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
i
Ó copyright 2020
By
Abdulla N. Ali
i
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.
ii 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
iii 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.
iv 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.
v 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
vi 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
vii 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.
viii 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
ix 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
x 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
xi 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
xii 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
xiii
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
xiv 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
xv 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
xvi 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
xvi
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.
xvii 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
1 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).
2 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
3 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.
4 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
5 (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.
6 References
Chailleux, A., Desneux, N., Seguret, J., Do Thi Khanh, H., Maignet, P., Tabone, E. 2012.
Assessing European egg parasitoids as a means of controlling the invasive South
American tomato pinworm Tuta absoluta. PLoS ONE 7: e48068.
Colazza, S., Peri, E., Salerno, G., Conti, E. 2010. Host searching by egg parasitoids:
exploitation of host chemical cues. In: Parra, J.R.P., Consoli, F.L., Zucchi, R.A.
editors. Egg parasitoids in agroecosystems with emphasis on Trichogramma.
Springer. 97-147.
Dicke, M., Van Loon, J.J., Soler, R. 2009. Chemical complexity of volatiles from plants
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10 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.
11 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).
12 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
13 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
14 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.
15 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
16 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).
17 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
18 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
19 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).
20 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 Statistical Estimate Stander Error F value p value Parameters Parameter (SEM) Emergence Intercept 92.4 0.85 108.20 0.0001 rate 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 Intercept 32.8 0.6 49.77 0.0001 eggs per female 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*
21 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).
a 100 a b 80
60
40
20 % Emergence rate
0 One Two Ten
No. of founder females
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
22
80 1 Female
60
40 CV%
20
0 0 2 4 6 8 10 Generation
80 2 Females
60
40 CV%
20
0 0 2 4 6 8 10 Generation
80 10 Females
60
40 CV%
20
0 0 2 4 6 8 10 Generation
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.
23 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).
100 b c 80 a
60
40
20 Sex ratio (% female)
0 One Two Ten No. of founder females
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.
24
86
84 a
82 b 80
78 Sex ratio (% female)
76 22°C 25°C
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).
25 40 a a a 30
20 per female 10
Mean number of eggs laid 0 One Two Ten No. of founder females
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.
26 80 1 Female
60
40 CV%
20
0 0 2 4 6 8 10 Generation
80 2 Females
60
40 CV%
20
0 0 2 4 6 8 10 Generation
80 10 Females
60
40 CV%
20
0 0 2 4 6 8 10 Generation
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
27 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
28 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).
29 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
30 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.
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40 CHAPTER 3
SEARCHING BEHAVIOR OF TRICHOGRAMMA PAPILIONIS IN RESPONSE
TO HOST EGGS, HOST PLANTS, AND INDUCED VOLATILE PLANT CUES
Abstract
Egg parasitoids have evolved various searching behaviors to facilitate finding and locating their hosts in nature. Plant volatiles and host cues induced by egg deposition may be used by female wasps to detect host eggs. Host seeking behavior in response to different infochemical cues was studied in Trichogramma papilionis (Hymenoptera: Trichogrammatidae) using Y-tube olfactometry. T. papilionis females preferred odors from Helicoverpa zea (Lepidoptera: Noctuidae) eggs compared to Ephestia kuehniella (Lepidoptera: Pyralidae) eggs. When plants were infested with H. zea eggs, T. papilionis preferred infested sunn hemp, Crotalaria juncea, over maize, Zea mays. No preference was observed in 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. The difference in plant volatile organic components with and without oviposition was investigated by head-space analysis. Compounds detected at notably higher concentrations in the egg plus plant material samples were found to be consistent with several compounds known to be pheromone-associated. Leaves with eggs oviposited on them produced some compounds that were unique compared to untreated plant material, and some of those compounds have known insect pheromone activity.
Keywords: Trichogramma papilionis; Helicoverpa zea, Ephestia kuehniella, sunn hemp, maize, searching behavior, olfactory bioassays.
41 3.1 Introduction
Parasitic organisms, especially parasitoids that are natural enemies of many phytophagous insects, are well known to exploit a blend of physical and chemical cues to find and locate a suitable host for their progeny in nature. Olfactory cues have a crucial role in host-natural enemy searching behavior in terms of host location and egg- deposition, particularly in parasitoids (Steidle and Van Loon, 2002).
Some plant species have been shown to release secondary metabolic compounds, 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).
Plant responses may be stimulated by chemicals present in the herbivore's regurgitation
(Hilker and Meiners, 2002), oviduct secretions (Hilker et al., 2005), or by some substances in oral secretions of larvae in lepidopteran species (Colazza et al., 2004;
Tumlinson and Lait, 2005), that may act as elicitors. Various factors, such as fertilization rate (Gouinguene and Turlings, 2002), genetic diversity (Degen et al., 2004), and the effect of plant pathogens (Rostás et al., 2006), have been found to have an impact on the induction of plant volatiles. Parasitoids are known to take advantage of plant-emitted volatiles as cues to find and locate their hosts or prey (Dicke and Sabelis, 1988; Turlings et al., 1990). Plants can benefit from the response of natural enemies of phytophagous insects to chemical volatiles as a defense mechanism (Vet and Dicke, 1992). Early defensive warning of egg deposition, before eggs hatch to produce the damaging larval stages could be particularly beneficial to plants (Tamiru et al., 2012). Chemical cues can influence the foraging strategies of natural insect enemies. Parasitoids can use chemical cues emitted by the plant to locate hosts within the habitat over long distances. Over
42 shorter distances, they can typically detect cues directly from their prey or hosts (Vet and
Dicke, 1992). Hypersensitive plant responses to egg deposition have been reported in
Brassica nigra (L.) to Pieris butterflies (Lepidoptera: Pieridae) (Shapiro and
DeVay,1987) and in potato, Solanum spp., to the Colorado potato beetle Leptinotarsa decemlineata (Say) (Balbyshev and Lorenzen, 1997). Plant response to egg deposition has attracted relatively little interest in comparison to the effects of feeding activity of herbivores (Schroder et al., 2005). However, recent studies on the role of oviposition induced plant volatiles (OIPVs) have opened novel perspectives in host location by egg parasitoids (Fatouros et al., 2008; Colazza et al., 2010). Several studies have revealed that parasitoid searching behavior can be affected by the combination of insect host chemical cues and OIPVs. These cues can originate from a range of sources, such as adult insect footprints to wing scales near oviposition sites, and their combination with OIPVs. These different host cues are often reliable signals for host location by parasitoids (Hilker and
Fatouros, 2015). Chemical cues can be a crucial point in host selection and searching behavior for a wide range of parasitic wasps including Trichogramma species (Lewis and
Martin, 1990; Schmidt, 1994; Fatouros et al., 2005; Vet and Dicke 1992; Vinson 1998;
Colazza et al., 2010). However, there have been very few studies on Trichogramma spp. searching behavior at all, particularly in relation to chemical cues.
The mechanisms of volatile plant emission resulting from egg deposition are varied, which suggests that plants can respond in different ways to egg deposition by different insect species. Hilker and Fatouros (2015) indicate that in many insect taxa, egg-derived elicitors such as exocrine secretions covering egg-shells were positively involved in the induction of plant defensive response, especially when the egg secretions
43 not only coat the egg shells but also include the area of plant tissue between egg and leaf surface. An application of egg-derived elicitors caused plants to respond similarly to normal oviposition (Fatouros et al., 2008). Oviposition by herbivorous insects can change the blend of plant volatiles released, and egg deposition induced plant volatiles have been shown to lure egg parasitoids, with terpenoid emissions being a key attractant (Hilker and
Meiners, 2010). Insect egg deposition can also change gene expression that moderates plant primary and secondary metabolic functions, for example, the modification of hundreds of genes by the oviposition of the large white P. brassicae (L.) on Arabidopsis thaliana (L.) leaves (Bruessow and Reymond, 2007). Egg deposition can induce changes in plant leaves and elicit the release of a diversity of plant synomones such as terpenoids, isothiocyanates, and green leaf components; these volatiles are not induced by a specific species or egg deposition behavior (Wegener et al., 2001; Schroder et al., 2007; Bruce et al., 2010; Buechel et al., 2011).
Trichogramma wasps (Hymenoptera; Trichogrammatidae) are egg parasitoids of
Lepidoptera, and they are often used in augmentative biological control programs against a diversity of phytophagous pests in many economically important crops. These egg parasitoids occur in different cropping systems and natural habitats worldwide, and their host searching behavior has been studied by some authors (Hassan, 1993; Wajnberg and
Hassan, 1994; Smith, 1996; Parra and Zucchi, 2004; Fatouros et al., 2005).
Trichogramma papilionis (Nagargatti,1974) has been observed to produce high levels of parasitism in cover crops such as sunn hemp (Crotalaria juncea) yet is an ineffective natural enemy of Helicoverpa zea in adjacent maize plants (Zea mays)
(Wright, unpublished data). Discovering ways to attract Trichogramma to target crops
44 using semiochemicals could significantly improve augmentative biological control using these parasitoids. This study addresses the following key questions, (1) do female wasps show a different response to stimuli from different egg hosts? (2) Do female wasps respond differently to different plant habitats? (3) Do chemical volatiles contribute to their responses? (4) What volatiles, if any, are involved in these interactions? The ability of T. papilionis females to respond to host cues and plant volatile cues was investigated using olfactometer bioassays. Female preference for different host eggs (H. zea and E. kuehniella), and different plant habitats (sunn hemp and maize) was examined. The effect of oviposition by H. zea on sunn hemp leaves, on T. papilionis host searching was also analyzed. Volatile organic compounds (VOCs) that might be induced by egg deposition of H. zea moths on sunn hemp plants, as well as the volatile emissions from the eggs per se, were identified.
3.2 Materials and Methods
3.2.1 Behavioral response of parasitoids
Y-tube olfactometer experiments with plants and/or egg host material were conducted in the laboratory to evaluate parasitoid responses to volatile emissions from the test materials (Yang et al., 2008; Faturos et al., 2012).
3.2.2 Dynamic Y-tube olfactory bioassays
A Y-tube olfactometer apparatus, as described by numerous researchers
(Takabayashi and Dicke, 1992, Peñaflor et al., 2011a, Fatouros et al., 2012, Graziosi and
Rieske, 2013, Fatouros et al., 2014) was used in these experiments. The glass
45 olfactometer system was obtained from Volatile Collection System Co. (FL, USA). This apparatus is appropriate for small insects like Trichogramma spp. and consists of a Y- tube with a 115 mm long central stem (i.d., 14mm) and two 150 mm arms set at a 65º angle (Graziosi and Rieske, 2013) as well as two (i.d., 19mm) trapping bulbs linked to the terminal end of each arm (Figure 1). Groups of 10 T. papilionis females were released into the Y-tube olfactometer for each trial (Fatouros et al., 2012). Air was pulled through silicone tubing from a vacuum pump (Model: SA55JXGTD-4144, Emerson, St. Louis,
MO. USA) into the system, purified and humidified by passing through two 180 mL
Pyrex vials containing activated charcoal and tap water respectively, then passed through a flow meter. Airflow at 300 mL/min was supplied to each arm of the Y-tube from each odor source container. The odor source containers used were dependent on the experiment: for plant experiments, airflow was passed through two 2000 mL flasks, each one containing a control plant (either sunn hemp or corn plants) or plant with H. zea eggs; on average six plants were used. For egg experiments, two 250 mL flasks were used, each containing either eggs (H. zea or Ephestia kuehniella eggs) or clean air. Eggs were placed directly in the container with a fine brush. All experiments were conducted at ambient temperature (21± 2ºC). The glass Y-tube system was placed horizontally into a plastic box, the walls of the box were covered with black tape to block external stimuli, and white paper was put on the bottom. The apparatus was illuminated from above with a
60W light bulb. Prior to testing, plants were gently removed from their pots, the roots soaked in water for 15 minutes, and then covered with aluminum foil, then placed in the odor source container (Fatouros et al. 2012).
46 Ten gravid female 72 h old T. papilionis were released simultaneously into the olfactometer by using an insect inlet adaptor. Female wasps, which were naive and had no previous contact with the odor sources, were obtained from a colony maintained on E. kuhniella eggs in the laboratory. According to Fatouros et al. (2012, 2014), wasps simultaneously released in a group have no impact on the behavioral response of one another in an olfactometer bioassay; each wasp acts individually in terms of searching and host location. As described in Peñaflor et al. (2011a), the behavioral response of the female wasps was monitored for 30 minutes, or until they traveled up either Y-tube arm past the "threshold line" located in the center of each arm. Wasps that passed this threshold were scored as having made a "choice," while those that did not leave the entrance or make a choice for one arm were considered to have made "no choice" and excluded from the statistical analysis. In total, six replicates were carried out per group, and each group of wasps was used only once. The olfactometer apparatus was disassembled after each replicate and washed with detergent liquid, tap water, and 70%
(v/v) ethyl alcohol, then dried using a heat source. After reassembling the device, the position of the odor sources and the Y-tube arms were switched to avoid any bias.
47
Figure 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.
3.2.3 Plants and Insects
Plants
Maize (Zea mays L.) and sunn hemp plants (Crotalaria juncea L.) were grown in a greenhouse (22±3°C, R.H. 70 –78%, L16: D8). Maize/sweet corn, variety Supersweet
#10, U. (commercial sources, Hamakua seed, and Supply Co.), and Sunn hemp, variety
Tropic Sun (Crop Care Hawaii LLC), were grown from seed. Seeds were germinated in a
Petri dish then individually transferred to plastic pots containing about 250 g potting soil.
Maize plants used in the olfactometer bioassay were grown until they had three fully expanded leaves (Peñaflor et al., 2011b). Sunn hemp plants were grown for 2-3 weeks and had 12-14 leaves.
Insects
48 Helicoverpa zea eggs
Corn earworm (H. zea) eggs were collected from cornfields at Waipahu, West
Oʻahu, Hawaiʻi. After emergence, H. zea caterpillars were placed individually in sterilized plastic vials containing soy-wheat germ (general artificial diet for lepidoptera,
Frontier Scientific Services, Inc.). The diet trays with the caterpillars were kept in a cage at 25 ± 2°C; 16L:8D. Emerging pupae were separated by sex and placed into rearing cages. For experiments using plants, intact plants (maize and sunn hemp) grown in the greenhouse were used as oviposition surfaces. For experiments using only eggs, the cages were lined with wax paper (Waxtex®), upon which moths deposited eggs, which were subsequently collected for experimental use. Adults were fed a 10% honey solution with moist cotton wool that was renewed every two days (Peñaflor et al., 2011a).
3.2.4 Egg deposition experiments
3.2.4.1 Egg-infested plants
As described by Peñaflor et al. (2011a) and Fatouros et al. (2012), 80-100 pairs of adult moths of H. zea (females – males 1:1) were offered both maize and sunn hemp plants in separate cages for one night to lay eggs. Female moths laid on average 41.5± 2 eggs per plant on both sunn hemp and maize plants. Plants were exposed for oviposition overnight and used for experiments the next day. Moths laid eggs predominantly on the lower leaves but also on other parts of the plant such as the stem, terminal bud, and petiole. Control plants were treated the same as infested plants but were not exposed to
H. zea moths or any other insects. Each plant (control and egg-bearing) was used only once and then removed from the experiment.
49
3.2.4.2 Helicoverpa zea and Ephestia kuehniella eggs
To obtain corn earworm eggs, about 50 pairs of adult moths were placed in cages
(30cm x 30cm). Sheets of folded wax paper (Waxtex®) were hung from the top of the cages to serve as a surface for egg deposition overnight, and eggs were collected using a fine brush the next day. E. kuehniella eggs were obtained from Beneficial Insectary Inc.
(Redding, CA, USA) and kept in the refrigerator before the olfactory experiments. On average, about 2,000 H. zea eggs and 0.5 g of E. Kuehniella eggs were used in the olfactometer and replaced after every two sets of wasps were exposed in the olfactometer.
3.2.5 Trichogramma Wasps
A T. papilionis colony was started with wasps emerging from Lampides boeticus
(Lycaenidae) eggs collected on sunn hemp, Waialua, Oʻahu, Hawaiʻi, USA. T. papilioinis wasps were maintained on E. kuehniella (Zeller) 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. Parasitized eggs were held in Plexiglas cages (15 cm x 15 cm x 15 cm) under the environmental conditions mentioned above. Upon emergence, adult parasitoids were provided clusters of E. kuehniella eggs with droplets of pure honey as an energy source. Glue (Elmers Glue®) was used to attach the factitious host eggs onto the yellow cardboard strips (10 cm x 5 cm). T. papilionis females used in experiments were 72 h old and allowed to mate with males before trials. Male T. papilionis emerge about 12 h before females, and they wait on parasitized eggs until females emerge. Copulation happens soon after emergence.
50 Both sexes remained alive for 4-6 days when provided with honey droplets.
3.2.6 Volatile collection and headspace analysis
Dynamic headspace analysis was conducted to collect sunn hemp plant volatiles and corn earworm egg volatiles. Only sunn hemp was investigated for semiochemicals as there was no significant increase in attraction of the wasps to maize leaves with eggs in the olfactometer experiments. Volatile compounds were collected by pulling air through
Pyrex containers containing the odor sources at a rate of 200 mL/min for 4h, through a preconditioned glass cartridge filled with approximately 150 mg of sorbent material,
Tenax TA (35/60 mesh; Restek, USA). The containers were connected to a vacuum pump. Air was pulled through silicone tubing into the system using a vacuum pump, purified and humidified by passing through two different 180 mL Pyrex vials containing activated charcoal and tap water, respectively, then passed through a flow meter. A 300 ml/min rate of airflow was applied using a vacuum pump. Prior to sampling, plants were taken from pots, the roots wrapped in aluminum foil, and placed in a 500 mL Pyrex jar for 15 minutes to avoid volatile compounds that could be emitted from the roots. For trace level (low ppb or ppt level) analyses, blank desorptions were run before trapping commenced. Containers were disassembled after each run and washed with detergent liquid, tap water, ethyl alcohol 70%, then dried with a heat source at 45ºC for 20 min. In total, 12 plant samples (treatment vs. control) were taken on 12 different days, similar to
Fatouros et al. (2012). Volatiles were sampled at 12, 24, and 48 h after oviposition by H. zea. Each plant was only used once. Upon finishing the volatile collection, the sorbent tubes were capped with 1/4in brass storage caps, tightened finger-tight plus a quarter
51 turn, and stored at ambient temperature until shipped to Jordi Labs (Mansfield, MA) for analysis. Also, corn earworm egg volatiles were collected under similar controlled conditions in the lab from more than 2000 eggs, using the same procedure as for plant volatile collections.
To qualitatively investigate the difference in volatiles, Desorption Gas
Chromatography-Mass Spectrometry (DMS) was carried out at Jordi Labs (Jordi Labs,
MA, USA). The sample was analyzed using a Gerstel Thermal Desorption Unit, Gersel
Cooled Injection system, Agilent 7890A gas chromatograph, and a 5975C mass selective detector using gas injection. A majority of the sorbent material in the headspace trap was removed from each sample and placed in DMS vials for analysis without further preparation. The run conditions of Thermal Desorption (TD) were initial temperature
50°C, ramp rate 350°C/min, ramp end temperature 300 °C, and ramp hold time 3 min.
The cooled injection system was standard heater mode, initial temperature -100 °C, equilibration time 0.40 min, initial time 0.00 min, ramp rate 12.00 °C/s, ramp end temp.
310°C and ramp hold time 3.00 min. Finally, the run conditions of GC analysis had an initial delay of 0.10 min, initial oven temp. 50 °C, hold time 1:5 min, final hold time 10 min, detector temp. 28 °C, mass range (low mass 29, high mass 550), and column HP-5
30M x 0.25 x 0.25 µm film. Data were collected using Chemstation software (Agilent
Technologies, Germany). Sample peaks were compared with over 796,613 reference compounds using the NIST/EPA/NIH mass spectral search program (Jordi Labs protocols). For precise details of the TD-GCMS(DMS) process, see (Fatouros et al.,
2012; Wong et al., 2013; Maceira et al., 2017 and Marsol-Vall et al., 2018).
52 3.2.7 Statistical analysis
The behavioral response of T. papilionis females to stimuli was assessed using
Fisher's exact test. JMP13 Pro® (SAS Institute, Cary, NC) was used to evaluate the statistical significance of differences in the choice of females to olfactory cues as following: 1) H. zea eggs versus clean air, and E. kuehniella eggs versus clean air; 2) H. zea eggs versus E. kuehniella eggs); 3) Egg-infested corn plant versus control plant (non- treated plant) and egg-infested sunn hemp plant versus control plant; 4) Egg-infested maize plant versus egg-infested sunn hemp plant. Female wasps that did not make a choice were excluded from the statistical analysis.
3.3. Results
3.3.1 H. zea and E. kuehneilla eggs versus blank air
In this assay, the female wasps showed a statistically significant positive response to H. zea eggs vs. clean air (control) in the Y-tube olfactometer experiment (Fisher's exact test p = 0.012, Figure 2-A), with 53% making a choice for corn earworm eggs and about 38% choosing the blank air arm. A non-significant response was observed for E. kuehniella eggs against control (Fisher's exact test p = 0.10, Figure 2-A).
3.3.2 H. zea eggs versus E. kuehneilla eggs
The female wasps responded significantly more frequently (Fisher’s exact test p =
0.002, Figure 2-B) to the cues from fresh H. zea eggs than to E. keuhneilla eggs. While
* 53 54% of the wasps chose H. zea eggs, about 22% selected the E. kuehneilla side in the y- tube olfactometer.
3.3.3 H. zea egg-infested sunn hemp plant and maize plant versus uninfested plant
T. papilionis female wasps demonstrated a highly significant response to sunn hemp plants infested with H. zea eggs compared to uninfested sunn hemp plants (control)
(Fisher's exact test p = 0.001). Approximately 59% of the tested wasps preferred the egg- infested sunn hemp plants over non-infested sunn hemp plants (Figure 2-C). Similarly, female wasps were significantly more attracted to maize plants with H. zea eggs over intact maize plants (Fisher's exact test p = 0.02, Figure2-C).
3.3.4 H. zea egg-infested sunn hemp versus H. zea egg-infested maize
The olfactometer assay results show strong evidence that the wasps responded positively to the sunn hemp plant substrate with eggs compared to Z. mays (Fisher exact test p = 0.006). While sunn hemp plants with H. zea eggs attracted approximately 48% of the female wasps, about 33% of the wasps were attracted to egg-infested maize plants in the olfactometer (Figure 2-D).
54 A B 60 ** Treated 60 Blank air ** Helicoverpa zea Ephestia kuehneilla 40 ns 40
20 20 % Response of female wasps
0 % Response of female wasps 0 Helicoverpa zea Ephestia Kuehneilla Choice 80
C Treated D ** Control 60
60 ** * Egg- infested sunn hemp plant 40 Egg- infested maize plant 40
20 20 % Response of female wasps
0 0 Sunn hemp plant Maize plant % Response of female wasps Choice
Figure 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 3.3.5 Headspace volatile collection from sunn hemp plants.
The DMS chromatogram for the Treatment (sunn hemp with eggs) and Control (sunn hemp without eggs) samples is presented in Figure 2, and a summary of identifications of the major peaks detected in the samples are shown in Table 1. A large number of peaks were detected across both samples. These included compounds consistent with linear alkanes, aldehydes, aromatics, polyterpene-related compounds, naphthalene derivatives, and ester-related compounds. Notable peaks detected at higher concentrations in the Treatment sample were found to be consistent with anisole, β-myrcene, cis-butyric acid, trans-isoeugenol, and bis(2-ethylhexyl) phthalate. In total, 55 potential semiochemical compounds were detected, and eight volatile components were unique to treatment samples (3 non-pheromone related, five pheromone- related) (Appendix A). However, it is not certain whether the volatile emissions were induced in the plant by egg-deposition of H. zea moths, or if they were kairomones directly from the eggs.
In this regard, it is worth considering the relative abundance inferred from the DMS overlays
(Figure 2, 3) of anisole is about 0.5x 107 in the corn earworm egg-infested sunn hemp plant DMS chromatograms analysis, for approximately 200 eggs plus plant material. In the corn earworm
DMS overlay (Figure 3), the abundance is approximately 3.5x107, for 2,000 eggs. Similarly, the relative abundance of bis(2-ethylhexyl) phthalate is approximately 1.5x107 in both sunn hemp with corn earworm eggs and with corn earworm eggs alone. Correcting the abundance of each compound for the number of eggs in the plant plus eggs assay compared to corn earworm egg assay indicates that the concentration of the compounds released in the sunn hemp plus egg assays was substantially higher than would be expected if they were released from the eggs included in the assays, alone. Oviposition thus appears to have induced higher release of these compounds from the sunn hemp plants.
56
Figure 3: Overlay of DMS chromatograms for uninfested Crotalaria juncea plants - Control
(blue), C. juncea with Helicoveropa zea eggs - Treatment (green), and a control blank (red).
Numbers in parentheses above peaks identify compounds (see Table 1) that were determined to be of particular interest.
3.3.6 Dynamic headspace analysis of H. zea eggs
A majority of peaks detected were found to be 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 noted to have also been observed in corn earworm egg-infested sunn hemp plants, including anisole and myrcene, which were the two most significant peaks in the corn earworm egg spectrum (Figure 3). Furthermore, a summary of the major peaks detected in the DMS chromatogram is presented in Table 1. Also,
57 some volatile compounds were observed to be common to the sunn hemp control and treatment samples as well as to the corn earworm eggs sample (Table1, Appendix B).
Figure 4: Overlay of DMS chromatograms of corn earworm (Helicoverpa zea) eggs and a control blank. Numbers in parentheses above peaks identify compounds listed in Table 1.
58 Table 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.
No. Chemical volatiles Corn Sunn hemp+ Corn Earworm Sunn hemp Earworm Eggs (treatment) plant (control) Eggs 1 anisole + Greater peak area in the + treatment sample Pheromone-related 2 β-Myrcene + + - 3 cis-3-Hexenyl butyric - Unique - acid 4 bis(2-ethylhexyl) + Greater peak area in the + phthalate treatment sample
5 trans-Isoeugenol - Unique - Pheromone-related 6 acetophenone + + - 7 isobutyl tetradecyl - - Unique carbonate 8 nonanal + Unique - 9 nonanol - - Unique 10 5-Hexen-2-one3- - - Greater peak acetyl-4-methyl area in the control sample 11 α-Farnesene Unique - - Pheromone-related 12 benzene, (1- + - - methylethyl)
59 3.4. Discussion
T. papilionis females showed variable searching behavior in response to the range of cues to which they were exposed in this study. Trichogrammatidae wasps demonstrate different searching behaviors and responses when they are provided with different infochemical cues, such as oviposition induced plant volatiles. Egg parasitoids can be arrested by plant volatiles when they enter the habitat and experience arrestment cues. Some studies have shown that
Trichogramma spp. respond to either herbivore infested plants (feeding activity or egg- deposition) or healthy plant volatiles, and have been shown to respond to sex pheromones and kairomones from phytophagous insects eggs or moth wing scales (Lewis et al., 1982; Noldus and
Van Lenteren, 1983; Noldus et al., 1991; Frenoy et al.,1992; Romeis et al., 1997; Fatouros et al.,
2005, 2007, 2008; Alsaedi et al., 2016). This study evaluated T. papilionis response in olfactometer assays to different egg hosts and oviposition induced plant volatiles that could impact the searching behavior efficacy of T. papilionis as a potential augmentative biocontrol agent in crop fields (e.g., maize), possibly deployed as attractants or arrestants in crop fields to concentrate the host-seeking efforts of the wasps in the target crop.
3.4.1 The response of T. papilionis female wasps to egg hosts
T. papilionis responded positively to H. zea eggs over clean air. In contrast, the wasps did not react significantly to E. kuehneilla compared with blank air control, demonstrating a distinct response to volatile emissions from H. zea eggs compared to E. kuehniella eggs. The significant response to H. zea eggs shows that infochemical (semiochemical) signals were still being emitted from eggs that were 24 hours old. These chemical cues from the eggs alone were adequate to illicit a response from the wasps in the olfactometer, in contrast with other studies that have
60 reported a response in trichogrammatid wasps to host egg only in the presence of moth scales, hairs, and ovipository secretions (for details see Fatouros et al., 2008; Colazza et al., 2010). The non-significant response in this study of T. papilionis females to the factitious host (E. keuhneilla) was due to either a lack of chemical cues or the age of the eggs. The wasps did not respond to olfactory stimuli from these eggs even though the wasps were reared on E. kuehneilla eggs for several generations in the laboratory colony, where the eggs are readily parasitized under high-density rearing conditions. Rearing host and egg deposition behaviors were shown by
Kaiser et al. (1989) and Fatouros et al. (2005) to influence the behavioral response of
Trichogramma wasps, so this result is somewhat surprising as it would appear that our colony insects have not become habituated to the factitious host eggs. Evaluation of T. ostriniae in Y- tube olfactometer assays demonstrated an innate positive response to the egg mass volatiles, scale volatiles, and synthetic sex pheromones of Ostrinia nubilalis (Hübner), and that by exploiting these host cues, T. ostriniae could efficiently find and locate O. nubilalis eggs in the field (Yong et al., 2007). Furthermore, trichogrammatid wasps have been shown to exhibit a positive behavioral response in terms of host egg kairomones (Colazza et al., 2010). Chemical compounds from moth scales and ovipository secretions might be involved in the response of T. papilionis to H. zea eggs.
3.4.2 The response of T. papilionis to plant volatiles
The response of T. papilionis females varied between the two different plants (habitats) offered as choices to the wasps. The wasps were highly responsive to sunn hemp plants with H. zea eggs, compared with control plants (intact sunn hemp plants), and preferred egg-infested maize plants over the control (intact uninfested corn plants), This suggests that there was an
61 interaction between the eggs and the sunn hemp plants and between the maize and the eggs as well, producing a stimulus that attracted the wasps, although to a substantially lesser extent than in the case of sunn hemp. Studies have shown that egg-deposition may induce plant volatiles as a defensive response. For example, Shapiro and DeVay (1987) showed that oviposition of P. brassicae and Pieris rapae (L.) eggs caused a hypersensitive response in mustard leaves
Brassica nigra (L.). Meiners and Hilker (1997, 2000) investigated the plant volatiles induced by egg- deposition activity on elm trees, Ulmus minor (Miller). They found that the elm leaf beetle
Xanthogaleruca luteola (Müller) (Coleoptera: Chrysomelidae) can induce the trees to release chemical volatiles that are detected by the egg parasitoid Oomyzus gallerucae (Fonscolombe)
(Hymenoptera: Eulophidae). The much stronger response observed in the current study, of T. papilionis to corn earworm egg infested sunn hemp over corn earworm egg infested maize might be due to the absence of maize plant defensive substances induced by H. zea ovipostion on a commercial maize variety. Open-pollinated varieties (maize landraces) originating from Latin
America (central Mexico) can react to spotted stemborer Chilo partellus (Swinhoe) egg deposition and release semiochemical volatiles including HIPVs. In contrast, some commercial hybrid maize varieties lack this adaptation. This may occur due to the loss of some defensive traits during selective crop breeding (Tamiru et al., 2011, 2012). It has previously been shown that egg deposition on Z. mays does not always result in the attraction of parasitoids. Peñaflor et al. (2011b) showed that egg deposition by the moth Spodoptera frugiperda (J.E. Smith)
(Lepidoptera: Noctuidae) on maize leaves could result in limited plant volatile emissions or a low level of plant synomones in comparison to egg-free maize. These factors may help explain the strong response of T. papilionis to sunn hemp with eggs compared to the relatively small response to the maize with eggs in this study.
62 In this headspace analysis study, volatile profiles of H. zea egg-infested sunn hemp plants
(Treatment) and clean sunn hemp plants (Control) showed a diversity of semiochemical compounds ranging from alkanes, aldehydes, aromatics, polytepene-related compounds to naphthalene derivatives and ester-related compounds (Table 1). Notably, some volatile emissions
(peaks) were found at higher concentrations in the Treatment sample, which means that egg- deposition of corn earworm might trigger sunn hemp plants to release some early warning secondary metabolic-compounds in response to the oviposition of corn earworm moths.
Our dynamic headspace analysis of corn earworm eggs revealed that the eggs per se release a diversity of semiochemical compounds. These chemical volatiles were mostly phosphates, pheromone-related compounds, various natural products, a series of glycol-related compounds, and a series of fatty acid ester-related compounds. Many of these compounds were also observed in sunn hemp plant samples, including anisole and myrcene, which were the most significant peaks in the GC analyses. There is a possibility that degradation products of chemical compounds originally collected, resulted from heat treatment during the GC-DMS process.
These compounds may be breakdown products of the parent molecules coming from the eggs and/or plants due to heating to achieve headspace analysis, but this is unlikely to have resulted in major deviations in the identification of volatile products from the samples in this study.
In conclusion, this is the first study to document the response of T. papilionis to host egg and plant volatile emissions. While the egg parasitoids showed a positive response to both H. zea eggs and egg-laden sunn hemp plants, significant responses were not observed for either the factitious host (E. kuehneilla) or egg-infested maize. Identifying the compounds that attracted the wasps to plants with eggs may be useful in developing means to attract the parasitoids to an atypical crop-pest system, and thus enhance augmentative- and conservation biological control
63 options. Chapter four addresses the effect of chemical volatiles (using synthetic compounds) that were found to be released by the stimulated sunn hemp plant infested with corn earworm eggs, on the behavior of T. papilionis.
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70 CHAPTER 4
RESPONSE OF TRICHOGRAMMA PAPILIONIS WASPS TO BLENDS OF SYNTHETIC
SEMIOCHEMICALS
Abstract
Parasitoid wasps may rely on chemical volatiles from herbivorous insects and their plant hosts as infochemical cues for finding their preferred host. This study was conducted to test the reaction of Trichogramma papilionis Nagarkatti (Hymenoptera: Trichogrammatidae) to chemical volatiles that were qualitatively detected from host eggs and egg infested plants using headspace analysis. Specific blends of semiochemicals may be highly significant in terms of the effect they have on insects. Several volatile compounds were selected, singly, and as combinations of chemicals to determine whether these volatile compound blends could enhance orientation responses of T. papilionis wasps toward the source of the stimulus. In a Y-tube olfactometer, female wasps were significantly attracted to only two chemical volatiles among the seven compounds selected from the headspace analysis. Only anisole and bis(2-ethylhexyl) phthalate were attractive to T. papilionis wasps when used separately. Additionally, the wasps showed a significant attraction toward a blend of anisole and bis(2-ethylhexyl) phthalate at a ratio of 25 μL/100 μL. There was no significant response to the other concentration ratios of that blend. This study aimed to find specific compounds or blend of compounds to potentially manipulate the performance of T. papilionis wasps under field conditions fields and increase their efficacy.
Key Words: Trichogramma papilioins, Y-tube olfactometer, headspace analysis, chemical volatiles, anisole, bis(2-ethylhexyl) phthalate.
71 4.1 Introduction
In general, plant volatiles are considered to have a significant role in tri-trophic interactions or multitrophic interactions. This is considered especially significant in regard to plant-insect interactions, where semiochemicals can perform as cues in insect host finding or as indirect plant defenses, such as by attracting natural enemies of herbivores (Shrivastava et al.,
2010). Natural enemies of many herbivores can be attracted to a blend of chemical volatiles emitted from the infected plant (Dicke et al., 2003; Dicke, 2009). Volatile chemicals are well known to attract parasitoid wasps toward their hosts. Therefore, the biggest challenge for the wasps is to exploit and detect the right volatile compound or blend of volatiles that can assist in distinguishing host cues among a tremendous variety of odors of many origins, that may present at the same time and place (Hilker and McNeil, 2008). Studies of olfactory cues have been used over the last few decades to elucidate the searching behaviors of beneficial insects; for example, parasitoids are drawn toward cues originating from the host and its plant host (Avila et al., 2016).
The specific compound or blend of compounds that will be attractive to the insect species is a crucial aspect in mediating searching behavior (Turlings and Ton, 2006). In addition, Kaplan
(2012) suggested that studies can definitively identify which volatile should be used when we seek to enhance the efficiency of a single natural enemy, which can be easier/more effective than enhancing the performance of multiple natural enemies. The suggested course of studies is first volatile collection and identification, followed by studies of the olfactory response of natural enemies to the proposed volatiles in the laboratory. In that way, the most attractive compound chosen under in vitro conditions could be assessed as a prospective attractant in the field
(Kaplan, 2012). Despite that, there are few studies blends of inducible plant volatiles responsible for the response of natural enemies. A study by Gols et al. (2009) showed different
72 semiochemical compounds alone or as a mixture can be used as cues by the parasitoid wasps to find their hosts and can also affect their behavioral preferences. The specific compound(s) within a blend of semiochemical volatiles responsible for the response of natural enemies is the question posed by many studies (e.g., Scutareanu et al. (1997). A study by De Baor and Dicke (2004) was among the first to address this, by testing the variation in responses of the predatory mite
Phytoseiulus persimilis to two different blends of volatiles. Synthetic plant volatiles have been studied in laboratories regarding natural enemy attraction, but not much has been done under field conditions (Simpson et al., 2011).
Understanding the searching behavior of beneficial insects can provide useful information to improve the efficacy of natural enemies, for example, determining their habitats, or release strategies would be helpful as augmentative biological control agents (Landis et al.,
2000). In this context, studies of searching behavior have been done for many insects, examining how they use the chemical compounds to communicate with each other. These studies have improved our understanding of how natural enemies, particularly parasitic wasps, perceive the cues and exploit them in host finding (Wajnberg and Colazza, 2013). However, parasitoid wasps show different degrees of performance on different insect hosts or the same host in different crops, due to the differences in multi-trophic interactions. A unique blend of volatiles can be released from different plant parts when eaten by the same herbivore on the same plant
(Tumlinson et al., 1993). Furthermore, Trichogramma searching behavior and parasitism rate can be manipulated using plant volatiles (Altieri et al., 1982). Ravi et al., (2006) mentioned that different parasitism rates by Trichogramma wasps had been measured for the same host from different crops due to the variation in info-chemicals influencing the parasitoids searching effectiveness.
73 Chemical ecology can be applied in augmentative and conservation biological control efforts that seek to enhance and increase natural enemy activity by attracting beneficial insects
(predators and parasitoids) into the pest region and providing resources that sustain them there.
Many semiochemical compounds are commercially available, and these artificial compounds are used to attract natural enemies or trigger plants to produce their volatiles as attractants (Khan et al., 2008). Allison et al. (2009) point out that olfactometer bioassays alone do not clearly demonstrate how natural enemies respond to volatile organic compounds (VOCs), they may simply be useful tools to identify and illustrate the response of beneficial insects to VOCs.
Different techniques have been used to explore which chemicals are essential in the interactions between parasitoid insects, plants, and herbivorous hosts, and how the insects can maximize useful information from VOCs (Wilson et al., 2015). These techniques quantify behavioral, physiological, and neurological aspects of these interactions. Wilson and his colleagues (2015) defined two broad mechanisms explaining how insects decipher the infochemical cues. This includes species-specific semiochemicals, where insects can use a specific chemical compound(s) emitted by an individual plant or herbivorous host; the second broad mechanism is where insects respond to a particular ratio of concentrations of chemical compounds within a blend. Both general scenarios may be highly significant in influencing parasitoid behavior and should be elucidated if efforts to use semiochemicals to manipulate parasitoids under field conditions are planned.
In this study, chemical compounds of interest were selected from a previous study (see
Chapter 3). These chemical compounds were tested synthetically to find the cues that attracted
Trichogramma papilionis females to sunn hemp and maize plants infested with Helicoverpa zea eggs (Chapter 3). The objective was to find a promising volatile or a blend of volatiles that
74 maximize Trichogramma papilionis response and could potentially be used to enhance and increase the efficiency and performance of T. papilionis in the field.
4.2 Materials and Methods
4.2.1 Test insects
The egg parasitoid T. papilioins used in these olfactory bioassays was laboratory-reared using irradiated Mediterranean flour moth Ephestia kuehniella (Zeller) (see Chapter 2 and 3 for more details).
4.2.2 Y-tube olfactometer bioassays
The same Y-tube olfactometer apparatus described in Chapter 3 was used in these experiments.
4.2.3 Compounds of interest
Bis(2-ethylhexyl) phthalate (DEHP):
Chemical and physical data (ECHA, 2008; HSDB, 2010; Lide, 2010).
Chemical abstract service registration number (CAS) No.: 117-81-7.
Synonyms: DEHP, 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester, bis(2-ethylhexyl) 1,2- benzenedicarboxylate, bis(2-ethylhexyl) o-phthalate, bis(2-ethylhexyl) phthalate, di(2- ethylhexyl) phthalate, dioctyl phthalate.
75 Chemical structure:
Molecular formula: C24H38O4
Relative molecular mass: 390.56 g/mol.
Description: Colorless liquid with almost no odor.
Boiling point: 384 °C.
Melting-point: –55 °C.
Density: 0.981 g/cm3 at 25 °C.
Solubility: Sparingly soluble in water (0.27 mg/L at 25 °C); slightly soluble in carbon tetrachloride; soluble in blood and fluids containing lipoproteins; miscible with mineral oil and hexane.
Volatility: Vapor pressure, 1.42 × 10−7 mm
Hg at 25 °C
3 Octanol/water partition coefficient: log Kow , 7.6
Conversion factor in air: 1ppm = 15.94 mg/m3 ppm = 15.94 mg/m3
76 Phthalate derivatives compounds are detected in secondary metabolites of organisms, including plants, animals, and microorganisms (Zhang at el., 2018). DEHP has been detected in several different sources such as water, air, soil, and living organisms (ATSDR, 1993). Also,
DEHP has been shown to be acquired by the plants from the environment, in four vegetable crops including Brassica chinensis L. (bok choy), Brassica campestris L. (field mustard), Vigna unguiculata Walp. (cowpea), and Solanum melongena L. (eggplant) (Du et al., 2010). A study by
Al-Bari et al. (2005) reported that bis(2-ethylhexyl) phthalate is produced by the thermophilic bacterium species Streptomyces bangladeshensis as an antimicrobial agent. As a pollutant,
DEHP is an endocrine disrupting chemical that causes a significant drop in the activity of EcR transcription gene of the heterodimeric ecdysone receptor complex on the aquatic larvae of
Chironomus riparius (Planelló et al., 2011). There are suggestions that DEHP is produced as a metabolite by plants as well as microorganisms, and that the compound may have some beneficial impacts (Ortizt and Sansinenea. 2018). DEHP has been isolated from crown flowers
(Calotropis sp.), and it is reputed to have antimicrobial effects (Ortizt and Sansinenea 2018). It has been suggested to provide allelopathic properties to crofton weed (Ageratina adenophora) which may contribute to seedling mortality in competitor species (Yang et al. 2016). It thus seems potentially viable that DEHP has evolved in some plants as a defense mechanism against pathogens and competing plants; selection as a metabolite that confers defense against herbivorous insects may a further ecological function of DEHP. Aviles et al. (2019) demonstrated ecdysteroid function and possible antagonist effects in Spodoptera littoralis
(Noctuidae), and prolonged larval/pupal stages, with delayed in emergence caused by DHEP.
77 Anisole
Chemical and physical data (NIST, 2020)
Molecular formula: C7H8O
Molecular weight: 108.1378 g/mol.
CAS Registry Number: 100-66-3
Chemical structure:
Anisole Properties
Melting point: -37 °C
Boiling point:154 °C(lit.)
Density 0.995 g/mL at 25 °C(lit.) vapor density 3.7 (vs air) vapor pressure 10 mm Hg (42.2 °C)
The chemical compound anisole was identified from Stachys riederi var. japonica
(Family: Lamiaceae) and it was found to have fumigant toxicity against maize weevils
(Sitophilus zeamais) and booklice (Liposcelis bostrychophila) (Quan et al., 2018). This chemical also found in mature green berries of Coffea arabica and it has strong attraction (solely and in a blend) to nymphs of the variegated coffee bug Antestiopsis thunbergii Gmelin (Heteroptera:
Pentatomidae). A study by Steenhuisen et al., (2013) showed that flower heads of Protea species
78 (Proteaceae) released the anisole, and that it attracts pollinators, cetoniid beetles (Atrichelaphinis tigrina). the role of anisole in plant physiology appears to be largely unexplored – no searches in abstracting systems returned any citations for ‘anisole’ and ‘plant physiology’.
4.2.4 Tests of Trichogramma response to volatile compounds
For volatile chemical testing, all synthetic chemical compounds were delivered as a solution formula from Sigma Aldrich Inc. Seven semiochemical compounds identified as potentially influencing host detection in T. papilionis in previous work (Chapter 3) were tested as attractants of T. papilionis females, initially each one alone. These volatiles were trans-isoeugenol (> 99% purity), anisole (> 99.7% purity) , β-myrcene (> 95% purity , cis-3-Hexenyl butyric acid (bis(2- ethylhexyl) phthalate (> 98% purity), acetophenone (> 98% purity), and α-farnesene (> 95% purity). A dose of 100 μL was applied as the treatment for each compound tested. Compounds were selected specifically because of their substantial or unique presence in treatment samples
(e.g.) greater gas chromatograph (GC) peak areas with the presence of eggs on plant material, or pheromone-related compounds and unique to plant material with eggs suggesting a possible release of semiochemicals in response to oviposition (Chapter 3: Table1). In addition, there are some interesting interactions known among those chemicals and insect taxa (see Appendix C).
GC peaks were used per the suggestions in the Agilent user’s manual (Agilent ChemStation,
2004, p.114) to estimate the ratio of chemical volatiles to each other. The ratio of anisole to bis(2-ethylhexyl) phthalate (0.26:1) was estimated by comparing the heights of peaks from gas chromatography (GC-DMS) chromatograms on volatiles released from sunn hemp leaves with
H. zea eggs (Chapter 3). Further, the response of female wasps to blends with varying ratios of these chemical compounds was tested. The wasps were provided with a choice between a single
79 chemical compound or combination of compounds in one olfactometer arm and filtered air only in the other arm. A micropipette was used to place individual compounds or a blend of chemicals at different volumes or volume ratios of each chemical placed onto the filter paper.
Concentrations of the chemicals are as follows: anisole: 9.201 mol/L; bis(2-ethylhexyl) phthalate: 2.522 mol/L; trans-isoeugenol: 6.601 mol/L; β-Myrcene: 5.806 mol/L; cis-3-Hexenyl butyric acid: 5.198 mol/L; acetophenone: 8.572 mol/L; α-Farnesene: 4.301. Treated samples were inserted into one arm of the Y-tube olfactometer (Zhu et al., 2017). The other arm was clean air (control).
As described before (see Chapter 3), three-day-old mated female T. papilionis were released into the olfactometer in groups of 10 individuals. Female wasps were naive and had no previous contact with the odor sources. Wasps were observed for 15 minutes or until they traveled up either Y-tube arm past the "threshold line" located in the center of each arm (Peñaflor et al., 2011). Wasp choices were categorized depending on their behavioral response. Wasps were scored as having made a "choice" when they passed the threshold line mentioned above, while those that did not leave the entrance or make a choice for one arm were considered to have made "no choice" and excluded from the statistical analysis. In total, three or four replicates were carried out for each concentration of a chemical, and each group of wasps was used only once.
The risk of conditioning/ desensitizing the test animals to the chemicals being tested was avoided by using new batches of wasps each time. The olfactometer apparatus was disassembled after each replicate and washed with detergent liquid, tap water, and 70% (v/v) ethyl alcohol, then dried using a heat source. After reassembling the device, the position of the odor sources and the
Y-tube arms were switched to avoid any bias.
80 4.2.5 Statistical analysis
The behavioral response of T. papilionis females to selected stimuli was assessed using
Chi-Square Goodness of Fit tests and compared with a two-tailed exact binomial test. JMP13®
(SAS Institute, Cary, NC) was used to evaluate differences in the choice of T. papilionis females to olfactory volatiles. Female wasps that did not choose any side of the olfactometer (“no choice”) were excluded from the statistical analysis.
4.3 Results
4.3.1 Response of Trichogramma wasps to volatile compounds
Trichogramma papilionis female responses to each of the seven semiochemical compounds are shown in Figure 1. Wasps were preferentially attracted to the olfactometer arm containing anisole and bis(2-ethylhexyl) phthalate (χ2 = 5.76, d.f. = 1, p = 0.016 and χ2 = 7.36, d.f. = 1, p = 0.0067 respectively) over the blank air (control) arm. In contrast, female wasps showed a significant preference for the blank air (control) over cis-3-hexenyl butyric acid (χ2 =
4.54, d.f = 1, p = 0.032). β-myrcene (χ2 = 0.57,d.f = 1, p = 0.449), trans-isoeugenol (χ2 =
0.64,d.f. =1, p = 0.423), acetophenone (χ2 = 1.19, d.f. =1, p = 0.273) and α-farnesene (χ2 =
0.23,d.f. =1, p = 0.408) were also tested but were not found to influence the wasps behavior significantly (Figure 1). Also, the wasps' response to different concentrations of each compound varied (Figure 2). The most attractive concentration for each chemical compound is shown in
Figure 3: 43.3% of wasps responded to anisole at 100 μL and 150 μL over the control (Figure 2-
B), with 10% and 23.3% responding to clean air, respectively. In the treatment with 100 μL of bis(2-ethylhexyl) phthalate over blank air 52.5% of female wasps chose the chemical cue compared with 27.5% that selected the control arm of the Y-tube (Figure 2-C). Female wasps
81 also were shown to have a significantly increased positive response to the combination of anisole and bis(2-ethylhexyl) phthalate only at a blend ratio of 25 μL /100 μL (χ2 = 4.65, d.f. = 1, p =
0.031) (Figure 3). On the other hand, no significant response was found to anisole and bis(2- ethylhexyl) phthalate in the following ratios: 100 μL/25 μL (χ2 = 0.46, d.f.= 1, p = 0.5001); 25
μL/50 μL (χ2 = 0.24, d.f.=1, p = 0.587); 25 μL/25 μL (χ2 = 0.14, d.f.=1, p = 0.705) (Figure 3).
Control 60 ** Treated ** ns ns ** 40 ns ns
20 % Respone of female wasps 0
Anisole -Myrcene β -Farnesene α Acetophenone trans- IsoEugenol
Bis(2-ethylhexyl) phthalate Cis-3- Hexenyl butyric acid
Figure 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 A B C Acetophenone Anisole Bis(2-ethylhexyl) phthalate 60 60 ** ** ** ** 60
40 40 40
20 20 20
0 0 0 % Respone of female wasps % Respone of female wasps % Respone of female wasps 25µL 50µL 100µL 150µL 25µL 50µL 100µL 150µL 25µL 50µL 100µL 150µL
D E F β-myrcene 60 60 Cis-3- Hexenyl butyric acid Eugenol 60
40 40 40
20 20 20
0 0 0 % Respone of female wasps % Respone of female wasps 25µL 50µL 100µL 150µL 25µL 50µL 100µL 150µL % Respone of female wasps 25µL 50µL 100µL 150µL
G
60 α- Farnesene
40
20 Treatment 0 Control % Respone of female wasps 25µL 50µL 100µL 150µL
Figure 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
30.0 ns Ani 100µL Bis 50µL 20.0
25.0 ns Ani100µL Bis 25µL 30.0
33.3 ** Ani 25µL Bis 100µL 53.3
40.0 ns Ani 25µL Bis 50µL 45.0
30.0 ns Ani 100µL Bis 100µL 23.3 Blend ratio
40.0 ns Ani 75µL Bis 75µL 30.0
40.0 Ani 50µL Bis 50µL 40.0
30.0 ns Ani 25µl Bis 25µl 33.3
0 20 40 60 % Response of female wasps Blank air Treatment
Figure 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 4.4. Discussion
This study was an attempt to identify which volatile compounds or blends of compounds were involved in T. papilionis positive responses to H. zea eggs and sunn hemp (C. juncea) leaves described in a previous study (see Chapter 3). Testing the response of female wasps to chemical volatiles showed varied responses. The wasps showed the highest preference for anisole and bis(2-ethylhexyl) phthalate individually over clean air. On the other hand, wasps were not shown to respond to trans-isoeugenol significantly, β-myrcene, Cis-3-hexenyl butyric acid, acetophenone, and α-farnesene. Female wasps were in fact repelled by some concentrations of these chemical compounds. Significantly, females showed preference to a blend of anisole and bis(2-ethylhexyl) phthalate with the ratio of 25 μL anisole:/100 μL bis(2-ethylhexyl) phthalate and showed no significant difference in behavior to any other ratios of anisole and bis(2- ethylhexyl) phthalate blends tested. The preferred ratio closely matches the measured approximate ratio of these volatiles emitted by C. juncea plants with eggs (Chapter 3). This may be explained by the fact that some volatiles can synergize with each other and act as one unit when combined in a mixture. Their synergistic effect may cease if any compound is removed, or a threshold concentration is exceeded (Tasin et al., 2007). Other chemicals may not have a synergistic effect (Bruce et al. 2005; Tasin et al., 2007; Sun et al., 2016).
Furthermore, the quality and quantity of chemical volatiles in a blend can make a difference in the responses of natural enemies (De Boer and Dicke, 2004). Result reported in this study does not mean that the ideal ratio of these two chemical volatiles has been definitively determined, and further formal quantitation of the concentration of the compounds in volatile blends released form plants should be conducted. Some tested concentrations of the blend of anisole and bis(2-ethylhexyl) phthalate did not attract T. papilionis females. Herbivore-infested
85 plants may release a blend of volatiles, many of which are considered to be minor constituents
(Dicke and van Loon, 2000), and they may interact with major constituents (Najar-Rodriguez et al., 2010). These minor components are believed to influence insect behavior (Dicke, 1999). This was shown in Cotesia wasps, which had a different response to a natural chemical blend compared with a synthetic mimic containing only the major components and lacking minor ones
(Turlings et al.,1991). In contrast, while excluding major components can affect host preferences, in some cases, a minor constituent can be eliminated without any dramatic change
(Bruce and Pickett, 2011). Semiochemical blend ratios can be varied within a certain range of ratios and remain effective attractants (Najar-Rodriguez et al., 2010). Plant species can qualitatively and quantitatively release different blends of volatiles under various circumstances.
The concentration of these semiochemicals released may differ in their amount and ratios, which may affect the searching behavior of insects. The importance of species-specific and ratio- specific plant volatiles can be seen clearly in a review by Bruce et al. (2005). They hypothesized that insects might recognize plant volatiles in two possible ways: unique plant volatiles that do not occur in other closely related plant species or certain blend ratios of compounds. For example, when an insect perceives a diverse mix of chemicals that are not unique or specific to the host plant, the insect must recognize the specific olfactory cues within a blend of chemical compounds for it to be effective (Bruce et al., 2005). Selecting particular single chemicals over blends of compounds would likely reduce the amount of information that the insect olfactory system can detect among the diversity of chemical compounds in the environment (Wilson et al.,
2015). In addition to the specific chemical compounds, the appropriate semiochemical blend would 'cancel or mitigate' the 'noise,' interference from non-specific signals in the environment, that Wilson et al. (2015) discuss in their review. This 'noise' has been found to have a significant
86 impact on the ability of insects to respond to plant signals or cues, and any adaptations to improve the efficiency of the parasitoids in responding to plant signals should provide fitness benefits for the parasitoids and the plants attacked by insect hosts (Wilson et al., 2015). It is possible in this study that some significant minor components of the volatile semiochemical blend that influence the efficacy of some major components were missed in the analyses.
Trichogramma wasps react differently to a spectrum of synthetic semiochemicals (e.g.)
Lewis et al.,1982; Boo and Yang, 2000; Romeis et al., 2005; Fatouros et al., 2008; Wilson and
Woods, 2016; Laxmi and Rani, 2019). A study by Peñaflor et al. (2011) showed a significant behavioral response in T. pretiosum females to a synthetic mixture of green leaf volatiles at a range of tested concentrations. Still, T. pretiosum was not attracted to a major component, (E)-β- farnesene, at 5 and 50 μL/mL, and the wasps were repelled (preferred the control) at 500 and
1,000 μL/mL concentrations. This is consistent with the results of the present study where T. paplionis females did not show any attraction to a closely related sesquiterpene compound (α- farnesene) over clean air (control). Sen et al. (2005) showed that beta-myrcene was among chemicals that did not elicit a significantly higher amplitude response in an electroantennogram study (EAG) in T. chilonis (Ishii). Similarly, T. papilionis did not show a significant response to any concentrations of myrcene. A study by Ngi-Song (1995) showed Cotesia flavipes (Cameron) females responded positively to synthetic anisole, (E)-β-farnesene, and (Z)-3-hexenyl acetate.
These chemicals were identified from maize plants infested with Chilo partellus (Swinhoe) using gas chromatography-mass spectrometry (GC-MS) analysis. In addition, gas chromatography- electroantennography (GC-EAD) analysis of the antennal receptors of C. flavipes females showed a significant response to these three compounds when tested singly or in a mixture.
Anisole was attractive to C. flavipes females at all the doses tested in a T-tube olfactory bioassay
87 when tested alone or in a combination as well as being electroantennography (EAG) active.
These results are consistent with the present study, where T. papilionis females showed a significant positive response to anisole.
In conclusion, the importance of studies of searching behavior for natural enemies in biological control programs is evident, and numerous papers have been published addressing the significance of semiochemicals in host- and habitat location. This study showed a variety of responses to different semiochemical compounds, with some chemical compounds preferred by the wasps over clean air. These preferred compounds may be promising as attractants for T. papilionis in cropping systems. Our knowledge of how chemical cues can positively or negatively affect the response of beneficial insects such as parasitic wasps may provide the best understanding for achieving successful field parasitism (Aliva et al., 2016). Further work on the
T. papilionis system should include field trials to determine whether compounds such as anisole, or blends of anisole and bis(2-ethylhexyl) phthalate facilitate increased host-finding by the wasps. For this purpose, Chapter 5 will address the field study of the effect of semiochemical compounds on the searching behavior of T. papilionis depending on the result of this study.
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98 CHAPTER 5
THE EFFECT OF PLANT DERIVED SEMIOCHEMICALS ON SEARCHING
BEHAVIOR OF TRICHOGRAMMA PAPILONIS IN DIFFERENT ENVIRONMENTS
Abstract
This study investigated the behavioral responses of the egg parasitoid Trichogramma papilionis to semiochemicals originating from plants upon which host eggs were laid, in an effort to find a way to enhance the efficacy of these wasps in different habitats. The response of T. papilionis wasps to a combination of volatiles previously identified to influence T. papilionis behavior in olfactometer studies was studied under greenhouse and field conditions. Initial experiments indicated that anisole alone did not make a difference in the parasitism rate of sentinel eggs in cornfields in comparison to the control plots. Greenhouse results revealed that there was a significant difference in the attraction of wasps to a blend of volatiles over the control. The parasitism rate was 9.21 (± 2.35) %, for sentinel eggs with a blend of volatiles (anisole + bis(2- ethylhexyl) phthalate), whereas control parasitism rate was 1.44 (± 0.90) %. The distance of the released wasps from the cue source was shown to play a significant role in the response to the chemical blend. The closer the sentinel egg traps with volatiles, the higher the parasitism rate was. Trials in open and covered habitat with the optimal volatile blend showed variable outcomes. The parasitism rate with the optimal blend of chemical compounds (anisole and bis(2- ethylhexyl) phthalate) was higher in closed-canopy habitat compared to the control. At the same time, there was no significant difference in open habitat. This suggests that different environmental conditions have a crucial role in influencing the function of volatile organic compounds as an attractant to egg parasitoids.
Keywords: Trichogramma papilionis, optimal blend, single chemical volatile, greenhouse, open, and covered habitat.
99 5.1 Introduction
Adult female parasitoids have a limited amount of time after emerging to find a suitable host for their progeny (Aartsma et al., 2017). They typically have to locate small hosts, sometimes at low population densities, in complex habitats, and often over relatively large distances. The adult wasps rely on a range of chemical and visual cues to locate appropriate habitat and hosts therein. A review by Turlings and Ton (2006) discussed how specific plant signals could be targeted with the intent to use them the attraction of beneficial insects, such as parasitoid wasps, to target habitats. They emphasized that specific insects may respond to unique combinations of volatiles released by plants to facilitate searching for their hosts upon those plants. They suggest that the manipulation of plant volatiles that act as semiochemicals to natural insect enemies can be used to enhance biological control under field conditions. Many more laboratory studies regarding the use of plant volatiles to manipulate the searching behavior of beneficial insects have been done in comparison to the number of field studies (Hunter, 2002).
The role that induced plant volatiles play in the searching behavior of natural enemies has been ascertained mostly from laboratory studies such as olfactory bioassays and wind tunnels. Larger- scale studies are important in clarifying the role of plant volatiles as semiochemicals for insects under field conditions (Bernasconi et al., 2001).
Plants can release a wide variety of volatile compounds in response to insect oviposition
(Fatouros et al., 2008; Colazza et al., 2010; Hilker and Fatouros, 2015) or feeding activities by herbivorous larvae (Kessler and Baldwin, 2001; Hilker and Meiners, 2002). Many of these volatile compounds are considered to be useful olfactory cues to attract parasitic wasps (Romeis et al., 1997; Peñaflor et al., 2011), while others are just "noise" that can create interference between plant stimuli and natural enemies (Wilson et al., 2015). Plant stimuli can attract
100 parasitoid wasps to suitable hosts in the habitat over long distances or short distances (Vet and
Dicke, 1992). Parasitoid wasps can find and locate their preferred hosts in complex environments by exploiting plant stimuli such as volatile organic compounds (VOCs) (Wilson and Woods,
2016). Synthetic semiochemical compounds that can be successfully used as an attractant to natural enemies in crop production systems (James and Grasswitz, 2005) appear as a potential alternative to manipulate the emission of induced plant volatiles to cope with the stress of the variable environment (Becker et al., 2015).
Spatial scales such as plant, patch, and landscape should be taken into consideration when testing the response of parasitoid insects to a blend of chemical volatiles, as these different scales may change the searching behavior of parasitic insects or have an effect on the concentration of chemical volatiles in the environment. A review by Aartsma et al. (2017) underscored the importance of spatial scales in the interaction between plant (odor sources) and the parasitoids (the receivers).
Trichogrammatid species are among the most widely used biological control agents in augmentative release programs. They have been used extensively in many economic crops as a successful augmentative biocontrol agent against many major pests (Smith, 1996). Studies have demonstrated that Trichogramma species can be responsive to plant volatiles (Romeis et al.,
1999; Reddy et al., 2002; Fatouros et al., 2008; Peñaflor et al., 2011). Trichogramma parasitism rate is significantly influenced by different habitats, including variables such as plant host structure and egg host location (Romeis et al., 2005). Plant volatiles have been found to play an essential role in increasing parasitism levels as shown in some studies (e.g., Altieri et al., 1981;
Thaler, 1999; Liu and Jiang, 2003), while others suggest an interaction between olfaction and visual cues (Gardner and Hoffmann, 2020).
101 The key question addressed in this study whether Trichogramma papilionis respond to semiochemicals from plants identified as attractants in olfactometer studies (Chapter 3 and 4), to the cues in less constrained (greenhouse) and open-air environments.
5.2 Material and Methods
5.2.1 Trichogramma wasp culture
The egg parasitoid T. papilioins used in this study was laboratory-reared using irradiated
Mediterranean flour moth Ephestia kuehniella (Zeller) eggs as a factitious host (see Chapter 2 and 3 for more details).
5.2.2 Single volatile chemical trial: Preliminary Field experiments.
Preliminary field trials were conducted in corn plots at Corteva Agriscience™ on the north shore of Oahu, Hawaii, in August 2019. Field experiments were prepared in a randomized complete block design with each treatment replicated four times, as in Manandhar and Wright
(2015), with some modifications. Corn Plots were 12 m by 12 m, separated by 6 m within a block and each block was separated by 10 m. Three treatments were compared in the cornfields at the corn silking stage as follows: control block (no T. papilionis release), single stimulus
(anisole) plus T. papilionis release, and T. papilionis-only release. About 2 g of parasitized eggs
(approximately 80,000 wasps), were released in each plot within different distances (1, 2, 4, 6, and 8 m) from the chemical source.
Battery-powered SpaRoom® Scentifier™ portable Essential Oil & Fragrance Diffusers, charged with 5 mL of anisole, were continuously deployed in each corn plot simultaneously with
102 the emergence of the parasitoid wasps. Two diffusers were used for each plot. Sentinel Ephestia eggs were placed in the field on yellow cards and left for three days before retrieval for analysis.
5.2.3 Greenhouse experiments
Based on the preliminary field trails results (see below), it was decided to conduct experiments in more confined environments using an optimized blend of semiochemicals
(Chapter 4). Greenhouse trials were conducted at the greenhouses of the College of Tropical
Agriculture & Human Resources (CTAHR), University of Hawaii in March 2020. Greenhouses were set up for testing the response of T. papilionis wasps to a blend of semiochemicals (anisole and bis(2-ethylhexyl) phthalate) in the optimized ratio (26:1 by volume) that was shown to have a significant positive attractant effect to T. papilionis (Chapter 4). Two greenhouses (control and a volatile semiochemical treatment) were used for this purpose. The same greenhouse was used repeatedly, as there was no way to clean them of volatiles between trials. All Trials were conducted around 10 am and lasted for 24 h. Eight cards of E. kuhniella eggs parasitized by T. papilionis per trial were placed into each greenhouse using a cardboard container containing approximately 0.5 g of parasitized eggs, with approximately 80-85 % parasitism. The chemical blend was placed in a piece of cotton wick using a micropipette. Each piece of cotton wick contained 26 μL anisole: 100 μL bis(2-ethylhexyl) phthalate) blend. Sentinel E. kuehniella eggs were glued to a small piece of yellow card. A cotton wick saturated with the mix of volatiles was placed together inside an inverted clear plastic cup with no lid, to prevent water entry from the sprinklers in the greenhouse, and suspended by stringing copper wire through a hole in the middle of the cup. The greenhouses had air movement of approximately 1 m.s-1, which provided a wind tunnel effect within each greenhouse; wasps were released downwind from the semiochemical treatments or controls. The air handling system in the greenhouse is located >2 m
103 above the floor, thus more or less level with the height that the wasps were released, and the sentinel vessels were deployed. The elution rate of the chemicals was measured in the laboratory and and they would be present briefly in the environment for about 2-3 days. The wasps were emerging in mass from the cards at release, and all emerged typically within two days of release.
In total, five replicates were performed on multiple days. The distances between the sentinel egg containers and the wasp release containers were measured for both the chemical and control treatments and included in the statistical analysis as a factor. All treatments (chemical volatiles and control) were left for three days in the greenhouse and then returned to the lab for examination. Percent parasitism was measured by counting the number of parasitized eggs on each card and dividing by the total number of sentinel eggs of the same card. Each greenhouse had a variety of plant species, which were a mix of native Hawaiian species and some cucurbits.
These plant species belonged to the following genera: Kadua sp., Myoporum sp., Sesbania sp.,
Brighammia sp., Delissea sp., Cyanea sp., Psydrax sp., Bidens sp., Osteomeles sp., Polyscias sp.,
Solanum sp., Acacia sp., Pepperomia sp., Dodonea sp., Nototrichium sp. and Sapindus sp..
Plants were at least 1.5 m below the chemical sources and the release points. The insects and sentinel eggs were high above any plants where the airflow occurred, so there was unlikely to be any interference from the plants below
5.2.4 Open field optimal volatile blend trial
The same protocol as in the greenhouse trials was used at the Oahu Urban Garden Center
- CTAHR (UGC), Pearl City, Oahu, Hawaii in March 2020. A blend of anisole and bis(2- ethylhexyl) phthalate) (26:1) was used for the semiochemical treatment. Three treatments were
104 compared: control (no wasps or chemicals introduced), wasp release only, and chemical volatiles plus wasp release with 1.2 g of parasitized eggs (approx. 80,000 parasitoids) were released per plot. These trials used the same apparatus to suspend the sentinel Ephestia egg cards as in the greenhouse experiment. A total of ten such sentinel egg cards, five at 50 cm and five at 2 m from each wasp release point, were placed around the location of each wasp release. Four release locations, two treatment, and two control were prepared in a randomized complete block design with each treatment replicated two times. The sentinel eggs were left exposed for three days in an open environment with only low-growing plants (approximately 50 cm).
5.2.5 Open and covered habitat optimal volatile blend trial
The same protocol as the UGC trials was used at the University of Hawaii Campus. A blend of anisole and bis(2-ethylhexyl) phthalate) (26:1) was used for the semiochemical treatment. Three treatments were compared: control (no wasps or chemicals introduced); wasp release only, and chemical volatiles plus wasp release with 0.5 g of parasitized eggs
(approximately 40,000 wasps) were released per plot. The open habitat areas had only short grasses, while the covered habitat plots had a continuous overstory of milo tree (Thespesia populnea) and Plumeria sp..
5.2.6 Statistical analysis
JMP15 Pro® (SAS Institute, Cary, NC) was used to evaluate the statistical significance of differences in the greenhouse and field trials with the chemical blend. Two-way ANOVA was used in the greenhouse and UGC and UH campus experiments, the chemical blend and control
105 treatments, and the distance between sentinel eggs and the location of wasp release container, as main factors. One-way ANOVA was used for the single stimulus trial.
5.3 Results
Single compound trials: The experiments with anisole only, in cornfields, returned non- significant results (F (2,11) = 3.12, p = 0.092; Figure 1). Statistically, the plots with T. papilionis releases had similar egg parasitism levels (Figure 1). These negative results were the incentive for continuing further experiments with a blend of compounds shown to be more attractive than anisole alone, initially in the greenhouse, and then in open-air plots.
Single chemical (anisole)
Control
Wasp release only
Anisole
0 10 20 30 40 % Parasitism
Figure 1. Mean parasitism rate (± SEM) by Trichogramma papilionis in cornfields comparing anisole as an attractant, to untreated release plots, and control plots with no anisole and no wasps released (p = 0.092).
106
The overall results from the greenhouse trials showed that there was a significant difference between the chemical volatile treatment and control at different distances (F (7,32) =
7.9, p <0.001). The overall parasitism rate of sentinel eggs by T. papilionis in the chemical blend treatment (anisole and bis(2-ethylhexyl) phthalate) was 9.21 (± 2.35) %, and the overall parasitism rate in the control population was 1.44 (± 0.90) % (Figure 2-A). Additionally, the parasitism rate in the volatile blend trial was shown to be affected by the distance between wasp release containers and sentinel egg traps (F (1,24) = 8.5, p = 0.0003, Figure 1-B). The highest parasitism rate was 21.89 (± 4.82) % for the chemical treatment, compared with 3.75 (± 2.97) % for control at the same distance (0.5 m) (Figure 1).
The UGC trials showed no difference between the volatile chemical blend and wasp- release only treatments F (3,15) = 2.07, p = 0.146 (Figure 3-A), possibly owing to the high numbers of wasps released. Lower wasp release densities were used for the UH trials. The outcomes of the UH Campus trials demonstrated that there was a significant difference between treatments (volatile chemical blend vs. the control) for T. papilionis parasitism rate in covered habitat F (3,36) = 3.4, p = 0.0286. In contrast, no statistically significant difference between the treatments was observed for T. papilionis parasitism in open habitat F (3,36) =1.2, p = 0.320. In general, the trials in covered habitat showed a higher parasitism rate of T. papilionis wasps in the chemical blend 30.72 (± 5.842) % over the control treatment 12.32 (± 4.30) %. On the other hand, the overall parasitism rate of T. papilionis in an open-air trial using the chemical blend was
16.91 (± 4.626) % vs. 27.02 (± 6.273) % in the control treatment (Figure 3-B).
107
15 A
10
5 % Parasitism
0
Control Chemical
B 30 Chemical Control
20
10 % Parasitism
0 0 2 4 6 Distance (m)
Figure 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
108 of up to 6 m; A) overall percentage of parasitism; B) percentage parasitism at different distances from the release point, treatment, and control.
A B Chemical Control
80 ns ✱✱ ns 40
60 30
40 20 % Parasitism 20 % Parasitism 10
0 0 Chemical Control Covered habitat Open habitat
Figure 3. Mean parasitism rate (± SEM) by Trichogramma papilionis: A) open habitat B). covered habitat, comparing the optimal blend (anisole + bis(2-ethylhexyl) phthalate) as an attractant, to untreated releases. ** = significant at a = 0.05, ns = non- significant.
109 5.4 Discussion
The results from the cornfield experiment showed that there was no significant attractive effect of the single chemical volatile used (anisole) under the trial conditions. The experiments with the blend of semiochemicals were more encouraging. The results of the greenhouse trials showed that the parasitism rate by T. papilionis was significantly increased when the wasps were exposed to the blend of volatiles (anisole and bis(2-ethylhexyl) phthalate). Parasitism was considerably higher in the semiochemical treatment compare to the control. There was a significant reduction in wasp attraction to the semiochemicals over a 6 m distance. Open-air results at UGC revealed that statistically there was no significant difference in T. papilionis attraction between the volatile chemical treatment and the control (Figure 3-A). This is likely because of the high release density (>80,000 wasps per plot) at the UGC experiments, which likely resulted in a flush of wasps that simply blundered into the sentinels, hence the lack of any significant effect for the treatments. The UH campus results (with reduced wasp release density) demonstrated that while there was a significant difference between treatments in the covered habitat, statistically no significant difference occurred between treatments in the open habitat.
The outcomes of the UH campus trials appear to be consistent with the findings of the greenhouse trials. It was noted that the parasitism rate of T. papilionis increased using the optimal blend under tree canopies, similar to the greenhouse studies. In contrast, the open habitat results showed no difference between the chemical treatments and the control. This may be the result of several factors. First of all, chemical degradation or dissipation of volatile compounds in an open environment is likely to increase rapidly (Aartsma et al., 2017). In an open environment, the temperature may rapidly impact the vapor pressure of volatile compounds, and even light intensity and radiation intensity could result in rapid loss of volatile cues (Becker et al., 2015).
110 Moreover, the ratio of chemical volatiles in response to chemical degradation of volatile compounds may change dramatically and then result in new modified blends that differ from the original combination (Blande et al., 2014; Simpraga et al., 2016). Air current is the main, if not the only, carrier of the odor from source to insect (the receiver), where it mainly affects the direction and the speed of airborne chemical volatiles (Riffell et al., 2008). From this, the optimal blend of chemical volatile may have drifted away from the chemical traps due to the high wind speeds and resulted in a non-attraction of T. papilionis in open, unprotected environments. On the other hand, the opposite can be seen in covered habitats (greenhouse and
UH campus covered trial) where the parasitism rate of T. papilionis increased using the same blend of chemicals used in the open habitat. This could be since abiotic factors have been limited under the protected conditions provided by covered habitats in comparison with open ones.
Consequently, chemical degradation and wind speed may have decreased in the covered habitat.
Landscape variables have also been proven to play a role in the effective exploitation of chemical cues by insects (Aartsma et al., 2017). In this regard, Voskamp et al. (1998) found that electroantennogram responses of tsetse flies (Glossina pallidipes) to host odors were varied.
Tsetse flies respond to an odor source up to over 60 m in woodlands in comparison with only
20m in open fields, suggesting that a wooded area kept chemical volatiles intact over a larger area than a flat open area; dissipation of the compounds was likely reduced in more protected environments.
There may ultimately be applications of these results for improved field augmentative biological control using T. papilionis. A field study by James (2003) showed evidence that using synthetic plant volatiles in appropriate blends could attract some beneficial insects (parasitoids and predators) under field conditions. Similarly, the current study showed that an appropriate
111 combination of chemical volatiles provided an attractive semiochemical effect to T. papilionis.
Bernasconi et al., (2001), found that induced maize volatiles produced with Spodoptera littoralis caterpillar regurgitant attracted parasitic and predatory insects under field conditions. When
Kessler and Baldwin, (2001) mimicked the release of three chemical compounds (cis-3-hexen-1- ol, linalool, and cis-a-bergamotene), that were emitted naturally by Nicotiana attenuata plants during the attack by three species of leaf-feeding herbivores, they found that a generalist predator increased predation rates.
The single chemical volatile field experiments of this current study did not show a higher parasitism rate in the anisole treatments. This may be explained by the fact that anisole alone may not make a difference in the searching behavior of T. papilionis under the field conditions prevailing. It is possible that the positive attractant effect of anisole observed in laboratory experiments (Chapter 3) could be lost with the influence of background ‘noise’ from other volatile compounds in the environment (Wilson et al., 2015). The quality and quantity of blends of chemical volatiles in the environment can influence the response of natural enemies (De Boer and Dicke, 2004). The synergism between chemical volatiles has also been found to play a crucial role in a natural enemy response regarding localizing and locating their preferred host or host plant (Tasin et al., 2007). Thaler (1999) showed that inducing tomato plants with jasmonic acid increased parasitism of sentinel Spodoptera exigua caterpillars by Hyposoter exiguae by
37%. This suggested that the crops in the field would benefit from induced plant defense.
Parasitoids wasps are able to recognize induced plant volatiles that are associated with their host.
This ability may vary from specialist to generalist parasitoids, where generalist wasps can learn to recognize plant volatiles even among a mixture blend of volatiles. In contrast, specialist wasps need to use their inherent ability to do so. In addition, the optimal combination of volatiles is
112 necessary for helping generalist wasps to focus on specific attractants that associated with their hosts or their host plants (Turlings and Erb, 2018). A laboratory and field study by Wilson and
Woods, (2016) was one of a few studies that showed two species of generalist egg parasitoids (T. deion and T. sathon) could learn and respond innately to olfactory cues.
In the current experiments, the distribution of T. papilionis wasps was affected by the distance between the wasps’ launch containers and the chemical traps with the sentinel eggs, where the closest the chemical traps had significantly higher the parasitism. This may occur since chemical volatiles have a specific travel distance, or the ability of the wasps to exploit the chemical cues was affected by the reliability-detectability theory (Vet and Dicke, 1992). This suggests that some cues can be reliable but less easily detected at low concentrations, and may not predictably indicate the host presence, especially from a long distance. Aartsma et al., (2017) showed that, with increasing the distance between the source of plant volatiles (sender) and the parasitoids (receiver), there is increased degradation of the plant volatile compounds as a result of reacting with other chemical compounds in the environment and their dilution in air currents.
This process will influence the ability of the insect parasitoids to use the odor source and find the host reliably.
In conclusion, good evidence exists that VOCs positively influence the attraction of parasitoids to habitats and hosts (Heil, 2008). The findings of this present study, and example studies cited, emphasize the importance of improving our understanding of how tri-trophic interactions (natural enemies- herbivores and host plant), as well as the real 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.
113 References
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induced plant volatiles and tritrophic interactions across spatial scales. New Phytologist
216: 1054-1063.
Altieri, M.A., Lewis, W.J., Nordlund, D.A., Gueldner, R.C., Todd, J. W. 1981. Chemical
interactions between plants and Trichogramma wasps in Georgia soybean fields. Journal
of Environmental Protection and Ecology Protection Ecology 3: 259-263.
Bernasconi, O.M.L., Turlings, T.C.J., Edwards, P.J., Fritzsche‐Hoballah, M.E., Ambrosetti, L.,
Bassetti, P., Dorn, S. 2001. Response of natural populations of predators and parasitoids
to artificially induced volatile emissions in maize plants (Zea mays L.). Agricultural and
Forest Entomology 3: 201-209.
Blande, J.D., Holopainen, J.K., Niinemets, U. 2014. Plant volatiles in polluted atmospheres:
stress responses and signal degradation. Plant, Cell and Environment 37: 1892-1904.
Becker, C., Desneux, N., Monticelli, L., Fernandez, X., Michel, T., Lavoir, A. V. 2015. Effects
of Abiotic Factors on HIPV-Mediated Interactions between Plants and Parasitoids.
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De Boer, J.G., Dicke, M. 2004. The role of methyl salicylate in prey searching behavior of the
predatory mite Phytoseiulus persimilis. Journal of Chemical Ecology 30:255-271.
Gardner, J., Hoffmann, M.P. 2020. How important is vision in short-rage host finding by
Trichogramma ostriniae used for augmentative biological control? Biocontrol Science
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114 Heil, M. 2008. Indirect defense via tritrophic interactions. New phytologist 178: 41-61
Hilker, M., Fatouros, N. E. 2015. Plant responses to insect egg deposition. Annual Review of
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Hilker, M., Meiners, T. 2002. Induction of plant responses to oviposition and feeding by
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181-192.
Hunter, M. D. 2002. A breath of fresh air: beyond laboratory studies of plant volatile-natural
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James, D.G. 2003. Synthetic herbivore‐induced plant volatiles as field attractants for beneficial
insects. Environmental Entomology 32: 977-982.
James, D. G., Grasswitz, T. R. 2005. Synthetic herbivore-induced plant volatiles increase field
captures of parasitic wasps. Biocontrol 50: 871-880.
Kessler A, Baldwin IT. 2001. Defensive function of herbivore‐induced plant volatile emissions
in nature. Science 291: 2141-2144.
Liu, S.S. Jiang, L.H. 2003. Differential parasitism of Plutella xylostella (Lepidoptera:
plutellidae) larvae by the parasitoid Cotesia plutellae (Hymenoptera: braconidae) on two
host plant species. Bulletin of Entomological Research 93: 65-72.
Manandhar, R., M. G. Wright. 2016. Effects of Interplanting Flowering Plants on the Biological
Control of Corn Earworm (Lepidoptera: Noctuidae) and Thrips (Thysanoptera:
Thripidae) in Sweet Corn. Journal of Economic Entomology 109: 113-119.
Peñaflor, M. F. G. V., Erb, M., Miranda, L. A., Werneburg, A. G., Bento, J. M. S. 2011.
Herbivore-induced plant volatiles can serve as host location cues for a generalist and a
specialist egg parasitoid. Journal of Chemical Ecology 37: 1304-1313.
115 Romeis J., Shanower T.G., Zebitz, C.P.W. 1997. Volatile plant infochemicals mediate plant
preference of Trichogramma chilonis. Journal of Chemical Ecology 23: 2455-2465.
Romeis, J., Shanower, T.G., Zebitz, C.P.W. 1999. Why Trichogramma (Hymenoptera:
Trichogrammatidae) egg parasitoids of Helicoverpa armigera (Lepidoptera: Noctuidae)
fail on chickpea. Bulletin of Entomological Research 89: 89-95.
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Trichogramma egg parasitoids - Underlying mechanisms and implications. Basic and
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Smith, S.M., 1996. Biological control with Trichogramma: advances, successes, and potential of
their use. Annual Review of Entomology 41: 375-406.
Thaler JS. 1999. Jasmonate-inducible plant defences cause increased parasitism of herbivores.
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Turlings, T. C. J., Erb, M. 2018. Tritrophic interactions mediated by herbivore-induced plant
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Turlings, T.C.J., Ton, J. 2006. Exploiting scents of distress: the prospect of manipulating
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116 Voskamp, K., Den Otter, C.J., Noorman, N. 1998. Electroantennogram responses of tsetse flies
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117 CHAPTER 6
GENERAL CONCLUSTION AND RECOMMENDATIONS
6.1 General Conclusions
This dissertation addressed the potential impacts of severe inbreeding on captive mass rearing of Trichogramma papilionis, as well as aspects of the chemical ecology of this parasitoid.
The initial intention was to continue field studies on the possible impacts of inbreeding in mass rearing (with a range of colony founder numbers), on wasp searching efficiency and overall field efficacy as an augmentative biological control agent in corn (Zea mays), where Helicoverpa zea is a significant pest, and the intended target of this wasp. However, preliminary field trials indicated that T. papilionis did not search for and parasitize H. zea eggs at all efficiently in cornfields, and the work was redirected to understanding the searching behavior of the wasp, concentrating on its potential use of semiochemicals in host location.
Examining the effect of colony founder size, it was clear that relatively high wasp fitness could be achieved with 10 female founders, and in for some fitness measures, even as few as two. No significant difference in fecundity (eggs laid per female) over 10 generations was detected even from a single founder female, suggesting that the imposed bottleneck did not result in reduced female. Founder number did affect both emergence rate and sex ratio of T. papilionis, which was lowest in the most severely inbred colonies, with a single founder. Rearing temperature and humidity affected some fitness aspects of founder progeny over 10 generations such as sex ratio and parasitized eggs per female, with reduced fitness under cooler conditions.
Under field conditions, T. papilionis readily parasitizes eggs on sunn hemp, Crotalaria juncea. The searching behavior work thus compared the wasps' behavior in response to Z. mays
118 and C. juncea, with and without H. zea eggs. In Y-olfactometer experiments to examine the response of female T. papilionis females to semiochemicals from host eggs, plant substrates, and plants with or without eggs, the following was observed: The wasps respond significantly to
Helicoverpa zea eggs over clear air. The wasps showed a positive significant response to sunn hemp infested with H. zea eggs as well as to corn plants infested with corn earworm moth eggs.
Furthermore, T. papilionis females preferred odors from H. zea eggs compared to Ephestia kuehniella eggs. The wasps showed no response to E. kuehniella eggs compared with clear air.
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; the wasps preferred infested sun hemp plant with H. zea eggs over infested maize plants. Chemical volatile collection and headspace analysis to detect potential semiochemicals emitted by plants with H. zea eggs, showed that sunn hemp samples emitted volatile compounds consistent with linear alkanes, aldehydes, aromatics, polyterpene-related compounds, naphthalene derivatives, and ester-related compounds across both H. zea egg-infested and uninfested sunn hemp plants. Higher concentrations of some chemical compounds were found in H. zea egg-infested sunn hemp compared to un-infested sunn hemp. These were consistent with anisole, β-myrcene, cis-butyric acid, trans-isoeugenol, and bis(2-ethylhexyl) phthalate
Single chemicals and combinations of chemicals identified form the plants and eggs were tested to determine T. papilionis wasp response. Female wasps were significantly attracted to only two chemical volatiles (anisole and bis(2-ethylhexyl) phthalate) among the seven compounds selected from the headspace analysis study. The wasps had a significant positive response to some concentrations of anisole and bis(2-ethylhexyl) phthalate, while some other concentrations of the other chemical compounds repelled the wasps. Female wasps responded
119 significantly to a combination of anisole and bis (2-ethylhexyl) phthalate at 25μL /100μL concentration which is quite nearly the ratio of anisole to bis (2-ethylhexyl) phthalate in the
(GC_DMS) chromatography (Chapter 3).
Finally, larger scale experiments were conducted to assess the attractiveness of the semiochemicals identified as attractive in the olfactometer studies under greenhouse and field conditions. Preliminary experiments in cornfields with anisole as the sole hypothesized attractant, showed no significant attractive effect. Greenhouse trials were conducted to determine whether the optimal blend identified in the laboratory would attract T. papilionis. Parasitism rate by T. papilionis was significantly increased when the wasps were exposed to the blend of volatiles (anisole and bis (2-ethylhexyl) phthalate) in a greenhouse. Trials in open air conditions showed a significant, albeit short distance attraction, difference between treatments in habitat with closed tree canopies, while no significant difference between treatments was observed in open habitat.
Parasitoid host location can be achieved through the insects using a range of different host-finding strategies, ranging from random searches, through using visual and chemical cues.
Many trichogrammatid egg parasitoids are considered to be fairly generalist in their host use, but often habitat specialists, limiting their ability to use a broad range of habitats in many species.
The results of this work indicate that this is in fact the case, demonstrating the existence of oviposition-induced cues from certain plants. This has broader implications, in that oviposition induced chemical cues may be more widespread than currently know, and the searching behaviors of various Trichogramma species may be influenced by this. There may be broad global interest among biological control researchers in these findings. Trichogramma host- searching behavior has typically been considered to be unsophisticated, relying essentially upon
120 undirected searching. The results of this dissertation suggest that this may be incorrect, and that there may be highly evolved host searching behaviors among some Trichogramma species.
Recent research (Gardner and Hoffmann 2020) starts to suggest that Trichogramma search behavior includes visual and olfactory cues, but has not investigated the synergy between oviposition and the release of semiochemicals form plants. In some crop systems, particular
Trichogramma species are considered to be more efficient than other, for example T. ostriniae is a highly efficient parasitoid of Ostrina nubilalis in cornfields (Wright et al., 2001, 2002), but not in solanaceous crops (Kuhar et al., 2004). Encouraging a wider understanding of the types of cues that these wasps respond to may improve their performance in suppressing pests in many agroecosystems.
In screening parasitoids as prospective biological control agents in classical biological control programs, the procedures are typically conservative (Van Driesche and Bellows 1996;
Hajek 2004; Hajek et al., 2016), depending on captive no-choice exposures of potential non- target hosts to parasitoids. This likely results in some false-positive parasitism records on non- target species, and ignores ecological impacts on host selection by the parasitoids. Some studies
(e.g., Wright et al., 2005) show that habitat selection plays a significant role in Trichogramma host detection. This dissertation goes further and shows that subtle chemical cues may be released by plants upon which Lepidoptera eggs have been laid – this has significant implications for non-target potential of a parasitoid being considered for classical biological control. It is possible that including an understanding of the chemical ecology of the wasps’ host location behavior could significantly impact studies of potential non-target impacts.
121 6.2 Recommendations and further work:
1. Establishing a new colony of wasps with more founder numbers is best in order to avoid
any significant loss in the quality of biological characteristics as long as they are offered
appropriate conditions such as (25 ± 2 ◦C, 60 –70% RH, LD 16: 8 h), but relatively small
numbers of founders may be effective.
2. Identifying the semiochemical compounds that attracted the wasps to plants with eggs
may be useful in developing means to attract the parasitoids to an atypical crop-pest
system. These preferred compounds may be promising as attractants for T. papilionis in
cropping systems, as well as other Trichogramma species in different parasitoid-host-
habitat systems.
3. This study emphasizes the importance of improving our understanding of how tri-trophic
interactions (natural enemies, herbivores and host plants), as well as the real impact of
variable environments, impact parasitoid wasps. These studies may offer ways to
improve natural enemy efficacy in augmentative and conservation biocontrol efforts.
4. Extending the field study is essential in order to determine whether T. papilionis can be
manipulated to search more effectively in non- preferred habitat (e.g., cornfields),
through the use of chemical attractants to enhance their performance. The potential for
plant-derived semiochemical cues (e.g., from sunn hemp) that were shown to be
attractive to T. papilionis wasps in olfactory trials (Chapter 4 and 5), simultaneous with
T. papilionis releases, should be further examined.
122 References
Hajek, A.E. 2004. Natural enemies: an introduction to biological control. Cambridge University
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Hajek, A.E, Hurley, B.P., Kenis, M., Garnas, J.R., Bush, S.J., Wingfield, M.J., van Lenteren,
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123 Appendices
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
125
126
127
128
129 Appendix B: Summary of DMS result for corn earworm Helicoverpa zea eggs and a control blank.
130
131
132
133
134
135 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. References are listed in Chapter 4.
Chemical compound Plant, insect taxon- derived Functional activity References chemicals anisole Stachys riederi var. japonica Fumigant toxicity against maize weevils (Sitophilus Quan et al., 2018. (Family: Lamiaceae) zeamais) and booklice (Liposcelis bostrychophila).
Mature green berries of Coffea Strong attractive, solely and in a blend) to nymphs of the Njihia et al., 2017. arabica variegated coffee bug Antestiopsis thunbergii Gmelin (Heteroptera: Pentatomidae).
Flower heads of Pollinator attraction Steenhuisen et al., Protea species (Proteaceae) in cetoniid beetles 2013. (Atrichelaphinis tigrina).
Volatiles of older male desert Aggregation pheromone in adult male desert locust. Torto et al., 1994. locust Schistocerca gregaria (Forskål)
trans- isoeugenol Colombian tree barks Croton Lethal effect Jaramillo-Colorado et malambo (Karst) against Artemia franciscana al., 2014. + repellent activity against Tribolium castaneum Herbst
136 Leaves of Clausena anisata Insecticidal activity against filariasis vector, Culex Pavela et al., 2018. (Willd.) quinquefasciatus, and Musca domestica
Betel leaves (Piper betle) Repellent to Aedes aegyptii Alighiri et al. 2018.
Synthetic Ambrostoma quadriimpressum (Chrysemolidae) attractant Wang et al. 2017.
Green leaf volatiles Attractive to Phyllopertha horticola (Scarabaeidae) Ruther & Mayer 2005.
Basil (Ocimim sp.) Pesticidal activity, beetles, mosquitoes Vasudevan et al., 1999.
Ecdysteroid function and possible antagonist effects in Aviles et al., 2019. bis(2-ethylhexyl) Synthetic Spodoptera littoralis (Noctuidae), prolonged larval/pupal phthalate stages and delay in adult emergence). (DEHP)
Endocrine Disrupting Endocrine disruptor chemicals Planelló et al., 2011. Chemical significant drop in the activity of EcR transcription gene of the heterodimeric ecdysone receptor complex.
Pollutant Toxic to Drosophilidae, negative impacts on Chen et al., 2018. neurotransmission.
acetophenone Pigeon pea Attractive to rice pest Sogatella furcifera Nebapure, 2020 , Hu et al., 2019.
Volatile organic compounds Attractant to Spodoptera littoralis caterpillars de Fouchier et al., (VOCs) 2018.
137 Adult male moth Female Conogethes punctiferalis (Pyralidae) attractant Stanley et al., 2018. pheromone.
Tea plant (Camellia sinensis) Electroantennogram sensitivity in Ectrops obliqua Zhang et al., 2018. (Geometridae).
White spruce (Picea glauca) Plant resistance to spruce budworm (Choristoneura Mageroy et al., 2017. fumiferiana).
Mammals, human sweat Attractive to Simulium vittatum (Simuliidae) seeking hosts. Verocai et al., 2017.
Floral volatile blend, Vigna Host location by Maruca vitrata (Crambidae). Wang eta l., 2014. unguiculate, Lablab purpureus
Bark beetle (Scolytinae) Anti-aggregation pheromone in Scolytinae. Erbiligin et al., 2008.
Alfalfa (Medicago sativa) Attractive to alfalfa seed chalcid (Bruchophagus roddi, Light et al., 1992. Eurytomida).
138 alpha-farnesene Synthetic Attractive to the rice pest Sogatella furcifera. Hu et al., 2019.
Brewer’s yeast Insect attractant. Ljunggren et al., 2019. (Saccharomyces cerevisiae)
Fruit fly, Ceratitus capitate Male pheromone blend component, C. capitata Falchetto et al., 2019. (Tephritidae).
Coffee fruit (Cofea arabica) Modifies attractiveness of fruit to Hypothenemus hampei Blassioli- Moraes et (Scoytinae). al., 2019.
Tessaratoma papillosa Attracts parasitoid, Anastatus japonicus (Eupelimdae), to Wang et al., 2017. (Tessaratomidae) host eggs.
β-myrcene Brewer’s yeast Insect attractant. (Yeasts emit insect attracting volatiles Ljunggren et al., 2019. (Saccharomyces cerevisiae) typically associated with flowers.)
Mammals Olfactory response in naïve mosquitoes (Aedes aegypti, Culicidae). Vinauger et al., 2014.
Synthetic No attractive response in invasive moths in Hawaii, Noctuidae, Crambidae. Landolt et al. 2011.
Orchid flowers Mimic aphid alarm pheromones to attract hoverflies (Orchidaceae) (Syphidae) for pollination. Stokl, J., et al., 2011.
Viburnum spp. (Adoxaceae) Strong EAG response in Batocera horsfieldi (Cerambycidae) Yang et al., 2011.
139