implementation of en masse pesticide ap plication in the forties, become a growing problem for agriculturalists worldwide (Learmount et al. 2002). Hogsette (1998) documents that house fly resistance to pyrethroids emerged within ten years of implementation; resistance of pest filth flies to ave rmectins such as abamectin (Clark et al. 1995) developed swiftly in insect populations and established readily in the gene pool. In the future, agriculturists may find that relying solely on pesticidal methods to prevent insect damage to livestock and cro ps will have become wholly ineffective, even as currently it proves problematic (Hogsette et al. 1991 ) to maintain livestock pests such as horn flies at or below their damage thresholds with registered pesticides. Treating agricultural pest problems with b iocontrol organisms, however, does not incur resistance to that treatment method as do pesticide applications. Most commercially utilized biocontrol organisms are wasps which are pupal parasitoids parasitoid in the family Pteromalidae (Hymenoptera: Chalc idoidea). Biological control of pest flies involves releasing either adult parasitoids of the pest fly, or fly pupae with developing parasitoids within, en masse at an infested location (Geden and Hogsette 2006). Adult parasitoids increase the mortality of the host insect at a specific point in development by killing the host, usually prior to adult eclosion or adult emergence from the puparia (Rueda and Axtell 1985, Quicke 1997). Little is known about the efficacy of other parasitoid families in controllin g pest flies. Trichopria nigra (Hymenoptera: Diapriidae) is a gregarious endoparasitoid of pest fly pupae that has been shown to significantly reduce population levels of house fly and stable fly pupae in the research conducted within this body of work. It demonstrate that T. nigra a novel and little known parasitoid wasp is an efficient biocontrol

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21 organism for the s t able fly S. calcitrans and potentially of other pest fly species within its host repertoire via selective condi tioning. Family Diapriidae Biology The family Diapriidae (superfamily Proctotrupoidea) consists of minute wasps, all of which are parasitoids of other insect taxa (Nixon 1980). Diapriid wasps are known endoparasitoids of members of Diptera, Coleoptera (Ma sner 1993), and in the unique case of genus Ismarus, other Hymenoptera ( Loiacono 1987). Diapriidae is divided into four subfamilies: Ismarinae, Ambositrinae, Belytinae and Diapriinae. Some members of Diapriidae are solitary; many, such as Trichopria nigra are gregarious endoparasitoids (Nixon 1980). Solitary parasitoids are defined as those insects which lay one egg per host; gregarious parasitoids lay multiple eggs per host (Johnson and Triplehorn 2004). Subfamily Diapriinae, to which T. nigra belongs, sh ares the fewest traits and is often considered by insect taxonomists as the subfamily to which parasitoids not fitting the other three subfamilies are assigned (Buss, personal correspondence). Medvedev (1988) provides a thorough catalog of characteristics shared among members of Diapriidae. Among these features include a dark and glossy exoskeleton, a small and rounded head with long antennae, and a markedly narrow first abdominal segment (petiole). Despite most members of Diapriidae having large wings rel ative to absolute body size, they are poor fliers compared to many other Hymenoptera (Medvedev 19 8 8). Distribution While genera of Diapriidae are distributed worldwide, (Masner 1976, Masner 1993), the exact distribution of most of the approximately four t housand species placed within Diapriidae is largely unknown (Masner and Garca 2002).

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22 Trichopria nigra Origin and Distribution Roger Moon (University of Minnesota) collected pupae of Stomoxys calcitrans on dairy farms near the cities of Almaty, Kazakhstan and Kraznodar, Russia in 1999 (C.J. Geden, USDA/ARS, personal correspondence). The purpose of such collections was to discover novel parasitoids of the stable fly and house fly. Pupae collected by Dr. Moon were transported to the United States and the fi rst documented emergence and colony establishment of T. nigra occurred in 2000 at the USDA/ARS CMAVE facility in Gainesville, Florida. This is the first documented laboratory colony of T. nigra. The distribution of T. nigra is not completely known; its dis covery in Eastern European and Eurasian sites by D r. Moon suggests that distribution is at least partially Palearctic ; Medvedev (1988) cites the distribution as Romania (Moldavia) The most recent record of T. nigra in the wild is a 2002 collection of Tric hopria spp. taken from sites in Kalambaka, Greece (Petrov 2002); the collector lists the known distribution of T. nigra as Germany north to Sweden and east to Moldova. Given the proximity of Moldova and Sweden to Russia, it is likely that the distribution of T. nigra extends from central Europe into northern Asia. Related species in the genus Trichopria are distributed worldwide; collections have been made in the eastern (Bradley et al. 1984) and western United States (Krombein et al. 1979a; Krombein et al 1 979b) as well as European countries such as Hungary (Hogsette et al. 1994). In Africa, Trichopria spp. have been observed in countries such as Zimbabwe (Huggert and Morgan 1993; Morgan et al. 1990) and Ethiopia (Huggert 1977). Given that many discoveries of Trichopria species are made in and near poultry houses on all continents the genus has been discovered (Hogsette et al. 1994; Morgan et al. 1990), and that T. nigra emerged from S. calcitrans pupae collected by Dr. Moon on dairy farms, it is likely tha t

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23 T. nigra may live and reproduce at least in the proximity of livestock and poultry rearing facilities. Biology The first entomological description of Trichopria nigra is attributed to Nees (1834); this reatise on Diapriidae (Kieffer 1916), and more recently in (2002) description of Diapriidae Few taxonomic keys provide an adequately detailed identification of the genus. Medvedev (1988) offers the following couplet for identification of members of Trichopria : than 1 st antennal segment, and these hairs discumbent. Temples with white or silvery hairs. Longitudinal axis of eye usually not shorter than length of middle coxa. Head roundish or rectangular on dorsal surface. Females: Antennae at least with dark clava. Antennal segments, commencing from the 3 rd In addition to the above couplet, T. nigra adults possess features that distinguish the species from sister spec ies; the 2 nd and 3 rd antennal segments are approximately equal in length and the legs, in contrast to the glossy black body color, are a dark yellow color and nearly translucent (Medvedev 1988). Males and females are dimorphic with respect to antennal shap e; females possess a clubbed terminus at the distal end of each antenna whereas males possess comparatively more uniform antennae. The sexes are not dimorphic with respect to size, although a larger host ( e.g., S. bullata is larger than S. calcitrans ) will usually produce larger parasitoid offspring of both sexes (see Chapter 3: Effect of Host Size on Parasitoid Size). Food and Hosts T. nigra species, T. stomoxydis which is a gregarious endoparasitoid of S. calcitrans shares a similar life history to T. nigra. It was determined that T. stomoxydis does not host feed prior to, or following,

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24 host location and oviposition; without a food source such as plant nectar, longevity wa s calculated to be approximately three days (Morgan et al. 1990). Other shared life history features include the endoparasitic nature of larval development; both T. nigra and T. stomoxydis immatures feed and develop within the body of the pupating host. Un like T. nigra however, T. stomoxydis is host specific to S. calcitrans (Nash 2005). In colony at the USDA/ARS CMAVE facility, adult T. nigra are provisioned with honey on an ad libitum basis. Despite being a koinobiont species, adult female T. nigra do no t host feed and thusly do not require a meal to complete ovariole maturation (Chan and Godfray 2005). Water is also provided ad libitum Although it is likely that this insect procures moisture from nectar food sources in the wild; the moisture content of most commercial honeys, at 20 40% is considerably lower than that of plant nectars due to the concentration of sugars (Bijlsma et al. 2006). For a detailed description of rearing methodology, see Chapter 3.

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25 CHAPTER 3 NOTES ON SPECIES REC OGNITION AND RE ARING METHODS Introduction Very few references to Trichopria nigra (Nees) exist in the scientific literature. A 1984 collection of seaw eed fly pupae from two species collected from Kieler Forde, Germany (Heitland 1988) Coelopa frigida (F.) and Fucellia t ergina (Zett.) yielded specimens identified as T. nigra Petrov (2002) mentions T. nigra in a catalogue of Diapriid parasitoids found in Greece, although first identification of this species is attributed to Nees in 1834 (noted in the Hymenoptera collect ion of the Zoological Museum, University of Copenhagen) A field survey by Hogsette et al. (1994) in central Hungary lists two species of Trichopria which were found in proximity to pupae of Musca domestica L. and Stomoxys calcitrans (L.) ; given the discov ery of T. nigra in Russia and Kazakhstan by Moon (personal communication) as well as in China by Yujie et al (1997), it is possible that this species has a broadly Palearctic range. The lack of a definitive and readily available species description, in add ition to the lack of known photographs and images of this insect, indicate the need for a detailed analysis of T. nigra morphology and differentiation from related species. Furthermore, rearing methodology of this insect is included for the benefit of sc ientists who wish to rear T. nigra in a laboratory setting T. nigra (the Russian collected population and the Kazakhstan collected population) are morphologically and behaviorally similar, and are reared ide ntically. Positive Identification of Species Adult Insect T. nigra adults share many of the general traits of family Diapriidae, being described as small (1 6 mm), black parasitoid wasps with large eyes and geniculate antennae possessing 11

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26 15 segments, a nd an ovipositor that is not visibly protruding (Masner and Garca 2002) The insect is readily identified as a wasp, with three well defined body segments, a narrow waist, and two pairs of wings. The sexes are not sexually dimorphic with regard to size, a s both male and female adults grow to a maximum mean length of 2 3 mm from head capsule to terminal abdominal segment. It has been documented (Ueno 2004) that the size of many pupal parasitoids, as well as the sex ratio of progeny produced, varies in direc t relation to the size of their hosts. The sizes of T. nigra determine if size plasticity with regard to host size is correlated in this parasitoid species. Adult T. nigra reared on pupae of Stomoxys calcitran s (L.) we re on average one third lighter (mean mass 0.069 0.001m g .) than individuals reared on pupae of Sarcophaha bullata Parker (mean mass 0.106 0.001m g.), with a head capsule diameter that is ~80 % that of indiv iduals reared on S. bullata ( Table 3 2 ). The head is rounded and possessing of large, black eyes that are the most prominent feature of that segment. A ntennae attach superiorly and medially to the eyes, and sexing of the adult insect is most easily perfor med by examining the shape of the antennae. In female insects the antennal segments enlarge dista lly into a distinct club ( Figure 3 2). Male T. nigra adults, however, possess antennae that are near ly uniformly filamentous ( Figure 3 3). Both male and female insects possess, as do all Diapriine wasps (Masner 1976), t hirteen antennal segments. S econd and third antennal segments are equal in length; this feature along with the unique coloration of the legs is considered the defining trait for T. nigra, separat ing it from T. socia in a dichotomous key (Medvedev 1988).

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27 The exoskeleton is uniformly black in color and highly reflective, almost lustrous; the legs are of dark amber to golden yellow color, with femoral and coxal segments distinctly d arker than the tar sal segments. The entire insect is sparsely covered with slender bristles. The cephalic point of the first thoracic segment, as well as thoracic segments 2, 3 and the thoracic abdominal juncture, are covered in dense collars of short, golden hairs. T. nigr a ovipositor size is small in relation to body length, and markedly short in comparison to ovipositor length in taxa such as Ichneumonidae. T he length of the ovipositor is retracted except during probing and oviposition ( Figure 3 4). This feature is shared among Diapriidae (Medvedev 1988). Females are gregarious in oviposition behavior, and adult T. nigra chew multiple exit holes in the host puparia ( Figure 3 5 A ). Developing parasitoids require most of the room inside the developing fly host, and dissected cuticle which protected the par asitoid larvae inside ( Figure 3 5B ). Female Reproductive Potential with Respect to Body Size Female reproductive anatomy of T. nigra consists of two bi lobed ovarioles ( Figure 3 6) of the m eroistic type, germ cells differentiating into both oocytes as well as nurse cells. Like most koinobionts, f emale T. nigra emerge with competent (autogenous) oocytes (Waage and Greathead 1986) and do not nee d to feed in order to oviposit, and oocytes are l ikely not replenished as they are used. As demonstrated in Figure 3 1 3 2, and 3 3 adult body size can vary greatly in this species, to the extent that it is questioned whether the size of adult female T. nigra is indicative of their reproductive potenti al. To determine whether a correlation exists between adult female size and fecundity (egg load), dissections of female T. nigra reared on two hosts were conducted. Twenty 2 5 day old female T. nigra reared on Stomoxys calcitrans and twenty 2 5 day old fe male T. nigra reared on S. bullata were ovariectomized. The number of eggs for each female was counted ( Figure 3 6)

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28 and the width of the head capsule measured. The mean ( SE) head widths and number of eggs are listed i n Table 3 1 A regressional analysis was conducted for each treatment as well as both treatments combined, with head capsule width and number of eggs as variables ( Figure 3 7) Furthermore, PROC GLM was conducted (SAS 2000) to determine whether rearing host has an effect on egg load. For fema les reared on S. calcitrans pupae, the average head capsule width was 0.320 mm, approximately 84% of the head capsule width of females reared on S. bullata pupae (0.380 mm) (Table 3 1) This difference was s tatistically s ignificant (SAS, 2000) and was cons istent with hypotheses about the T. nigra size/fitness relationship, and previous parasitoid research (see Quicke 1997; Cohen et al. 2005). Because head capsule width is an indicator of overall body size and often, of fecundity and adult fitness (Liu 1985 Visser 1994 respectively ) it was expected that larger females ( i.e., females reared on S. bullata the larger host) would possess more eggs. This was found to be the case as well, with T. nigra reared on S. calcitrans possessing on average approximatel y 76% of the egg load their S. bullata cohorts developed. The observed differences in body size and fecundity of T. nigra when reared on two hosts of differing host sizes agrees with literature on parasitoid fitness with respect to host fitn ess. In gener al, parasitoids whose hosts are larger have more food available and therefore can acquire more nutrition for growth and development ( e.g., of eggs). Some literature suggest that for koinobiont parasitoids, who develop internally and allow their hosts to co ntinue physiological development, the correlation between host size and parasitoid fitness is not necessarily valid (see Jenner and Kuhlmann 2005). For T. nigra however, which is an unusual koinobiont in that it parasitizes its hosts during their pupal st age, host growth in a spatial context has mostly ceased and in fact is constrained by the puparium containing the host. It may be assumed that a clearly

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29 lineated relationship between body size and size of the host is a characteristic more commonly associat ed with idiobionts and those koinobionts which parasitize later stages of their hosts. Rearing Methods At 25 C, development requires ~26 d from oviposition for adult emergence (personal observation). Males and females emerge from puparia at the same time. It is not known whether adults are competent to oviposit immediately following adult emergence. It is also not known whether any mating occurs inside host puparia although such mating would prove unlikely given the spatial constraints of adult parasitoids inside the puparia. A related species, Trichopria stomoxydis, mates shortly after emergence of adults from host puparia, with male insects emerging approximately 24 h p rior to female insects (Morgan et al. 1990) It is likely that T. nigra follows a similar mating behavior. For a detailed life history of T. nigra see Chapter 4. T. nigra has successfully parasitized pupae of Stomoxys calcitrans (L.) Ophyra aenesce n s ( Wiedemann) Haemotobia irritans (L.) and Sarcophaga bullata Parker Colonies of T. nigra have been successfully maintained utilizing S. bullat a and S. calcitran s as hosts. Additionally, T. nigra females readily examine and probe with their ovipositors the pupae of Musca domestica and have been observed ovipositing inside M. domestica puparia; regardless of a high pupal mortality in the host, no adult parasitoids have emerged in numerous laboratory rearing attempts (personal observation). Other instances of T. nigra parasitizing a dipteran host include the cheese skipper, Piophila casei L. (Teodorescu and Ursu 1979). All of the above hosts are flies in the infraor der Muscamorpha. Additionally, T. nigra has been reared on the small pest fly Sturmia bella (Meigen) (Diptera: Tachinidae) (Nash 2005). The majority of demonstrated T. nigra hosts are commonly considered pest flies in close association with human habitats; as data on this species is highly incomplete a detailed list of all hosts is not available.

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30 Containers C lear plastic storage containers ( ca. 25 x 14 x 12 cm) were utilized as rearing containers. Container tops were discarded and the containers sealed acro ss their opening with a white cotton tube and two rubber bands to prevent wasp escape and to allow for periodic access to the container ( Figure 3 8 ) Rearing containers were placed on one side to that access via cotton tube was from the side rather than th e top of the container. Water was provided ad libitum on cotton balls placed in one ounce plastic cups (Sweetheart Cup co., Owings Mills, MD); one or two water soaked cotton balls in cups were placed in each rearing container. When access to the interior o f the container was not required, the end of each cotton tube was tied securely into a single knot. Diet Commercially purchased (human consumption grade) ho ney was provided to adult wasps on cotton balls placed in two ounce plastic food service container s (Sweetheart Cup co., Owings Mills, MD). Additionally, a square piece of muslin cloth (ca. 10 x 6 cm) was covered in a thin layer of honey and placed on the inside superior wall of the rearing container. This was done to prevent drowning of parasitoids i n the honey due to their small size. Since T. nigra does not host feed, no pupae as a protein food source were required for colony maintenance. See Chapter 4 for a discussion on whether honey is required for colony maintenance. This researcher has also suc cessfully reared T. nigra on a 50% v/v solution of honey and water provided on cotton balls in one ounce plastic food service containers (Sweetheart Cup co., Owings Mills, MD). Host Provisioning Both strains of T. nigra (Russian and Kazakhi,) were reared on ~2 day old pupae of Sarcophaga bullata in ~300 cc paper containers covered loosely with a piece of muslin cloth

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31 and a rubber band. Paper containers of stung pupae were covered to prevent accidental hyperparasitism from other species reared in the labo ratory, and kept in an incubator at 25 C until adult emergence was first noted Time required for development from stinging of pupae to emergence of adults was approximately 26 days at a temperature of 25 C. At first sign of emergence from puparia adu lts were transferred to rearing containers (see above) by placing the paper food service containers containing pupae and new adults, uncovered, in the rearing containers. Adult wasps, beginning at 5 6 d post emergence, were provisioned with ~ 250 cc fresh unstung 2 to 3 day old S. bullata pupae for 5 6 days on a weekly basis, to establish the subsequent generation. For the purpose of experimentation requiring T. nigra reared on hosts other than S. bullata, host provisioning methods were identical except th at ~250 cc of pupae from S. calcitrans or M. domestica were utilized. An interesting aspect of host provisioning for T. nigra is that, while adult females will readily attempt to parasitize M. domestica pupae, and very few host pupae survive to adult emerg ence, no parasitoids emerge d from puparia. The mechanism for this phenomenon is unknown, although it is hypothesized that rapid melanization of wounds caused by probing kill parasitoid eggs, if any are laid (personal observation). Dissections of house fly puparia stung by T. nigra at 2, 5 and 10 days post sting have not yielded any parasitoid larvae.

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32 Table 3 1. Head capsule widths and egg numbers of Trichopria nigra females and males (50/50 sex ratio) reared on pupae of Sarcophaga bullata and Stomoxys calc itrans For S. bullata reared, F=8.99; df= 1 P<0.05. For S. calcitrans reared, F=8.60; df= 1 P<0.05. Rearing Host Head Capsule Width (mm) Egg Load n S. bullata emerged 0. 382 (.007)a 114.3 (6.4)a 20 S. calcitrans emerged 0.320 (.010)b 87.2 (4.5)b 20 Means in the same column followed by the same letter are not significantly different (SAS 2000).

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3 3 Table 3 2. Head capsule widths and weights for female and male Trichopria nigra (50/50 sex ratio) reared on pupae of Sar cophaga bullata and Stomoxys calcitrans __________________________________________________________________________ ____ Rearing Host Head Capsule Width (mm) Weight (mg) S. bullata emerged 0.384 (.005) 0.106 (.001) S. calcitrans emerged 0.322 (.005) 0.069 (.001)

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34 Figure 3 1 Adult f emale Trichopria nigra lateral view

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35 Figure 3 2 Adult male Trichopria nigra, lateral view.

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36 Figure 3 3. Probing female Trichopria nigra The ovipositor is extended and inserted into the puparium of Stomoxys calcitrans

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37 Figure 3 4: Dissection of T. nigra abdomen reveals the retracted ovipositor in close relation to the ovarioles.

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38 A) B) Figure 3 5. A) Emp ty puparium of Sarcophaga bullata Multiple exit holes created by Trichopria nigra are visible. B) Dissected puparium of S. bullata. B) Dissected host remnants. During parasitoid development, the interior of the host fly is consumed.

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39 Figure 3 6. Microscopy image (400x) of 3 day old Trichopria nigra ovarioles Small dark circles at the base of the ovarioles (arrow) are the spermathecae.

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40 Figure 3 7. Regression analysis for body size of female Trichopria nigra (dire ctly correlated to head capsule width in mm) and egg load size (in eggs/female)

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41 Figure 3 8 Rearing containers used for colonies of Trichopria nigra at the USDA/ARS Center for Medical, Agricultural and Veterinary Entomology in Gainesv ille, FL.

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42 CHAPTER 4 LONGEVITY AND FECUND ITY EXPERIMENTS Introduction Reproduction is nutritionally costly in all animals; insects prove no exception. Female parasitoids that oviposit must have competent eggs, and must be competent themselves to lay eggs. Many parasitoids, before laying eggs inside or on a host, will utilize a host as a food source (Godfray 1994). This feeding strategy of utilizing a potential host to feed both an adult parasitoid and its offspring is widespread among idiobionts. Females o f idiobiont parasitoid species emerge without competent eggs and must host feed to acquire protein for egg maturation. Additionally, odopbionts parasitize their hosts in later host life stages (Quicke 1997). In contrast, koinobionts parasitoids which par asitize their hosts early in development and allow hosts to continue development rarely host feed. The life history of Trichopria nigra (Nees) is largely unknown. It has been observed that T. nigra does parasitize multiple species of higher flies, and th at it is not observed to feed on potential hosts before or during oviposition. From an evolutionary perspective, these behaviors are unusual. T. nigra is a koinobiont parasitoid. Its hosts continue to develop post oviposition (see chapter 3 for experiments and observations on this topic). Many of the characteristics of T. reproductive behavior and physiology however, reflect those of idiobiont parasitoids (Hawkins et al. 1990); for example, idiobionts tend to have a relatively wide host range that e ncompasses multiple host species. Preliminary data indicate that T. nigra readily parasitizes several closely related host species. Additionally, laboratory rearing of this insect has demonstrated a low female fecundity, despite gregarious immature develop ment within the host (Geden, personal observation). Lastly, T. nigra parasitizes its hosts later in their development, a

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43 trait normally ascribed to idiobiont species (Quicke 1997), yet is an endoparasitoid with larval wasps demonstrating a koinobiont trai t by feeding and developing inside the host viscera. When a parasitoid develops inside a host that is growing (such as a larva), nutritional resources are continually acquired by the host. Less need exists for a well yolked, larger egg, because continual growth provides a renewed food source (Giron et al. 2002). Because of this, koinobionts tend to possess smaller eggs and are proovigenic ( i.e., eggs are chorionated and the female insect does not require a protein meal to produce yolk). In genera l, it is assumed that the main nutritional components gleaned from feeding on host hemolymph are sugars such as trehalose and amino acids (Giron et al. 2002, Quicke 1997), although results from longevity experiments (Ferracini et al. 2006, Jervis and Kidd 1991 ) offer inconclusive evidence that host feeding imparts any benefits such as longevity to the maternal parasitoid. Because the purpose of host feeding is solely to develop the ovaries and produce competent eggs (Quicke 1997), it would not be expecteded that host feeding incurs any lifespan lengthening effects to female parasitoids. Therefore, T. nigra a parasitoid which has never been documented host feeding, should not benefit from the presence of host pupae with regard to lifespan because it would n ot be expected that such hosts would be utilized as a food source. However, it is not known whether T. nigra males and females, when provided with a carbohydrate food source such as honey or plant nectar, live longer or produce more progeny than individual s maintained solely with a source of water. Additionally, it is not known whether the presence of host pupae for the purpose of oviposition appreciably lengthens or shortens the lifespan of male and female T. nigra. The effects of providing food and hosts to parasitoid wasps have been studied in many non diapriid parasitoids. In a n early experiment conducted on Nasonia vitripennis (Walker)

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44 (Hymenoptera: Pteromalidae) it was determined that the longevity of unfed parasitoids was approximately 7 days for fema les and less than two days for males (Nagel and Pimintel 1963). It is generally assumed that for many insects the addition of an energy source ( i.e., food) extends the lifespan of individuals. Various parasitoid species in the families Pteromalidae and Dia priidae which are reared at CMAVE, for example, live as long as four or five weeks in a colony situation, where rearing containers include a food source in the form of honey (Ferrero, personal observation). Irvin et al. (2007) determined that when three s pecies of egg parasitoids in the genus Gonatocerus were given a honey water solution, the longevity of male and female parasitoids increased between 1400% and 1800% in all three species, compared to parasitoids given only water. Furthermore, studies which involved feeding parasitoids a variety of potential carbohydrate food sources in the form of insect derived sugars (such as trehalose) or plant derived sugars (such as fructose), indicate that plant derived sugars increase the longevity of parasitoids more appreciably (Jacob and Evans 2000, 2004). The Diapriid parasitoid Trichopria s tomoxydis Huggert provides compelling evidence that feeding on a carbohydrate source suc h as a sugar to parasitoids in a laboratory setting changes the lifespan of the adult insect Morgan et al. (1990) claimed that T. stomoxydis presented little potential as a biological control agent in part because of its short adult lifespan. In a laboratory setting, provided water but no food source, the maximum longevity for all parasit Similar results were obtained in rearing of the Encyrtid parasitoid Tachinaephagus zealandicus Ashmead (De Almeida et al. 2002 a ) where at temperatures from 16 and female parasitoids given food and water lived up to two t imes longer than those with access to water alone. Interestingly, this effect diminished with increasing temperatures at which adults

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45 were held. T. zealandicus was later observed (De Almeida et al. 2002) to attack significantly more pupae when females had access to both food (provided as honey) and water than when given only water; females given neither food nor wat er had the lowest attack rates Additionally, no correlation could be made between feeding treatment and fecundity, except in the instance of f emale s given neither food nor water. Since sugar metabolism is not required for maturation of parasitoid eggs (Quicke 1997), it is logical to assume that providing most parasitoids with a sugar based food source would not substantially affect the number, i f not the quality, of progeny produced. The number of offspring produced by many parasitoids is dependent on adult body size and food availability. It has been demonstrated (Ueno and Ueno 2007) that providing the Ichneumonid parasitoid Itoplectis naranyae Ashmead with hosts increases their fecundity; while I. naranyae females could mature (chorionate) eggs without host feeding, feeding produced an increase in future egg clutch size. Egg clutch size appears to be at least partially connected to the geograph ical regions in which strains develop, as is documented in the case of Muscidifurax raptor Girault and Sanders strains (Legner 1969; Legner 1979). M. raptor strains from Peru are almost completely solitary in laying of egg clutches, whereas the Chilean st rain of this same insect demonstrates a higher instance of gregariousness. If tendency toward solitary oviposition is favored with smaller hosts, and gregarious oviposition more common in larger hosts (Godfray 1994), then it could be hypothesized that T. n igra is more likely a gregarious parasitoid. The objectives of these experiments were twofold. The first objective, with regard to adult parasitoids, was to establish how long male and female T. nigra parasitoids live, respectively, when provisioned solely with water, with water and a carbohydrate food source, with water and a potential host, and with water, a carbohydrate food source plus potential hosts. The second

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46 objective, with regard to the F 1 generation produced by adults in Objective (1), was to exa mine how the above four treatments affect female fecundity with emphasis on parasitism rate, number of progeny produced by female parasitoids, and the sex ratio of progeny produced in the F 1 generation. Materials and Methods Study Site Male and female T. n igra specimens for the bioassay were combined from two strains reared at the USDA/ARS Center for Medical, Agricultural and Veterinary Entomology facility. All host flies i ncluding Sarcophaga bullata Parker and Stomoxys calcitrans (L.), were reared Parasitoids To provide adequate numbers of insects for experimentation, a Kazakh strain (KzTn) and a Russian strain (RuTn) of T. nigra were combined. Both strains were reared on ~2 day old pupae of Sarcophaga bullata Newly emerged T. nigra adults were transferred to clear plastic containers (25W x 14L x 12H cm). Honey was ap plied to muslin strips (approx. 15 x 8 cm) and placed on the upper interior wall of the containers, and to cotton balls placed inside ~30ml plastic cups (Sweetheart Cup co., Owings Mills, MD). Water was provided ad libitum on cotton balls in 30ml plastic c ups. Adult wasps were provisioned with ~250 cc fresh, unstung 2 to 3 day old S. bullata pupae. Parasitoids were exposed to the pupae for 5 6 days on a weekly basis until the death of adult wasps, to establish an F 1 generation, which was used in experiment ation.

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47 Longevity Arena Arenas consisted of translucent 60 ml plastic containers (Sweetheart Cup co., Owings Mills, MD) measuring 5.5 cm in height and 7 cm in diameter with matching snap lock plastic lids. To provide air flow and exposure to honey and wate r, and to prevent parasitoids from escaping, a 5 cm dia. circular piece was removed from the center of each lid with a scalpel and replaced with circles of fine copper mesh (#50 size) fastened over the holes with a hot glue gun. Twenty arenas were construc ted to test four treatments with five repetitions per treatment. Food and water were provided to wasps by placing a cotton ball saturated with either a honey water solution or water placed atop the mesh cover, allowing a portion of the mesh to remain uncov ered for air flow (Figure 4 1) Experimental Design Longevity determination The experiment was designed to assess the survivorship of male and female adult T. nigra when provided solely with water, with water and a carbohydrate food source (honey) with w ater and a host, and with honey and water as well as a host. Food consisted of a 50% w/w solution of clover honey and water kept refrigerated at 12 obtained from the tap. Hosts consisted of 2 day old pupae of S. calcitrans ( L. ). Prior to exposure to T nigra in arenas, S. calcitrans pupae were washed, dried and so rted into groups of 200 for ease of replacement on a daily basis. Host pupae were presented to the pupae and water and pupae food and water treatments for a period of 24 h by gently pouring 2 00 pu pae into the rearing container Exposed pupae were removed from the arenas at the end of each 24 h period. Adult parasitoids were manually separated from the pupae by gently shaking them through a metal sieve (#12 mesh size) Dead wasps were removed, counted and sexed. The remaining, live wasps were returned to the

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48 appropriate arenas with 200 fresh S. calcitrans pupae. Ten sets of 200 pupae (five repetitions of two treatments) were recovered from the arenas after each 24 h period. Fecundity estimation Fecundity of parasitoids with respect to treatment was determined by holding exposed S. calcitrans pupae from each day of the longevity experiment. The number of pupae which did not produce adult flies and the number of pupae which produced adult parasitoi ds were counted. Pupae collected daily from the longevity experiment were placed in ~60 ml translucent plastic cups (Sweetheart Cup co., Owings Mills, MD) with plastic lids and retained in an incubation chamber at 25 C until S. calcitrans adults emerged (emergence time was between two and four days post wasp exposure). Adult flies were not given food or water, and died within 48 h. Dead flies and empty puparia were removed by hand. Pupae which did not produce adult flies were placed individually into siz tran slucent plastic cups (Figure 4 2 ) sorted by treatment ( i.e., food, water and hosts or water and hosts). Cups of encapsulated pupae were returned to the 25 C (0.4C, 70% RH) incubator until a dult wasps emerged. The numbers of wasps that emerged per pupa per day were counted for each treatment, and total numbers of male and female progeny counted to observe the sex ratio of the F 1 generation. Pupae which produced neither flies or parasitoids we and Meyer 1985). Ten of these pupae were dissected per treatment daily to determine whether they had been parasitized by T. nigra but the developing parasitoids did not survive to adult o observed parasitism occurred. The total number of mummy parasitoids for each day was estimated with the equation (Ferrero) T M = (M/10) (T N P P ) where

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49 T M = total number of pupae which did not produce adult flies or parasitoids, M = total number of pupa e from ten dissections which contained dead immature parasitoids, T N = total number of pupae which did not produce adult flies, and P P = total number of pupae which did not produce flies but did produce adult parasitoids. The total number of parasitized S calcitrans pupae for each day and treatment was calculated as T P = P P + T M where T M = total number of parasitized pupae which did not produce adult flies or parasitoids, and P P = total number of pupae which did not produce flies but did produce adult par asitoids. Statistical Analysis The adult longevity data for each treatment and for each sex were analyzed via two way analysis of variance (Proc GLM; SAS Institute 2000) using honey, pupae, and honey pupae as model effects. Treatment means were separate Unless otherwise stated, P values of less than 0.05 were considered statistically significant. For fecundity dat a (i.e., fly pupae that were parasitized, and the resultant parasitoid progeny) statistical tests f actored in sex of T. nigra progeny when noted; for all other progeny data analyzed, male and female numbers were combined. One way analysis of variance (ANOVA) was performed (SAS Institute 2000) with the total number of parasitized pupae, the total number of mummy pupae, the cumulative number of male progeny, the cumulative number of female progeny, the percentage of female progeny, and the total number of parasitoid progeny, respectively, as dependent variables. This was done to determine if the water trea tment only versus the water and honey treatment produced significant differences in any of the above

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50 parameters. Data analyzed in the one way ANOVA were subjected to arcsine transformation and analyzed as proportions. Regression analyses were performed on the numbers of T. nigra progeny that emerged per pupa for both of the pupae included treatments to determine whether a relationship existed between the presence or absence of parental feeding and resultant progeny. Regression analyses were also performed to derermine whether a relationship existed between parental feeding and number of progeny (and whether T. nigra fecundity could be influenced by parental feeding) Results Longevity Determination The longest lifespan for both sexes was observed in the t reatment in which both honey and pupae were available to parasitoids, and the shortest lifespan for both sexes was observed in the treatment providing pupae and water but no honey. Mean longevity for females was between 11 and 13 days for the water only tr eatment, the water and honey treatment, and for the water, honey and pupae treatment, respectively (Table 4 1). For females given only water, the lifespan was less (under ten days). Mean longevity for males (in days) was between five and ten days for all t reatments (Table 4 1). Mean mortalities for all four treatments are plotted for males ( Figure 4 3 A) and females (Figure 4 3B). The provisioning of parasitoid females with honey resulted in a significant increase in longevity (F = 11.73, df = 1, 367; P = 0.007) (Table 4 2). There was, however, a significant pupae honey interaction (F = 5.91; df = 1, 367; P < 0.05), which indicated that parasitoids given pupae and honey lived significantly longer than parasitoids given pupae alone (T able 4 1). Female paras itoids given pupae and water did not express a significant increase in lifespan (F = 1.83; df = 1, 367; P > 0.05) ove r females given only water. Additionally females with access to pupae but not honey died sooner than females which were provided only wate r (Table 4 1).

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51 Results with male parasitoids were similar to those with females. Provisioning male T. nigra with honey significantly lengthened lifespan (F = 81.96; df = 1, 386; P < 0.0001); providing males with pupae did not lengthen lifespan compared to the water only males however (F = 0.05; df = 1, 386; P > 0.05). Overall the effect of providing parasitoids with either honey, or honey and pupae, was stronger in males than in females, with the mean lifespan of males provided with honey and pupae nearl y double that of the control males which received neither (Table 4 1). The interaction of honey and pupae on males wa s statistically significant, with males provided both living the longest of the four treatments (Tables 4 3 ). Fecundity Estimation Because only two of the four experimental treatments in the Longevity Estimation involved pupae and therefore produced T. nigra progeny, only two treatments (water and pupae; water, pupae and honey) are discussed below. The mean number of pupae that were parasiti zed (killed) by T. nigra adults provisioned with water did not differ significantly from the mean number of pupae parasitized by adults provisioned with honey and water (Table 4 4). The number of mummy pupae le 4 4) produced did not differ significantly between treatments. The cumulative number of male and female progeny, respectively, did not differ between treatments, although the percentage of female parasitoids that emerged from successfully parasitized pu pae did differ significantly (F = 6. 2; df = 1, 8; P < 0.05), with slightly more female progeny emerging from pupae which were stung by females provisioned with honey as well as water (Table 4 5). The cumulative number of pupae parasitized over time is pres ented in Figure 4 4 Females began parasitizing hosts immediately, and within 2 days had parasitized over half of the number of hosts that they would parasitize during their lives. By day 5, females in both treatment groups had parasitized over 90% of the total hosts that they would parasitize. When progeny production

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52 is viewed in terms of successful production of adult progeny per day, a similar pattern is evident ( Figure 4 6 ), with a maximum progeny production occurring in the first 2 3 days of adulthood. The number of adult progeny produced per parasitized host in the honey fed group showed a noticeable decline over time, with pupae stung by young females producing about twice as many parasitoids (ca. 6 per host) as older females ( Figure 4 6 B ) (R 2 =0.5876) In contrast, pupae stung by female parasitoids given only water showed no significant trend in parasitoid progeny produced over their reproductive lifetime ( Figure 4 6 A ) (R 2 =0.0175). Discussion Longevity Determination Because T. nigra is proovigenic, i ts eggs are fully chorionated upon emergence of adult female parasitoids from host puparia (Quicke 1997) and, as such, female parasitoids do not need to feed on their hosts in order to obtain amino acids for yolking (Heimpel et al. 1996; Heimpel and Rosenh eim 1998). It was not expected that the presence of S. calcitrans pupae, in the absence of food, would significantly increase the lifespan of either female or male parasitoids. Likewise, because from a physiological perspective little incentive remains for female parasitoids to stay alive once they have utilized all eggs, it was predicted that giving T. nigra females access to host pupae would decrease their life expectancies compared to withholding pupae In this study, female T. nigra lived approximately two day s longer in the absence of pupae than when given free access to fresh pupae on a daily basis (Table 4 1), but this difference was not statistically significant. In invertebrate models, the correlation between bearing young and life expectancy in fem ales has been studied extensively, with data demonstrating that producing offspring has a negative impact on female longevity in the post reproductive phase ( Chapman et al. 1998 ). Mukhopadhyay and Tissenbaum (2006) suggest that the energy required to produ ce gametes and

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53 develop offspring may be taken from energy that would otherwise be utilized to maintain somatic cells (those which will not produce gametic cells); if a tradeoff occurs when an animal attempts reproduction, a shortening of life expectancy wo uld be, and indeed is, observed. This phenomenon may explain why male T. nigra despite not producing or laying eggs, experienced a shortened l ifespan when provided with pupae but not honey. If production of gametes is, for both sexes, energetically costly then males that mated with females who had potential hosts available (as unstung pupae) would have utilized their gametes and no longer have any requirements to remain alive. The increase in longevity for T. nigra females and males which were provisioned with both honey and pupae is unusual. It was hypothesized that the presence of a food source would increase the mean lifespan of all parasitoids because of an input of energy for physiological maintenance as well as providing additional energy to locate mates (male and female parasitoids) or hosts (female parasitoids only) (Wckers 199 4 ; Wckers 199 8 ). However, while the presence of honey does significantly affect male parasitoid longevity for T. nigra the presence of pupae should not because male parasi toids neither feed nor oviposit inside pupae. The analysis indicated that the effect of honey on longevity was stronger when pupae were present than when they were absent. This was likely due to the continued presence of pupae allowing females to oviposit fewer eggs each day over a longer period of time, and therefore be more selective in their choice of host pupae. When provided with pupae and water but no honey both male and female parasitoids did not demonstrate a significant difference in longevity com pared to conspecifics that were only given water (Table 4 1). It was not expected that providing hosts to parasitoids in the absence of a food source would extend lifespan for males. Because most female parasitoids control the

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54 timing of fertilization of e ggs and oviposition (Quicke 1997), there is little pressure on males to mate with females as quickly as possible. It was predicted that providing pupae to female T. nigra adults in the absence of a carbohydrate food source such as honey would decrease thei r lifespan due to a greater pressure on females to oviposit before depleting the energy allocated for host seeking and oviposition; however, the greater mean lifespan of females provided with water alone was not significantly different from the mean lifesp an of those females provided with water and pupae (Table 4 1). Indeed, this indicates that, as with males, the life lengthening effects of honey were greater when host pupae were present than when they were absent. This may be because the energetic demands required for drilling and oviposition by female T. nigra were lessened with the availability of energy via honey. Fecundity Estimation Of the parameters analyzed via two way ANOVA (Table 4 4), only the cumulative numbers of killed pupae differed significa ntly between treatments (pupae only, versus pupae and honey). The increase in number of pupae killed by female parasitoids given both honey and pupae (856.8 30.3) compared to females provided pupae and no honey is because a greater number of pupae were kil led between d 4 and 9 ( Figure 4 4 ). Both treatments demonstrated greatly reduced killing of host pupae by d 11, with the majority (>90%) of cumulative pupae killed by d 10. Since many parasitoids have demonstrated a higher fecundity rate and produce longer lived progeny whe n provided with older hosts (de Almeida et al. 2002; Bellows, Jr. 1985), the influence of host age on data collected from parasitoid progeny in this experiment was controlled for by utilizing host pupae of a strictly defined age. On avera ge, each female of T. nigra produced about 23 adult progeny over her lifetime. This is substantially higher than the 10.5 and 3.6 progeny produced per female of T. stomoxydis (Morgan et al. 1990) and T. painteri (Huggert and Morgan 1993), respectively. It is similar to

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55 the lifelong fecundity figure of 21.4 progeny per female of the encyrtid larval parasitoid Tachinaephagus zealandicus Ashmead when T. zealandicus is supplied with house fly pupae (Geden et al. 2003). Most of the research on filth fly par asitoids has concentrated on pupal ectoparasitoids in the family Pteromalidae. Spalangia endius Walker which has been used successfully as a fly biological control agent, produces only about half as many progeny per female as T. nigra (Morgan et al. 197 6). Spalangia cameroni Perkins another commonly used fly parasitoid, has a longer lifespan than S. endius and produces about 30 40 progeny per female (Legner and Gerling 1967, Moon et al. 1982). Perhaps the most widely used parasitoi d for fly control are Muscidifurax raptor and related species in the same genus. Lifelong fecundity estimates for M. raptor vary from 26 185 progeny per female over her lifetime depending on the strain and health of the colonies under consideration (Morgan et al. 1979, Zchor i Fein et al. 1992). Fecundity of T. nigra therefore is squarely within the range of several other economically important species of muscoid fly parasitoids. The geographic distribution of T. nigra (Geden and Moon, unpublished data ) across eastern Europe is also within the distribution of other common filth fly parasitoids, such as S. endius Walker, S. cameroni Perkins and M. raptor (Hogsette et al. 1994 2000). Having identical host milieus and overlapping geographical distributions indicate that in the wild, these species likely inhabit complementary niches It would be interesting to determine in the future whether introduction of T. nigra in conjunction with other commercially important p arasitoids of filth flies ( e.g., S. endius, S. cameroni and M. ra ptor ) increases control of flies that both species are known to parasitize, such as M. domestica and S. calcitrans Future experimentation involving the life history of Trichopria nigra will utilize a larger sample size for longevity studies. It would also be prudent to feed adult T. nigra particularly

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56 females, on a variety of sugar based food sources (such as plant nectar, honey, glucose solution), to more accurately determine whether an artificial (laboratory) diet significantly extends or shortens the l ifespan of this parasitoid, compared to the lifespan when fed on a diet more accurately mimicking their natural diet. Additionally, it is not known at this time whether feeding females before allowing them access to host pupae, rather than providing both a t the same time, a ffects the number of progeny that are produced successfully. In this experiment, T. nigra in the water, honey and pupae treatment were given access to all three factors from day 0; perhaps withholding pupae and feeding parasitoids first would incur a benefit of some sort.

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57 Table 4 1. Mean lifespan s (SE) (d) of Trichopria nigra adults under different feeding treatments at 25 C. Treatment n lifespan (d) n lifespan (d) Water only 90 11.94 (0.51)ab 102 6.71 (0.33)b Water + honey 102 12.53 (0.52)a 97 8.97 (0.35)a Water + pupae 80 9. 85 (0.52)b 94 5.46 (0.30)b Water + honey + pupae 99 12.99 ( 0.52)a 97 10.05 (0.49)a on analysis (SAS 2000) For females, F =6.49; df=3, 367; P <0.001. For males, F =30.54; df=3, 386; P <0.001.

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58 Table 4 2 Female T. nigra t wo way ANOVA results for influence of presence/absence of honey and host pupae on survival time. Numbers marked with an asterisk are considered significant (P < 0.05). Water alone was considered the control, therefore water is not included as a model effect. Model effect df Sum of Squares Mean Square F P Honey 1 297.5 297.5 11.73 0.0007* Hosts 1 46.4 46.4 1.83 0.1769 Honey x Hosts 1 150.0 150.0 5.91 0.0155* Error 367 9309.3 25.4

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59 Table 4 3 Two way ANOVA results for influence of presence/absence of honey and host pupae on survival time of male T. nigra Water alone was considered the control, therefore water is not included as a model effect. Model effect df Sum of Squares Mean Square F P Honey 1 1129.2 1129.2 81.96 <0.0001* Hosts 1 0.8 0.8 0.05 0.8151 Honey x Hosts 1 132.3 132.0 9.60 0.0021* Error 386 5318.2 13.8 Numbers marked with an asterisk are considered significant ( P <0.05).

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60 Table 4 4 Effect of diet on mean number (SE) of pupae killed by female T. nigra pupae producing wasps, having dead wasps, and total number of parasitized pupae Treatment Nu mber of Pupae Number of Pupae Number of Pupae with Cumulative Number of Killed Producing Wasps dead immature wasps Pupae Parasitized Pupae Only 715.4 ( 48.1) 47.0 ( 6.0) 163.6 ( 19.9) 210.6 ( 15.5) Honey and Pupae 856.8 ( 30.3) 46.4 ( 7.5) 172.1 ( 21.4) 218.5 ( 27.7) ANOV A F 6.2* <0.1ns 0.09ns df = 1, 8 ; P < 0.05

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61 Table 4 5 Cumulative ( SE) numbers of male and female progeny, respectively, produced by parasitoids given only water and pupae, or water, honey and pupae T reatment Cumulative Number of Cumulative Number of Cumulative Number Percent Male Progeny Female Progeny Progeny Produced Female Hosts Only 141.8 ( 42.9) 83.3 ( 22.1) 225.0 ( 27.2) 37.0 ( 1.9) Honey and Pupae 229.4 ( 41 .3) 107.8 ( 43.3) 237.2 ( 36.7) 45.4 ( 2.6)

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62 Figure 4 1. Rearing containe rs for longevity determination Air flow and access to water and/or food was provided via mesh screen on container lids. Five repetitions were provided with water, five with water and 200 S. calcitrans pupae, five with a 50% V/V water and honey solution, and five with a 50% V/V water and honey solution and 200 S. calcitrans pupae. Cotton ball saturated with either water or 50% V/V water and honey solution S. calcitrans pupae, when provided

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63 A) B) Fig ure 4 2 60 ml cups containing individually encapsulated S. calcitrans pupae. A) Following emergence of flies from unparasitized pupae, those pupae which did not produce flies were each placed int o a gelatin capsule so that the number of wasps emerged from each successfully parasitized pupae could be observed. B) Successfully parasitized pupa.

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64 A) B) Figure 4 3 Mean adult A) female and B) male Trichopria nigra mortality for all treatments.

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65 Figur e 4 4 Total number of S. calcitrans pupae parasitized by female parasitoids for each of two treatments (water and hosts, food and hosts, respectively). The total number of parasitized pupae was defined as the sum of pupae which successfully produced adult para sitoids and pupae which contained immature parasitoids that never emerged. After days 9 10, no adult parasitoids emerged from pupae.

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66 Figure 4 5 Mean number of adult parasitoids that emerged daily for each of two treatments (water a nd hosts, food and hosts, respectively). After days 6 7, very few parasitoids emerged. The highest numbers of adult emergence were observed from day 1 through day 3. 1 2 3 4 5 6 7 8 9 10 11 12

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67 A B Figure 4 6 The average number of parasitoids that emerged per pupa per day A) Water and hosts only. B) Food, water and hosts. The average number of parasitoids per pupa was calculated for each replicate, and averages plotted. The R 2 value for the regression line in A) = 0.5876. The R 2 value for the regression line in B) = 0.0175.

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68 Figure 4 7 Cumulative mean number of adult parasitoids that emerged from S. calcitrans pupae for each of two treatments (water and hosts, food and hosts, respectively). After days 6 7, very few adult par asitoids emerged.

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69 CHAPTER 5 ARENA CHOICE EXPERIMENTS Introduction Host specificity is a common feature found in many parasitoid genera. In general, koinobionts (insects which parasitize a host in the early stages of host development, and in which the ho st continues to develop) demonstrate a higher level of host specificity than do idiobionts (Althoff 2003); this aspect of parasitoid evolution is most likely due to the necessity of a koinobiont parasitoid being able to develop and mature in confluence wit h a host with a continuously changing endocrine milieu Idiobionts, by contrast, parasitize their hosts in later stages of development and, because the host insect may go through few if any endocrinological changes such as change of instar that influence p arasitoid development, the host is often Because the nutritional and physiological dependence of koinobiont parasitoids on their hosts is greater compared to externally living idiobiont par asitoids, host range s of koinobionts tend to be narrower than those of idiobionts (Hochberg and Ives 2000; Godfray 1994). Parasitoid species possess specific and innate range s of hosts they can successfully parasitize. Askew and Shaw (1986) hypothesized th at koinobiont parasitoids would be more host specific than idobionts because of the internal development and nutritional and endocrinological dependencies that koinobionts have on their hosts. Other Trichopria species have been found to parasitize Stomoxys calcitrans (L) ; T. stomoxydis Huggert and T. painteri Huggert and Morgan are known only to parasitize that species ( Mo r g an et al. 1990 ; Huggert and Morgan 1993 ). Another closely related species, T. anastrephae Lima, was found to parasitize pupae of Anastr epha species in western Brazil (Garcia and Corseuil 2004)

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70 The question of whether parasitoids are innately competent to locate hosts, or whether eggs, is a n important one. Little is known about the effects of conditioning on fly pupal parasitoids, and even less about conditioning on parasitoid species in the family Diapriidae, such as T. nigra (Nees) More generally, it is known that for many parasitoids, as sociative learning is Selection Principle has been observed in many insects (Craighead 1921; Smith and Cornell 1979; Davis and Stamps 2004). According to this principle, first observed in he rbivorous insects (Craighead 1921), the host an insect develops within provides the first visual and olfactory conditioning, so that adult insects can later locate the same host they developed within, for laying their eggs. The odors and visual presence as sociated with pupae often serve as stimuli (see Jandt and Jeanne 2004 for example) that parasitoids associate with a reward ( i.e., oviposition, or feeding prior to oviposition) Odor stimuli can be either compounds from either potential host pupae or their surrounding environment (Sullivan et al. 2000; de Jong and Kaiser 1992). In some instances, host loc ation stimuli are visual rather than olfactory as is the case for the phorid fly parasitoid of fire an ts, Apocephalus paraponera Borgmeier (Morehead and F eener 2000). Regardless of the type of stimulus, l earning i s an adaptive behavior. O nce conditioned to seek out olfactory or visual cues the foraging time for future oviposition events is often shortened, maximizing the number of hosts parasitized and egg s laid by the parasitoid (Stireman III 2002) Diadromus pulchell us Wesmael (Hymenoptera: Ichneumonidae), a parasitoid wasp which attacks larvae of the moth Acrolepiopsis assectella Zeller appears to utilize volatile compounds emitted from A. assectella l arvae and their frass to locate possible hosts for oviposition (Lecomte and Thibout 1993). When adult female parasitoids are exposed to either frass or larval

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71 hosts prior to placement in an olfactometer, the number of turns made before choosing the host/fr ass odor over a control was fewer than those made by females never exposed to hosts or frass post emergence (Lecomte and Thibout 1993). However when nave D. pulchell us females were exposed to volatiles from A. assectella larvae and a control in an olfact ometer, they did not show any significant differences in behavior when compared to female D. pulchell us reared on an atypical host, Plodia interpunctella (Hubner). These findings indicate that for some species, post developmental exposure to a host is a c rit ical step for females to learn host selection In the laboratory, Trichopria nigra (Nees) successfully parasitizes pupae of a number of nuisance flies, including Stomoxys calcitrans L. Ophyra aenescen s (Wiedemann) Haemotobia irritan s (L.) and Sarcop haga bullata (Parker) in the laboratory ( Geden and Ferrero, personal observation ) Additionally, c olonies of T. nigra have been successfully maintained utilizing these same fly species as hosts. T. nigra readily probes pupae of Musca domestica and appears to oviposit; although pupal mortality in the host is high no adult parasitoids have emerged in numerous laboratory rearing attempts (Geden, unpublished data) Other dipteran hosts parasitized by T. nigra include Piophila casei (L.) (Teodorescu and Ursu 19 79) and tachinid flies (Nash 2005) T. nigra demonstrate s an unusual life history with respect to the interplay of physiology and behavior of parasitic wasps Unlike many other endo parasitoids, it develops inside a pupal host rather than a larval one Fu rthermore, the ability of T. nigra to develop successf ully in a number of hosts is atypical of endoparasitoids Conditioning studies involving T. nigra cannot be found in the literature and the effects of conditioning on this parasitoid are unknown. The ref ore, the first objective of these experiments was to determine if conditioning T. nigra to a particular host via exposure would bias T. nigra

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72 presented with multiple host choices. The second objective w as to determine if rearing T. nigra on two different host sp ecies affects their likelihood of choosing the same species wh en newly emerged F 1 parasitoids are presented with a choice of multiple hosts Materials and Methods Study Site Parasitoids and ho st flies were reared at the USDA/ARS Center for Medical, Agricultural and Veterinary Entomology (CMAVE), Gainesville, FL. The following experiment was Hosts The host fly species Stomoxys calcitrans (L.) Musca domestica L. and Sarcophaga bullata Parker were taken from USDA colonies. Pupae uti lized for both experiments were colle cted from rearing containers 2 3 days following pupation and either used immediately, or stored in chambers at 14 C (70% RH) for up to four days to retard physiological development and emergence of adult flies. It has been demonstrated that holding pupae of these species at similar temperatures (see Moribayashi et al. 1999, Leopold et al. 1997) does not significantly affect quality or sur vivorship of the insect. Parasitoid strains The T. nigra adults used for the bioassay were combine d from two strains reared separately and were later pooled to provide adequate numbers of parasitoids. A Kazakhi strain and a Russian strain, both maintained on S. bullata pupae and assumed to be identical in physiology and behavio r were used for experimentation For details on rearing of T. nigra, see Chapter 3. Conditioning p arasitoids for short term assay Three days after emergence from S. bullata pupae [t o provide adult wasps with adequate time for wing and exoskeleton hardening, maturation of ovarioles, and opportunities to mate]

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73 male and female T. nigra were removed from their rearing container s with an aspirator The wa sps were combined then divided int o fourths and placed into 250 ml paper cup s containing 140 cm 3 of either S. bullat a S. calcitrans or M. domestica pupae or no pupae ( control) for the purpose of conditioning was ps to pupae of one species The 250 ml paper cup s containing wasps and tre atments were secured across their openings with cotton cloth to allow air flow and prevent wasp escape. Atop each of t he four cotton cloth covers was placed a cotton ball saturated with distilled water and a streak of honey was applied to the cotton to pr ovis ion wasps with food C up s were then placed in an incubator (25 C 0.4 C, 70 % RH) for 48 h, after which the wasps were separat ed from the pupae by placing both in a #12 mesh screen sieve and shaking gently so T. nigra adults would fall onto a clean pi ece of white paper for collection Wasps were then immediately introduced to arenas for experimentation. Arena for short term assay Arenas consisted of clear plastic containers measuring ca. 24 x 24 x 10 cm (L x W x H) with plastic airtight snap closure t ops (Glad Products company, Oakland CA). Into each arena were placed four ~ 5 cm dia. plastic Petri dish bottoms each containing 2.0 g. S. bullata pupae, 2.0 g. S. calcitrans pupae, 2.0 g. M. domestica pupae, or an empty (blank) Petri plate serving as a co ntrol (Figure 5 1). Petri plates were arranged in a randomized block design between repetitions. Five repetitions were conducted for each of four treatments, and the assay was repeated twice for a total of ten repetitions per treatment. Arrangement of Petr i plates was to provide maximum distance between choices (approx. 8 cm between plates). The wasps which had been conditioned to different fly species were introduced to arenas by aspirating them following sieving procedure, and then gently blowing a pea s ized ball of parasitoids into the center of each arena Plastic lids were immediately secured tightly on

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74 containers to prevent wasp escape during the assay. Containers were left for 2 hours to provide wasps with adequate time to choose a host species and i nitiate probing and/or oviposition At 2 h plastic lids were removed from the arenas, covers were placed on Petri plates to contain wasps, and the Petri plates were frozen for 12h to kill wasps. Dead male and female wasps in each of the Petri plates were counted. Parasitoid s for rearing assay Adult ( 5 day old ) T. nigra from the combined strains were divided into two plastic rearing containers (25 cm L x 14 cm W x 12 cm H see Chapter 3 for furt her details on rearing methods). O ne container of wasps was pr ovisioned with 150 cm 3 of S. bullata pupae, and the other with 150 cm 3 of 2 3 d old S. calcitrans pupae, in 250 ml paper cups The layer of pupae was approximately 3 5 cm deep. The cups of pupae were held inside their respective T. nigra rearing containers for 6 d to allow time for females to oviposit in the puparia. After 6 d, the paper food cups of pupae were removed from the T. nigra rearing containers, adult parasitoids shaken off with a #12 mesh sieve and discarded and cloth covers secured across the opening of the containers (now containing only potentially stung pupae) The containers were placed in an incubator (25 0.4 C 70% RH ) and held until the emergence of the next generation of parasitoids Upon emergence of parasitoids, a cotton ball saturated with distilled water and a streak of honey was placed atop the cloth cover to provide parasitoids with moisture an d a food source. Wasps were introduced to the e xperimental arena following a 3 d maturation period. Arena for rearing assay Arenas consisted of cle ar plastic containers measuring 24 x 24 x 10 cm (L x W x H) with plastic airtight snap closure tops (Glad P roducts company, Oakland CA), of similar design to those in the short term host exposure assay. Into each arena were placed three ~5 cm dia. plastic

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75 Petri dish bottoms each containing 2.0 g. of S. bullata pupae 2.0 g. S. calcitrans pupae, or an empty (bl an k) Petri plate serving as a control (Figure 5 2). Petri plates were placed in a randomized block design between repetitions. Five repetitions were conducted for each of two treatments Petri plates were arranged to provide maximum distance between choice s (approx. 10 cm between plates). The w asps reared on either S. bullata pupae or S. calcitrans pupae were introduced in pea sized balls to arena s via a manual aspirator, one group for each arena. Plastic lids were immediately secured tightly on containers to prevent wasp escape during the assay. Containers were left for 2 h as in the previous test At 2 h, plastic lids were removed from the arenas, covers were placed on Petri plates to contain wasps, and all arenas were frozen for 12 h Dead male and femal e wasps in each of the Pe tri plates were counted. Statistical Analysis For the short term host exposure assay, numbers of wasp s in each Petri plate were separated and analyzed by sex The numbers for the five r epetitions were averaged for each treatment an d the mean raw numbers of male and female wasps exposed to each treatment ned wasps were compared with unconditioned parasitoids for host preference vi a one way analysis of variance (SAS Institute 2000) Additionally, an arcsin transformation was performed on mean numbers of wasps to correct for significant variation in total numbers of wasps among repetitions, and the 0). For the rearing assay, numbers of wasps in each Petri plate were separated and analyzed by sex Following sexing of parasitoids in each of the Petri plates, numbers for repetitions were averaged for each treatment and the mean numbers of male and femal e wasps exposed to each treatment were t (P=0.05; SAS Institute 2000). Additionally, an

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76 arcsin transformation was performed on mean numbers of wasps to correct for significant variation in total numbers of wasps among repetiti ons, and the proportions analyzed using Results No significant differ ences were observed between the proportions of female parasitoids conditioned on S. calcitrans pupae which were counted in ( e.g., assumed t o have oviposited in) Petri plates containing different pupal types (Table 5 1 ) T he Petri plate of M. domestica pupae lured a significant ly greater proportion of female parasitoids (df=1 ; P<0.0001) 6 than the pupae of the oth er two species (Table 5 1) Alt hough numerically more female parasitoids which were conditioned with S. bullata than did female parasitoids conditioned to other hosts, the difference was not significant (0.051) Male parasitoids conditioned to S. bullat a pupae demonst rated no significant preference to any of the Petri plates of pupae, regardless of conditioning (Table 5 2). Unconditioned parasitoids of either sex did not demonstrate any significant preference for a part T h e host in which the f emale parasitoids were reared had a significant impact on the ir behavior in the experiment (df=1 ; P<0.0001), with approximately twice as many females choosing their natal host rather than a non natal host (Table 5 3). This preference f or natal host w as not observed in males (df=1 ; P=0.57) reared on either S. calcitrans or S. bullata pupae (Table 5 4). For the host exposure experiment, post experiment counts (total number of wasps were counted for five of the twenty containers) determine d that the groups introduced to the arenas experiment, post experiment counts (total number of wasps were counted for five of the twenty

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77 containers) determine d that the groups introduced to the arenas averaged 432 21 wasps of mixed sex per arena. Discussion The large proportion of female T. nigra responding to the visual and olfactory cues provided in the experimental arena, as well as females observed and c ounted while attempting to T. nigra may attempt to oviposit inside any of these species and use them as hosts. The results of these experiments indicate that t he host range of T. nigra is fairly broad, especially for a koinobiont (Whitfield 1998; Quicke 1997). Additionally, other species of Trichopria have been shown to have wide host ranges (Morgan et al. 1990 ; Garcia and Coseuil 2004) Such an assumption must be made with caution however, as it has been demonstrated that hymenopterous insects can be readily conditioned to artificial stimuli (Jong and Kaise r 1991; Jandt and Jeanne 2004). Another reason for caution is the role of host habitats in directing sear ching behavior in T. nigra Parasitoids typically follow a series of steps that begin with host habitat location (Vinson 1976). If T. nigra is conditioned to search for carrion rather than dung, then carrion inhabiting flies, e.g., calliphorids and sarcoph agids s hould be parasitized by T. nigra more often than stable flies. Field research in the home range of this species is needed to determine whether parasitism of stable flies is common, or the result of chance encounters. The experiment conducted utili zing parasitoids which had successfully emerged from both S. bullata and S. calcitrans pupae, indicates that this species utilizes both host species where they both occur in the wild. Moreover, laboratory studies can sometimes suggest stronger host prefere nces than are evident in the field, where parasitoids are subject to more complex sets of stimuli (Mandeville and Mullen s 1990 a, b ).

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78 Learning among parasitoid hymenoptera is well documented and follows the general pattern of insect reward driven associati on Females of the fruit fly larval parasitoid Leptopilina boulardi Barbotin et al. (Hymenoptera: Eucoilidae) for example, were exposed to an artificial odor ( i.e., one not naturally produced by the host or plants fed on by the host) in conjunction with a n oviposition experience in a laboratory ( de Jong and Kaiser 1992 ). The exposed females would demonstrat e a much stronger preference for the scent they were exposed to during stimul us/reward conditioning compa red to nave female parasitoids. Conditioned fe males also demonstrated a much stronger preference than did their control counterparts, which were not classically conditioned to any stimulus Selection of a suitable host at close range is essential and the availability of visual or olfactory cues may that parasitoid has learned that certain cues signal host availability (Hochberg and Ives 2000). Most parasitoids whose host range is greater than one host species will often ch oose the most abundant host for the greatest number of oviposition event s (Hastings and Godfray 1999 ); while possibly the result of a female parasitoid accidentally encountering the most abundant host species at a greater statistical rate, may also be expl ained by a greater number of visual or olfactory cues pres ented by the most abundant host. The number of viable host pupae available is often an indicator of host fecundity (Hochberg and Ives 2000), and is perhaps as well, an indicator of host attractiven ess to parasitoids. To this end, an abundance of all potential host species was presented in the experiments, to prevent one potential host being more available than the others. The stronger response of female T. nigra tested in the rearing assay comp are d to those conditioned for 48 h indicate that rearing of this wasp on a particular host increases the fidelity

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79 of adult females to forage for and oviposit in the same host as their natal species. Hopkin s Host Selection principle has often been cited as a strong motivator for insects to search for and utilize hosts which resemble their own natal hosts (Michaud and Grant 2005 ) although the life stage at which this induction occurs remains somewhat controversial (Barron 2001; Davis and Stamps 2004) It would seem logical that, for parasitoids with more than one potential host, initial exposure to host kairomones predisposes female parasitoids to forage for that particular host. T his preference for natal host appears to be established when newly emerged parasi toids exit their host puparia and encounter the odors associated with that species and location (Vet 1983; Vet and Groenewald 1990). Such a preference would be particularly adaptive in relatively constant habitats that support sequential generations of hos ts (Vet 1983; Turlings et al. 1992). Although the literature on insect learning is voluminous, little is known about conditioning and host selection in parasitoids of muscoid flies and even less is known about host selection behavior in the family Diapr iidae Nasonia vitripennis (Walker) shows a moderate preference for the host species on which is reared, but it is not clear whether this preference represents pre imaginal conditioning or an induction event in which newly emerged adults experience host r emains and empty puparia at the time of eclosion (Oghushi 1960, Smith and Cornell 1979). In contrast, there is little evidence for rearing host effects on host preference in parasitoids in the genera Muscidufurax, Dirhinus or Spalangia (Mandeville and Mu llens 1990b, Oghushi 1960). The only known report of adult conditioning of fly parasitoids is that of Mandeville and Mullens (1990b). In this study, the strong innate preference of Muscidifurax zaraptor for house fly over Fannia cannicularis (L.) pupae w as shifted in favor of the latter species by 2 days of experience ovipositing on that host. This shift occurred in spite of the fact that M. zaraptor is substantially more successful on M. domestica than on F. canicularis hosts

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80 (Mandeville and Mullens 199 0b). The observation that T. nigra can be conditioned to favor a host on which it can not develop (house fly [Geden and Moon 2008]) is a curious parallel to the host shift seen in M. zaraptor Due to the inability to rear T. nigra on pupae of M. domestica choices for the rearing assay were limited to S. bullata and S. calcitrans pupae (see Chapter 2). The host selection behavior of T. nigra when presented with multiple potential host insects, and the shift in that behavior (in most instances) to preferent ially seek pupae of the host previously conditioned to is consistent with the current literature on associative learning in other parasitic Hymenoptera. While of parasitoids reared on multiple host species (as opposed to a brief post emergent exposure to a particular host), certainly initial exposure of female parasitoids to host kairomones presents a case for de facto hat, in the absence of later associative learning that exposes parasitoids to different host species, establishes a default mechanism for seeking out hosts that are native to a certain geographical area. Further research should be performed to examine the means by which T. nigra might learn to seek out a particular host, and seeking behavior. Further research conducted involving host choice with T. nigra should utilize a much higher sample size, especi ally with regards to the rearing assay.

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81 Table 5 1. P roportion of female T. nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single host species. Means in rows followed by different letters are significantly differ ent according to test (P=0.05; SAS Institute 2000). Mean (SE) p roportion of parasitoids recovered on host S. calcitrans M. domestica S. bullata ANOVA P Conditioning Host pupae pupae pupae F S. calcitrans 0.34( 0.10) a 0.36( 0.12) a 0.30( 0.05) a 0.81 0.50 M. domestica 0.27( 0.07) a 0.43( 0.10) b 0.30( 0.10) a 9.81 0.0002 S. bullata 0 .29( 0.07)a 0.31( 0.09)a 0.40( 0.10) a 2.95 0.0512 No conditioning 0.32( 0.07) a 0.35( 0.13) a 0.34( 0.07) a 0.80 0.50

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82 Table 5 2 P roportion of male T. nigra responding to pupae of 3 host species after prior conditioning for 48 h on pupae of a single host species. Means in rows test (P=0.05; SAS Inst itute 2000). Mean (SE) p roportion of parasitoids recovered on host S. calcitrans M. domestica S. bullata ANOVA P Conditioning Host pupae pupae pupae F S. calcitrans 0.45 ( 0.05 )a 0.23 ( 0.04 ) b 0.34( 0.04 ) a b 6.36 0.0056 M. domestica 0.40 ( 0.03 )a 0.31 ( 0.02 )a 0.29 ( 0.03 ) a 2.83 0.0771 S. bullata 0.38 ( 0.04 )a 0.31 ( 0.05 )a 0.30 ( 0.03 )a 0.78 0.4697 No conditioning 0.37( 0.04 ) a 0.35( 0.05 ) a 0.29( 0.02 ) a 1.07 0.3593

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83 Table 5 3. Mean proportions o f female parasitoids reared on either S. calcitrans or S. bullata pupae who selected either S. calcitrans or S. bullata pupae as a first host choice. Means followed by the same letter are not statistically significant by PROC GLM ( P =0.05). Emerged from On S. calcitrans pupae On S. bullata pupae S. calcitrans 0.70 a 0.30 b S. bullata 0.36 b 0.64 a Df=1 8 ; F = 33.25; P < 0 .0001

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84 Table 5 4. Mean proportions of m ale parasitoids reared on either S. calcitrans or S. bullata pupae who selected either S. calcitrans or S. bullata pupae as a first host choice. Means followed by the same letter are not statistically significant by PROC GLM ( P =0.05). Emerged from On S. calcitrans pupae On S. bullata pupae S. calcitrans 0.45 a 0.55 a S. bullata 0.60 a 0.40 a Df=1 8 ; F =1.72; P =0.202

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85 A B Figure 5 1. Containers for parasitoids conditioned for 48 h on different host pupal types, with Petri plates containing three choices of pupal host as well as a blank control Petri plate. A) Containers were arrang ed in a randomized block pattern to control for variations in light intensity, temperature and surroundings. B) During the experiment, plastic tops were securely fastened onto containers to prevent escape of insects.

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86 Figure 5 2. Containers for parasitoids that emerged from different host pupal types with Petri plates containing two choices of pupal host as well as a blank control Petri plate. Containers were arranged in a randomized block pattern to control for variations in lig ht intensity, temperature and surroundings

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87 CHAPTER 6 Y TUBE OLFACTOMETER EX PERIMENTS Introduction For most insects, olfaction serves as a vital form of communication. The necessity of locating and identifying odors is important for insects that cannot ut ilize visual or auditory cues to locate mates, food, hosts and water (Hallem et al. 2006). Female insects, especially those whose mates must fly great distances for mating opportunities, may g ive off pheromone compounds to signal their location to males, e .g., the tobacco hornworm moth Manduca sexta L. (Daly et al. 2001) and silkworm moths (Lepidoptera: Saturniidae) (Hansson 1995). Olfactory c ues are provided by different insect species, vertebrates and plants as we species The use of kairomone compounds is well documented in entomology (Johnson and Triplehorn 2004), mainly to attract insects to a source. The gall wasp Antistrophus rufus Gillette, for example, has demonstrated a sex mediated development schedule in which adult males em erge from galls pri or to female emergence to increase their chances of mating with a virgin female (Tooker et al. 2002). Male s of A rufus locate unemerged f emales by following olfactory cues produc ed by st ems of their host plants ( genus Silphium ) rather t han from olfactory cues provided by the female s of A rufus. Honey bees (Hymenoptera: Apidae) provide another classic example of olfaction based resource location (Reinhard et al. 2004), in which a scent ( e.g., from food) is a ssociated with a location. Bee s exposed to a scent specific to a location will return again to that location. Research on the relationship between associative learning and olfaction in insects has yielded interesting examples of how strongly an initial exposure to a scent that is tied to a reward ( e.g., food or host source) can affect the future behavior of that insect. Foraging members of the yellowjacket species Vespula germanica (Fab.) will, when simultaneously exposed in their nest

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88 to a food source and a scent, will preferentially v isit a source outside the nest with that particular scent profile, compared to a control (Ja ndt and Jeanne 2004). While much entomological data suggest that associative learning is a n important component of behavior for many insects, those species with a l ife history strate gy dependent on a host ( e.g., parasitoids) present the question of whether prior exposure to a host will affect the later behavior of that insect. Specifically, it is unknown for many parasitoids whether close contact with a host during t he larval stages will affect the adult behavior of that insect with regards to locating and ovipositing in that same host. Many species of parasitoid wasps have demonstrated measurable c hanges in behavior following exposure to chemicals associated with hos ts or host fostering habitats. In many cases, the surroundings of the host may provide the olfactory stimuli, rather than the host itself. Such odors may be produced by plants that a host feeds on, decaying plant or animal matter that host larvae develop in, or host frass. Both of the parasitoid wasps Dibrachys cavus (Walker) and Roptrocerus xylophagorum (Ratzeburg) (Hymenoptera: Pteromalidae), for example, demonstrated a greater preference for the frass of their respective host insects than for the hosts (Chuche et al. 2006; Sull ivan et al. 2000, respectively). The host insect itself may be the source of volatile chemicals that attract parasitoids rather than the surroundings of the host The Diapriid parasitoid Trichopria drosophilae Perkins was found t o be attracted to kairomones emitted by the anterior spiracles of its host, Drosophila melanogaster L. (Romani et al. 2002). Host recognition by Muscidifurax raptor Girault and Sanders involves chemicals emitted by its host Musca domestica L. rather than from the feeding and rearing medium of the host (in this case, manure) (McKay and Broce 2003). In an olfactometer, M. raptor preferred the odor of host pupae alo ne over the odor of manure. W hen

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89 both manure and pupae were p resented in one arm of the olfa cto meter, M. raptor was not attracted to the odors from that arm ( McKay and Broce 2003). Utilizing an olfactometer to document insect choice making behavior is a common technique in associative learning research with parasitoids. de Jong and Kai ser (1991), f or example, demonstrated that fruit fly larval parasitoid Leptopilina boulardi Barbotin et al. (Hymenoptera: Eucoilidae) female s exp osed to an artificial odor in conjunction with an oviposition experience would demonstrat e a much stronger choice in an olfa ctometer for the scent they were exposed to duri ng stimulus/reward conditioning compared to nave female parasitoids, as well as to controls which were not classically conditioned. In this study, artificial odors were considered ones not naturally produced by the host or plants fed on by the host. Later studies with L. boulardi indic ated that when female s were exposed to multiple od ors at different times they demonstrated a strong preference for all odors compared to nave parasitoids, with the strongest p reference for the most recently learned host ( d e Jong and Kaiser 1992). Learning by parasitoid s to prefentially follow an odor has been documented in Cotesia marginiventris Cresson (Turlings et al. 1989), Leptopilina heterotoma (Thomson) (Papaj and Vet 19 90) and Microplitis croceipes (Cresson) (Lewis and Tumlinson 1988). In the case of M. croceipes which is a parasitoid of Heliothis zea (Boddie), female wasps could be successfully conditioned to locate odors which are not naturally associated with their h ost, and in fact are unattractive, such as vanilla ( Lewis and Tumlinson 1988). Because Trichopria nigra (Nees) has demonstrated a host range consisting of multiple fly species (chapter 3) as well as significant change s in behavior following conditioning t o a host pupal type (chapter 5 ), the first objective was to determine whether conditioning female s of T. nigra to a host pu pae create s a stronger preference to odors from that host they were

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90 exposed to, compa red to nave female parasitoids ( e.g., females not previously exposed to pupal odors). The second objective was to determine whether the length of time females of T. nigra are exposed to a potential host influe nces their host seeking behavior. Lastly, it was unknown whether rearing T. nigra on different host species affects female host preference, and whether the speed and strength of nave female T. nigra response to host odors change with age. Materials and Methods Study Site All experiments were conducted in a glass Y tube olfactometer (Agric ultural Research Systems, Gainesville FL ) ( Figure 6 1) approximately 24 cm in length, with a tube diameter of approximately 5 cm. Parasitoids were prevented from escaping during experiments by closing the main arm of the Y tube with a glass entry capsule c ontaining a mesh opening (for air escape). F low rate was 0.3 L per minute ( LPM ) for all experiments A clean air source was provided by the air system at the CMAVE facility, which was supplied via a nalgene tube and filtered by bubbling through H 2 O before flowing through the arms of the olfactometer To negate the possible effect of phototaxis, l ighting was diffuse, being provided by four 32 W fluorescent light s positioned centrally a pproximately 1.2 m a bove the olfactometer R oom temperature was 22 24C Parasitoids For all experiments except 6 3 (rearing effect on host choice), female T. nigra adults from both the Russian and Kazakh strains were pooled immediately following emergence Only female parasitoids were utilized in experiments since Chapter 5 data suggest that male parasitoids do not react significantly in a closed arena to volatiles from host pupae. Strains were pooled by then aspirating females from the bo ttom of both containers with a small handheld aspirator. All

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91 aspirated females were gently blown into a Petri plat e bottom to pool both strains, and gently dropped into gelatin capsules for holding prior to experiments. For each repetition, five females we were introduced to the olfactometer by gently tapping the gelatin capsule against a hard surface to stun the parasitoids and prevent them from flying away before placement in the olfactomet er, and twisting it open with the parasitoid containing half shaken in to the terminal end of the olfactometer The entry capsule was then placed into the terminal end of the olfactometer, preventing escape. Controls were run prior to each experiment, cons isting of five replicates of five nave female parasitoids introduced to the olfactometer with an air flow of 0.3 LPM. Both arms were empty and parasitoids were monitored for bias toward either arm of the olfactometer. The Y tube apparatus and associated stimulus capsules ( Figure 6 2) were not washed between replicates, as one arm of the Y tube was designated for one of two stimuli, and attraction to that arm did not change between replicates. Between experiments, all glass parts of the Y tube apparatus we re washed with detergent and rinsed with tap and deionized water, respectively. All pieces were allowed to air dry, to avoid accidental marking of glass pieces with volatiles from drying materials and fabrics. For all experiments, s tr ength of response was determined as being the proportion of females which made a choice by traveling at least halfway down one arm of the olfactometer. Experiment 6 1 : Effect of Three D ays of Conditioning on Host C hoice T. nigra adults (both strains) were aspirated from rearin g containers within 12 h of adul t emergence and divided into three groups. A surplus (<1000) of parasitoids was collected to ensure an adequate number of surviving females. Approximately 150 cc of 2 d old Sarcophaga bullata Parker pupae and 150 cc of Stom oxys calcitrans (L.) pupae were each placed into ca. 500 ml paper cups. One group of parasitoids introduced to each container for conditioning. The third

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92 group was placed in to an empty paper cup as a control. C ont ainers were covered with muslin and secured with rubber bands. A streak of honey and a water dampened cotton ball place d atop the muslin cover provided food and water The paper containers were placed in a 25C incubator ( 0.4C, 70% RH) and T. nigra females were allowed 3 d to associate host pupa l odors with oviposition ( i.e., conditioning) After the 3 d conditioning interval parasitoids were knocked down by refrigerating the paper cups for several minutes (~ ) and then a spirating females from the inside of the cups Four replicates of five females each ( i.e., five females at a time) conditioned to S. bullata pupae, were introduced for 3 min into the olfactometer with ca. 1.0 g of either S. bullata or S. calcitrans pupae ( ca. 2 d old ) in the arms, respectively. Pupae had, immediately prior to experimentation, into the attractant chambers at the ends of the arms. Four replicates of five females each ( e.g., five at a time) conditioned to S. calcitrans pupae, were introduced for 3 min into the olfactometer with ca. 1.0 g of S. bullata and S. calcitrans pupae ( ca. 2 d old) in the arms, respectively. Additionally, four replicates of five unconditioned females each were introduced to the olfactometer with c a. 1.0 g each of S. bullata and S. calcitrans pupae ( ca. 2 d old) in the arms. First choice was noted for each female within a replicate, as well as time required for each female to make a first choice. Females which did not choose either arm of the olfac tometer within 3 min were considered non responders and not included in data analysis. Experiment 6 2 : Host Choice R espo nse after 1 d, 3 d, and 5 d of C onditioning on S. bullata P upae T. nigra adults (both strains) were aspirated from rearing containers w ithin 12 h of adul t emergence and divided into two groups. A surplus (<1000) of parasitoids was collected to ensure

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93 an adequate number of surviving females. Approximately 150 cc of 2 d old S. calcitrans pupae were placed into a ca. 500 ml paper cup One gr oup of parasitoids was placed in the container with S. calcitrans pupae, and the other was pl aced in an empty paper cup as a control. Containers were covered with muslin and secured with rubber bands. A streak of honey and a water dampened cotton ball plac ed atop the muslin cover provided food and water. The paper containers were placed in a 25C incubator ( 0.4C, 70% RH). After 1 d females were aspirated from each container in groups of five (eight groups total) with a manual aspirator and placed in gel atin capsules for experimentation and the remaining parasitoids returned to the incubator Eight replicates of five females each, conditioned to S. calcitrans pupae, were introduced for 3 min into the olfactometer with ca. 1.0 g of S. calcitrans pupae i n one arm and a control arm not containing a stimulus First choice was noted for each female within a replicate, as well as time required for each female to make a first choice. Females which did not choose either arm of the olfactometer within 3 min were considered non responders and not included in data analysis. Eight more sets of five females were collected at 3 d and 5 d after parasitoids were exposed to pupae, and the above experimental procedure repeated. Experiment 6 3 : Host Choice by Parasitoids R eared on Different H osts T. nigra adults were reared on pupae of S. bullata and S. calcitrans respectively (for detailed rearing methodology, see Chapter 3). Within 12 h of parasitoid emergence, the ~500 ml paper cups of pupae and newly emerged parasitoi ds were p rovided with food and water via a streak of honey and a water dampened cotton ball placed atop the secured muslin cover s. The paper cup s were placed in a 25C incubator ( 0.4C, 70% RH) for 48 h to allow for cuticle hardening and sexual maturity of parasitoids.

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94 Six replicates of five females each, reared on S. bullata pupae, were introduced for 3 min into the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca. 2 d old) in the arms, respectively. Six replicates of five fema les each, reared on S. calcitrans pupae, were introduced for 3 min into the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca. 2 d old) in the arms Additionally, four replicates of five unconditioned females each were introduced t o the olfactometer with ca. 1.0 g each of S. bullata and S. calcitrans pupae (ca. 2 d old) in the arms. First choice was noted for each female within a replicate, as well as time required for each female to make a first choice. Experiment 6 4 : Response of Unconditioned Parasitoids to Hosts During Five Days Post E mergence T. nigra adults were aspirated from rearing containers within 12 h of adult emergence and placed into a ca. 500 ml paper cup which was then covered with muslin, secured with rubber bands, and food and water provided via a streak of honey and a water dampened cotton ball placed atop the muslin cover. A surplus (<1000) of parasitoids was collected to ensure an adequate number of surviving females. The paper cup was placed in a 25C incub ato r ( 0.4C, 70% RH) for 1 d to allow for cuticle hardening and sexual mat urity of parasitoids. After 1 d six replicates of five females were aspirated from the paper container and introduced for 3 min into the olfactometer with ca. 1.0 g of S. calcitrans pupae (ca. 2 d old) in one arm and a control (empty) arm. First choice was noted for each female within a replicate, as well as time required for each female to make a first choice. The above procedure was repeated on days two, three, four and five after e mergence. Statistical Analysis For Experiment 6 1, G tests were conducted to determine if the sampling distribution of frequency of choices female parasitoid s made in the olfactometer was random, or if there were

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95 significant differences between the distri butions of the three treatments. Parasitoids conditioned to S. calcitrans were compared to parasitoids conditioned to S. bullata Additionally, parasitoids conditioned to S. calcitrans as well as parasitoids conditioned to S. bullata were individually comp ared with the controls. G test statistics were rounded to two significant figures, and an alpha level of P<0.05 was considered significant. For Experiment 6 2, two sets of G tests wer e conducted to test for variance in the frequency of choices made by unc onditioned and S. calcitrans conditioned parasitoids with respect to time and treatment. S. calcitrans conditioned parasitoids were compared to unconditioned controls at one day, three days and five days. Individually, both S. calcitrans conditioned parasi toids and unconditioned controls were subjected to two G tests: one comparing day one choices for that treatment with day three choices, and another comparing day one choices with day five choices. G test statistics were rounded to two significant figures, and an alpha level of P<0.05 was considered significant. For Experiment 6 3, a single G test was conducted to test for variance in the frequency of choices made by S. calcitrans c onditioned parasitoids versus S. bullata conditioned parasitoids. Additiona lly, for parasitoids that did respond to a stimulus in the olfactometer, a two way ANOVA was conducted on the mean response times of each treatment to the two choices, to determine if any significant differences in response time emerged as a result of rear ing host (SAS Institute 2000) For Experiment 6 4, G tests were conducted to test for differences in response strength ( e.g., number of parasitoids that chose pupae or the control arm) over ti me. Four comparisons were made, examining cumulative choices ma de by female parasitoids on days one and two, days one and three, days one and four, and days one and five. G test statistics were rounded to

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96 two significant figures, and an alpha level of P<0.05 w as considered significant. A two way ANOVA was conducted to test for any significant differences in mean response time and mean response strength for the five days the experiment was conducted. Response strength was determined as the proportion of five females that made a decision within 3 min for each repetition. Results Unconditioned female parasitoids preferred S. calcitrans pupae over S. bullata pupae (Table 6 1). Host conditioning made a significant difference in the number of T. nigra females which responded to their conditioning host versus an unfamiliar ho st (Table 6 5). Comparing S. calcitrans conditioned parasitoids to unconditioned controls (G=0.02; df=1) did not demonstrate any significant difference in strength of response, nor did S. bullata conditioned parasitoids compared to the unconditioned ones (G=2.16; df=1). Comparing S. calcitrans conditioned parasitoids with their unconditioned counterparts did not yield any significant differences on day one, day three or day five (Table 6 2). Comparing the cumul ative numbers of S. calcitrans conditioned pa rasitoids responding to pupal odors on days one and three yielded a significantly greater response on day three (G=4.70; df=1), although comparing day one and day five responses of S. calcitrans conditioned parasitoids did not (G=3.42; df=1). Additionally no significant changes were observed between responses of unconditioned parasitoids when days one and three and one and five were compared (G=0.75; 0.27 respectively; df=1). The greatest increase in response rate was seen in S. calcitrans conditioned par asitoids between day one and day three (Table 6 2). For parasitoids reared on two different hosts, there was no significant correlation between host rearing type and pupal choice in the olfactometer (Table 6 5). While a greater number of S.

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97 calcitrans cond itioned parasitoids chose their natal pupal type over an unfam iliar stimulus (Table 6 3), S. bullata conditioned parasitoids did not c hoose their natal pupal type. The number of nave parasitoids responding to odors from S. calcitrans pupae increased from day one to day three, then decreased slightly to day five (Table 6 4). While response strength did not change with respe ct to time in Experiment 6 4, the mean response time did change significantly (P=0.04; df=4) over the five days. Comparing the strength of unconditioned T. nigra female response during the first five days after adult emergence yielded no significant differences in G tests (Table 6 5). The fastest mean response time for female parasitoids to S. calcitrans pupae was on day three. Discussion The results of Experiment 6 1 agree with the first arena experiment conducted in Chapter 4; i.e., that short term conditioning of T. nigra females to volatiles produced by host pupae does indeed influence their host seeking behavior to preferentially sear ch for hosts of that exposure type. Interestingly enough, while 48 h of female T. nigra exposure to one of two pupal types created a preference for conditioning host, experiment 6 3 does not corroborate with the second arena experiment conducted in Chapter 4. While in Chapter 4 we found that rearing does influence host selection in an enclosed arena, the female parasitoids who were reared on S. calcitrans and S. bullata pupae did not demonstrate a significant preference for their rearing host when presente d with those pupal types as choices in a Y tube olfactometer. d e Jong and Kaiser (1992) s uggest that the most recent exposure to positive stimulus in association with an odor has the greatest influence on insect behavior, compared to earlier conditioning e xperiences. From an ecological perspective, this assumption makes sense because, in the wild, a female parasitoid who encounters multiple positive stimuli ( e.g., an odor in association with an oviposition event) is most likely to receive a reward if she se eks out the most recen t location or type of stimulus.

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98 Several explanations exist for the lack of differences in the behavior of unconditioned parasitoids compared to those conditioned in S. calcitrans pupae. One possibility is that utilizing clean, dried p upae does not provide a strong enough attractant for parasitoids, who are instead seeking out odors of the environment associated with that host, such as odors associated with larvae, their frass, or their larval rearing substrate (Sullivan et al. 2000). Additional research is needed to determine whether T. nigra utilizes a hierarchy of stimuli which includes host habitat and proximity of host larvae in addition to cues that direct them to host pupae. For many parasitoids, a combination of cues is necessar y to successfully locate hosts. This may be a combination of olfactory cues, of which some are attractive at a distance and others at close range ( Morehead and Feener 2000 ), or a combination of visual and olfactory cues. Diachasmimorpha juglandis for exam ple, a parasitoid of Rhagoletis flies, locates its host via the Rhagoletis food source, walnut fruit husks (Henneman et al. 2004). In an olfactometer, D. juglandis can discern between intact walnuts and thos e which have been damaged by Rhagoletis. However, when presented with visual rather than olfactory cues, such as when both mechanically damaged and host damaged walnut husks were presented, D. juglandis had little success discerning between husks with hosts and husks without ( Henneman et al. 2004). Clear ly, visual clues are often important for host location at a distance, luring parasitoids close enough to locate olfactory cues. For pupal parasitoid s it is possible that a combination of odors contributes to host seeking behavior. However, for parasitoid s with a broad host range, such as T. nigra, the question arises of whether parasitoids naturally seek out multiple, and possibly quite different, odors. McKay and Broce (2003) found that, for Muscidifurax zaraptor Kogan and Legner, the odors emitted from house fly puparia were much more attractive than the manure house fly

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99 larvae naturally occur in. Furthermore, female parasitoids could not differentiate manure with a combination of pupae and manure as a stimulus, when presented with both choices in a y t ube olfactometer. T. nigra with a host range which encompasses flies that develop in a variety of substrates and media ( e.g., manure and carrion), the smaller and more closely related milieu of odors emitted from related host species may be the primary a ttractant, rather than a broad spectrum of complicated odors. The increase in response time to S. calcitrans pupae by unconditioned parasitoids in a Y tube olfactometer through day three, followed by a decrease in both response rate and response time, indi cates that the optimal oviposition time for this species is likely around three days post emergence. If T. nigra does not mate i nside the host (as is suggested due to spatial constraints ), the delay in optimal host location behavior may be explained by T. nigra requiring this amount of time to mate and complete hardening of the cuticle. Clearly, further research is required to examine whether conditioning to host pupae does indeed override preference for natal host type, as well as whether post emergent con ditioning to the same pupal type as the natal host type strengthens the response of female T. nigra searching for a host to oviposit in. In addition, it remains to be determined whether olfactory cues associated with a host, such as the media in which S. c alcitrans and S. bullata develop, are stronger attractants for female T. nigra compared to the odors associated with pupae alone.

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100 Table 6 1. First choices made by female parasitoids condit ioned for 3 d on either S. calcitrans or S. bullata pupae in a Y tu be olfactometer. The two choices presented in the olfactometer arms were S. calcitrans and S. bullata pupae, respectively. Conditioning Cumulative number of parasitoids attracted to host S. calcitrans S. bullata No conditioning 30 17 S. calcitrans 45 16 S. bullata 26 28

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101 Table 6 2. First choices made by female parasitoids conditioned on S. calcitrans pupae and tested at 1 d, 3 d and 5 d against unconditioned female parasitoids of the same age. The two choices presented in the olfa ctometer arms were S. calcitrans pupae and a blank arm respectively. Conditioning Day after Cumulative number of parasitoids attracted to host emergence S. calcitrans blank No conditioning 1 19 15 No conditioni ng 3 28 7 No conditioning 5 24 7 S. calcitrans 1 1 4 6 S. calcitrans 3 25 6 S. calcitrans 5 26 8

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102 Table 6 3. First turn choices made by 3 d old female parasitoids reared on either S. calcitrans or S. bullata pupae and r un in a Y tube olfactometer. The two choices presented in the olfactometer arms were S. calcitrans and S. bullata pupae, respectively. Rearing Cumulative number of parasitoids attracted to host S. calcitrans S. bullata S. calcitrans 13 6 S. bullata 12 11

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103 Table 6 4. First turn choices made by female parasitoids run in a Y tube olfactometer at 24 h intervals after emergence. Day 1 was defined as 24 h post emergence. ANOVA F values are listed below means. Numbers marked with an aste risk are considered significant ( P <0.05). Day Cumulative number of parasitoids attracted to Response time to S. calcitrans blank S. calcitrans (s) 1 19 9 44.0 (4.9) 2 23 6 52.6 (5.6) 3 25 6 35.7 (3.9) 4 32 7 36.5 (3.7) 5 23 10 49.2 (6.1) ANOVA F 2.54* df=4,1; P <0.05

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104 Table 6 5 Results of G tests for the four olfactometer experiments comparing likelihood ratios between variables. Numbers marked with one asterisk are considered significant at P<0.05, while numbers marked with two asterisks are considered significant at P <0.01. Experiment Variables Comparison: conditioning, time G ________ _____________________________________________________________________ 6 1 Host conditioning on S. calcitrans v. S. bullata 8.04** S. calcitrans or S. bullata S. calcitrans v. no conditioning 1.23ns S. bullata v. no condi tioning 2.52ns 6 2 Host conditioning on Day 1: S. calcitrans v. no conditioning 1.07ns S. calcitrans Day 3: S. calcitrans v. no conditioning <0.01ns Day 5: S. calcitrans v. no conditioning <0.01ns Host conditioning on Unconditioned, day 1 v. day 3 4.69* S. calcitrans with time Unconditioned, day 1 v. day 5 3.42ns S. calcitrans day 1 v. day 3 0.75ns S. calcitrans day 1 v. day 5 0.27ns 6 3 Rearing on S. calcitrans S. calcitrans v. S. bullata 1.15ns or S. bullata pupae 6 4 Unconditioned, per day Unconditioned, day 1 v. day 2 0.97ns post emergence Unconditioned, day 1 v. day 3 1.27ns Unconditioned, day 1 v. day 4 1.79ns Unconditioned, day 1 v. day 5 0.02ns At P < 0.05, G 3.8, and at P < 0.01, G

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105 A) B) Figure 6 1. Y tube olfactometer u sed in experimentation. A) Air flow and humidity were controlled for by a regulator bubbling air through distilled water. B) Up to two choices were presented to parasitoids in screened chambers that allowed air flow of volatiles. Choice was counted as the first arm a parasitoid moved halfway down, regardless of future turns or time spent in that arm.

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106 Figure 6 2. Y tube olfactometer arms and entry capsule. Between experiments, all glass pieces were washed, rinse d t wice and allowed to air dry.

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107 LIST OF REFERENCES Althoff, D. M. 2003. Does parasitoid attack strategy influence host specificity? A test with New World braconids. Econ. Entomol. 28: 500 502. Anderson T. E. and N. C Leppla. 1992. Advances in Insect Rear ing for Research and Pest Management. Textbook. Westview Press, New York NY. Askew, R. R. and M. R Shaw 1986. Parasitoid communities: their size, structure and development. In J. K. Waage and D. Greathead, eds. Parasitoid Community Ecology 177 202. O xford University Press, New York NY. Barron, A. B. 2001. selection principle. J Ins Behav 14: 725 737. Bellows Jr. T. S. 1985. Effects of host age and host availability on developmental period, adult size, sex ratio, longevity and fecundity in Lariophagus distinguendus F urster (Hymenoptera: Pteromalidae). Res Pop Ecolog 27: 55 64. Bijlsma, L ., L. L. M Bruijn, E. P. Martens and M. J. Sommeijer n 2006 Water content of stingless bee honeys (Apidae, Meliponini): in terspecific variation and comparison with honey of Apis mellifera Apidologie 37: 480 486. Borror, D. J., D. M. DeLong and C. A. Triplehorn. 1974. An Introduction to the Study of Insects (4 th Ed). Holt, Rinehart and Winston. Bradley, S. W., D. C. Boot h and D. C. Sheppard. 1984 Parasitism of the Black Soldier Fly by Trichopria sp (Hymenotpera, Diapriidae) in Poultry Houses. Environ Entomol 13: 451 454. Bush, S. E. and S. H. Clay ton. 2006. The role of body size in host specificity : Reciprocal transfer experiments with feather lice. Evolution 60: 2158 2167. Campbell, J. B. and I. L. Berry. 1989 Economic threshold for stable flies on confined livestock, pp. 18 22. In J. J. Petersen and G. L. Greene (eds.), Current status of stable fly (Diptera: Muscida e) research. Misc. Pub. No. 74, Entomol. Soc. Am., College Park, MD. Chan, M. S. and H. C. J. Godfray. 2005. Host feeding strategies of parasitoid wasps. Evol. Ecology 7: 593 604. Chapman, T., T. Miyatake, H.K. Smith and L. Partridge. 1998 Interactions of mating, egg production and death rates in females of the Mediterranean fruit fly, Ceratitis capitata Proc Royal Soc London 265: 1879 1894 Chuche J., A. Xureb and D. Thiry 2006. Attraction of Dibrachys cavus (Hymenoptera: Pteromalidae) to its host frass volatiles. J Chem Ecol 32: 2721 2731.

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108 Clark, J. M., J. G. Scott F. Campos and J. R. Bloomquist 1995. Resistance to avermectins extent, mechanisms and management implications. Ann Rev Entomol 40: 1 30. Cohen J. E., T. Jonsson, C. B. Mller, H. C. J. Godfray and V. M. Savage 2005. Body sizes of hosts and parasitoids in individual feeding relationships. Proc Nat Acad Sci 102: 684 689. Consoli, F. L. and S. B. Vinson 2004. Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenop tera: Braconidae). Comp Biochem Physio B (Biochem and Molec Bio) 137: 463 473. Craighead, F. C. 1921. Hopkins host selection principle as related to certain Cerambycid beetles. Journ Agri Res 22: 189 220. Daly K. C., M. L Durtschi and B. H. Smith. 2001 Olfactory based discrimination learning in the moth, Manduca sexta J Insect Physiol 47:375 384. Davis J. M. and J. A. Stamps. 2004. The effect of natal experience on habitat preferences Trends Ecol Evo 19: 411 416. De Almeida, M. A. F., A. P Do Prado and C. J. Geden. 2002. Influence of Temperature on Development Time and Longevity of Tachinaephagus zealandicus (Hymenoptera: Encyrtidae), and Effe cts of Nutrition and Emergence Order on Longevity Environ Entomol 31: 375 380. De Almeida M. A. F., C. J. Geden and A. P. Do Prado 2002 Influence of Feeding Treatment, Host Density, Temperature, and Cool Storage on Attack Rates of Tachinaephagus zeala ndicus (Hymenoptera: Encyrtidae) Environ Entomol 31: 732 738. Decker G. C. and W. N. Bruce. 1952. House Fly Resistance to Chemicals. Am. J. Trop. Med. Hyg. 1: 395 403. De Jong, R. and L. Kaiser. 1992. Odor learning by Leptopilina boulardi a speciali st parasitoid (Hymenoptera: Eucoilidae). Journ Insect Beh 4: 743 750. De Jong R. and L. Kaiser. 1991. Odor preference of a parasitic wasp depends on order of learning. Experientia 48: 902 904. Eggleton, B and R. Belshaw. 1992. Insect Parasitoids : An E volutionary Overview Philosophical Transactions: Biological Sciences 337: 1 20. Eggleton, B. and R. Belshaw. 1993. Differences between Diptera, Hymenoptera and Coleoptera parasitoids: provisional phylogenetic explanations. Biol Journ Linn Soc 48 : 213 226 Eldridge, B. F. and J. D. Edman 2003. Medical Entomology: A Textbook on Public Health and Veterinary Problems Caused by Arthropods. Kluwer Academic Publishers, Norwell, MA

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109 Ellers, J ., J. J. M Van Alphen and J. G Sevenster 1998. A field study of size fitness relationships in the parasitoid Asobara tabida Journ A nimal Ecol 67: 318 324. Ferracini C ., G. Boivin and A. Alma 2006. Costs and benefits of host feeding in the parasitoid wasp Trichogramma turkestanica Ent Exp et Appl 121 (3): 229 234. Garcia, F. R. M and E. Corseuil 2004. Native hymenopteran parasitoids associated with fruit flies (Diptera : Tephritidae) in San ta Catarina State, Brazil Florid Entomol 87: 517 521. Geden, C. J., M. Almeida and A. P. do Prado. 2003. Effects of Nosema Disease on Fitness of the Parasitoid Tachinaephagus z ealandicus (Hymenoptera: Encyrtidae). Environ Entomol 32: 1139 1145. Geden, C. J. and J. A. Hogsette (eds.). 1994. Research and Extension Needs for Integrated Pest Management of Arthropods of Veterinary Importance. Proceedings of a Workshop in Lincoln, Nebraska. Geden, C. J. and J. A. Hogsette. 2006. Suppression of house flies (Diptera : Muscidae) in Florida poultry houses by sustained releases of Muscidifurax raptorellus and Spalangia cameroni (Hymenoptera : Pte romalidae) Environ Entomol 35: 75 82. Gelman, D. B ., D. Gerling and M. B. Blackburn 2005. Host parasitoid interactions relating to penetration of the whitefly, Bemisia tabaci by the parasitoid wasp, Eretmocerus mundus J Insect Sci. 5: 46. Gelman, D. B ., D. Gerling, M. B. Blackburn and J. S. Hu 2005. Host parasite interactions between whiteflies and their parasitoids. Archives Insect Physio Bioch em 60: 209 222. Giron D., A. Rivero N. Mandon E. Darrouzet and J. Casas 2002. The physiology of host feeding in parasitic wasps: implications for survival. Funct Ecol 16. Godfray, H. C. J. ed. 1994. Parasitoids: Behavioral and Evolutionary Ecology. P rinceton University Press, Princeton NJ. Hallem E A A. Dahanukar and J. R. Carlson 2006 Insect odors and taste receptors. Ann Rev Entomol 51: 113 135. Hansson, B. S. 1995. Olfaction in Lepidoptera. Experientia 51: 1003 1027. Hastings, A.. and H. C. J Godfray. 1999 Learning, host fidelity, and the stability of host parasitoid communities. Am Nat 153: 295 301. Hawkins, B. A., R. R. Askew and M. R. Shaw 1990. Influences of host feeding niche and foodplant type on generalist and specialist parasitoids. Ecol Entomol 15: 275 280.

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116 Wcke rs, F. L. 1998. Food supplements to enhance biological control in storage systems: effects of hosts and honey on the longevity of the bruchid parasitoids Anisopteromalus calandrae and Heterospilus prosopidis. Proc Exper Appl Entomol 9: 47 52. Whitfield, J B. 1992 Phylogeny of the non aculeate Apocrita and the evolution of parasitism in the Hymenoptera. Journ of Hymenopt Research 1: 3 14. Whitfield J B. 1998. Phylogeny and Evolution of Host Parasitoid Interactions in Hymenoptera. Ann Review Entomol 43: 129 151. Yujie, G., J. A. Hogsette, G. L. Green and C. J. Jones. 1997. Survey Report on Pupal Parasitoids in Livestock and Poultry Facilities in China. Chin Journ Biol Contol 13: 106 109. Zchori Fein, E., R. T. Roush and M. S. Hunter 1992. Male production induced by antibiotic treatment in Encarsia formosa an asexual species. Experientia 48: 102 105.

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117 BIOGRAPHICAL SKETCH Kimberly Marie Ferrero was born on March 18, 1983, the first child of Patti A. Ferrero and Dr. Frank A. Ferrero. Her fat her delivered her at Miami Mercy Hospital, where he was a heart surgeon. Kimberly is the oldest of five children, and has two younger sisters, Ashley and Francesca, as well as two younger brothers, Christopher and Shawn. A first generation Italian American she grew up in Miami, Florida and attended high school at Lake Mary High in Orlando, Florida. As a young child her love of science blossomed under the guidance of many wonderful teachers, so that at the age of seventeen she beca me one of only two studen t s to teach honors biology at her high school. Following high school, she began attendance at the University of Florida and in 2001 evolutionary biology of primates. Her yea rs at the University of Florida allowed her to take part in research travel to places such as Indiana University in the United States, St. Andrews University in Scotland, and Suriname in South America. A summer internship in the final year of her undergrad medical and veterinary entomology, with an emphasis on control of medically important Diptera (flies and mosquitoes). Kimberly subsequently spent the next two years working at the United States Department campus Center for Medical, Agricultural and Veterinary Entomology, and her love of public health issues was rewarded when sh research on a little known parasitoid from Eastern Europe that attacks and kills filth flies that are common worldwide. She has given numerous talks and lectures on the importance of control of arthropod disease vect ors, and has taught Introductory Entomology at the University of Florida

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118 as a Graduate Teaching Assistant. She spends much of her free time collecting insects in the wilderness of north central Florida, and enjoys swimming, kayaking, sailing, rock climbing and gourmet cooking on a graduate student budget. to her father, Dr. Ferrero. A gifted doctor and scientist with degrees in biology and physics, his unwavering sup port of her education and insistence that women can be influential figures in the sciences has allowed her to complete this thesis. Although Dr. Ferrero passed away one year before seeing his daughter obtain the first of her graduate degrees, his passion f or helping people through a greater understanding of the natural world lives on in his daughter.