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CAPTIVE REARING AND SEMIOCHEMCIAL ECOLOGY OF TRICHOGRAMMA

PAPILIONIS (HYMENOPTERA: TRICHOGRAMMATIDAE)

A DISSERTATION SUBMITTED TO THE

GRADUTE DIVISION OF THE UNIVERSITY OF HAWAIʻI AT MĀNOA IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR IN PHILOSOPHY

IN

ENTOMOLOGY

MAY 2020

BY

ABDULLA N. ALI

DISSERTATION COMMITTEE:

MARK G. WRIGHT, CHAIRPERSON

JON- PAUL BINGHAM (UNIVERSITY REP)

LEYLA KAUFMAN

PETER FOLLETT

GORDON BENNETT

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Ó copyright 2020

By

Abdulla N. Ali

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DEDICATION

At first dedicating this dissertation to Almighty Allah, without his mercy and sympathy, I was

not able to accomplish this study. Almighty Allah gave me the power and confidence to done

project work and also holy prophet Muhammed and his family (Peace Be Upon Them) who are a

light for my life. I also dedicate this dissertation to my lovely parents with the deepest gratitude

whose love and prayers have always been a source of strength for me. To my father who did not

live long enough to see me complete this successful mission.

My dedicated also goes to my lovely wife Maha and my children Wisam and Taim who fill my

heart with hope and happiness. To my siblings and friends, their continuous support and advice

have helped me to pursue my goals.

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ACKNOWLEDGMENTS

Apart from my efforts, the success of my project depends largely on the encouragement

and guidelines of many others. I take this opportunity to express my gratitude towards them.

First of all, I would like to thank my major professor Dr. Mark G. Wright for his continual and

valuable guidance, huge support that I believe without his supervision I would not finish any of

my research. It is not enough to express myself for all the great things he has done to me. Dr.

Wright is more than an advisor. I have to be humble with all the things I learned from him in his

laboratory. I learned from him how to be a professional; how do I think critically and link ideas.

I would like to express my serious thank you to my dissertation committee members, Drs. Leyla

Kaufman, Jon-Paul Bingham, Peter Follett, and Gordon Bennett. I appreciate all thoughtful

feedback and criticism of my chapters that were very deliberate and directed me through my

Ph.D. study. I am truly thankful for all useful inputs. My family (Mom, Dad, brothers and

sisters). To my sweetheart wife Maha A. Najm and my kids (Wisam and Taim Ali), my depth

thank you to you all for your prayers and immersive support. Thank you Maha for being very

penitent and genuinely supportive to me. Thank you for giving me such a beautiful gift (Wisam

and Taim). I would also like to thank the Ministry of Higher Education & Scientific Research,

University of Kufa, Iraq for granting me a scholarship and an opportunity to study abroad. Iraqi

cultural office in Washington D.C., they are appreciated for their genuine support and financial

aid through five and a half year.

To my all student colleagues, it was such a blessing time to be with you. I am indebted to

David Honsberger for his assistance with fields work, critical reading and editing on the first

drafts of my thesis manuscripts. Finally, it is my pleasure to be indebted to all people who

believed in me and supported me even without my awareness. There are definitely many people

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whose names have not mentioned, helped me to accomplish this important mission in my life.

This work was funded by Hatch Project 919-H, administered by CTAHR, and the Ministry of

Higher Education & Scientific Research, University of Kufa, provided support to me.

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LIST OF PUBLICATIONS

Ali, A.N. and Wright, M.G. 2020. Behavior response of Trichogramma papilionis in response to

host eggs, host plants, and induced plant cues. Biological Control. (Accepted)

Ali, A.N. and Wright, M.G. Fitness effects of founder female number of Trichogramma

papilionis reared on a factituous host Ephestia kuehniella (Zeller). Proceedings of the Hawaiian

Entomological Society (In review)

Ali, A.N. and Wright, M.G. 2018. Response of Trichogramma papilionis to plant volatiles

associated with Lepidoptera oviposition. Biological Control in Pest management Systems of

Plants Annual Meeting. Whitefish, Montana, October 2018. (Oral presentation)

Papers in Preparation:

Ali, A.N. and Wright, M.G. Response of Trichogramma papilionis wasps to blends of synthetic

semiochemicals.

Ali, A.N. and Wright, M.G. The effect of plant derived semiochemicals on searching behavior of

Trichogramma papilionis in different environments.

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ABSTRACT

This study addressed aspects of mass rearing of Trichogramma papilionis (Hymenoptera;

Trichogrammatidae), including the effects of varied colony founder size on wasp fitness, and the

exploitation of the wasps to locate egg hosts in which to deposit thereof progeny. Effects of

initial founder female number of T. papilionis were investigated using fitness parameters

(emergency rate, sex ratio and fecundity) to quantify the effects of a severe bottleneck (single

founder female) on 10 subsequent generations. Results showed that no significant difference for

eggs laid per female over ten generations, suggesting that the imposed bottleneck did not result

in reduced female fecundity for any founder population size. However, founder numbers did

affect both the emergence rate and sex ratio of T. papilionis. Further investigation of the impacts

of inbreeding on field performance of the wasps was discontinued as extremely limited host

finding ability of the wasps was observed in some habitats. The emphasis of the work was thus

shifted to elucidating the searching behaviors of T. papilionis in relation to chemical cues. The

hypothesis that T. papilionis are attracted to host habitat by host plant or egg-associated volatile

chemicals was tested.

The response of T. papilionis females to olfactory cues from host eggs, host plants and

induced plant volatiles were studied. The response of T. papilionis females to different info-

chemical cues was tested in Y-tube olfactory assays. Wasps made a positive response to odors

from corn earworm (CEW) eggs Helicoverpa zea (Lepidoptera: Noctuidae) compared with blank

air, while there was a negative response to Ephestia kuehniella eggs (Lepidoptera: Pyralidae)

compared blank air: T. papilionis females thus preferred odors from corn earworm eggs over the

Mediterranean flour moth eggs. Further, the wasps were attracted to volatile emissions from sunn

hemp Crotalaria juncea (L.) over maize Zea mays (L.), despite both plants infested with H. zea

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eggs. No preference was observed for plants not infested with H. zea eggs, suggesting T.

papilionis showed a positive response to stimuli from sunn hemp plants that might be induced by

H. zea oviposition. Chemical volatile collection and headspace analysis was conducted.

Headspace analysis and thermal desorption and gas chromatography–mass spectrometry (TD-

GCMS) was used to qualitatively and semi-quantitatively determine the difference in plant

volatile organic components (VOCs) from Helicoverpa zea egg infested sunn hemp plants

compared with intact sunn hemp plants and H. zea eggs only. TD is used as a preconcentration

technique of VOCs for gas chromatography-mass spectrometry (GC–MS), making it useful to

detect low-concentration analytes that would otherwise be undetectable. Results demonstrated

that sunn hemp plants released 55 chemical volatiles with five compounds that were unique, or

were emitted in higher concentrations, for plants infested with CEW eggs. These volatile

compounds were consistent with linear alkanes, aldehydes, aromatics, polyterpene-related

compounds, naphthalene derivatives, and ester-related compounds. High concentrations of

anisole, β-myrcene, cis-butyric acid, trans-isoeugenol, and bis(2-ethylhexyl) phthalate were

found in infested sunn hemp. The majority of GCMS peaks detected from H. zea eggs were

consistent with phosphates, pheromone-related compounds, various natural products, a series of

glycol-related compounds, and a series of fatty acid ester-related compounds. Several

compounds were shared in sunn hemp samples and corn earworm eggs: anisole, β-myrcene, and

bis (2-ethylhexyl) phthalate, but were detected in higher concentrations from the plants with H.

zea eggs.

Evaluation of the response and the performance of T. papilionis females in y-tube

olfactory bioassays to single compounds, and blends of synthetic chemical showed that the

wasps were significantly attracted to only two of the assayed chemical volatiles (anisole and

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bis(2-ethylhexyl) phthalate). Some concentrations of anisole and bis (2-ethylhexyl) phthalate

were attractant to the wasps, whereas some concentrations of the other tested chemical

compounds repelled the wasps. Wasps were attracted to a blend of anisole and bis(2-ethylhexyl)

phthalate (25μL /100μL ratio) which is similar to the ratio of anisole to bis(2-ethylhexyl)

phthalate detected in the (GC-DMS) chromatograph for C. juncea plants infested with H. zea

eggs. No significant attraction to any other blend ratios of anisole and bis(2-ethylhexyl) phthalate

was observed.

Greenhouse and field experiments were conducted to determine whether the patterns

observed in the y-olfactometer were consistent under less constrained conditions. The optimal

blend identified above was initially tested in a greenhouse, and later in closed-canopy

environments (under trees) and open habitat with no trees. The parasitism rate by T. papilionis

wasps was significantly increased when the wasps were exposed to anisole and bis(2-ethylhexyl)

phthalate blend in both greenhouse and outdoor trials (covered habitat), at least over short

distance (up to 2m from the volatile sources).

The findings presented in this dissertation underscore the importance of improving our

understanding of how tri-trophic interactions (natural enemies- herbivores and host plants)

interact to influence insect behavior, as well as the impact of variable environments, impact

parasitoid wasps. The results may also contribute to finding a way to improve natural enemy

efficacy in augmentative and conservation biocontrol efforts. Semiochemical cues can positively

or negatively affect the response of parasitic wasps. This may provide an understanding of

ecology that could facilitate achieving successful field parasitism and thus enhanced pest

management.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS……………………………………………………………..................iii

LIST OF PUBLICATIO........................................…………………………………......................v

ABSTRACT...................................................................................................................................vi

LIST OF TABLES…………………………………………………………………....................xiv

LIST OF FIGURES…………………………………………………………………...................xv

LIST OF ABBREVIATIONS.....................................................................................................xvii

CHAPTER 1: GENERAL INTRODUCTION AND DISSERTATION STRUCTUR...................1

1.1 Background ……………………………………………………………………..................... 1

1.2 Aims of this dissertation...........................................................................................................4

1.3 Dissertation organization..........................................................................................................5

References........................................................................................................................................7

CHAPTER 2: FITNESS EFFECTS OF FOUNDER FEMALE NUMBER OF

TRICHOGRAMMA PAPILIONIS REARED ON A FACTITIUOS HOST EPHESTIA

KUEHIELLA (ZELLE)..................................................................................................................11

Abstract…………………………………………………………………………………..............11

2.1 Introduction………………………………………………………………………..................12

2.2 Materials and Methods ……………………………………………………………................16

2.2.1 Egg parasitoid colony...............................................................................................16

2.2.2 Experimental population...........................................................................................16

2.2.2.1 Evaluation of the effects of number of founder females.........................................16

2.2.2.2 Establishing isofemales and other lines.................................................................17

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2.2.2.3 Evaluating fitness-proxies of the founder females over successive

generations.........................................................................................................................18

2.2.3 Statistical analyses...................................................................................................19

2.3 Results……………………………………………………………………….........................20

2.3.1 Fitness measures of Trichogramma papilionis.............................................20

2.3.2 Response of emergence rate..........................................................................22

2.3.3 The response of sex ratio..............................................................................24

2.3.4 The response of parasitized eggs per female................................................25

2.4 Discussion................................................................................................................................28

2.4.1 Variability among the fitness parameters.....................................................28

Conclusion.............................................................................................................31

References……………………...…………………………….......................................................32

CHAPTER 3: SEARCHING BEHAVOR OF TRICHGRAMMA PAPILIONIS IN RESPONSE

TO HOST EGGS, HOST PLANTS, AND INDUCED PLANT

CUES……………………………………………….....................................................................41

Abstract…………...……………………………….......................................................................41

3.1 Introduction………………………………………………………………………..................42

3.2 Materials and Methods……………………………………………………………….............45

3.2.1 Behavioral response of parasitoids……………………………………......45

3.2.2 Dynamic Y-tube olfactory bioassays……………………………………….45

3.2.3 Plants and Insects……………………………………………….................48

3.2.4 Egg deposition experiments…………………………………………….….49

3.2.4.1 Egg-infested plants……………………………………………….............49

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3.2.4.2 Helicoverpa zea and Ephestia kuehniella eggs………………………….50

3.2.5 Trichogramma wasps………………………………………………............50

3.2.6 Volatile collection and headspace analysis..................................................51

3.2.7 Statistical analysis………………………………………………................53

3.3. Results………………………………………………………………………….....................53

3.3.1 H. zea and E. kuehneilla eggs versus blank air…………………................53

3.3.2 H. zea eggs versus E. kuehneilla eggs…………………………………......54

3.3.3 H. zea egg-infested sunn hemp plant and maize plant versus uninfested

plant……………………………………...............................................................54

3.3.4 H. zea egg-infested sunn hemp versus H. zea egg-infested maize…………54

3.3.5 Headspace volatile collection from sunn hemp plants.................................56

3.3.6 Dynamic headspace analysis of H. zea eggs................................................57

3.4 Discussion................................................................................................................................60

3.4.1 Response of T. papilionis female wasps to egg hosts………………………60

3.4.2 Response of T. papilionis to plant volatiles……………………………......61

Conclusion………………………………………..................................................63

References.....................................................................................................................................64

CHAPTER 4: RESPONSE OF TRICHOGRAMMA PAPILIONIS WASPS TO BLENDS OF

SYNTHETIC SEMIOCHEMICALS.............................................................................................71

Abstract…………………………………………………………………………………..............71

4.1 Introduction………………………………………………………………………..................72

4.2 Materials and Methods………………………………………………………….....................75

4.2.1 Test insects ...................................................................................................75

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4.2.2 Y-tube olfactometer bioassays......................................................................75

4.2.3 Compounds of interest .................................................................................75

4.2.4 Tests of Trichogramma response to volatile compounds..............................79

4.2.5 Statistical analysis .......................................................................................81

4.3. Results……………………………………………………………………….........................81

4.3.1 Response of Trichogramma wasps to volatile compounds...........................81

4.4. Discussion………………………………………………………………………...................85

Conclusion.............................................................................................................88

References .....................................................................................................................................89

CHAPTER 5: THE EFFECT OF PLANT DERIVED SEMIOCHEMICALS ON SEARCHING

BEHAVIOR OF TRICHOGRAMMA PAPILONIS IN DIFFERENT

ENVIRONMENTS........................................................................................................................99

Abstract……………………………………………………..........................................................99

5.1 Introduction………………………………………………………………………................100

5.2 Materials and Methods………………………………………………………………...........102

5.2.1 Trichogramma wasp culture.......................................................................102

5.2.2 Single volatile chemical trial: Preliminary Field experiments...................102

5.2.3 Greenhouse experiments.............................................................................103

5.2.4 Open field optimal volatile blend trial........................................................104

5.2.5 Open and covered habitat optimal volatile blend trial...............................105

5.2.6 Statistical analysis......................................................................................105

5.3 Results……………………………………...........................................................................106

5.4 Discussion……………………………………………………………..................................110

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Conclusion...........................................................................................................113

References....................................................................................................................................114

CHAPTER 6: GENERAL CONCLUSTION AND RECOMMENDATIONS...........................118

6.1 General Conclusions...............................................................................................................118

6.2 Recommendations and further works....................................................................................122

References....................................................................................................................................123

Appendices...................................................................................................................................124

Appendix A. Summary of the major peaks (VOCs) emitted by sunn hemp plant in

response to H. zea egg-deposition (Treatment) and healthy sunn hemp plant

(Control)....................................................................................................................124

Appendix B. Summary of DMS result for corn earworm Helicoverpa zea eggs and a

control blank..............................................................................................................130

Appendix C. A brief summary literature overview of the chemical compounds tested in

Chapter 4, emphasizing interactions with various insects. Citations were obtained

from Web of Science®, searching for the specific compound in association with

insects. The biological origin (plant or insect) and functional activity (in insects) for

each are summarized..................................................................................................136

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LIST OF TABLES

Table 2.1. Analysis of variance of Trichogramma papilionis fitness parameters as affected by

founder population and environmental conditions (25°C vs 22°C) over 10

successive generations in captivity. Laboratory line: 22 ± 2ºC, 60 –70% RH, LD

16:8 h. photoperiod. Mass-rearing line: 25 ± 1ºC, 75-88% RH, LD 16:8 h.

photoperiod. * Indicates a significant effect, P < 0.050, LSD

test.….....................................................................................................................21

Table 3.1. Summary of volatile chemicals collected, showing the compounds with the largest

peaks, from corn earworm eggs, corn earworm infested sunn hemp plant, and

healthy sunn hemp plants. (+) = Present; (-) = Not present...................................59

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LIST OF FIGURES

Fig. 2.1. Response (Mean ± SEM) in the emergence rate of three founder population sizes (1, 2,

and 10 founder females) of Trichogramma papilionis over ten serially bottlenecked

generations. Treatments not connected by the same letter were significantly

different......................................................................................................................22

Fig. 2.2. Coefficient of variation in the emergence rate of Trichogramma papilionis from three

founder population sizes (1, 2, 10 females) over ten generations..............................23

Fig. 2.3. Trichogramma papilionis sex ratios of progeny from three founder female treatments

(1, 2, and 10 founder females). Values are estimated mean (± SEM). Bars not connected by the

same letter are significantly different............................................................................................24

Fig. 2.4. Mean estimates (± SEM) of the sex ratio of Trichogramma papilionis in two

experimental lines (22°C, L- line, and 25°C, M- line). Treatments not connected by

the same letter were significantly different................................................................25

Fig. 2.5. Mean (± SEM) number of Ephestia eggs parasitized per Trichogramma papilionis

female for the three founder treatments (1, 2 and 10 founder females). Bars not

connected by the same letter are significantly different.............................................26

Fig. 2.6. Coefficient of variation in percentage parasitized Ephestia eggs for three different

founder population size colonies (1, 2, and 10 founder females) of Trichogramma

papilionis over 10 successive generations..................................................................27

Fig. 3.1. A schematic representation of a Y-tube olfactometer apparatus illustrating the direction

of airflow, odor sources, and site of introduction of the study animals. This figure is

adapted from http://www.chromforum.org..................................................................48

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Fig. 3.2. Response of Trichogramma papilionis females to various olfactory cues in a y-tube

olfactometer. A: H. zea eggs and E. kuehneilla eggs vs. Blank air; ** significant positive

response to H. zea eggs vs. blank air (Fisher’s exact test p = 0.012), non-significant

Fisher’s exact test p = 0.10; B: to H. zea eggs vs. E. Kuehneilla eggs; ** Fisher’s exact

test p = 0.002; C: to H. zea egg-infested sunn hemp vs. intact sunn hemp (control), and

egg-infested maize vs. intact maize (control); ** Fisher’s exact test p = 0.001, p = 0.020;

D: to H. zea egg-infested sunn hemp vs. H. zea egg-infested maize; ** Fisher's exact test

p = 0.006..............................................................................................................................55

Fig. 3.3. Overlay of DMS chromatograms of Control (blue), Treatment (green), and a control

blank (red). Numbers in parentheses above peaks identify compounds (see Table 3)

that were identified to be a special interest..................................................................57

Fig. 3.4. Overlay of DMS chromatograms of corn earworm eggs and a control blank. Overlay of

DMS chromatograms of corn earworm eggs and a control blank. Numbers in

parentheses above peaks identify compounds (see Table 3) ......................................58

Fig. 4.1. Olfactory behavioral response of Trichogramma papilionis females in a y-tube

olfactometer bioassay to select volatile compounds, measured as the percentage of

wasps choosing the chemical cue over the control. The difference of the insects

choosing an odor was determined by a χ2 goodness of fit test. ** = significant at a =

0.05 and ns = non- significant......................................................................................82

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Fig. 4.2 Percentage response of Trichogramma papilionis females to different ratios of

semiochemical volatiles in a y-tube olfactometer to select volatile compounds,

measured as the percentage of wasps choosing the chemical cue over the

control. ** = significant preference for treatment over control at a = 0.05, χ2

goodness of fit tests. Bars without connectors were not significantly different

for positive responses to the cues.....................................................................83

Fig. 4.3. The percentage positive response of female Trichogramma papilionis wasps to a

range of blend ratios of volatile compounds in a y-tube olfactometer to select

volatile compounds, measured as the percentage of wasps choosing the

chemical cue over the control. ** = significant, a = 0.05 and ns = non-

significant response, χ2 goodness of fit tests....................................................84

Fig. 5.1. Mean parasitism rate (±SEM) by Trichogramma papilionis in cornfields

comparing anisole as an attractant, to untreated release plots

(p = 0.092) ..................................................................................................106

Fig. 5.2. Parasitism rate of Trichogramma papilionis from the greenhouse (mean ±SEM),

with and without chemical attractants (anisole + bis(2-ethylhexyl) phthalate),

and over a distance of up to 6m; a) overall percentage of parasitism; b)

percentage parasitism at different distances from the release point, treatment

and control.....................................................................................................108

Fig. 5.3. Mean parasitism rate (±SEM) by Trichogramma papilionis: a) open habitat b).

covered and open habitat) comparing the optimal blend (anisole + bis(2-

ethylhexyl) phthalate) as an attractant, to untreated release trial. ** significant

at a = 0.05 and ns = non- significant.............................................................109

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LIST OF ABBREVIATIONS

L line, Laboratory line; M line, Mass-rearing room line; CEW, corn earworm moth

Helicoverpa zea; GC-DMS chromatography, Desorption Gas Chromatography Mass

Spectrometry. UGC, urban garden center; UH campus, University of Hawaiʻi at Mānoa

campus. Ani, anisole; Bis, bis(2-ethylhexyl) phthalate.

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CHAPTER 1

GENERAL INTRODUCTION AND DISSERTATION STRUCTURE

1.1 Background

Egg parasitoids are extensively used in biological control programs. Their impact in

suppressing pests is expected to be realized by decreasing the number of emerging larvae

(van Lenetern, 2003). The genus Trichogramma (Hymenoptera: Trichogrammatidae)

contains a wide range of species that are widely applied and studied natural enemies

because they are used in augmentative biological control, albeit with varying degrees of

success (Bueno et al., 2009; Smith, 1996). Trichogramma spp. are used to target

lepidopteran pests (Suckling and Brockerhoff, 2010), since they can be mass-produced

inexpensively and released at inundative densities in crop systems (Mansour, 2010;

Chailleux et al., 2012). Many species (e.g., T. pretiosum, T. ostriniae) have been

extensively researched (e.g., Hoffmann et al., 1995; Upadhyay et al., 2001; Knutson,

2005). Trichogramma papilionis has received limited research attention in general,

despite occurring in areas where it might be a valuable natural enemy of some critical

pest species. The first record of T. papilionis from the Hawaiian Islands was reported in

(Oatman et al., 1982). Trichogramma spp., which are egg parasitoids of a variety of

insect pests, especially Lepidoptera, are often mass-reared in large numbers for use in

augmentative biological control.

Ease of mass-rearing is a significant benefit of Trichogramma spp., but may also

result in reductions in insect quality and fitness. There are many issues with captive

rearing of parasitoids that may impact their effectiveness as biological control agents,

including inbreeding, adaptation to captive rearing conditions, and loss of fitness within

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colonies. The number of individuals used to start captive colonies has the potential to

influence a number of these factors.

The efficient location of hosts is fundamentally essential for parasitoid success in the

field (Wang et al., 2016). Egg parasitoid females have evolved various searching

behavior tactics to find their hosts in nature the cues they use range from visual to

olfactory, or semiochemical, ones. Many species use multiple types of cues, often at

different scales. For example, T. ostriniae has been shown to use visual and olfactory

cues in searching, as well as some degree of random searching (Gardner and Hoffmann,

2020). Inducible plant volatiles and host cues that are induced by the interactions of

herbivorous insects and plants are among the most effective semiochemical compounds

that a female wasp can exploit to discriminate its hosts under complex environmental

conditions. Chemical cues could be a critical point in host selection and searching

behavior in many parasitoid Hymenoptera, including Trichogramma species (Lewis and

Martin, 1990; Schmidt, 1994; Fatouros et al., 2005). Host-specific cues may be very

related to Trichogramma searching behavior and may compete with many other habitat-

related cues (Wright, 2019). Olfactory cues have a crucial role in host-natural enemy

searching behavior in terms of host location and egg- deposition and have been studied

more extensively for parasitoids than predators (Steidle and Van Loon, 2002). Some

plant species have been shown to release secondary-metabolic compounds, often as

volatiles from the leaves or the roots, into their environment as a response to insect

feeding and oviposition (Dicke et al., 2003; Rasmann et al., 2005; Dicke et al., 2009).

Herbivore induced plant volatiles (HIPV) have attracted numerous studies, while few

have been done on oviposition induced plant volatiles (OIPVs) (Schroder et al., 2005).

3

Hilker and Meiners (2006) reviewed the most recent studies on egg-deposition and

inducible plant volatiles that plants may use as defensive strategies against herbivorous

attack. They highlighted mechanisms of the defensive strategy that might be responsible

for the elicitation of plant synomones. These include the interaction between plant cells

and egg-deposition on the plant's surface and the effect of endosymbiotic microorganisms

on both plant tissue and the host insect. Wajnberg and Colazza (2013) highlight elements

of parasitoid behavior and focus on strategies of manipulating the behavior of parasitoids

to maximize the effects of these beneficial insects in pest management.

As part of the natural ecosystem, parasitoids have intimate interactions with other

organisms in their environment. It is crucial to elucidate parasitoid-herbivore-plant

relationships among trophic levels (Ode, 2013). Studying this relationship allows us to

achieve a better understanding of how parasitoid wasps perform. With this, we may be

able to increase the effectiveness of parasitoid wasps against target pests (Meiners and

Peri, 2013). According to Nordlund et al. (1988), host-habitat location in nature, host

location (e.g., host position on the plant), and host acceptance are considered to be the

most critical steps for successful parasitism.

Similarly, Vet and Dicke (1992) distinguished three different searching strategies

used by female wasps: infochemical (semiochemical) trails or plumes from different life

stages of the host, herbivore-induced plant volatiles, and associative learning. Some cues

can be reliable but less easily detected due to their minute quantities in the environment

and may not predictably indicate the host presence, especially over a long distance, this

approach is known as the reliability-detectability theory (Vet and Dicke, 1992). For

example, cues derived from an insect host can be extremely reliable. Still, they may not

4

be as easily detected as plant volatiles because of the huge biomass of plant material

relative to insect hosts (Colazza et al., 2010). Some stimuli are considered to offer

limited information about the host quantitatively but are more reliable because they come

directly from the host eggs, and can be important for generalist species. These stimuli

include egg contact kairomones, egg volatile kairomones, and plant synomones induced

by egg deposition (Colazza et al., 2010). Other stimuli originate from different life stages

of the host or from the plants, where plant stimuli can be more detectable as they are

released in higher quantities. These include cues from scales from adult lepidoptera, adult

traces, host pheromones, and allomones, as well as plant synomones induced by the

feeding activities of immature stages of herbivores, herbivores induced plant volatiles

(HIPVs), but HIPVs may not be as reliable an indicator of the existence of host eggs (Vet

and Dicke, 1992; Fatouros et al., 2008; Colazza et al., 2010).

1.2 Aims of this dissertation: Central hypothesis:

This dissertation study was conducted to address the central hypothesis that

fitness of Trichogramma papilionis under captive mass rearing conditions may be

optimized by avoiding intensive inbreeding, and that T. papilionis depends on chemical

cues in the environment to locate their hosts, and hence understanding of searching

ecology of T. papilionis can be used to improve augmentative biocontrol agents'

performance.

5

1.3 Dissertation organization

This dissertation is divided into six chapters as follows:

1) Chapter one: General introduction and dissertation structure. This

chapter describes the research problem and outlines the importance of egg

parasitoid wasps in augmentative biological control.

2) Chapter two: Effect of founder colony size. The goal of this study was to

test the effect of female founder population size on the fitness of progeny of T.

papilionis (Hymenoptera: Trichogrammatidae) over successive generations

and highlight any change in their biological performance. Three fitness

parameters were considered: fecundity (number of eggs per female),

emergence rate, and sex ratio, as well as the influence of different

environmental conditions (temperature range and humidity) on the fitness of

the wasps.

3) Chapter three: Searching behavior in captivity, chemical collection, and

headspace analysis. Searching behavior of T. papilionis in response to host

eggs, host plants, and induced volatile plant cues was examined using Y- tube

olfactometry. The chapter investigated the ability of T. papilionis females to

respond to egg host cues (Helicoverpa zea and Ephestia kuehniella), and

different plant habitats (sunn hemp and maize) and examined the effect of

oviposition by H. zea on sunn hemp leaves on T. papilionis host searching.

Headspace analysis and thermal desorption and gas chromatography-mass

spectrometry (TD-GCMS) were used to determine the volatile organic

components from sunn hemp Crotalaria juncea (L.) and Helicoverpa zea

6

(Boddie) (Noctuidae) eggs that might influence T. papilionis searching

responses.

4) Chapter Four: Olfactory bioassays. This chapter evaluated the response of

T. papilionis wasps, in Y-tube olfactory bioassays, to blends of synthetic

semiochemicals identified in Chapter 3 as potential attractants. The primary

objective was to identify an optimal combination of compounds that serve as

attractants to T. papilionis.

5) Chapter Five: Semiochemical trials (greenhouse and field conditions).

The effect of synthetic plant-derived semiochemicals on the searching

behavior of T. papilonis in different environments was analyzed. The response

of T. papilionis to a combination of volatiles previously identified in

olfactometer studies was studied under greenhouse and field conditions.

6) Chapter Six: General conclusions and recommendations. This chapter

provides brief concluding comments and recommendations for further studies.

7

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11

CHAPTER 2

FITNESS EFFECTS OF FOUNDER FEMALE NUMBER OF TRICHOGRAMMA

PAPILIONIS REARED ON A FACTITIOUS HOST EPHESTIA KUEHNIELLA

(ZELLER).

Abstract

Trichogramma species (Hymenoptera: Trichogrammatidae) are egg parasitoids of

a variety of insect pests, especially Lepidoptera. Trichogramma are often mass-reared

in large numbers for use in augmentative biological control. There are many issues

with captive rearing of parasitoids that may impact their effectiveness as biological

control agents, including inbreeding and loss of fitness within colonies. The goal of

this study was to test the effect of female founder population size on the fitness of

progeny of T. papilionis over successive generations and highlight any change in

their biological performance. Two parasitoid lines were started from 1, 2, and 10

inseminated founder females from wasps that were initially collected from Lampides

boeticus L. (Lycaenidea) eggs in sunn hemp Crotalaria juncea fields. The progeny of

these founder females was tracked for ten generations, to evaluate their fitness and

performance. Fitness parameters were considered: fecundity (number of eggs per

female), emergence rate, and sex ratio, as well as the influence of different

environmental conditions (temperature range and humidity) on the fitness of the

wasps. The results showed no significant difference for eggs laid per female over ten

generations, suggesting that the imposed bottleneck did not result in reduced female

fecundity for any founder population size. However, low founder numbers did affect

both the emergence rate and sex ratio of T. papilionis. These results suggest that

establishing a new colony of wasps with at least moderate founder numbers is better

to avoid any significant loss in quality of biological characteristics as long as rearing

is done under appropriate conditions (25 ± 2◦C, 60 –70% RH, LD 16: 8 h).

Keywords: Trichogramma papilionis, Factitious host, Ephestia kuehniella, Founder

female, Population fitness.

12

2.1 Introduction

Several species of egg parasitoids are commonly used in biological control

programs, and can potentially play a valuable role in suppressing pests through

decreasing the number of emerging larvae (Van Driesche and Bellows, 1996; van

Lenetern, 2003). Trichogramma spp. (Hymenoptera: Trichogrammatidae) are among the

most widely applied and studied augmentative biological control agents. These parasitoid

wasps are used in biological control programs against a diversity of phytophagous pests

in many economically important crops (Hassan, 1993; Wajnberg and Hassan, 1994;

Smith, 1996; Parra and Zucchi, 2004), in part because they can be inexpensively reared

on factitious hosts (Li, 1994; Smith, 1996; Parra, 1997; Haji et al., 1998; van Lenteren,

2003; Parra and Zucchi, 2004). Trichogramma wasps primarily parasitized the eggs of

Lepidoptera species (moth and butterflies). Some Trichogramma species can also attack

hosts in a broad range of habitats and parasitized the eggs of different insects such as

lacewings, flies, true bugs, beetles, other wasps (Knutson, 1998). Trichogramma species

were mass-produced initially in the early 1900s after entomologists discovered the

potential successes of using them in biological control programs, despite the few

commercial attempts to produce these wasps in the U.S. (Knutson, 1998).

Trichogramma are mostly reared in laboratory circumstances on a factitious host

such as Mediterranean flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae)

(Parra, 1997; Bertin et al., 2017), which is not the intended target host of the wasps

(Bertin et al., 2017). Factitious hosts are used as they are typically less costly, and high

quality of mass-reared parasitoids can be achieved, even though rearing the egg

parasitoids in a factitious host may result in an evolutionary interaction over successive

generations, selecting for a captive condition strain of the wasps (Hoffmann et al., 2001).

13

After mass-rearing, the egg parasitoids are ready to be liberated as biocontrol agents,

usually in crop fields under varied environmental conditions. There are however

drawbacks associated with mass-rearing insect in captivity, that may lead to a decrease in

the quality of insects produced (Bertin et al., 2017) and most of these potential problems

are related to the genetic diversity of the colonies, inbreeding depression, accumulation of

detrimental mutations, and genetic adaptation to captive conditions (Frankham et al.,

2002). Many desirable aspects in egg parasitoids (e.g., physiological, phenotypic, and

behavioral characteristics) may vary in vitro when the parasitoids are reared on

alternative hosts, and that may result in undesirable changes in the performance of the

parasitoids under field environments (Leppla and Fisher, 1989; Hopper et al., 1993).

Genetic drift, an evolutionary process in which the frequency of an existing gene variant

(allele) in a population changes over a period of time or generations in small populations

is considered a primary cause of the loss of genetic diversity of species (Joslyn, 1984;

Allendorf, 1986; Hopper et al., 1993; Gabriel et al., 1991; Gabriel and Bürger, 1994;

Oostermeijer et al., 2003; Frankham, 2005; Grueber et al., 2013).

Genetic drift has the most significant negative impact on small populations

(Prezotti et al., 2004), which should be taken under consideration when establishing

laboratory colonies of insects, to ensure that an adequate number of individuals are used

to create the colony, and in the long-term, to sustain a genetically viable captive

population (Wajnberg, 1991). There are, however, no generally agreed-upon rules about

the ideal number of individuals required to create a new viable population in vitro, which

may be started from less than ten individuals, to hundreds or even thousands of

individuals (Mackauer, 1976; Bartlett, 1985). Many researchers have shown or suggested

14

that rearing egg parasitoids on alternative hosts over many generations can cause the

parasitoids to deviate either in host preference, or ability to effectively locate and

parasitize target hosts under field conditions. Ultimately this might lead to adverse

impacts on parasitoid fitness and declining field performance with continued captive

rearing (Kaiser et al., 1989; van Bergeijk et al., 1989; Hassan and Guo, 1991; Woodworth

et al., 2002; Antolin et al., 2006; Araki et al., 2007; Henry et al., 2008; Li et al., 2010).

Accumulation of deleterious recessive alleles is also more likely in captive colonies,

probably more so in those started from very few founders (Bartlett, 1984; Stephens et al.,

1999). A population comprises a set of variable genotypes compounded by the number of

different individuals (Luque et al., 2016). Population size is considered one of the

determinants of genetic structure (Nielsen and Slatkin, 2013). Founder number may thus

affect newly established colonies by demographic and genetic mechanisms such as

recessive allele expression, and demographic stochasticity (Lande, 1993; Boyce et al.,

2006).

Genetic mechanisms have received intense attention in terms of inbreeding

depression, which can significantly increase the short-term potential for extinction during

the process of colonization of a habitat (Newman and Pilson, 1997; Bijlsma et al., 2000;

Reed et al., 2002, 2003). Individual founders that have heterogenic genes and phenotypic

variation are more likely to be successful colonizers (Drake, 2006; Wagner et al., 2017;

Forsman, 2014; Szűcs et al., 2017). When establishing new populations of bio-control

agents, researchers typically seek to maximize the fitness of the individuals by avoiding

the adverse impacts of inbreeding for the founder and early generations in captivity

(Castañé et al., 2014). Genetic heterogeneity of colony founders has an effect on the

15

potential for inbreeding depression, and the number of individuals that are used to

establish a new colony significantly affect the heterogeneity. It may be a simple matter

for some species, for researchers to find an adequately large number of individuals to

establish a new colony, while for other species, this may be a limiting factor (Castañé et

al., 2014). Regardless of the genetic mechanisms, the number of founders is persistently

identified as a fundamental determinant of colonization success across a diverse range of

taxa (Lockwood et al., 2005; Colautti et al., 2006; Blackburn et al., 2015).

In seeking to identify species that may have potential for positive impacts in

augmentative and conservation biocontrol programs in Hawaii, Trichogramma papilionis

was identified as a species that may possess valuable characteristics. Trichogramma

papilionis has received limited research attention in general. The first record of T.

papilionis from the Hawaiian Islands was reported in Oatman et al., (1982). It is readily

reared in captivity on factitious hosts (Wright unpublished data).

This study aimed to test the effect of the initial number of founder females on life-

history characteristics and leading fitness-proxies of resulting populations, including

fecundity of females, adult emergence rate, sex ratio, mean parasitized eggs per female.

16

2.2 Materials and Methods

2.2.1 Egg parasitoid colony

A T. papilionis colony was established from Lampides boeticus L. (Lycaenidae)

eggs collected on sunn hemp plants, Crotalaria juncea, Waialua, O’ahu Island, Hawaii,

USA. The wasps were maintained on E. kuehniella eggs in a climate-controlled room (25

± 1◦C, 50–70% RH, LD 16: 8 h) (Huigens et al., 2009, 2010) for multiple generations

over fifteen months, until the experiment started in the laboratory. E. kuehniella eggs

parasitized by T. papilionis were held in Plexiglas cages (15x15x15cm) under the

environmental conditions mentioned above. Upon emergence, adult parasitoids were

provided clusters of Ephestia eggs glued to the surface of a sheet of paper using “Elmer’s

glue-all®”, with droplets of pure honey as an energy source. Wasps of both genders lived

for 8-10 days when they were provided with honey droplets. T. papilionis females used in

experiments were 24 h old and were left to mate with males during that period. Males

emerged about 12 h before females and waited on other parasitized eggs for female

emergence. Copulation occurs immediately after female emergence. All experiments

were conducted in the laboratory.

2.2.2 Experimental populations

2.2.2.1 Evaluation of the effects of the number of founder females

To test the effects of the number of founder females upon starting a new colony in

the laboratory, three different founder population sizes (1, 2 and 10 mated female wasps)

were used: 1, 2 and 10 gravid females were isolated from the original colony into small

17

glass vials (4.5 cm length, 0.5 cm diameter), these vials were closed with perforated

screened lids for ventilation. The glass vials and their covers were reused for repeated

generations after being washed with detergent liquid and tap water then autoclaved at

121°C at 100 kPa for 15 minutes. Ten replicates were used for each founder population

size - each vial equaled one replicate. The vials were placed in a tube rack. A droplet of

pure honey was placed into each vial with a small needle, which was connected to a 5-cc

syringe to feed the wasps throughout the experiment.

2.2.2.2 Establishing isofemales and other lines.

To establish lines from generations produced from the original founders, the

females were placed individually into glass vials and offered clusters of E. kuehniella

eggs for parasitism. These clusters of E. kuehniella eggs were glued on small strips of

yellow paper using “Elmer’s glue-all®”. The strips of factitious host eggs were UV

irradiated for 30 min. before being introduced into the glass tube, to reduce the likelihood

of fungal contamination (Stein and Parra, 1987). Ephestia egg patches were observed

from being parasitized after exposure to female wasps until adult emergence. Four trails

were created.

To estimate the effects of different environmental conditions on the colonies,

combined with the sizes of the founder-female number of wasps, two experimental lines

were established. One line was kept in a climate room for insect rearing (25 ± 2◦C, 60 –

70% RH, LD 16: 8 h), which represented the “Mass-rearing room line” (M) and another

line was kept in a laboratory with different ambient conditions (22 ± 1◦C, 75-88% RH,

LD 16:8h), the so-called “Laboratory line” (L).

18

After the adult wasps emerged from each generation, the population lines were

continued, through allowing females to mate with males from the same replicate for 24-

48 h, after which individual females were randomly chosen and isolated + in new glass

tubes that contained droplets of pure honey as a nutrient and energy source (Bertin et al.,

2017). The same number of the founder females (1, 2 and 10 female wasps) were isolated

from each respective founder colony used to create the original replicate populations

(subpopulations). Thus, one mated female was taken from the one-female founder

colony, two mated females from the two founder-female colony, and ten mated females

were taken from the ten-founder colony. These females were then placed gently into the

glass tubes as described above. Females were distinguished from the males based on the

sexual dimorphism in the antennae (Bowen and Stern, 1966). Antennal dimorphism was

used to identify sexes, using the antennomere number, which is higher in males, as well

as males have different antennae shape, filiform and rarely forming an apical club,

whereas, in females, the last flagellomeres are enlarged and swollen, (Romani et al.,

2010). A dissecting microscope was used to visually confirm the sex of the wasps.

2.2.2.3 Evaluating fitness-proxies of the founder females over successive generations

Three parameters were used for measuring the fitness for each founder population

over 10 generations: fecundity (the number of parasitized eggs per female), emergence

rate of progeny, and sex ratio of progeny. These fitness parameters were traced for the

founder treatments from the initial founder females through the tenth generation. To

quantify the number of eggs laid per female after each parasitism period, the numbers of

19

parasitized eggs in each vial were separately counted aided by a laboratory counter

(Fisher Scientific), using the dissecting microscope. The number of blackened eggs,

which denoted that the egg was parasitized and that the parasitoid larvae are developing,

was used to distinguish parasitized eggs from unparasitized ones. The percentage rate of

subsequent emergence of adult wasps was estimated by counting the emergence holes on

parasitized eggs, divided by the total number of darkened parasitized eggs (with and

without emergence holes) (Pratissoli et al., 2005). The sex ratio of adult wasps was

quantified as the number of males and females emerging from eggs in each vial for each

generation. Parasitized eggs per female was estimated by dividing the total number of

parasitized eggs of Ephestia per vial by the number of founder females (Bowen and

Stern, 1966; Bertin et al., 2017).

This experimental design aims to test the following hypothesis: rearing

Trichogramma wasps in a factitious host with variable numbers of founders (1, 2 and 10

mated females), and under a variety of rearing conditions will result in inbreeding

depression and might result in a subsequent reduction in fitness of the progeny.

2.2.3 Statistical analyses

Analysis of variance (ANOVA) was conducted using JMP13 Pro (SAS Institute,

Carey, NC). Female fecundity (production of eggs, and percentage of eggs per female),

the emergence of adults, and sex ratio were fitted to a mixed linear model with treatment

and generations as fixed factors, replicates as random factors, and overlapping

generations. Least square means differences Tukey (HSD) were used to compare means

20

for parasitized eggs per female, sex ratio, emergence rate, and generations among

treatments. The full data set was used to estimate p-values for founder-size effects (Szűcs

et al., 2017). Comparisons of sex ratio and parasitized eggs per female versus lines and

those of the 10th generation were analyzed by a Student’s t-test (Castañé et al., 2014).

2.3 Results

2.3.1 Fitness measures of Trichogramma papilionis

The outcomes of this experiment revealed significant differences for both

emergence rate and sex ratio among the three founder-population sizes for T. papilionis

(F(4,639) = 25.3, p < 0.0001 and F(4,639) = 27.1, p < 0.0001) respectively, whereas, no

significant difference for number of eggs laid per female was found among treatments.

There were substantial significant differences between the breeding cohorts in the

laboratory colony and the mass-rearing room colony, in terms of sex ratio and parasitized

eggs per female (F (4,639) = 5.93, p < 0.015 and F (4,639) = 8.18, p < 0.0044 respectively

(Table 1).

21

Table 1: Analysis of variance of Trichogramma papilionis fitness parameters as affected

by founder population and environmental conditions (25°C vs 22°C) over 10 successive

generations in captivity. Laboratory line: 22 ± 2 ºC, 60 –70% RH, LD 16:8 h. photoperiod.

Mass-rearing line: 25 ± 1ºC, 75-88% RH, LD 16:8 h. photoperiod. * Indicates a significant

effect, P < 0.050, LSD test.

Fitness

Parameters Statistical

Parameter Estimate Stander Error

(SEM) F value p value

Emergence

rate

Intercept 92.4 0.85 108.20 0.0001

Experiment 0.67 0.85 0.59 0.44

Treatments 2.67 1.19 25.56 0.0001 *

Lines -1.5 0.85 3.16 0.075

Sex ratio

Intercept 81.1 0.77 104.15 0.0001*

Experiment 0.5 0.77 0.41 0.51

Treatments 1.54 1.08 27.2 0.0001 *

Lines -1.9 0.77 5.93 0.015 *

Parasitized

eggs

per female

Intercept 32.8 0.6 49.77 0.0001

Experiment 1.1 0.6 3.0 0.083

Treatments 0.8 0.92 1.30 0.272

Lines -1.8 0.65 8.18 0.0044*

22

2.3.2 Emergence rate response

The results show that there are statistical differences in the mean emergence rate

among the female founder colony sizes. There was no significant difference in emergence

rate of progeny between ten and two founder females. However, ten and two female

founders have a significantly higher progeny emergence rate compared to one female

founder colonies (Figure 1). Emergence rates had different patterns among treatments

over generations. Founder populations of ten and two females showed consistent patterns

of adult emergence over multiple generations, whereas founder populations with just one

female had substantial variation among generations, and 2 founders was intermediate,

with 10 founders most consistent (Figure 2). No significant difference in the emergence

rate between the two experimental lines (M and L) was detected (data not shown).

Figure 1: Response (Mean ± SEM) in the emergence rate of three founder

population sizes (1, 2, and 10 founder females) of Trichogramma papilionis over ten

serially bottlenecked generations. Treatments not connected by the same letter were

significantly different

One Two Ten0

20

40

60

80

100

% E

mer

genc

e ra

te

aab

No. of founder females

23

Figure 2: Coefficient of variation in the emergence rate of Trichogramma papilionis

from three founder population sizes (Treatments, 1, 2, 10 females) over ten generations.

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

1 Female

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

2 Females

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

10 Females

24

2.3.3 The response of sex ratio

Overall, the sex ratio of the offspring was generally female- biased among

treatments, with significant differences among all treatments (Figure 3). Ten founder

females consistently produced a greater ratio of females than two- and one founders.

There was a significant difference in sex ratio between the two experimental lines (L and

M), where the mass rearing room (M) line produced significantly, albeit slightly, more

females (83.0 ± 1.1%) than the lab line (L) (79.2 ± 1.1%) (Figure 4; t = 2.32; d.f. = 631; p

= 0.010).

Figure 3: Trichogramma papilionis sex ratios of progeny from three founder female

treatments (1, 2, and 10 founder females). Values are estimated mean (± SEM). Bars not

connected by the same letter are significantly different.

One Two Ten 0

20

40

60

80

100

ab c

No. of founder females

Sex

ratio

(% fe

mal

e)

25

Figure 4: Mean estimates (± SEM) of the sex ratio of Trichogramma papilionis in two

experimental lines (22°C, L-line, and 25°C, M-line). Treatments not connected by the

same letter were significantly different.

2.3.4 Parasitized eggs per female

There were no statistically significant differences among treatments for

parasitized eggs per female (Figure 5). Additionally, founder population sizes of ten and

two females showed less variation over the 10 generations than the one founder-female

colonies, with the single female colonies showing consistent high variability in fecundity

(Figure 6). The results showed a small difference between the two experimental lines (L

and M), with significantly higher (t = 2.79; d.f. = 631; p = 0.0027), numbers of

parasitized eggs in the warmer mass rearing room (34.7 ± 0.92 eggs per female)

compared to the 3°C cooler laboratory-reared line (30.98 ± 0.94).

22°C 25°C76

78

80

82

84

86

a

bSe

x ra

tio (%

fem

ale)

26

Figure 5: Mean (± SEM) number of Ephestia eggs parasitized per Trichogramma

papilionis female for the three founder treatments (1, 2 and 10 founder females). Bars not

connected by the same letter are significantly different.

One Two Ten0

10

20

30

40

Mea

n nu

mbe

r of e

ggs

laid

pe

r fem

ale

aaa

No. of founder females

27

Figure 6: Coefficient of variation in percentage parasitized Ephestia eggs for three

different founder population size colonies (Treatments, 1, 2, and 10 founder females) of

Trichogramma papilionis over 10 successive generations

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

1 Female

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

2 Females

0 2 4 6 8 100

20

40

60

80

Generation

CV

%

10 Females

28

2.4 Discussion

2.4.1 Variability among the fitness parameters

Overall, there was considerable variation in the essential characteristics of

parasitoid fitness across generations as a result of different founder population sizes.

There were significant differences in sex ratio and adult wasp emergence rate among

three different founder population sizes of T. papilionis, with the smallest, single female

founder populations showing the lowest fitness. Additionally, the wasps exhibited

different performances within two different environmental conditions in terms of their

fecundity, the mean number of parasitized host eggs per female. These results strongly

suggest that the number of founder females impacts multiple aspects of the progeny

fitness over generations, reducing fitness with female isolines, but remaining surprisingly

fit in colonies initiated even with only two females.

This study showed that there was no significant difference in the mean number of eggs

laid per female across generations irrespective of founder number. The level of eggs laid

per female was more consistent in the ten and two founder-female treatments than the

single-founder treatment, which suggests that increasing founder size has benefits in

terms of consistency of productivity in the wasps over multiple generations. Using a

factitious host with a low number of founder females might be responsible for

undesirable consequence when establishing a new colony of parasitoids. Hoffmann et al.

(2001) showed that T. ostriniae, a parasitoid of Ostrinia spp. (Pyralidae), reared on E.

kuhniella had typical longevity, but had lower fecundity compared with wasps that were

raised in different hosts, such as Ostrinia nubilalis and Citotroga cerealella. Moreover,

they suggested that wasps emerging from the inferior-quality hosts performed poorly

29

even when offered a higher-quality host, implying that Ephestia eggs produced lower

quality wasps, possibly with epigenetic effects that produce multiple reduced-quality

generations. A study by Prezotti et al. (2004) compared the effect of three founder sizes

on the quality of sexual populations of Trichogramma pretiosum under controlled

conditions. Their results indicated that the fecundity (mean number of eggs parasitized)

of progeny of one, five, and ten pairs of T. pretiosum varied significantly, where a

negative regression was observed between the mean number of laid eggs per female and

the inbreeding coefficient. Single-pair colonies showed a 14% reduction in the rate of

parasitism compared to the 10-pair colony. The results of this present study contrast with

the findings of Prezotti et al. (2004), where there was no significant difference in the

mean number of laid eggs per female for 1, 2, and 10 founder females. In addition, all

other studied traits (emergence rate, sex ratio, longevity, and percentage of deformed

adults), in the Prezotti et al. (2004) study, were not significantly different among all

parental population sizes. The results of the current study also have some similarities to

Prezotti et al. (2004), in how the performance of founder size significantly varied over

generations, with lower numbers of parental insects producing progeny that had more

variable performance. Trichogrammatidae in general, are considered to be arrhenotokous

(Hamilton, 1967; Stouthamer and Kazmer, 1994; Russell and Sothermare, 2011),

resulting in extreme female-biased sex ratios (Suzuki and Hiehata, 1985). The sex ratio of

T. papilionis was significantly affected by the initial number of founder females in the

colony, albeit possibly because some of these females were not inseminated prior to

removal from the emergence sites, where the high sex ratios of female wasps are closely

related to fertilization rate (Suzuki and Hiehata,1985).

30

Furthermore, the influence of various rearing conditions on the biological

characteristics of T. papilionis progeny was considered. In this study, founder wasps

showed varied performance in terms of reproduction and sex ratio of progeny when

reared under different environmental conditions. Similarly, Pratissoli et al. (2005)

showed that the sex ratio of T. pretiosum and T. acacioi progeny was affected by

temperature, while the number of individual wasps per parasitized egg was not affected.

The emergence rate (viability) of the progeny varied among treatments in the

experiments reported here. Ten and two founder wasps showed a higher level of viability

over multiple generations; single-founder colonies had an inconsistent pattern in

emergence rate across generations. Pratissoli et al. (2005) found that there was an effect

of varying temperatures on the emergence (eclosion) rate of T. pretiosum and T. acacioi

adults where they found a higher emergence rate for both species at 20, 25 and 30 ºC,

which is not consistent with the results presented here, where the emergence rate was

similar in both lines. Inbreeding between closely related individuals may result in

expression of recessive traits, due to the similarity between the pair mates’ genomes.

Inbreeding issues are known to have negative effects on fitness traits (Frankham, 2005).

inbred individuals are more likely to be sensitive to environmental stress than outbred

individuals, perhaps because environmental stress promotes the expression of detrimental

recessive alleles (Fox et al., 2011). However, this is not always the case, some studies

showed a significant correlation between inbreeding depression and extreme

environments, while other studies have shown the opposite (Fox et al., 2011; Frank and

Fischer, 2013). When Frank and Fischer (2013) studied the effect of the three

temperature treatments and the interaction with inbreeding in the tropical butterfly

31

Bicyclus anynana they found that in spite of even low inbreeding level, temperature

significantly affected some fitness-related traits including fecundity and egg hatching

success. However, they concluded that the results of their study did not support the

hypothesis that sensitivity to environmental stress is more likely to occur in inbred

individuals than outbred ones, despite the significant effect of temperature treatment on

some fitness measures. Fox et al., (2011) found good evidence for the effect of varying

temperatures on the larval development of the seed-feeding beetle, Callosobruchus

maculatus; rearing at 20°C did not impose significant stress on the outbred beetles, yet it

did impose the most stressful environment for inbred larvae. Here in the present study,

two different rearing temperatures showed varied effects on some fitness-related traits.

Sex ratio and female fecundity of T. papilionis progeny over multiple generations were

more impacted at the lowest rearing temperature. I suspect that the effects of inbreeding

depression become more evident at the lower, and possibly more stressful rearing

temperatures tested, as showed by Fox et al., (2011).

In conclusion, it is clear that larger numbers of founder insects are likely to

produce higher quality colonies in the long term. My data showed that as few as two

founder females provided acceptable emergence rate of wasp progeny, despite some

increased variability overall. A single founder female produced poor colonies as

measured by most fitness proxies measured. Interestingly, the three different founder size

treatments almost had the same parasitism rate. Rearing conditions impacted the

performance of the wasps. Additional work is needed to determine why more female

progeny and eggs per female were produced under the slightly warmer rearing

conditions.

32

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