The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

HORMONAL PRODUCTS IN COCKROACH EMBRYOS;

HOST SPECIFICITY OF ENTOMOPATHOGENIC NEMATODES

AND THE EFFECT OF SURFACE COAT PROTEINS FROM

NEMATODES ON INSECT IMMUNITY

A Thesis in

Entomology

by

Xinyi Li

© 2005 Xinyi Li

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

August 2005

The thesis of Xinyi Li was reviewed and approved* by the following:

Diana L. Cox-Foster Professor of Entomology Thesis Advisor Chair of Committee

A. Daniel Jones Senior Scientist of Chemistry

Kelli Hoover Associate Professor of Entomology

Liwang Cui Assistant Professor of Entomology

Stephen L. Rathbun Associate Professor of Statistics

Gary Felton Professor of Entomology Head of the Department of Entomology

*Signatures are on file in the Graduate School

iii ABSTRACT

My thesis is composed of two parts. One part is the research on hormonal products in cockroach embryos. This part was conducted under the guidiance of Dr. Glenn L.

Holbrook. The other part is focused on the effects of surface coat proteins of entomopathogenic nematodes on insect immunity. Dr. Diana L. Cox-Foster guided me through the second part of my research and has served as my thesis advisor.

Abstract I

Juvenile hormone (JH), produced by the corpora allata (CA), regulates molting and reproduction in many insects, including cockroaches. It is known, however, that JH is produced only after dorsal closure, a conspicuous event in embryogenesis.

Dorsal closure is an important physiological change in embryos. I found that the ratio of dorsal closure to embryo development time was consistent (about 45% of total embryo development) across most cockroach species. This conservation was linked to reproductive biology of the cockroaches. The only viviparous cockroach, Diploptera punctata, completed dorsal closure at 20.8 % of embryo development time. Blattella germanica, whose reproductive mode is different from other oviparity cockroaches, finished its dorsal closure at 38.5 % of embryo development time. Other oviparous and ovoviviparous cockroaches completed dorsal closure at similar percentages of the embryo development time.

It is reported that embryonic CA produce both JH and its immediate precursor methyl farnesoate (MF) in Nauphoeta cinerea. Using a radiochemical assay, the present

iv research found that cockroach embryos produced and released both JH and MF across all

three reproductive modes. These include Periplaneta americana, Eurycotis floridana,

Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, and

Diploptera punctata. I also found that the control of conversion of MF into JH by

epoxidase, the last step of biosynthesis of JH, is species dependent. These results suggest that the conversion of MF into JH is a rate-limiting step and was species specific.

Abstract II

Entomopathogenic nematodes (EPNs) are good candidates for biological-control agents for soil-dwelling insects. Infective juveniles (IJ) of EPNs enter insect hosts and

release symbiotic bacteria that kill the hosts. Insects defend against EPNs by a rapid

cellular immune response that includes encapsulation and melanization, which kills

EPNs. EPNs have to overcome insects’ innate immunity to survive and reproduce.

This study was designed to understand host immune responses to two species of

nematodes, Heterorhabditis bacteriophora and Steinernema glaseri, and the relationship

of immunity to host specificity. I hypothesized that EPNs induce or suppress the immune

responses in their host based on their surface coat proteins (SCPs). The insect hosts I

tested were immature stages of wax worm Galleria mellonella, oriental beetle larvae

Exomala orientalis, Japanese beetle larvae Popillia japonica, tobacco horn worm larvae

Manduca sexta, northern masked chafer larvae Cyclocephala borealis, and adult house cricket Acheta domesticus.

I found that H. bacteriophora and S. glaseri infected and reproduced in their

susceptible hosts well. In these hosts, EPNs were melanized and encapsulated at low

v percentages and a high percentage of EPNs were free-moving. In the resistant hosts,

most EPNs were melanized and encapsulated and few EPNs were free moving. S. glaseri

NC strain was more successful compared to the S. glaseri FL strain in the same hosts.

These results suggest the nematodes elicited immune responses in hosts that correlated

with their infectivity. I also found that hemocytes from M. sexta, a susceptible host,

recognized S. glaseri at a low percentage during the first hour post nematode

introduction. After 24 hours, H. bacteriophora escaped recognition of hemocytes from

G. mellonella, a susceptible host.

I demonstrated that different species and strains of EPNs had different SCPs. I

isolated and characterized the SCPs from S. glaseri NC strain. These SCPs suppressed

immune responses in the oriental beetle larva, a susceptible host for S. glaseri, thus

protecting H. bacteriophora from being killed in the same host, as it normally would be.

Immuno-suppression was dose-dependent. Also, multiple injections of the SCPs

protected H. bacteriophora better in Oriental beetle larvae. In a nondenatured state, two

isolated SCPs in the SCPs of S. glaseri each conveyed this immuno-suppressive effect.

The two SCPs were composed of smaller proteins when separated on two dimensional

PAGE. Hemocytes of oriental beetle larvae started degrading after exposure to the proteins for 3 hours. Some of the SCPs from S. glaseri NC strain were sequenced and

one of them was enolase, which is also secreted by other parasites.

vi TABLE OF CONTENTS

List of figures ...... ix

List of tables ...... xi

Acknowledgements ...... xii

Chapter 1: Overview of hormonal products in cockroach embryos ...... 1

1.1 Introduction ...... 2

1.1.1 Overview of Juvenile hormone in insects and cockroaches ...... 2

1.1.1.1 The insect corpora allata and juvenile hormone ...... 2

1.1.1.2 Juvenile hormone in cockroach embryos ...... 4

1.1.1.3 Methyl farnesoate, the JH precursor in cockroach embryos ...... 5

1.1.1.4 Variation in JH and MF in embryos ...... 6

1.1.2 Overview of cocoroach reproductive modes and phylogeny ...... 8

1.1.2.1 Reproductive biology of cockroaches ...... 8

1.1.2.2 Phylogeny of cockroaches studied ...... 8

1.1.3 Overview of radio chemical assay ...... 10

1.2 Hormonal products of cockroach embryos ...... 11

1.2.1 Dorsal closure and cockroach embryo development ...... 11

1.2.2 Methyl farnesoate and Juvenile hormone production in cockroach embryos ...... 13

1.2.3 Farnesol Stimulation of hormonal production in cockroach embryos ...... 15

Refereneces ...... 23

Chapter 2 Dorsal closure, reproduction, juvenile hormone and methyl farnesoate production in cockroach embryos ...... 27

Abstract ...... 28

Introduction ...... 29

vii Material and methods ...... 35

Results ...... 42

Discussion ...... 56

Acknowledgement ...... 60

References ...... 61

Chapter 3: Overview of insect immunity and entomopathogenic nematodes ...... 66

3.1 Introduction ...... 67

3.1.1 Overview of insect innate immunity ...... 67

3.1.1.1 Insect hemocytes and cellular immunity ...... 69

3.1.1.2 Insect humoral immunity, antimicrobial peptides and signalling pathways ...... 74

3.1.1.3 Recognition of nonself ...... 84

3.1.2 Overview of Entomopathogenic nematodes ...... 83

3.1.2.1 Biology of Entomopathogenic nematodes ...... 87

3.1.2.2 Epicuticle and extracellular matrices of Entomopathogenic nematodes ...... 90

3.1.2.3 Noncollagenous surface coat proteins ...... 92

3.1.3 Overview of Steinernema glaseri, Heterorhabditis bacteriophora, host immune response and surface coat proteins ...... 92

References ...... 96

Chapter 4: Infectivity of Entomopathogenic Nematodes and Immune Responses of Their Insect Hosts ...... 101

Abstract ...... 102

Introduction ...... 104

Material and methods ...... 108

Results ...... 114

Discussion ...... 130

viii Acknowledgement ...... 132

References ...... 133

Chapter 5: Characterization of surface coat proteins from Steinernema glaseri that suppress immune responses in Oriental beetle larvae ...... 137

Abstract ...... 138

Introduction ...... 139

Material and methods ...... 142

Results ...... 149

Discussion ...... 163

Acknowledgement ...... 165

References ...... 166

Chapter 6: Characterization of surface coat proteins from Steinernema glaseri ...... 169

Abstract ...... 170

Introduction ...... 171

Material and methods ...... 173

Results ...... 176

Discussion ...... 185

Acknowledgement ...... 187

References ...... 188

Summary ...... 190

Appendice A: ...... 194

Appendice B: ...... 210

Appendice C: ...... 215

ix LIST OF FIGURES

Fig. 1.1 Molecular structure of juvenile hormone homologs and the JH III precursor, methyl farnesoate...... 18

Fig. 1.2 Terminal steps in the JH III biosynthetic pathway in cockroaches...... 18

Fig. 1.3 Phylogenetic tree of cockroach species investigated in this study, derived from Kambhampati, 1995...... 19

Fig. 1.4 Reproduction and development chart...... 20

Fig. 1.5 Flow chart of extraction of released and synthesized MF and JH from CA...... 21

Fig. 1.6 Flow chart of the split CA experiment ...... 22

Fig. 2.1 Phylogenetic tree of cockroach species studied derived from Kambhampati, 1995...... 34

Fig. 2.2 Parameters of embryo development in different cockroach species...... 45

Fig. 2.3 Hormonal products in different cockroach species...... 47

Fig. 2.4 A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes...... 50

Fig. 2.5 Hormonal products in different cockroach species by single member of corporal allatum with and without farnesol stimulation...... 51

Fig. 3.1 Toll and IMD pathways ...... 79

Fig. 3.2 Phenoloxidase activation system ...... 83

Fig. 4.1 The degree of recognition by hemocytes from hosts to the IJs of EPNs after 60 mins of exposure...... 116

Fig. 4.2 Percentages of EPNs recognized by hemocytes from different hosts at 1st and 24th hour...... 117

Fig. 4.3 Immune responses of P. japonica to different EPNs after 16 to 18 hours after injection of 10 EPNs...... 121

Fig. 4.4 Immune responses of different hosts to different EPNs after 16 to 18 hours after injection of 10 EPNs...... 122

Fig. 4.5 SCPs from S. glaseri NC strain (A), S. glaseri FL strain (B), and H. bacteriophora(C) separated first on an urea-IEF gel (pH 3.5-10) and then on a 2D PAGE (10% acrylamide SDS-PAGE)...... 124

Fig. 4.6 Surface coat proteins from S. glaseri NC strain failed to protect H. bacteriophora from host immune responses in M. sexta...... 126

Fig. 4.7 Surface coat proteins from S. glaseri NC strain protected H. bacteriophora from host immune responses in E. orientalis larvae...... 127

x Fig. 5.1 Comparison of effect of suppression of melanization by SCPs from S. glaseri NC strain among different extraction methods ...... 155

Fig. 5.2 Dosage effect of SCPs from S. glaseri NC strain on immune responses in E. orientalis larvae ...... 156

Fig. 5.3 Effect of exposure time of SCPs from S. glaseri NC strain on immune responses in E. orientalis larvae ...... 157

Fig. 5.4 Separation of SCPs from S. glaseri NC strain on 8% Nondenaturing PAGE and 8% SDS PAGE ...... 158

Fig. 5.5 Test electroeluted fractions of SCPs from S. glaseri NC strain on immune responses of E. orientalis larvae ...... 159

Fig. 5.6. Separation SCPs from Band A and Band B on 8% Nondenaturing PAGE and SDS PAGE ...... 160

Fig. 5.7 Separation of SCPs from Band A and Band B on 2D PAGE ...... 161

Fig.5.8. Effect of SCP A and B on hemocytes of E. orientalis larvae ...... 162

Fig. 6.1 Second dimension separation on 10% SDS PAGE gel of surface coat proteins of and H. bacteriophora (A) and S. glaseri (B) ...... 178

Fig. 6.2 The first dimension separation of surface coat proteins from S. glaseri on 10 % nondenaturing PAGE gel ...... 179

Fig. 6.3 The 2nd dimension separation of surface coat proteins from S. glaseri on 10 % nondenaturing PAGE gel ...... 180

Fig. 6.4 The 2nd dimension separation of surface coat proteins from S. glaseri on 10 % SDS- PAGE gel ...... 183

xi

LIST OF TABLES

Table 2.1 Ratios of JH and MF production by the embryonic CA at 40 % (65% for D. punctata) post-dorsal closure development time ...... 48

Table 2.2 Comparisons of JH/MF production between reported by us and Stay et al...... 58

Table 3.1 Summary of antimicrobial peptides and the pathways they involved in...... 81

Table 4.1 Infectivity of EPNs against insect hosts ...... 115

Table 5.1 Description methods of SCPs extractions used in Fig. 5.1 ...... 154

Table 6.1 Sequences of SCPs from S. glaseri NC strain ...... 181

Table 6.2 Protein Masses of SCPs from S. glaseri NC strain ...... 184

xii ACKNOWLEDGEMENTS

I sincerely thank my advisor Diana L. Cox-Foster for her guidance

and mentorship during the course of this study. Without her advice and

time, it would be impossible for me to finish my PhD program. I wish to

thank Dr. Glenn Holbrook who introduced me into cockroach researches. I

appreciate Dr. Kenneth Berger for his lab support and Dr. Sirimni

Kambhampati who confirmed cockroach phylogenic tree. I also want to

thank Dr. Liwang Cui and Dr. Kelli Hoover who kindly offered me the

studying opportunities at my transition time. I appreciated Dr. A. Daniel

Jones who helped me used LC-MS and Dr. Stephen L. Rathbun who

discussed data analysis with me. I also want to thank Dr. Richard Cowls,

Dr. Elizabeth Cowls, and Dr. Randy Gaugler for their advice and supporting

of material; and thank Dr. Yi Wang for his communication and advices.

Thanks Owen M. Thompson for his lab support.

Finally, I want to thank my parents, brothers and my wife for their love and support.

Chapter 1

Overview of hormonal products in cockroach embryos 2 1.1 Introduction

1.1.1 Overview of Juvenile hormone in insects and cockroaches

1.1.1.1 The insect corpora allata and juvenile hormone

The corpora allata (CA) are paired endocrine glands that lie posterior to the brain in

insects. The CA synthesize and release Juvenile Hormones (JH), as well as JH precursors

in some insects (Judy et al., 1973; Pratt and Tobe, 1974). Indeed, a wide variety of JHs

are produced by insect CA. For example, the CA of Lepidoptera release JH 0, JH I, JH II

and JH III, while in Hemiptera, Hymenoptera and Orthoptera, their primary product is JH

III (Cusson et al., 1991). As for the Blattaria, after reinvestigation, researchers have come to accept that cockroaches have no JH other than JH III (Schooley and Baker,

1985).

Juvenile hormone production is inhibited and stimulated, respectively, by allatostatins

and allatotropins, which are neuropeptides produced in the brain. In cockroaches, as rates

of JH synthesis change, the volume of the CA, as well as its ultrastructure and the number

of cells it contains, changes correspondingly (Chiang et al., 1996; Lee and Chiang, 1997).

However, whether rates of JH synthesis are regulated by these slow changes in cellular structures or through rapid modulation of rate-limiting step(s) by neuropeptides is still unknown (Lee and Chiang, 1997).

Juvenile hormones regulate larval development and, in many insects, adult

reproduction. In immature insects, the presence of a high JH titer brings about a

subsequent larval molt, whereas a low JH titer results in a metamorphic molt. In adult

cockroaches, cycles of JH biosynthesis correspond to reproductive cycles (Tobe and Stay,

1985). For example, in adult females of both the viviparous cockroach Diploptera 3 punctata and oviparous cockroach Blattella germanica, rates of JH synthesis increase during oocyte maturation, decrease before ovulation, and remain low through pregnancy until initiation of the next ovarian cycle (Lee and Chiang, 1997).

The functions of JH in insects were originally elucidated in experiments involving the

ablation of CA, followed by re-implantation of the glands. Removal of the CA during the

penultimate larval instar resulted in precocious metamorphosis, and implantation of CA

induced a supernumerary larval stage. Thus, it was demonstrated that JH inhibits

metamorphosis in larvae. Also, through similar experiments, researchers showed that the

presence of the CA was necessary for development of eggs in adult females, as well as

accessory reproductive glands in males (Homola and Chang, 1997). Beyond that, a vast

amount of research has been carried out to elucidate the functions of JH and the

mechanisms by which it acts, and to understand the development, function, and

regulation of CA in both larval and adult insects (reviewed in Holbrook et al., 1998).

The earliest research on in vivo JH biosynthesis was conducted by Roller and Dahm

(1971), who injected radio-labeled precursors of JH into adult male Hyalophora cecropia. Valuable information was garnered in these and subsequent injection experiments. However, the small size of the CA relative to the insect limited the potential of in vivo biosynthetic experiments, because injected precursors were quickly diluted as they became distributed uniformly throughout the animal. Competing metabolic pathways, outside the CA, diverted precursors to the formation of unrelated labeled intermediates, which greatly complicated the interpretation of results. The first in vitro study on JH was conducted by Roller and Dahm (1970), who incubated brain- 4 corpora cardiaca-CA complexes from H. cecropia male pupae in Grace's medium,

together with sterile, heat-treated hemolymph from pharate adult H. cecropia.

1.1.1.2 Juvenile hormone in cockroach embryos

All of the aforementioned investigations were performed on larval or adult insects,

but comparatively little research has been carried out on CA function in embryos. This is

the case despite the early discovery of JH activity in milkweed bug (Oncopeltus

fasciatus) embryos by Novak in 1951 (referred to in Holbrook et al., 1998).

Nevertheless, it is known that insect CA reach a structurally and physiologically

differentiated state during embryogenesis, near dorsal closure (Lanzerin et al., 1984).

Dorsal closure brings about a conspicuous change in the external appearance of the embryo—the ruptured embryonic membranes become enclosed within the anterior end of the midgut, forming a triangular opaque area just posterior to the head, which in itself is transparent since it contains no yolk and, at least early on, little brain tissue (Stay and

Coop, 1973). After dorsal closure, JH is produced in the embryo (Imboden et al., 1978).

Rates of JH biosynthesis by embryonic CA change dramatically from dorsal closure to hatch, and correspond with fine changes in the structure of the CA (Lee and Chiang,

1997). Interestingly, all research on CA function—that is, on JH synthesis—in embryos has been done in cockroaches.

The identity of JH has been determined in many cockroach species, and JH III has

been shown to be the JH homologue in adults of the beetle cockroach (D. punctata),

German cockroach (B. germanica), brown-banded cockroach (Supella longipalpa),

Madeira cockroach (Rhypharobia (Leucophaea) maderae), lobster cockroach (Nauphoeta

cinerea), and Oriental cockroach (Blatta orientalis) (Chiang et al., 1996). Juvenile 5 hormone III has also been found in embryos of D. punctata and N. cinerea; note that

Brüning and Lanzrein (1987) also had an unpublished observation on JH III in B. germanica embryos. Surprisingly, however, substantial methyl farnesoate (MF), the immediate precursor of JH III, has also been found in embryos of N. cinerea (Brüning

and Lanzrein, 1987).

Although it is now accepted that embryonic CA are biosynthetically active, the

function of JH in embryos is in debate (Lanzrein et al., 1984). In 1984, Lanzrein et al.

first reported that CA of embryos of N. cinerea produced a large amount of both JH and its precursor methyl farnesoate (MF); the techniques they used to determine this were thin layer chromatography (TLC) and gas-liquid chromatography mass-spectrometry

(GLC-MS) (Lanzrein et al., 1984). They went on to show that the titers of JH III and MF changed significantly during embryonic development (Brüning et al., 1985). Moreover, in a later investigation, they found that MF was released into the hemolymph in the embryo, instead of being retained in the CA, as it is in the adult (Brüning and Lanzrein,

1987). All these findings, together, indicated that MF was behaving as a hormone in cockroach embryos. Nevertheless, although it has been shown that JHs do function in embryos, the role of MF remains unclear (Brüning and Lanzrein, 1987).

1.1.1.3 Methyl farnesoate, the JH precursor in cockroach embryos

Methyl farnesoate differs from JH III only by the absence of an epoxide group (Fig.

1.1). In crustaceans, MF, which is suspected to play a similar role as JH III in insects

(Homola and Chang, 1997), is produced by the mandibular organ, a structure analogous

to the insect CA. MF circulates in the hemolymph and regulates protein metabolism, the

molt cycle and reproduction (Homola and Chang, 1997). Results in insects also provide 6 some evidence that MF has JH-like function. It induces vitellogenin synthesis and oocyte

growth in decapitated adult females of N. cinerea, and also prevents metamorphosis when

it is applied to last instars. Plus, farnesyl methyl ether and other farnesol derivatives,

which are precursors of MF, have a strong larvalizing effect when applied to embryos of

Schistocerca gregaria. It is still unknown whether this activity is owing to conversion of

MF into JH III by nonspecific epoxidases. However, preliminary experiments using 3H-

labelled methyl farnesoate indicated there is no, or only very little, conversion of injected

MF to JH III in N. cinerea adults, larvae and embryos (Lanzrein et al., 1984; Brüning and

Lanzrein, 1987).

Despite the large quantity of MF observed in N. cinerea embryos, this compound may

still simply be a by-product of JH III synthesis, having little biological function. It has

been speculated that the accumulation of MF could be the consequence of a limited

oxygen supply for conversion of MF to JH III (Lanzrein et al., 1984). It is also possible,

though, that MF acts as a hormone per se. This possibility was supported by the fact MF

exerts strong JH-like effects when injected into N. cinerea larvae and female adults

(Brüning and Lanzrein, 1987).

1.1.1.4 Variation in JH and MF in embryos

In 1991, JH III was reported to be the predominant product of embryonic CA during

late embryogenesis in the viviparous cockroach, D. punctata (Kikukawa and Tobe,

1991), an insect used as a model for studying CA development, function, and regulation.

Then in 1998, Holbrook et al. conducted a more comprehensive investigation and showed that throughout embryogenesis, D. punctata CA produced JH III but almost no MF. And most interestingly, at 49% embryo development time, D. punctata CA produced only JH, 7 while at a comparable stage the CA of N. cinerea produced several-fold more MF than

JH III (See discussion in Holbrook et al., 1998). The question remains why CA of N.

cinerea embryos release MF and those of D. punctata do not.

The D. punctata embryonic chorion is very thin and ruptures early in embryogenesis.

This means there is probably abundant free air exchange between embryos and their

surrounding environment. Therefore, embryonic CA of D. punctata are likely well-

oxygenated, and conversion of MF to JH III may very well be promoted (Holbrook et al.,

1998). This line of reasoning is in step with that of Burgin and Lanzrein (1998), who speculated that a well-developed chorion encompassing N. cinerea embryos inhibited oxygen penetration into embryonic tissues and thereby reduced the availability of oxygen for conversion of MF to JH III. This hypothesis, nevertheless, remains controversial because even under in vitro conditions, which should be oxygen-rich, the CA of N. cinerea embryos still produce substantial MF (Burgin and Lanzrein, 1998).

The CA of both larval and adult cockroaches, when incubated with late JH

precursors, show enhanced JH release in vitro (Holbrook et al., 1996). Juvenile hormone

biosynthesis by CA of embryos is, for example, increased by farnesol (see Fig. 1.2), the

second ante-penultimate precursor in the JH-biosynthetic pathway (Holbrook et al., 1996;

Feyereisen et al., 1983). This shows that in this species, in both pre-adults and adults,

none of the last four enzymes (including 10,11-epoxidase which is responsible for

conversion of MF to JH III) is the overall rate-limiting step in the JH-biosynthetic

pathway (Holbrook et al., 1996). This conclusion is widely accepted by researchers

(Schooley and Baker, 1985; Holbrook et al., 1996). However, all this research has been conducted either with CA of adult insects, which do not release MF, or with those from 8 D. punctata embryos, which also do not produce MF (Holbrook et al., 1996). The

discovery of considerable MF production by N. cinerea embryonic CA, however,

suggests that the last enzyme involved in JH biosynthesis, methyl farnesoate epoxidase,

might significantly limit JH production.

1.1.2 Overview of cockroach reproductive modes and phylogeny

1.1.2.1 Reproductive biology of cockroaches

Cockroaches are ideal subjects for studies on JH production. The CA have been

investigated in several species, with D. punctata and N. cinerea in particular serving as

model systems (Chiang et al., 1996, Holbrook, et al., 1998). The diversity in reproductive biology among cockroaches also makes them excellent model systems.

There are three basic types of reproduction in cockroaches.

1) Oviparity—Fertilized eggs develop outside the body of their mother. The ootheca

(egg case and its enclosed eggs) is carried externally for a time but is most often

deposited within 24 hours of its formation. The oviparous species I investigated include

two blattids, B. orientalis and Periplaneta americana, and two blattellids, S. longipalpa,

and B. germanica. In some species of a few genera, including Blattella, the ootheca is

carried for weeks, during the entire developmental period of the embryo. In such species,

the eggs obtain water from their mother (Roth, 1970).

2) Ovoviviparity—A mother first extrudes the eggs, as in oviparous cockroaches, but then retracts them into a uterine brood sac. There they remain until the embryos mature,

at which time the neonates break free from their embryonic membranes and the ootheca.

The embryos of ovoviviparous cockroaches, like those from oviparous ones, are endowed

with sufficient yolk to complete embryonic development, but must absorb water from 9 their mother to complete development (Roth, 1970). The ovoviviparous cockroaches

studied were Blaberus discoidalis, Phoetalia pallida, S. lanpyridiformis, R. maderae, N.

cinerea, and P. nivea, all of which are in the family Blaberidae.

3) Viviparity — Reproduction is similar to ovoviviparous forms, except that the yolk

provided to the eggs at oviposition is insufficient to support full development. The embryos take up water and other nutrients from their mother during gestation.

Diploptera punctata is the only known viviparous cockroach (Roth, 1970), and was investigated in my research.

Blatta orientalis, P. americana and S. longipalpa are oviparous species that produce

thick-walled egg cases. The ootheca of B. orientalis and P. americana have well-

developed serrated keels and distinct respiratory tubes (Roth, 1968). The ootheca of S.

longipalpa has a marked reduction in its keel, though ventilating devices still exist. The

oviparous B. germanica is considered a link between oviparous and ovoviviparous forms

(Roth, 1968). The anterior end of the ootheca in this species, held in the vestibulum, is

permeable, and water is taken up by the eggs from the female during embryogenesis. In

ovoviviparous cockroaches, the ootheca is comparatively thin, flexible, and absent of

both ventilating devices and calcium oxalate crystals which contribute to the rigidity of

the egg case (Roth, 1968). Compared to the egg case of oviparous cockroaches, that of B.

discoidalis is reduced. However, the oothecal membrane of this primitive ovoviviparous cockroach is fairly dark and rigid. Relatively thin and practically colorless, or amber, oothecae are found in N. cinerea and R. maderae, whereas very thin and lightly colored oothecae are found in P. nivea. In the only known viviparous cockroach, D. punctata, the 10 ootheca is so reduced that a considerable portion of the anterior eggs is not covered at all

(Roth, 1968).

I examined these species in order to address the hypothesis that MF is produced

because of insufficient oxygenation. In primitive species, the critical function of the egg case is to conserve water in the embryos. Hence, the ootheca is thick, which may inhibit

oxygen uptake. Since the conversion of MF into JH III is an epoxidation process that

requires oxygen, I hypothesized that MF will be most abundant in the embryos of

primitive species, whose egg cases are rather thick. So I surmised there will be no or

little MF in D. punctata and P. nivea, whose oothecae are thin to non-existent. In other

species—including B. orientalis, P. americana, S. longipalpa, B. germanica, Byrsotria

fumigata, B. discoidalis and R. maderae—there should be MF, because all these species

have oothecae thicker than that of N. cinerea. Moreover, the MF to JH III ratio should be

highest in the oviparous cockroach embryos, for they have the thickest oothecae of all.

And though, methyl farnesoate is present in embryos of N. cinerea, its titer may be lower

than in oviparous species.

1.1.2.2 Phylogeny of cockroaches studied

The phylogenetic relationship among these cockroaches was also established by

analyzing the DNA sequence of mitochondrial ribosomal RNA genes (Kambhampati,

1995). The results by and large support the reproductive biology of the cockroaches.

The cockroaches I studied were arranged based on this phylogeny (Fig. 1.3). (I

appreciate Dr. Kambhampati confirming this phylogenic tree).

11 1.1.3 Overview of radio chemical assay

Since a radiochemical assay (RCA) was developed by Pratt and Tobe in 1974, it has greatly facilitated research on JH biochemistry and physiology, including identification of secretory products of the CA and developmental variations in rates of JH (and MF) synthesis (Feyereisen, 1985; Kikukawa and Tobe, 1987; Yagi and Tobe, 2001). Pratt and

Tobe showed under RCA conditions that methionine was the exclusive source of the methyl ester group of JH III (Feyereisen, 1985). In the RCA, the S-methyl group of methionine is transferred enzymatically and stoichiometrically to a carboxylic acid precursor of JH. Because the pool of methionine in CA is extremely small, the radioactive methionine from the medium dilutes nonradioactive methionine within the

CA. Then, after a short lag period in which isotopic equilibrium is reached within CA, biosynthesized JH is stoichiometrically labeled. This RCA has been used in recent years with CA from locusts, cockroaches, and beetles, among other insect groups (Feyereisen,

1985; Yagi and Tobe, 2001).

1.2 Hormonal products of cockroach embryos

1.2.1 Dorsal closure and cockroach embryo development

Cockroach embryos take variable time to complete their development. For example, the pre-larval period lasts about 71 days at 27°C in the viviparous cockroach D. punctata

(Stay and Coup, 1970). At the same temperature, the time from oviposition to hatch in the cockroach B. discoidalis is 61 days, and in P. americana is 43 days (see below).

Correspondingly, the pre-dorsal closure period time also varies among species. In D. punctata, it lasts about 13 days (Stay and Coop, 1973), but 29 days in B. discoidalis and 12 21 days in P. americana (Chapter 2). Moreover, the percentage of total development

time occupied by the pre-dorsal closure period differs among cockroaches. In D.

punctata, the pre-dorsal closure period covers about 19 percent of total development

(Stay and Coop, 1973), whereas in B. discoidalis and P. americana, this percentage is 47 and 48, respectively (Chapter 2).

Before examining whether MF is ubiquitous in cockroaches, I must first characterize

their embryonic development. This will enable me to examine different species at similar

developmental stages, which is important, because between dorsal closure and hatch the number and structure of embryonic CA cells and rates of JH (and MF) synthesis undergo

dramatic changes. The cockroaches species I investigated were B. orientalis, P.

americana, S. longipalpa, B. germanica, Byrsotria fumigata, B. discoidalis, R. maderae,

N. cinerea, P. pallida, S. lanpyridiformis, P. nivea and. D. punctata (Fig. 1.3). These

cockroaches have been selected on the basis of their evolutionary relationships (Fig. 1.3;

Kambhampati, 1995) and reproductive modes.

Insect colonies were reared at 27 °C, under a 12 h light:12 h dark photoperiodic

regime, and provided rat chow and water ad libitum. Newly emerged adult females were collected from the colonies and maintained in groups of 2–5, along with males, whose number exceeds the female number by one. The insects were kept in large Petri dishes, or small cages, with egg carton, under the same conditions as the colonies. The females were examined every day until they had oviposited. Thereafter, the females were reared without males. If a female was oviparous, it was kept alone in a Petri dish with a piece of

egg carton. After it has deposited its egg case, almost invariably on the egg carton itself,

the female was removed. 13 In order to examine whether insects have undergone dorsal closure, embryos were dissected individually from egg cases. For oviparous insects, the egg cases were collected directly from the Petri dishes. For viviparous and ovoviviparous species, embryo broods were obtained from pregnant females by applying gentle posterior- directed pressure to the females' abdomens. Broods of all species were partitioned with blunt forceps into individual embryos under cockroach saline modified to 360 mOsm/l

(Holbrook et al., 1998). Healthy embryos were counted, and three of them were selected at random and examined to see if they have undergone dorsal closure. Other sets of females, or egg cases, were kept until hatch. Larval number was recorded at hatch along with length of embryo development.

The day of oviposition was recorded as day 0, and the mean day of dorsal closure

(DC) and hatch were calculated from collected data. Then, the percent time at which dorsal closure occurs during embryogensis was obtained by dividing mean dorsal closure time by mean length of embryogenesis, the average time from oviposition to hatch (see

Fig. 1.4 and calculations).

1.2.2 Methyl farnesoate and Juvenile hormone production in cockroach embryos

Characterize the presence of MF and JH III in cockroach embryos

Methyl farnesoate is present in N. cinerea embryos (Lanzrein et al., 1984), and rates of MF synthesis follow closely those of JH III for much of embryonic development. MF and JH III are low just after the CA develop shortly after dorsal closure. MF and JH production increase quickly and reach a summit at mid-embryogenesis, and then decline slowly toward hatch (Brüning et al., 1985). After I successfully characterized 14 embryogenesis in several cockroach species, I went on to examine hormone production

by the CA at developmental stages that are temporally and developmentally similar

across the species.

Developmental stages of embryos are normally described using percentage of total

development time (Stay and Coop, 1972). To identify CA of similar developmental

stage, I used a scale running from dorsal closure to hatch, not oviposition to hatch (See

Fig. 1.4). The scale had dorsal closure, not oviposition, as its starting point, and the best way to explain it is by examples. Dorsal closure occurs 26 days after oviposition

(fertilization) in B. orientalis, whose total embryo development time is 49 days. A 40-

day old embryo, then, would be 61% into the post-dorsal closure period: (40 days – 26 days) / (49 days –26 days) * 100% = 61%. And following from this, 61% post-dorsal

time would be at day 40, as calculated from the following: (49 days – 2 6 days) * 61% +

26 days = 40 days. Moreover, forty percent post-dorsal closure time would be at 35 days:

(49 days – 26 days) * 40% + 26 days = 35 days, and eighty percent post-dorsal closure time would be at 44.4 days: (49 days – 26 days) * 80% + 26 days = 44.4 days (Fig. 1.4).

To quantify MF and JH III, I used a radiochemical assay (RCA).

Embryo broods were obtained as described above. The hormonal products of CA

were examined at a time when the embryonic CA produce large amounts of both JH III

and MF in N. cinerea. Six to seven embryos were randomly selected and dissected from a brood. Embryos were individually dissected, and their heads were severed on a

dissecting plate under cockroach saline (Holbrook et al., 1998). Upon isolating a CA-CC 15 complex from other tissues (nerve, fat, etc.), a pair of CA (or a CA-CC complex) were

pre-incubated for at least one hour in L-15 B medium containing 100 µM L-[methyl-3H]- methionine; the complex were then transferred to 20 µl fresh radiolabeled medium in 6 ×

25 mm borosilicate glass culture tubes, where CA were incubated for 6 hr at 27˚C with shaking on a variable plane mixer. At the termination of an assay, 100 µl of isooctane was added to each culture tube and the medium was vortexed. Then, the culture tube was centrifuged at 2000 g for 5 minutes, and the isooctane was collected. The medium was re-extracted with another 100 µl of isooctane. The isooctane aliquots were pooled and stored in conical-bottomed tubes (Fig. 1.5).

To separate MF from JH III in samples, the solvent was first evaporated to a volume of less than 10 µl under a nitrogen stream. The sample then was spotted on a TLC plate, which was previously developed twice in 1:1 chloroform:methanol and then placed in an oven for a minimum of 24 h at 110 ˚C. After initially spotting the plate, the bottle from which the sample came was rinsed with 100 µl isooctane. Using nitrogen, the solvent volume was brought down to less than 5 µl, and this was then applied to the TLC plate.

Then, the TLC plate was developed in 85:15 hexane: ethyl acetate. Afterward, the plate was cut into pieces and these pieces were placed in scintillation vials. The scintillation vials were vortexed and examined with liquid scintillation spectrometry.

1.2.3 Farnesol Stimulation of hormonal production in cockroach embryos

Discovery of a large amount of MF released by the CA of N. cinerea embryo casts a new light on this topic. In my research, I asked if the final step, conversion of MF to JH

III, is rate-limiting. I hypothesized there must be some regulation methods in this final step in N. cinerea embryonic CA. To explore this issue, I used several species of 16 cockroaches whose embryonic CA produce MF as a system. At the very beginning, I added a certain radiolabeled precursor (farnesol in this case) into culture media and observed if it enhanced CA production of JH and (or) MF from embryos.

Two members of a corpus allatum pair produced similar amounts of hormonal output

(Holbrook et al., 1996), and splitting CA into two individual corpus allatum has been

used to study the effect of farnesol treatment (Holbrook et al, 1996). Embryos were

individually dissected, and their heads were severed on a dissecting plate under

cockroach saline (Holbrook et al., 1998). Upon isolating a CA-CC complex from other

tissues (nerve, fat, etc.), a pair of CA (or a CA-CC complex) was pre-incubated for at

least one hour in L-15 B medium containing (2Ci/mmol) 100µM L-[methyl-3H]- methionine (85 mCi/mmol, Amersham, Catalog number TRK705). After pre-incubation one pair of CA were split into two corporal allatum (Fig. 1.6). One corpora allatum then was transferred into 20 µl fresh radiolabeled medium of 100μM farnesol in 6 × 25 mm borosilicate glass culture tubes (marked with CF, contents of CA with farnesol stimulation), and the other one in the same culture settings but without farnesol (marked with C, contents of CA). The tubes were incubated for 6 hr at 27˚C with orbital shaking.

After incubation, I transferred 100 µl cold media into each tubes and vertexed and centrifuged them briefly. Then, 80 µl surface media was transferred into two clean 6 ×

50 mm tubes marked as RF (release with farnesol treatment) and R (release of CA) corresponding to CF and C, respectively. The corpora allatum were observed at the bottom of the C and CF tubes under a microscope. Then, 100 µl of isooctane was added to the CF and C tubes; and 200 µl of isooctane was added to the RF and R tubes. All the tubes were thoroughly vortexed and 90% of the isooctane (90 and 180, µl respectively) 17 was transferred into conical-bottomed vials marked with CF, C, R and RF corresponding to the culture tubes. The isooctane then was evaporated to 5 µl under nitrogen stream, and then the sample was spotted in lanes on a thin layer chromatography (TLC) plate

(silica gel IB2, J.T. Baker 4448-04), which was previously developed twice in 1:1 chloroform:methanol and then placed in an oven for a minimum of 24 h at 100 ˚C. The

JH III and MF standards were spotted on the plate to determine the fractions of JH III and

MF. Then, the TLC plate was developed in 85:15 hexane:ethyl acetate. Afterward, each lane of the plate was cut into 14 fractions and the fractions were placed in 5ml scintillation vials. Scintillation vials were vortexed with 5 ml Betamax scintillation cocktail (ICN 880020) and then sat overnight in dark then examined with liquid scintillation spectrometry. DPMs were recorded for each fraction (Fig. 1.6).

Using the technique described, I studied JH and MF synthesis and release with and without farnesol stimulation in each species of interest.

18 JH 0 JH I

JH II JH III

MF

Fig. 1.1 Molecular structures of juvenile hormone homologs and the JH III precursor, methyl farnesoate.

Farnesol

Farnesol dehydrogenase

Farnesal

Farnesal dehydrogenase

Farnesoic Acid

Methyl transferase

Methyl Farnesoate

Epoxidase

JH III

Fig. 1.2 Terminal steps in the JH III biosynthetic pathway in cockroaches.

19

P. pallidaA A S. lampyridiformis ovoviviparity B. discoidalis

B. fumigata D. punctata viviparity N. cinerea R. maderae ovoviviparity

P. niveaA A blattellids B. germanica* A S. longipalpa* oviparity B. orientalis blattids P. americana E. floridana*

Fig. 1.3 Phylogenetic tree of cockroach species investigated in this study, derived from

Kambhampati, 1995.

* Species are not included in Kambhampati, 1995

A JH and/or MF production could not be detected by our methods

20

adult female emergence oviposition dorsal closure hatch

pre-dorsal closure post-dorsal closure

embryogenesis (developmental time)

Fig. 1.4 Reproduction and development chart. Calculations shown below. DC = dorsal closure.

Formula 1

mean time to DC (days) percent time of DC during embryogenesis = ×100 mean length of embryogenesis (days) Formula 2

number of embryos undergone DC percentage of embryos undergone DC = ×100 number of embryos dissected Formula 3

embryo age – DC time (days) percentage of post-DC time (%) = ×100 mean length of embryogenesis (days)

21

release biosynthesis

Dissected out CA in 40% post DC time of embryos (in Diploptera, 65%). CA complex were pre-incubated in L-15B medium with L-[methyl-

3H]-Methoinine for 1 hour

CA were incubated in the same media for 6 hours.

To test biosynthesis of JH and MF, I extracted the media and CA with isooctane directly

To test release of JH and MF, I partitioned culture media into two parts after quick centrifuge

Extracted partition without CA with isooctane to test the release of JH and MF.

Extracts from at least 6 pairs of CA were pooled.

Fig. 1.5 Flow chart of extraction of released and synthesized MF and JH from CA.

22 Split CA experiment

Dissected out A pair of CA at 40% post DC time of embryos (in Diploptera, 65%)

CA complex were pre-incubated in L-15B medium

with L-[methyl-3H]-Methoinine for 1 hour.

Split and culture in two tubes

C/CF (20 µl media without/with Farnesol) C CF Incubate for 6 hours

Add 100 µl media into each tubes

Vertex, centrifuge C CF

Transfer 80µl into tubes marked R/RF

40µl remains in the tubes C/CF

RC RF CF Extract JH and MF with isooctane transfer 200 µl isooctane into R/RF transfer 100µl isooctane into C/CF RC RF CF Transfer isooctane from R/RF and C/CF

Extracts were separated in TLC, the fractions of TLC were tested using scintillation counter, and the amount of JH and MF in each tube was calculated.

C indicates contents of JH (or MF) retained in a Corporal allatum, R indicates JH

(or MF) released into the media, RF and CF indicates the corresponding with farnesol

(precursor) treatment.

Fig. 1.6 Flow chart of the split CA experiment 23 References:

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(1985) Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III throughout the life cycle of a hemimetabolous insect, the ovoviviparous cockroach, Nauphoeta cinerea. Experientia 41, 913–917. LANZREIN B., IMBODEN H., BURGIN C., BRUNING E., and GFELLER H. (1984) Biosynthesis, metabolism and mode of action of invertebrate hormones. III.11 On titers, origin and functions of juvenile hormone III, methyl farnesoate, and ecdysteroids in embryonic development of the ovoviviparous cockroach Nauphoeta cinerea. p454–465. Springer-veriag, Berlin Heidelberg. LAUFER H. and AHL J. S. B. (1995) Mating behavior and methyl farnesoate levels in male morphotypes of the spider crab, Libinia emarginata (Leach). J. Exp. Mar. Biol. Ecol. 193, 15– 20. MAUCHAMP B., LAFONT R., and KRIEN P. (1981) Juvenile Hormone Biochemistry Analysis of juvenile hormones by high-performance liquid chromatography coupled with mass spectrometry. p21–31. Elsevier/North-Holland Biomedical Press. MAUCHAMP B., ZANDER M., and WOLFF R. (1984) Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones. III.2, The qualitative and quantative determination of juvenile Hormones by Mass Spectrometry. p363–372. Springer-Verlag Berlin Heidelberg. MUNDERLOH U. G., and KURTTI T. J. (1989) Formulation of medium for tick cell culture. Exp. & Appl. Acar. 7, 219–229. PRATT, G.E., TOBE, S.S., WEAVER, R. J. and FINNEY, J. R. (1975). Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaneta americana. Gen. Comp. Endocrinol. 26,478 -484. PRATT, G.E. and TOBE, S.S. (1974). Spontaneous Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro. Life Sci. Feb 1;14(3),575-86. RACHINSKY A., TOBE S.S. and FELDLAUFER M.F. (2000). Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L.): developmental changes in sensitivity to JH precursor and allatotropin. Insect Biochem. Mol. Biol. 30, 729-737. ROTH L. M. (1968) Oothecae of the Blattaria. Ann. Entomol. Soc. Amer. 61, 83–111. ROTH L. M. (1970) Evolution and taxonomic significance of reproduction in Blattaria. SCHOOLEY D. A. and BAKER F. C. (1985) Juvenile hormone biosynthesis. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 7, 363–389. Pergaman Press, New York. 26 STAY B. and COOP A. (1973) Developmental stages and chemical composition in embryos of the cockroach, Diploptera punctata, with observations on the effect of diet. J. Insect Physiol. 19, 147–171. STAY B. and LIN H. L. (1981) The inhibition of milk synthesis by juvenile hormone in the viviparous cockroach, Diploptera punctata. J. Insect Physiol. 27, 551–557. STAY B., ZHANG, J.R. and TOBE, S.S. 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Chapter 2

Dorsal closure, reproduction, juvenile hormone and methyl farnesoate

production in cockroach embryos 28

Abstract

Juvenile hormone (JH), produced by the corpora allata (CA), regulates molting and reproduction in many insects, including cockroaches. It is known, however, that JH is produced only after dorsal closure, a conspicuous event in embryogenesis.

Dorsal closure is an important physiological change in embryos. I found that the ratio of dorsal closure to embryo development time was consistent (about 45 % of total embryo development) across most cockroach species. This conservation was linked to the reproductive biology of the cockroaches. The only viviparous cockroach Diploptera punctata completed dorsal closure at 20.8 % of embryo development time. Blattella germanica, whose reproductive mode is different from other oviparity cockroaches, finished its dorsal closure at 38.5 % of embryo development time. Other oviparous and ovoviviparous cockroaches completed dorsal closure at similar percentages of the embryo development time.

It is reported that embryonic CA produce both JH and its immediate precursor methyl farnesoate (MF) in N. cinerea. Using a radiochemical assay, I found that cockroach embryos produced and released both JH and MF across all three reproductive modes. These include Periplaneta americana, Eurycotis floridana, Blaberus discoidalis,

Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, and Diploptera punctata.

I also found that the control of conversion of MF into JH by epoxidase, the last step of biosynthesis of JH, is species dependent. These results suggest that whether the conversion of MF into JH is a rate-limiting step or not was species specific.

29 Introduction

The Corporal allata (CA) are a pair of endocrine glands that synthesize and release

Juvenile Hormones (JH), as well as JH precursors in many insects (Judy et al., 1973;

Pratt and Tobe, 1974). Juvenile hormones regulate larval development and adult

reproduction. In immature insects, the presence of a high JH titer brings about a

subsequent larval molt, whereas a low JH titer results in a metamorphic molt. In adult

cockroaches, cycles of JH biosynthesis correspond to reproductive cycles (Tobe and Stay,

1985).

Insect CA are biosynthetically active after the insect embryo reaches a structurally

and physiologically differentiated state called dorsal closure (Lanzerin et al., 1984).

However, few studies have been carried out to characterize dorsal closure in cockroach embryogenesis, the hallmark of the study of biosynthesis of JH by CA (Imboden et al.

1978; Stay and Coop, 1973). Besides early studies, very little work has been done to

characterize the reproduction biology of cockroaches (Guthrie, 1968; Cornwell, 1968).

Several studies of grasshoppers, true bugs and cockroaches show that in hemimetabolous insects, the absence or low JH titer in an embryo allows it to secrete a pronymphal cuticle around dorsal closure, and the JH level peaks before the pronymph molt to the nymph

(Truman and Riddiford, 1999, 2002). Information is limited on CA function in embryos.

The identity of JH has been determined in many cockroach species, and JH III is

the JH homologue in adults of the beetle cockroach (Diploptera punctata), German

cockroach (Blattella germanica), brown-banded cockroach (Suppella longipalpa),

Madeira cockroach (Rhyparobia maderae), lobster cockroach (Nauphoeta cinerea), and

Oriental cockroach (Blatta orientalis) (Chiang et al., 1996). Surprisingly, substantial

30 methyl farnesoate (MF), the immediate precursor of JH III, has also been found in

embryos of N. cinerea ((Lanzerin et al., 1984; Brüning and Lanzrein, 1987; Bürgin and

Lanzrein, 1988). However, JH III was reported to be the predominant product of

embryonic CA during late embryogenesis in the viviparous cockroach, D. punctata

(Kikukawa and Tobe, 1991).

Holbrook et al. (1998) conducted a comprehensive investigation and showed that

throughout embryogenesis, D. punctata CA produced JH III but no MF. However, by

using a higher specific activity radiolabeled precursor and investigating wider embryonic

developmental time than previous studies, a recent study showed that a small amount of

MF is synthesized and released by embryonic CA of D. punctata (Stay et al., 2002).

Besides the studies described above, nothing is known about the MF biosynthesis and

release profile in cockroaches.

In crustaceans, MF is suspected to play a similar role as JH III in insects (Homola

and Chang, 1997; Tobe and Bendena, 1999; Borst et al., 2001; Laufer and Biggers,

2001). In crustaceans, MF is produced by the mandibular organ, a structure analogous to

the insect CA. MF circulates in the hemolymph and regulates protein metabolism, the

molt cycle and reproduction (Homola and Chang, 1997; Borst et al., 2001; Laufer and

Biggers, 2001). Results in insects also provide some evidence that MF has JH-like

function. It induces vitellogenin synthesis and oocyte growth in decapitated adult

females of N. cinerea, and also prevents metamorphosis when it is applied to last instars

(Brüning and Lanzrein, 1987). However, preliminary experiments using 3H-labelled

methyl farnesoate indicated there is no, or only very little, conversion of injected MF to

JH III in N. cinerea adults, larvae and embryos (Lanzrein et al., 1984; Brüning and

31 Lanzrein, 1987). Nevertheless, although it has been shown that JHs do function in

embryos, it is also possible that MF acts as a hormone per se in cockroach embryos. This

possibility is supported by the fact that MF exerts strong JH-like effects when injected

into N. cinerea larvae and female adults (Brüning and Lanzrein, 1987). Whether MF is a hormone per se in insects is still unknown and how JH hormone evolved in insects is an

interesting question. A study of JH and MF production in insect embryos will help to

unravel this problem.

In D. punctata, the CA of embryo, larval and adult cockroaches, when incubated

with late JH precursors, enhance JH release in vitro (Holbrook et al., 1996; Feyereisen et

al., 1983). This indicates that none of the last four enzymes (including 10,11-epoxidase

which is responsible for conversion of MF to JH III) is the overall rate-limiting step in the

JH-biosynthetic pathway (Holbrook et al., 1996; Schooley and Baker, 1985). The

discovery of considerable MF production by N. cinerea embryonic CA, however,

suggests that the last enzyme involved in JH biosynthesis, methyl farnesoate epoxidase,

might significantly limit JH production. In cockroaches, whether rates of JH synthesis

are regulated by slow changes in cellular structures or through rapid modulation of rate-

limiting step(s) by neuropeptides is still unknown (Chiang et al., 1996; Lee and Chiang,

1997).

Cockroaches of three reproductive modes were involved in this study. Fertilized

eggs of Oviparous cockroaches develop outside the body of their mother. The ootheca

(egg case and its enclosed eggs) is most often deposited within 24 hours of its formation

(Roth, 1970). The oviparous species include three blattids, Blatta orientalis, Periplaneta

americana and Eurycotis floridana, and two blattellids, Suppella longipalpa, and

32 Blattella germanica. A female ovoviviparous cockroach first extrudes the eggs, as in

oviparous cockroaches, but then retracts them into a uterine brood sac. There they

remain until the embryos mature, at which time the neonates break free from their

embryonic membranes and the ootheca. The embryos of ovoviviparous cockroaches, like

those from oviparous ones, are endowed with sufficient yolk to complete embryonic

development, but must absorb water from their mother to complete development (Roth,

1970). The ovoviviparous cockroaches studied include Blaberus discoidalis, Byrsotria

fumigata, Phoetalia pallida, Schultesia lampyridiformis, Rhyparobia maderae,

Nauphoeta cinerea, and Panchlora nivea, all of which are in the family Blaberidae.

Viviparous cockroach reproduction is similar to ovoviviparous forms, except that the

yolk provided to the eggs at oviposition is insufficient to support full development. The

embryos take up water and nutrients from their mother during gestation. Diploptera

punctata, the only known viviparous cockroach (Roth, 1970), was investigated. The

phylogenetic relationship among these cockroaches was also established by analyzing the

DNA sequence of mitochondrial ribosomal RNA genes (Kambhampati, 1995) (Fig. 2.1).

Whether hormonal products in cockroach embryos are related to their production biology

or phylogenetic relationship is an interesting question.

A radiochemical assay (RCA) for JH was developed by Pratt and Tobe in 1974, and

it has greatly facilitated research on JH biochemistry and physiology, including

identification of secretory products of the CA and developmental variations in rates of JH

(and MF) synthesis (Feyereisen, 1985; Kikukawa and Tobe, 1987; Yagi and Tobe, 2001).

Under RCA conditions, methionine was the exclusive source of the methyl ester group of

JH III (Feyereisen, 1985). In the RCA, the S-methyl group of methionine is transferred

33 enzymatically and stoichiometrically to a carboxylic acid precursor of JH. Because the

pool of methionine in CA is extremely small, the radioactive methionine from the

medium dilutes nonradioactive methionine within the CA. Then, after a short lag period in which isotopic equilibrium is reached in the CA, biosynthesized JH is stoichiometrically labeled. This RCA has been used in recent years with CA from locusts, cockroaches, beetles, and among other insect groups (Feyereisen, 1985; Yagi and

Tobe, 2001).

34

P. pallidaA A S. lampyridiformis ovoviviparity B. discoidalis

B. fumigata D. punctata viviparity N. cinerea R. maderae ovoviviparity

P. niveaA A blattellids B. germanica* A S. longipalpa* oviparity B. orientalis blattids P. americana E. floridana*

Fig. 2.1 Phylogenetic tree of cockroach species studied derived from Kambhampati, 1995 * Species are not included in Kambhampati, 1995 A JH and/or MF production could not be detected by our methods

35 Materials and methods

Insects

Insect colonies were reared at 27 ± 1°C, under a 12 h light : 12 h dark photoperiod

and were provided rat chow and water ad libitum. Newly emerged adult females were

collected from the colonies and maintained in groups of 2–5, along with males, whose

number exceeds the female number by one. The insects were kept in large Petri dishes,

or small cages, with an egg carton, under the same conditions as the colonies. The

females were examined every day until they have oviposited (fertilized). Thereafter, the

females were reared without males. If a female was oviparous, it was kept alone in a

Petri dish with a piece of egg carton. After it deposited its egg case—almost invariably

on the egg carton itself—the female was removed and the egg cases were collected

directly from the Petri dishes. For viviparous and ovoviviparous species, embryo broods

were obtained from pregnant females by applying gentle posterior-directed pressure to

the females' abdomens.

Reproduction biology and dorsal closure

Broods of all species were partitioned with blunt forceps into individual embryos

under cockroach saline modified to 360 mOsm/l (Holbrook et al., 1998). The day of

oviposition was recorded as day 0. Three embryos were randomly selected and dissected

from each brood. The number of embryos examined and the number of embryos that

completed dorsal closure (DC) were recorded. The day of DC is the day when 100 % of

the embryos underwent dorsal closure. The number of healthy embryos in each brood was counted on day of DC. The day of hatch and the number of hatchlings in each broods were also recorded.

36 Radiochemical assay

Developmental stages of embryos are normally described using percentage of total

development time (Stay and Coop, 1972). To identify CA of similar developmental

stage, I used post-dorsal closure time, which was based on a scale from DC to hatch. For example, in B. orientalis DC occurs 26 days after oviposition and total embryo development time (embryogenesis, oviposition to hatch) is 49 days. Then 40 % post-

dorsal time would be at day 35, as calculated from the following: (49 days – 2 6 days) *

40 % + 26 days = 35.2 days.

The hormonal products of the CA were examined at the time when the embryonic

CA produced large amounts of both JH III and MF in N. cinerea (40 % post-dorsal

closure except 65 % for D. punctata) (Brüning et al., 1985, Holbrook et al., 1998). It was

reported that JH III titer peaks at 35 to 40 day of embryo development time (Short and

Edwards, 1992).

JH and MF release and synthesis were measured with a rapid partition radio-

chemical assay (Pratt and Tobe, 1974; Feyereisen and Tobe, 1981; Holbrook et al., 1996,

1997). Six to seven embryos were randomly selected and dissected from a brood. At least three broods were examined at one developmental stage for each species to characterize either synthesis or release of hormonal production. Embryos were individually dissected, and their heads were severed on a dissecting plate under cockroach saline (Holbrook et al., 1998). Upon isolating a CA-CC complex from other tissues (nerve, fat, etc.), a pair of CA (or a CA-CC complex) were pre-incubated for at least one hour in L-15 B medium containing (2Ci/mmol) 100 µM L-[methyl-3H]-

methionine (85 mCi/mmol, Amersham, Catalog number TRK705 ); the complex then

37 was transferred into 20 µl fresh radiolabeled medium in 6 × 25 mm borosilicate glass

culture tubes, where the CA were incubated for 6 hr at 27 ˚C with orbital shaking. To test

for synthesis of JH III and MF by insect CA, 100 µl of isooctane was added to each

culture tube and the medium was vortexed. Then the culture tube was centrifuged at

4000 g for 5 minutes and the isooctane was collected. The isooctane aliquots were pooled and stored in a conical-bottomed tube with nitrogen filling up the tube at –80°C.

To test the release of JH III and MF by CA, 100 µl of non-radiolabeled medium was add to each 6 × 25 mm borosilicate glass culture tube. The tubes were vortexed at a low speed and then centrifuged at 2000 g for 5 minutes to spin down the CA. Then 100 µl of mixed media without CA were collected carefully from the top with a capillary tube and transferred into 6 × 50 mm borosilicate glass culture tubes. After transferring 200 µl of isooctane to each of the tubes, the tubes were vortexed and then centrifuged at 4000 g for

5 minutes, and the isooctane was collected and pooled to store in the same condition as described above. The blanks for the test were generated using the same procedure without CA.

Analysis of products of embryonic CA by thin layer chromatography

To separate MF from JH III in samples, the solvent was first evaporated to a

volume of less than 10 µl under a nitrogen stream. The sample were spotted on a TLC

plate (Silica gel IB2, J.T. Baker 4448-04), which was previously developed twice in 1:1

chloroform : methanol and then placed in an oven for a minimum of 24 h at 100 ˚C. The

TLC plate was divided into nine 2-cm-wideth lanes with two 1-cm-wideth edges. The top edge is 2 cm and the bottom line was 4 cm from the edge. The samples were spotted

38 at the bottom line in the middle of each lane. After initially spotting the plate, the bottle

from which the sample came was rinsed with 100 µl of isooctane. Using nitrogen, the solvent volume was brought down to less than 5 µl, and the sample was applied to the

TLC plate also. The JH III and MF standards were spotted on the plate to determine the fractions of JH III and MF. The TLC plate was developed in 85:15 hexane : ethyl acetate and development was terminated when the solvent frontier reached the top edge.

Afterward, each lane of the plate was cut into 28 fractions and these were placed in 5ml scintillation vials. The scintillation vials were vortexed with 5 ml Betamax scintillation

cocktail (ICN 880020), stored overnight in the dark and examined with liquid

scintillation spectrometry. DPMs were recorded for each fraction. For analysis of

separating JH and MF production of embryonic CA on TLC plates, fraction 7 and 8 (or

two fractions in front of JH peaks) were combined as internal background for JH fraction,

and Fraction 26 and 27 (or two fractions after MF peaks) were used as internal

background for MF fraction (at least 8 fractions for one species). Fractions 9 to 16 were combined as a JH part and fraction 17 to 26 were combined as a MF part. DPMs of

released partition timed 6/5 to calculate the amount of hormonal products released

because only 100 µl of 120 µl media was used in the extraction procedure. For

calculation of rates of JH and MF biosynthesis and release, the following formula was used: 3H-dpm/(2.2 (dpm per pCi) × specific radioactivity of 3H-met (pCi per pmol) ×

incubation time (h)) (Yagi and Tobe, 2001). The results were hormonal products of 6

pairs of embryonic CA, which was the experimental unit in our case. To compare to the

results from previous study, the means were divided by 6 in discussion.

39 Split gland assay

Two members of a corpus allatum pair produced similar amounts of hormonal

output (Holbrook et al., 1996). Splitting the CA into two individual corpus allatum has been used to study the effect of farnesol treatment (Holbrook et al, 1996).

A CA-CC complex was pre-incubated in the same medium as described previously.

After pre-incubation, one pair of CA was split into two corporal allatum. One corpora

allatum was transferred into 20 µl of fresh radiolabeled medium of 100 μM farnesol in 6

× 25 mm borosilicate glass culture tubes (marked with CF, contents of CA with farnesol

stimulation), and the other one in the same culture settings but without farnesol (marked

with C, contents of CA). The tubes were incubated for 6 hr at 27˚C with orbital shaking.

After incubation, I transferred 100 µl of cold media into each tube and vortexed and

centrifuged them briefly. Then, 80 µl of surface media were transferred into two clean 6

× 50 mm tubes marked as RF (release with farnesol treatment) and R (release of CA) corresponding to CF and C, respectively. The CA were observed at the bottom of the C and CF tubes under a microscope. Then 100 µl of isooctane were added to the CF and C

tubes; and 200 µl of isooctane were added to the RF and R tubes. All of tubes were

thoroughly vortexed and 90 % of the isooctane (90 and 180 µl, respectively) was

transferred into conical-bottomed vials marked with CF, C, R and RF, corresponding to

the appropriate culture tubes. The isooctane was evaporated and applied to TLC plates as

described in the previous experiment. Each lane of the TLC plate was cut into 14

fractions. DPMs were recorded for each fraction.

Using the above techniques, I studied JH and MF synthesis and release with and

without farnesol stimulation in B. orientalis, B. fumigate, B. discoidalis, N. cinerea R.

40 maderae and D. punctata.

DPMs of fractions corresponding to JH and MF standard position were summed to

obtain peak fractions. While for analysis of JH and MF production of CA stimulated by

farnesol, two fractions at the end of each lane were combined (at least 8 fractions for one

species) to obtain internal background. DPMs of JH and MF retained in the CA without

(with) farnesol treatment were calculated as DPM of C (CF) minus half of the DPM of R

(RF), then times 10/9. DPMs of JH and MF released into media without (with) farnesol

treatment were calculated as DPMs of R (RF) times 1.5 and then 10/9. To calculate rates

of JH and MF biosynthesis and release, the following formula was used: 3H-dpm / (2.2

(dpm per pCi) × specific radioactivity of 3H-met (pCi per pmol) × incubation time (h))

(Yagi and Tobe, 2001). Negative numbers were recorded as zeros. Because micro

dissection can damage the tiny corpora allatum, only if each corpora allatum from the

same pair of CA had positive values were the data used in analysis,.

In results part, C and R indicate JH (MF) retained in corpora allatum and released

in media respectively. Similarly, CF and RF indicate JH (MF) retained in corpora

allatum and released after farnesol treatment.

Statistical analysis

Statistical analysis was carried out in MINITAB version 13.0 or SAS 8.0.

For days of embryogenesis, the number of eggs and the number of the hatchling, the data were transformed by square root. Species was considered as a factor nested in reproductive modes. DPMs of JH were transformed by logarithm. DPMs of MF were transformed by quadruple root. For JH and MF analysis, JH and MF data were analyzed in separate ANOVAs. Species was a factor nested in reproductive modes for the

41 ANOVAs. Releasing or synthesizing of JH and MF by CA was a fix effect, and the interaction between release/synthesis effect and species was included in the ANOVAs.

For farnesol stimulation experiment, species and C, R, CF, and RF were considered as fixed effects. The CA pairs were considered as a nested effect within species, and species and treatment were considered as a possible interaction. The data were transformed to meet the assumptions of ANOVA. Tukey pairwise comparisons were used if the ANOVA indicated significant results. The JH and MF production between ovoviviparous cockroaches and oviparous cockroaches were compared by contrasts.

42 Results

Number of eggs and number of hatchlings for each brood

Differences in the numbers of eggs (F = 267.93, df = 10, 397; p < 0.001) and the numbers of hatchlings (F = 71.56, df = 10, 397; p < 0.001) among 13 species of cockroaches were highly significant (Fig. 2.2). P. nivea had the highest number (71.2 ± 1.5) of eggs and the number of hatchlings (54.7 ± 3.4) per brood. D. punctata has the lowest number of eggs (11.8 ± 0.2) and hatchlings (9.6 ± 0.3) per brood. The number of eggs (F = 706.59, df = 2, 397; p < 0.001) and the number of hatchlings (F = 163.12, df = 2, 397; p < 0.001) were all very different among cockroaches of three reproductive modes. The number of hatchlings of ovoviviparity oviparity and viviparity were 31.5 ± 1.1, 18.9 ± 0.8 and 9.6 ± 0.3, respectively. The number of eggs of ovoviviparity (36.5 ± 0.8) was higher than oviparity (19.2 ± 0.7) and viviparity (11.8 ± 0.2). The average hatching rates was about 86.5 % which indicates hatching was highly successful in all cockroach species in this study. By regressing the average number of eggs against the average number of hatchlings in each brood, the ratio was very consistent across all species (F = 161.64, df = 1, 11; R2 adj. = 0.93 and p < 0.001). B. fumigata (ratio = 0.48) and P. nivea (ratio = 0.768) were very different from other species, indicating these species have low success in hatching. Among the ratios of the number of hatchlings to the number of eggs in one brood, P. americana had the highest ratio (1.03, higher than 1 because of experimental error). P. nivea had the significantly highest number of embryos (Tukey pairwise comparisons, p < 0.001) and the number of hatchlings (Tukey pairwise comparisons, p < 0.001) among all the species. The number of eggs of B. germanica was the second highest which was different from other cockroaches (Tukey pairwise comparisons, p < 0.001).

43 Dorsal closure and gestation time The gestation time of 13 species of cockroaches were significant different (F =

2721.15; df = 10, 397; p < 0.001). The gestation time among three reproductive mode

was significant different (F = 3052.50; df = 2, 397; p < 0.001). Viviparous D. punctata

had the longest gestation time (67.3 ± 0.2 day) compared to oviparous cockroaches (43.6

± 0.9 day) and ovoviviparous (44.0 ± 0.7 day). S. lampyridiformis and P. nivea had

similar gestation times (Tukey pairwise comparisons, p < 0.185), but gestation times of the other species were all different from each other (Tukey pairwise comparisons, p <

0.03). D. punctata had the longest gestation time (67.3 ± 0.2) and B. germanica had the shortest (20.8 ± 0.1 days).

In most species, more than 75 % of DC was occurred during a two-day period

indicating the embryos in each species developed at a similar rate. This suggests that not

only the embryos in the same brood but the embryos in the same species developed at a

similar pace.

When I performed a correlation analysis on averages of day of DC and day of

hatch, I found that the two were significantly correlated (F = 8.3; df = 1, 11; p = 0.015; R2 adj. = 37.8 %). In this analysis, D. punctata and B. germanica were the outliers.

Performing the analysis without outliers, the correlation was very significant (F = 145.85, df = 1, 9; p < 0.001; R2 adj. = 93.5 %). This indicated the time when DC occurred in

embryo development time was very consistent (around 45.0 % of embryo development time) in most the cockroach species studied. This suggested DC is probably a conserved developmental event in cockroaches. In other insect groups studied, DC occurs at about

48-60 % embryogenesis (Truman and Riddiford, 1999, 2002).

44 Conservation of time to DC might be controlled by nutrient availability of the

embryos. D. punctata is the only viviparous cockroach, whose embryos obtain nutrients from the mother. The DC day of D. punctata was the longest among the species studied.

However, it was relatively short (20.8 % at embryo development time), while time of embryogenesis in D. punctata was the longest among all species. The early DC in embryogenesis of the beetle cockroach might be related to the high amount of JH produced by the embryonic CA. Its embryonic CA become functional organs after DC.

B. germanica is unique between oviparous (egg-laying) and ovoviviparous (live-birth) roaches, because the mother keeps the egg case half inside of the body and half outside till they hatch. It is considered a link species between the two groups (Roth, 1970; Schal, et al., 1997). The DC day of B. germanica was the shortest among all species studied and it was 38.5 % of the total embryogenesis time. Its time of embryogenesis was the shortest among all cockroach species studied. B. germanica broods finished dorsal closure in just one day.

45

A 80 Number of eggs and hatchlings 71.2 70 eggs/brood nymphs/brood 60 54.7

50 46.3

39.3 35.8 40 34.9 33.4 32.6 32.8 number 30.1 28.1 30 27.3 25.2 23.4 23.6

14.4 14.7 17.6 15.3 20 11.8 14.1 15.0 13.813.8 11.3 9.6 10

800 a s a a a a a s B gestation time/day Gestatione time and DC day 70 DC day 67.30 63.53 60.77 60 58.15 52.71 49.04 50.46 50 43.32 40.21 40 36.05 33.81 day 32 33.06 29 30 30 27 26 25 21 20.77 2020 19 20 17 14

10 8

0

Blatta orientalis Panchlora nivea Pheotalia palluda Pheotalia Supella longipalpa Byrsotria fumigata Byrsotria Eurycotis floridana Eurycotis germanica Blattella Nauphoeta cinerea Diploptera punctata Diploptera Blaberus discoidalis

Rhyparobia maderae Periplaneta americana Schultesia landyriformis Figure 2.2 Parameters of embryo development in different cockroach species. Bars represent means ± S.E. A, average number of eggs and nymphs hatched per brood. B, average gestation day and dorsal closure day. The number of replicates ranged from 28 to 83.

46 Analysis of MF and JH of embryonic CA by thin layer chromatography

Using the technique described above, I could not detect any JH and MF biosynthesis in some species at 40 % post-dorsal closure development time. These species include S. longipalpa, and B. germanica P. pallida, S. lampyridiformis and P.

nivea. These species were small size cockroaches and their embryos were relative small.

It was possible that during the dissection, the small CA from these small cockroach

embryos were destroyed. It was also possible these small CA did not produce large

amount of JH an MF. They were not shown in Figure 2.3 and Figure 2.4. The results graphed were hormonal products of 6 pairs of embryonic CA, which was the experimental unit in our case. To compare with results from previous study, we divided the mean by 6 in discussion. Figure 2.3 is JH and MF production by embryonic CA at 40

% (in D. punctata, 65 %) post-dorsal closure development time. Table 2.1 presents the ratios of hormonal products in cockroach embryos.

47

0.25 A B MF production 0.20 MFsynthesis MF release 0.15 ) AB

pm/(h*CA 0.10

0.05 A A A A A A A A A A A 3.5 A A A 0.00 B 3.0 JH synthe sis JH release

2.5 F

2.0

1.5 pm/ (h*CA) CF

ECF AC 1.0 ACF ACF AF 0.5 AED ABE ABC ABE BD ABE AB ABE B

0.0

Blatta orientalis Eurycotis floridana Eurycotis fumigata Byrsotria Nauphoeta cinereaNauphoeta Diploptera punctata

Blaberus discoidalis Periplanta americana Rhyparobea maderea Rhyparobea Figure 2.3 Hormonal products in different cockroach species. Bars represent means ± S.E. A, MF production by embryonic CA. B, JH production by embryonic CA. Number of replicates ranged from 3 to 12 (6-pair of CA).

48 Table 2.1 Ratios of JH and MF production by the embryonic CA at 40 % (65 % for D. punctata) post-dorsal closure development time.

Embryo agea MF synthesis / JH MF release / JH MF release / JH release/

(days) synthesis* release MF synthesis JH synthesis Blatta orientalis 35 0.071 ± 0.05 (6) 0.123. ± 0.058 (4) 1.77C 1.15 Periplaneta americana 30 0.328 ± 0.18 (6) 0.270 ± 0.097 (3) 0.45 0.33 Eurycotis floridana 36 0.109 ± 0.03 (6) 0.079 ± 0.068 (2) 0.35 0.37 Blaberus discoidalis 42 0.055 ± 0.05 (6) 0.000 ± 0.000 (4) 0.00C 0.77 Byrsotria fumigata 41 0.058 ± 0.01 (7) 0.051 ± 0.038 (3) 0.57 0.63 Rhyparobia maderae 45 0.162 ± 0.08 (9) 0.099 ± 0.039 (6) 0.24 0.72 Nauphoeta cinerea 28 5.598 ± 0.99 (10) 0.718 ± 0.204 (8) 0.06 0.31 Diploptera punctatab 49b 0.013 ± 0.01 (3)C 0.014 ± 0.006 (3) 0.62 0.41 Data are rates of JH and MF biosynthesis and release by embryonic CA at 40 % post-dorsal closure time (For D. punctata, 65 % ). Data are shown as mean ± S.E.M. for 6 embryos in one brood. Numbers in the parenthesis are the number of broods. a Embryo age at 40 % post-dorsal closure development time. b Embryo age at 65 % post-dorsal closure development time. c Ratio is significantly different from other in the column (p < 0.01).

JH production by embryonic CA among 8 cockroach species studied was significantly different (F = 23.65; df = 5, 76; p < 0.001). The JH production among three reproduction modes was significantly different (F =27.52; df = 2, 76; p < 0.001). The amount of JH synthesized and released by CA was different (F = 5.99; df = 1, 76; p <

0.017). The interaction between the species and synthesis/release was not significant (F

= 0.09; df = 2, 76; p < 0.917). These results indicate that production of JH by cockroach embryonic CA is species dependent process. Embryonic CA of different cockroach species synthesizes and releases different amount of JH. However, when CA of a species of cockroach synthesized more JH hormone, they released more JH.

49 For MF production, the interaction between the species and synthesis/release was

significant (F = 2.73; df = 5, 78; p < 0.025). The interaction between the reproduction

modes and synthesis/release was significant (F = 4.79; df = 2, 78; p < 0.011). Theses

results indicate embryonic CA of different cockroaches synthesized and released MF

differently. This process is species dependent.

Three blattids, B. orientalis, P. americana and E. floridana, produced and released

appreciable amount of JH. Among these insects, the latter two species also produced and

released trace amounts of MF. I could not detect JH and MF in blattellids (S. longipalpa,

and B. germanica) with my method. In the only ovoviviparous cockroach, the titer of JH synthesized and released was very high, and MF was also produced and released. In P. pallida, S. lampyridiformis, and P. nivea, I did not detect any JH or MF. In the closely related species N. cinerea and R. maderae, I detected the same patterns between these two species, in which JH was produced and released and relatively large amount of MF were produced and released. While JH was produced and released by CA of B. discoidalis and B. fumigata, but little or no MF was produced at all (Fig. 2.4).

Contrast analysis found that ovoviviparous cockroaches (B. discoidalis, B.

fumigata, N. cinerea and R. maderae) produced more JH compared to oviparous

cockroaches (B. orientalis, P. americana and E. floridana) (p < 0.01). The ovoviviparous

cockroaches produced lower amounts of MF compared to oviparous cockroaches, but the

difference were not significant (p > 0.05).

50

P. pallidaA N

S. lampyridiformisA N

B. discoidalis JH JH-R MF B. fumigata JH JH-R MF MF-R

D. punctata JH JH-R MF MF-R N. cinerea JH JH-R MF MF-R R. maderae JH JH-R MF MF-R

P. niveaA N B. germanica*A N S. longipalpa*A N

B. orientalis JH JH-R MF MF-R

P. americana JH JH-R MF MF-R

E. floridana JH JH-R MF MF-R

Figure 2.4 A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes. (modified from Srinivas Kambhampati, Proc. Natl. Acad. Sci. USA. Vol. 92, pp. 2017-2020, March 1995).

51 0.035 A 0.03 R RF 0.025 C CF

0.02 MF [pmol/(h*CA)

0.015

0.01

0.003 ]

A) 0.002 l/(h*C o

F [pm M 0.001

0 0.35 Blaberus Byrsotria Rhyparobea Diploptera discoidalis fumigata maderea punctata R RF B 0.3 C CF

0.25 JH [pmol/(h*CA)

0.09

0.06

JH [pmol/(h*CA)] 0.03

0 Blaberus Byrsotria Rhyparobea Diploptera discoidalis fumigata maderea punctata Figure 2.5 Hormonal products in different cockroach species by single member of corporal allatum with and without farnesol stimulation. Bars represent means ± S.E. A, MF production by embryonic CA. B, JH production by embryonic CA. R, released products by corporal allatum; C, contents of products of corporal allatum, RF, released products after stimulation by farnesol; CF, contents of products of corporal allatum after stimulation by farnesol. The number of replicates ranged from 6 to 16 (pairs of CA).

52 Split gland assay

I could not detect JH or MF biosynthesis by single corporal allatum of B. orientalis

(N = 3). In N. cinerea (N = 20), I could not consistently detect JH or MF released by single corporal allatum.

ANOVAs were carried out for JH and MF production separately.

For JH biosynthesis, the amount of JH released (R), released with farnesol stimulation (RF), retained in CA (C) and retained in CA with farnesol treatment (CF) were significantly different (F = 39.86; df = 3, 106; p < 0.001) These values were very different among species studied (F = 15.99; df = 3, 106; p < 0.001), which indicates these values were species specific. The gland was a significant effect (F = 2.56; df = 37, 106; p

< 0.001) indicating there was individual variation within the same species. The interaction of treatment and species effect was not significant (F = 1.17; df = 9, 106; p <

0.319), which indicates as for JH synthesis and release, different species responded to farnesol treatment similarly (Figure 2.5).

For MF biosynthesis, the amount of MF released (R), released with farnesol treatment (RF), retained in CA (C) and retained in CA with farnesol treatment (CF) interacted with the species effect significantly (F = 4.36; df = 9, 111; p < 0.001). The gland was a significant factor (F = 17.63; df = 3, 111; p < 0.001). As in the JH analysis, the amount of MF retained in CA and released in media was different between the farnesol treatment and it was different among cockroach species. However, the interaction between treatment and species existed which indicates that cockroach species react with farnesol treatment in a species specific manner.

53 Across four species studied, the amount of JH released was higher than the amount

of JH retained in the CA (Tukey comparison, p < 0.001). With farnesol stimulation, the

amount of JH released by embryonic CA increased significantly (Tukey comparison, p <

0.001). There was no difference in the amount of JH retained in CA between with

farnesol treatment and without treatment (Tukey comparison, p < 0.259). For MF production, the amount of MF released was not different from the amount of MF retained in CA (Tukey comparison, p < 0.742). After farnesol incubation, the amount of MF retained CA was significant higher than that without treatment (Tukey comparison, p <

0.035). And the amount of MF released was not different between CA with and without

treatment (Tukey comparison, p < 0.980). . These results suggest that farnesol

stimulated MF production; however, CA did not release more MF. Instead, most of the

MF was converted into JH and most of the JH was released out of the CA. As for JH

biosynthesis across the species, B. fumigata produced the lowest amount of JH and D.

punctata produced the highest amount of JH. R. maderae produced the highest amount

of MF.

Figure 2.5 shows JH and MF production by embryonic CA at 40 % (in D. punctata,

65 %) post-dorsal closure development time with and without farnesol stimulation.

In the beetle cockroach, farnesol significantly stimulated the biosynthesis of MF,

and most of the MF was retained in the CA instead of being released into the media.

Without farnesol treatment, embryonic CA of beetle cockroach released 72 % of the JH it

produced. After farnesol treatment, D. punctata produced and released more JH than it

did without treatment. These results suggest that in beetle cockroach with farnesol

54 treatment embryonic CA produced huge amount of MF, and most MF in turn was

converted into JH and released into the media.

For R. maderae, after farnesol treatment, the amount of JH released by embryonic

CA increased to about 3 times of that without treatment. Without farnesol treatment, the

amount of JH released by embryonic CA was about 2 times of the amount of JH retained

in CA, while after the treatment, the ratio increased to about 3.

Without farnesol treatment, the amount of MF retained in embryonic CA was more than 10 fold higher than was released. After farnesol treatment the amount of MF retained in CA increased a lot but the amount released into the media did not. R. maderae produced significantly the highest amount of MF compared to other species studied. Results indicate that in R. maderae, farnesol stimulates production of MF, and most MF was retained in embryonic CA, but some of it was converted into JH and released into the media. This pattern of R. maderae was very similar to D. punctata.

B. fumigata and B. discoidalis showed a similar JH/MF release/ retention pattern, which is reasonable because these two species are phylogenically closer than the other species. Both of the species released about 80 % of the JH they produced with/without

farnesol treatment. After the farnesol treatment, both species released and retained more

JH than without treatment, but the difference was not significant. For MF biosynthesis,

both of species produced MF, but due to large variation among individuals, I could not

detect the difference among all treatment groups.

In D. punctata, there was 0.269 pmol JH released into the media with only 0.00072

pmol MF retained in CA, which suggests that most MF was converted into JH and

released into the media. However, in R. maderae, there was 0.075 pmol JH released into

55 the media with 0.0229 pmol MF retained in CA, which suggests that the step of

conversion of MF into JH is a rate-limiting step and MF was accumulated in CA.

Therefore, my results suggest that regulation of conversion of MF into JH is a species dependent step. Also, the amount of MF released depends on the species.

56 Discussion

For the only viviparous cockroach, D. punctata, DC occurred at 20.8 % of embryogenesis, which was similar to a previous report of 20 % (Stay et al., 2002). Stay et al. reported DC occurs on day 12 and the duration of embryogenesis is 63.0 days.

However, our results indicate that DC occurs on day 14 and embryogenesis is 67.3 respectively. The number of eggs/brood I obtained (11.8) was the same as that reported previously (Stay et al., 2002). For the JH/MF study, I used embryonic CA at 65 % post- dorsal closure time in D. punctata, and 40 % post-dorsal closure time for other species studied, because the time of dorsal closure occurred relative early in the embryogenesis in

D. punctata. This was a reasonable decision, because observation of the development of eye pigmentation and the development of the gut of embryos indicated D. punctata was at a comparable physiological stage to other species of cockroaches. For N. cinerea, my finding of the day of DC and the length of embryogenesis is consistent with a previous report (Brüning et al., 1985).

DC occurred at 47-57 % of embryogenesis across all cockroach species except D. punctata, which was similar to the 48-60 % reported for other hemimetablous insects

(Truman and Riddiford, 1999). The consistency of DC in embryogenesis is most likely related to how the embryos obtain nutrition.

When I studied JH/MF production by embryonic CA, I could not detect JH or MF production in several species. Most of them were smaller cockroaches, and their embryonic CA were relatively small. These CA probably produced titers of JH or MF below the level of detection in this study. The detection limits (background) for

TLC/RCA for this experiment were about 100-400 DPMs (equal to 0.004-0.016 pmol/hr)

57 for JH and 50-100 DPMs (equals to 0.002-0.004 pmol/hr) for JH. There were other possibilities for why I could not detect JH or MF. One possibility was that the small CA

were hard to clean, and the enzymes in tissue around CA-CC complex might degrade JH

and MF. The other possibility might be that I injured these small CA during the

dissection process. Another possibility could be for these cockroaches, embryonic CA

did not produce large amount of JH/MF at 40 % post dorsal closure time.

In the split glands (farnesol stimulation) experiment, I detected a small amount of

MF produced and released by embryonic CA of D. punctata. The detection limits

(background) for this assay were 30-50 DPMs (equal to 0.00114-0.00189 pmol/hr) for

both JH and MF. I could only detect JH and MF if DPMs of radiolabeled JH/MF were

higher than that of background. The JH/MF production data (at 65 % post-dorsal closure

time, 49 day of embryogenesis) obtained in this research was compared to the JH/MF

production data at 65 % post-dorsal closure time (45 days of embryogenesis) reported by

Stay et al. (2003) in Table 2.2. I used different culture media, incubation time, pre-

incubation design and different substrate to stimulate the CA, however the amount of JH

released was comparable. My data indicated that a higher percentage of JH and MF was

released (90 % and 24 %) after stimulation than that (39 % and 9 %) reported by Stay et

al. (2003). The underlying reasons may be that Stay used different strains of the D.

punctata or that Stay used chloroform in extracting JH/MF from CA. I used isooctane

which might have resulted in the low amount of MF detected. Stay et al. (2003) demonstrated that the amount of JH and MF produced by embryonic CA of D. punctata dropped dramatically after the major peak around 65 % of embryogenesis. Therefore, differences in staging of embryos may also have contributed to different results.

58 Table 2.2 Comparisons of JH/MF production in D. punctata reported by Stay et al. (2003) and Li (2005). Rates of JH/MF production Rates of JH/MF production after Source (pmol/pair CA/h) treatment (pmol / pair CA/h) * JH MF MF JH JH MF MF JH release biosynthesis Biosynthesis release biosynthesis release biosynthesis release Stay et 0.002- 0.50-1.50 0.15-0.40 0.03-0.06 1.18 0.455 0.325 0.029 al. 0.005 0.0015- Li (2005) 0.25-0.53 0.10-0.23 0.001-0.005 0.589 0.538 0.0019 0.00045 0.0018 • Stay et al. used 40 µm farnesoic acid to stimulate JH and MF production. They did not use pre- incubation and the incubation interval is 3 hours. I used 100 µM farnesol to stimulate JH and MF production with one hour pre-incubation and 6-hour interval. The culture media and radio activity (1-1.5Ci/mmol by Stay et al.) used were also different. The rates of JH/MF production were compared at 65 % post-dorsal closure time, which was 45 in Stay et al.’s study and 49 in present paper. • Li (2005), results found in current study, presented here.

For both of the experiments in which I used TLC/RCA techniques, there were high readings in front fractions on the TLC plates. These fractions were closer to JH thus the

background for JH was higher than that for MF. This increased level of radioactivity could be an indicator that MF (and JH) was degraded during the incubation. Potentially, degradation contributed to the lower amounts of MF produced by embryonic CA of D.

punctata in my experiments as compared to Stay et al.’s (2003) results.

R. maderae is phylogenetically close to N. cinerea. In 1984, Lanzrein et al.

reported that N. cinerea releases large amount of MF. I found the similar results in R.

maderae. However, in the previous report of N. cinerea, the results were reported as

radioactivity in CPM instead of the more widely used DPM or pmol/CA; therefore, the

results could not be compared qualitatively.

59 In crustaceans, MF is suspected to play a similar role as JH III in insects (Homola

and Chang, 1997). MF circulates in the hemolymph and regulates protein metabolism,

the molt cycle and reproduction (Homola and Chang, 1997). Moreover, MF exerts strong

JH-like effects when injected into N. cinerea larvae and female adults (Brüning and

Lanzrein, 1987). In the seven species of cockroaches representing the diversity and

evolutionary history of the order Blattaria, I found MF was released by embryonic CA,

which suggests that this compound may circulate in the blood and may act as a hormone

per se. Moreover, the presence of MF in cockroach embryos suggests this molecule

might be an ancient hormone in Arthropods.

It has been speculated that the accumulation of MF in the CA could be the

consequence of a limited oxygen supply for conversion of MF to JH III (Lanzrein et al.,

1984). Since I found MF in D. punctata whose embryonic chorion is thin and ruptures early in embryogenesis, it is clear that the production of MF is not caused by the limitation of oxygen supply of embryos.

In the JH biosynthesis pathway, farnesol is converted into farnesoic acid and then

into MF. In my experiments with farnesol treatment, large amount of MF accumulated in

the CA of R. maderae and D. punctata. However, the amount of JH released in the two

species increased significantly following the treatment, which indicated that MF

epoxidase converted most of the MF into JH. My results found in the two species

suggest that in embryonic CA, MF does not saturate MF epoxidase. This finding is

similar to that of female P. amaricana (Pratt et al. 1975). My results support the claim

that MF epoxidase is not a rate-limiting enzymes in the JH-biosynthetic pathway

(Holbrook et al., 1996; Schooley and Baker, 1985).

60 The ability of farnesoate treatment to up-regulate MF and JH biosynthesis was not

universal in all cockroach species. In two species, B. fumigata and B. discoidalis, there

was no accumulation of MF and the two species were not sensitive to farnesol treatment.

This suggests that embryonic CA of different cockroach species respond differently to the

farnesol treatment which has also been observed for CA of adult bees belonging to

different castes (Rachinsky, et al., 2000).

The role of MF in insects and evolution of JH in cockroaches will be elucidated by additional study of JH and MF biosynthesis and release using more sensitive techniques.

Potentially, a real time PCR study of epoxidase will reveal more information of regulation of conversion of MF into JH in cockroaches.

61

Acknowledgement I would like to thank Dr. Glenn Holbrook for his guidance on this paper. I also want to thank Dr. Kenneth Berger for his logistics support and his contribution to characterization of the hatch time in some of the cockroach species. I appreciate Dr. Kambhampati confirming the phylogenic tree used in present paper. I appreciate Dr. Stephen L. Rathbun who contributed opinions on statistical analysis. I appreciate help from Dr. Diana Cox-Foster who helped me put all the pieces into a manuscript. I wish to thank Dr. Coby Schal, who helped review the paper.

62

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Chapter 3

Overview of insect immunity and entomopathogenic nematodes 67 3.1 Introduction

3.1.1 Overview of insect innate immunity

Insects dominate almost all ecological niches and are confronted by an extremely

large variety of potentially harmful microorganisms (Hoffmann, 1995). To protect them

against various pathogens and parasites, insects have evolved effective humoral and

cellular immune systems (Gillespie et al., 1997; Carton and Nappi, 1997; Lackie, 1988).

The integumental defenses constitute the first line of defense against organisms

attempting to enter the insects’ hemocoel (Ashida and Brey, 1995; Carton and Nappi,

1997). The immune defenses also comprised of various cytotoxic integumental proteins

and antimicrobial peptides that are synthesized upon infection (Ashida and Brey, 1995;

Carton and Nappi, 1997). At the site of attack by pathogens or parasites, pro-phenol

oxidase is activated, which results in cuticular melanization and killing of the invaders.

However, the underlying mechanism is unclear (Carton and Nappi, 1997).

The epithelial surfaces of the insects (such as the epidermis of the digestive and genital tracts, tracheae and Malpighian tubules) produce antimicrobial peptides what inhibit microbial growth (Hoffman and Reichhart, 2002). Once a microorganism enters the hemocoel of an insect, it will face the innate immune responses.

Innate immunity is common to all metazoans and it is characterized by germline- encoded, non-rearranging receptors, and rapid effector mechanisms that involve phagocytosis, activation of proteolytic cascades and synthesis of potent antimicrobial peptides (Lavine and Strand, 2002; Hoffmann, 2003). In contrast, adaptive immunity, coexisting with innate immunity, is found in about 45,000 vertebrate species and depends on generation of complex repertoire of immune receptors in lymphocytes through 68 somatic gene rearrangement and on the clonal expansion of activated lymphocytes

(Hoffmann, 2003).

The innate immune response of insects is a rapid large spectrum host defense that

shares fundamental characteristics with the innate immune response of vertebrates.

However, insects produce no lymphocytes. Plus, the insects do not synthesize

immunoglobulins, such as gamma-globulin, and their immune system lacks the molecular specificity of antibodies from mammals. So the cellular immunity of insects is not

comparable to the cellular immunity of mammals in this sense.

Innate immune responses of insects comprise three tightly interconnected

reactions. The first is the induction of proteolytic cascades by wounding, the coagulation

cascade leading to localized blood clotting and of the prophenoloxidase (PPO) cascade

(Hoffmann, et al, 1996; Franc and White, 2000; Hoffman and Reichhart, 2002; Lavine

and Strand, 2002).

The second is cellular defense reactions, which consist predominantly of

phagocytosis of microorganisms by plasmatocytes, and/or nodulation by aggregation of hemocytes, and/or encapsulation of larger invading microorganisms (Hoffmann, et al,

1996; Hoffman and Reichhart, 2002; Lavine and Strand, 2002).

The third is humoral reaction, which is the rapid and transient induction of a

broad spectrum of antimicrobial peptides by microbial challenge of antimicrobial peptide genes in the fat body (a functional equivalent of mammalian liver), followed by the

secretion of these small-sized and cationic peptides into the hemolymph. Moreover, the humoral reaction includes the reactive intermediates of oxygen or nitrogen and the complex enzymatic cascades that regulate coagulation or melanization of hemolymph 69 (Vass and Nappi, 2001; Lavine and strand, 2002). Melanization also contributes to the

host defense and epithelial reactions which involve the local synthesis of antimicrobial

peptides in response to infection (Hoffmann, 2003; Hoffmann, et al, 1996).

3.1.1.1 Insect hemocytes and cellular immunity

Blood cells or hemocytes are suspended in the blood plasma. Individual cells can

have very different appearances under different conditions. Researchers in general consider there to be six main types: pro-hemocytes, plasmatocytes, granulocytes

(probably cystocytes or coagulocytes), spherule cells (spherulocytes), oenocytoids

(probably crystal cells in Drosophila) and adipohemocytes. The first five types of blood cells have been described from species in diverse orders including Lepidoptera, Diptera,

Orthoptera, Blattaria, Coleoptera, Hymmenoptera, Hemiptera and Collembola (reviewed by Lavine and Strand, 2002, Chapman, 1998).

The first population of blood cells appears during embryogenesis in anterior

mesoderm then spread throughout the whole embryo (Ratcliffe et al., 1985; Tzou et al.,

2002). The insect continues to produce hemocytes during larval or nymphal stages via

division of stem cells in lymph glands (larval hematopoietic organs) and/or cells in

circulation (Tzou et al., 2002; Lavine and Strand 2002).

There is evidence of conservation of the molecular basis for blood cell lineage in

mammalian and Drosophila hematopoeisis (Lebestky et al., 2000; Tzou et al., 2002).

Mammalian blood cells derive from hematopoietic stem cells that differentiate into different lineages under control hematopoietic transcription factors like GATA, Acute

Myeloid Leukemia-1 (AML1), and Friend of GATA (FOG), (Lebestky et al., 2000; Tzou

et al., 2002; Lavine and strand 2002). Four genes , serpent, lozenge, U-shape and glial 70 cells missing, have been identified in Drosophila which regulate key steps of

hematopoietic lineage development, and the first three are similar to mammalian GATA,

AML1 and FOG respectively.

Prohemocytes are stem cells that characterized by lack of organelles and high nuclear : cytoplasmic ratio (Chapman 1998). It was suggested that stem cells

(prohemocytes) in hematopoietic organs differentiate primarily into plasmatocytes;

whereas, other hemocytes types differentiate after release into circulation. This has not

been verified.

Plasmatocytes are the most abundant hemocytes (>30% usually) and vary in

shape. They are larger hemocytes and the cytoplasm may enclose granules. However,

they are usually amoeboid and their spreading and phagocytic capacities in vitro

differentiate them from other cell types (Ratcliffe 1986). They are involved in

phagocytosis and encapsulation of foreign organisms (Ratcliffe 1986; Chapman 1998).

Plasmatocytes are also the one of the two types of adhesive blood cells (the other is

granular cell) in larval stage Lepidoptera (Lavine and Strand, 2002).

Granulocytes typically have abundant endoplasmic reticulum and Golgi

complexes which contain large numbers of membrane-bound granules. They comprise

more than 30% of the blood cell population and discharge their contents (degranulate) on

the surface of intruding organisms as an early part of the defense response (Ratcliffe

1986; Chapman, 1998; Pech and Strand, 1996, 2000;).

Spherule cells, their function currently unknown, are named after the large

refractile spherules that comprise 90% or more of the cytoplasm (Chapman, 1998). They 71 are characterized by 1-3 μm inclusions which are not usually discharge in vitro. These

stable cells represent less than 5 % of the total hemocytes (Ratcliffe 1986).

Oenocytoids, discovered in Lepidoptera, have a complex array of microtubules.

They are large (15-30) cells with homogeneous cytoplasm and a small eccentric nucleus.

Oenocytoids are stable in vitro and they contain components of prophenoloxidases system which involved in melanization and cellular defense reaction (Ratcliffe 1986;

Ladendorff and Kanost, 1991). Adipohemocytes contain lipid droplets and well-

developed endoplasmic reticulum and Golgi complexes. (Chapman, 1998)

Phagocytosis

The main defense responses involving hemocytes are phagocytosis, nodulation

and encapsulation. Phagocytosis refers to the engulfment of entities by an individual cell.

Hemocytes phagocytose both biotic targets like bacteria, yeast and apoptotic bodies as

well as small abiotic targets like synthetic beads or particles of India ink (Lavine and

Strand, 2002).

When a small target binds with its cognate receptor, the process of phagocytosis is triggered. The binding signaling cascades then regulate formation of a phagosome and ingestion of target via an actin-polymerization-dependent mechanism. The phagosome matures into a phagolysosome when effector molecules are introduced by vesicle fusion events. These effector molecules then kill and/or degrade the target. Limited research of phagocytosis has been carried out in insects, however, the antibodies of mammalian signaling molecules that regulate phagocytosis cross-react with proteins in hemocytes lysates from a species of fruit fly. This suggests that the signal transduction pathways 72 regulating phagocytosis are conserved among insects and mammals (Foukas et al., 1998;

Lavine and Strand, 2002)

Mammalian phagocytes generate reactive oxygen intermediates (ROI) and

reactive nitrogen intermediates (RNI) which are released into the phagosome or

extracellularly, and which are toxic to a variety of microorganisms (Nathan and Hibbs,

1991). In insects, ROI and RNI also been detected in the hemolymph (Nappi and

Ottaviani 2000; Nappi et al., 2000; Lavine and Strand 2002). ROI and RNI also play a

role in immune-related signal transduction pathways besides direct cytotoxic effects on

parasites or pathogens. In mammals, ROI such as H2O2, and RNI such as NO, function as

secondary messengers in signal transduction pathways that include activation of NF-κB

(Lavine and Strand 2002).

Not only the fat body but hemocytes also produce antimicrobial peptides as well

(Lavine and Strand 2002; Ladendorff and Kanost, 1991;).

Nodulation and encapsulation

Nodulation is defined as multiple hemocytes binding to aggregations of bacteria

while encapsulation refers to the binding of hemocytes to larger targets like parasitoids,

nematodes and chromatography beads (Ratcliffe and Gagen, 1977; Ratcliffe, 1986). The

fact that nodule and capsule formation look nearly ultrastructurally identical indicates

that they are essentially the same process against different targets (Lavine and Strand,

2002; Vass and Nappi 2001). Granular cells and plasmatocytes in Lepidoptera (and lamellocytes in Drosophila) form an overlapping sheath in nodulation and encapsulation

(Vass and Nappi 2001) In some insects, the formation of nodule and capsule is highly

organized-- individual granular cells attach to the target, followed by adding layers of 73 plasmatocytes, and then end the nodulation process with a monolayer of granular cells

and apoptosis on the capsule periphery (Lackie, 1983; 1988; Pech and Strand, 1996,

2000; Lavine and Strand 2002).

The encapsulation and nodulation starts after recognition of non-self, when

circulating hemocytes change from non-adhesive to adhesive cells that are able to bind to

the target and one another (Lavine and Strand, 2002). This adhesion is probably

regulated by signal molecules, and the most potent plasmatocytes-activator identified to

date is a 23-amino-acid cytokine called plasmatocytes spreading peptide (PSP), which

induces plasmatocytes to aggregate or adhere to foreign surfaces within seconds at

concentration>=100pm (Clark et al., 1997). A follow-up study suggests that PSP

binding to its cognate receptor causes plasmatocytes to export cytoplasmically stored

adhesion molecules to their surface (Strand and Clark, 1999). Studies suggest that there

are other factors regulating plasmatocytes binding reaction in encapsulation, such as

integrins (Lavine and Strand, 2001). The encapsulation response is terminated when a

peripheral basal membrane-like layer is produced by granular cells, which probably

results from the IG-related protein hemolin inhibiting hemocyte aggregation (Ladendorff

and Kanost, 1991; Lavine and Strand, 2002). Studies suggest that several factors might

play a role as killing agents, which includes asphyxiation, toxic quinones or hydroquinones via the prophenoloxidase (PPO) cascade, and ROI and RNI and antibacterial peptides (Gillespie et al.; 1997 Nappi et al., 2000; Cox-Foster 1998).

74 3.1.1.2 Insect humoral immunity, antimicrobial peptides and signaling

pathways

Dividing the insect immune system into cellular and humoral responses is

somewhat arbitrary, as many humoral factors affect hemocyte function and hemocytes

are an important source of many humoral molecules. The hallmark of the humoral

reaction is the systemic antimicrobial response. Humoral immunity involves several

proteolytic cascades, of which the melanization cascade is the most important (Hoffmann

and Reichart, 2002).

Induced Antibacterial peptides

Wounding or injection of certain bacteria in some insects induces rapid and

transient de novo biosynthesis of antimicrobial (poly) peptides by the fat body (an

adipose tissue equivalent to the mammalian liver) and midgut (Hoffman et al., 1996;

Lehane et al., 1997). Some hemocytes, the heart, Maligning tubules, epidermis and

reproductive tract are also involved in production of antibacterial peptides (Hultmark,

1993).

At least 34 antimicrobial peptides in seven (or eight) distinct inducible

antimicrobial peptide families have been identified in Drosophila and other insects

(Hultmark, 2003; Hoffmann, 2003). Most of them are small, cationic and predominately

membrane active. Their activities are directed against various fungi, Gram-positive or

Gram-negative bacteria (Hoffmann and Reichart, 2003; Hoffmann, 2003).

Cecropin

Cecropins are first isolated from the hemolymph of Hyalophora cecropia, which

are induced by injection of certain bacteria and wounding (Hoffmann, 2003). 75 Homologous molecules have since been isolated from the waxmoth Galleria mellonella,

the tobacco horn worm Manduca sexta, several species of Diptera, from a beetle, and

others (Hultmark, 1993). H. cecropia produces cecropins A, B and D as well as several

minor cecropins C, E, and F, each with 35 to 37 amino acids. Cecropins are all 4 KD

cylindrical, amphipathic molecules with long hydrophobic regions on one end and act

like detergents. Cecropins lyse bacterial cell membranes; they also inhibit proline uptake

and cause leaky membranes. They are active against a large number of gram-positive

and gram-negative species (Hultmark, 1993). Cecropin-like molecules have also been

found in mammals (Hoffmann, et al., 1996).

Attacins (sarcotoxin II)

Attacins (sarcotoxin II) also have been isolated from H. cecropia and other

lepidopterans and dipterans. Attacins are gram-negative glycine-rich bacterial inducible

proteins of 20-28 kDa located in the alimentary canal. Their site of action is at the outer

bacterial membrane. These proteins prevent bacterial cell division by inhibiting

biosynthesis of outer membrane proteins (Gillespie et al., 1997; Bulet et al., 1999).

Diptericin

Diptericins are inducible 9 kDa glycine-rich antibacterial peptides that have parts

similar to the glycine-rich domains in the carboxyl-terminal region of attacins and sarcotoxin II. Like attacins, diptericins are only effective against a limited array of gram- negative bacteria (Gillespie et al., 1997).

Defensin

Defensins (formerly known as sapecin) are 4 kDa, anti-Gram-positive peptides

that are structurally similar to some scorpion venom toxins (Kart, 1990). Among all the 76 antimicrobial peptides, cysteine-rich defensins are found widespread in eukaryotic cells

from plants, insects and mammals (Borregaard, et al., 2000). Defensins show high

degrees of homology and strict structural conservation among insect groups (Hultmark,

1993). Defensins contain six conserved cysteine residues involved in three disulfide

bonds. They act at the bacterial cytoplasmic membrane and lyse cells by formation of membrane channels. They also inhibit Ca2+-activated K+ channels (Gillespie et al.,

1997).

Lysozyme

Peptidoglycan is a large, bag-shaped macromolecule that encases bacterial

membranes. It is universally present in bacteria. Lysozyme is an enzyme that hydrolyzes

β-1, 4 linkages between N-acetylglucosamine and N-acetylmuramic acid (glycosidic

bonds) in peptidoglycan in bacterial cell walls, and thereby causes lysis of bacterial cell

walls (Gillespie et al., 1997). This 14 kDa enzyme constitutively occurs in low levels in

hemolymph of most insects. Lysozyme is rapidly induced immediately upon bacterial

infection. Lysozymes are closely related to c-type lysozymes in vertebrates (Hultmark,

1993).

Other antibacterial proteins

A cysteine-rich antifungal peptide, drosomycin, has four intramolecular disulfides

and shows homology to plant antifungal peptides (Gillespie et al., 1997; Hoffmann, et al.,

1996). Drosomycin inhibits spore germination at high concentrations and delays growth of hyphae at lower concentrations. Other antifungal peptides identified to date are

tenecin, holtricin from Tenebrio molitor and Holotrichia diomphalia, respectively

(Gillespie et al., 1997; Bulet et al., 1999). 77 Andropin is a male-specific antibacterial product isolated from Drosophila melanogaster. It is thought to protect seminal fluid and the male reproductive tract against microbial infections (Bulet et al., 1999). Honeybees produce another family of such proteins known as apidaecins. Apidaecins (2kDa) do not appear to disturb bacterial membranes, and it is not yet clear how they work. It is speculated that these are bacteriostatic, rather than bacteriocidal, proteins. Other proline-rich inducible antibacterial peptides include lebocins in Bombyx mori, abaecin from a Hymenoptera, formaecins in Myrmecia gulos, drosocin and metchnikowin from drosophila, and pyrrhocoricin in a true bug (Gillespie et al., 1997; Bulet et al., 1999). Royalisin (5 kDa) is another antibacterial peptide found in the royal jelly of honeybees. This is also an amphipathic protein, and its mode of action may be similar to cecropins.

One of the most exciting findings in insect immunology is the discovery of an insect immune protein called hemolin in the hemolymph of the giant silkmoth

Hyalophora cecropia and tobacco hornworm Manduca sexta (Yu et al., 2002). Hemolin is a member of the immunoglobulin (Ig) superfamily that shares sequence homologies with mammalian immunoglobulins. Hemolin contains four Ig domains of the I-set type, which are most similar to those in neural cell adhesion molecules. This protein binds to the surface of bacteria, and it is likely that the binding may be the first step in the insect immune response (Ladendorff and Kanost 1990; Daffre and Faye 1997; Faye and Kanost

1998).

Toll – dorsal signaling pathway

The promoter regions of inducible antimicrobial peptide genes contain a nucleotide sequence similar to the mammalian binding sites for the mammalian member 78 of the Rel family of transcription factors NF-kB/Rel, which acts as an enhancer for genes

participating in inflammatory and acute phase responses (Fig. 3.1) (Hultmark, Engstrom;

Vilmos and Kurucz, 1998; Hoffmann, 2003).

The gene, dorsal is genetically identified as a regulator of dorsoventral patterning

in the early embryo in Drosophila (Imler and Hoffmann, 2001; Hoffmann, 2003). The

Toll signaling pathway is activated by Gram-positive bacteria and fungi. The Toll

signaling pathway in large part activates expression of antimicrobial peptides such as

Drosomycin (Hoffmann and Reichhart, 2002; Zasiloff, 2002).

Toll is a transmembrane receptor. Its extracellular domain contains leucine-rich

repeats and its intracytoplasmic region shows significant sequence similarity with the

corresponding part of interleukin 1 receptor (IL-1R). It is referred to as the Toll-IL-1R

(TIR) domain (Hoffmann and Reichhart, 2002). The TIR domain interacts with

intracytoplasmic molecules with a death domain, which includes myD88, Tube and Pelle.

The activation of Toll is triggered by a proteolytically processed form of the secreted

factor Spaetzle, which is thought be the Toll ligand. Spaetzle is a cystine-knot protein

that has similar structure to mammalian neurotrophins (Hoffman 1997; Hoffmann and

Reichhart, 2002; Gregorio et al., 2002).

The cleavage of Spaetzle is the end point of a complex extracellular proteolytic

cascade, which involves four distinct trypsin-like serine proteases (Gregorio et al., 2002).

Activated Toll signals via the Tube and Pelle proteins to the heterodimeric protein

complex Cactus-Dorsal, ultimately leading to the phosphorylation and degradation of

Cactus. The transcription factor Dorsal then translocates into the nucleus and regulates

the expression of selected set of genes (Imler and Hoffmann, 2001). In addition to 79

Gram Gram Fungi positive negative bacteria bacteria

Serine Nec Proteases

Spaetzle

Toll Pro-Spaetzle PGRP-LC

TIR MyD88

DD IMD DD DD DD DD FADD Tak1 Pelle Tube

Cytoplasm p IKK-γ IKK-γ Dredd Unknown IKK-β IKK-β

p Cactus Cactus Relish Dif Dorsal Relish homolog Rel Rel proteins Humoral defense Cellular defense

Dif Dorsal Drosomycin κB κB κB

Diptericin Cercropin Drosomycin Nucleus Rel Rel

Fig. 3.1 Toll and IMD pathways

80 Dorsal, Drosophila has two related Rel proteins, dorsal-related immune factor

(DIF) and Relish. In the mosquito, the homologue is Gambif-1 (Fig. 3.1) (Imler and

Hoffmann, 2001; Hoffmann, 2003; Vilmos and Kurucz, 1998).

IMD signaling pathway

Mutants defective in the Toll-Drosomycin circuit can still express antibacterial

peptides such as Cecropins and Diptericin. The alternative pathway is called IMD

(immune deficiency gene) pathway, which is activated by a DIF-related protein called

Relish (Fig. 3.1) (Zasloff, 2002). The IMD pathway responds mainly to Gram-negative bacterial infection (Tzou et al., 2002; Gregorio et al., 2002). The imd gene encodes a 25 kDa protein with a death domain that has strongest similarity to that of mammalian RIP

(TNF-receptor-interacting protein). Relish is not inhibited by Cactus, but carries its own inhibitory ankyrin-repeat sequences in its carboxyl-terminal region. Activation of Relish therefore, requires a signal-induced endoproteolytic cleavage that frees the Rel-homology domain. The downstream steps of the IMD pathway are still unclear. It has been suggested that FADD acts upstream of a mitogen-activated protein (MAP) kinase, a homologue of TAK 1. Then TAK1 probably activates a homologue of IKK- β and IKK-

γ/NEMO complex. The complex probably controls phosphorylation of Relish which

precedes its proteolytic cleavage (Imler and Hoffmann, 2001; Hoffmann, 2003).

The targets of antimicrobial peptides and the pathway they involved in are

summarized in Table 3.1:

81 Table 3.1 Summary of antimicrobial peptides and the pathways they involved in

Antigen (non-self Antimicrobial Pathways in Molecule recognized organism) peptides control Drosomycins Toll pathway fungi β1,3-glucan Metchnikowins Toll or IMD Attacins Toll and IMD lipopolysaccharides Cecropins Toll and IMD Gram-negative bacteria (LPS) Drosocins IMD Diptericins IMD Gram-positive bacteria peptidoglycan Defensin Toll and IMD (Engstrom, 1998; Hoffmann 2003)

JAK/STAT pathway

The Janus Kinase (JAK) / signal transducer and activator transcription (STAT) pathway also has been proposed to play a role in invertebrate immunity (Imler and

Hoffmann 2000, Vass and Nappi 2001). In Drosophila, the hopscotch gene encodes

JAK, and mutations cause hemoatopoietic defects, e.g., overproliferation of plasmatocytes and their premature differentiation into lamellocytes. Most important, the

Drosophila JAK/STAT pathway has been found to regulate normal embryonic and adult segmentation, cell proliferation and differentiation, and hyperactivation of pathways leading to tumor formation and hemocyte hyperproliferation (Vass and Nappi 2001).

The prophenoloxidase (proPO) activation system

The insect prophenoloxidase activation system involves a serine proteinase cascade with similarities to the blood clotting and complements systems of vertebrates

(Fig. 3.2). Pro-Phenoloxidase circulates in the hemolymph and is present in the cuticle of the insect body wall as an inactive zymogen (pro-enzyme). When injury occurred or 82 when microbial surface components such as lipopolysaccharide (LPS), lipoteichoic acid

(LTA), peptidoglycan, or beta-1,3-glucan, are recognized by pattern recognition receptors

(C-type lectins, glucan-recognition proteins, peptidoglycan-recognition protein), recognition somehow activates a serine proteinase, which then triggers activation of the serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase

(PAP). Recent studies demonstrate that the cuticular proPO of Bombyx mori is activated through a limited proteolysis by a serine proteinase, termed prophenol oxidase-activating enzyme (PPAE), which itself exists as a zymogen (Nappi and Ottaviani, 2000) PAP then converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO). PO can catalyze oxidation of monophenols to o-diphenols, and o-diphenols to the corresponding quinone. Non-enzymatic polymerization of quinone intermediates forms the black pigment called melanin. During melanin synthesis, the various quinones and superoxide formed are cytotoxic to invading microorganisms (Franc and White, 2000); therefore, the prophenoloxidase cascade is involved in nodule and capsule formation (Ashida and Brey,

1995).

83

Lipopolysaccharide (LPS), peptidoglycan, β 1, 3-glucan

C-type lectins, glucan-recognition proteins, peptidoglycan-recognition

prophenoloxidase -activating enzyme serpin

Prophenoloxidase phenoloxidase

Serine proteinase oxidation of phenol o-diphenol quinone melanin pro-prophenoloxidase -activating enzyme (pro-PPAE)

Fig. 3.2 Phenoloxidase activation system

84 3.1.1.3 Recognition of nonself

In the absence of acquired responses, the biggest challenge to the innate immune

system of insects is how to recognize pathogens and entities of nonself (Lavine and

Strand, 2002). Innate immune recognition relies on a growing number of receptors,

termed pattern recognition receptors (PRR) (McGuinness, et al., 2003). As Charles

Janeway originally hypothesized, PRRs recognize highly conserved and widely

distributed features of pathogens, especially features (patterns) that are not found on the

cells of multicellular organisms (Franc and White, 2000).

Several pathogen-associated molecular patterns (PAMP) that elicit immune

responses have been identified. Theses patterns are lipopolysaccharides (LPS) of Gram-

negative bacteria, the peptidoglycan of Gram-positive bacteria, the β1,3-glucan of yeast,

and the phosphoglycan of parasites (Lavine and Strand, 2002; Franc and White, 2000).

The striking common feature of microbial patterns is their polysaccharide chains, which

vary in length and carbohydrate composition. Therefore, lectins, a group of small protein molecules in insect blood which contain carbohydrate recognition domains that confer binding affinity to specific carbohydrates, have the ability to recognize a broad range of pathogens (Franc and White, 2000). Also, many lectins have multiple binding sites, resulting in lectin –linked aggregates of target cells (Gillespie et al., 1997). Groups of lectins have been identified from the flesh fly Sarcophaga peregrina, the American

cockroach Periplaneta americana, the silkworm Bombyx mori, the west Indian leaf

cockroach Blaberus discoidalis, and the fruit fly Drosophila melanogaster.

Developmental roles of lectins have been suggested in the Toll pathway (Franc and 85 White, 2000). A very different type of lectin, named hemocytin, which contains a lectin domain homologous to mammalian mannose-binding protein and a number of repeated sequences similar to repeated regions homologous to mammalian von Willebrand platelet aggregation factor, has been identified in B. mori (Gillespie et al., 1997). Hemocytin stimulates aggregation of hemocytes and is induced by the presence of Escherichia coli,

LPS, or peptidoglycan (Gillespie et al., 1997).

Hemolin, a secreted hemolymph protein, is composed of four repeated immunoglobulin (Ig)-like domains and is similar to neuroglian, a cell-adhesion molecule

(Franc and White, 2000; Gillespie et al., 1997). Hemolin is thought to have a role in immune recognition and in modulation of defensive responses in H. cecropia and M. sexta (Gillespie et al., 1997). Hemolin, induced by bacteria, contains four repeated domains homologous to C2-type immunoglobulin domains that are characteristic of cell adhesion molecules from vertebrates. Hemolin, which binds to the lipid A core of LPS on the bacterial surface and surfaces of hemocytes, can stimulate phagocytic activity toward Gram-negative bacteria of hemocytes and is suggested to modulate hemocyte adhesion (Gillespie et al., 1997).

Granulocytin, a C-type lectin from flesh fly S. peregrina hemolymph, can agglutinate of rabbit red blood cells (Fujita et al., 1998). Granulocytin is exclusively synthesized by a specific type of blood cell, the large granulocyte. The lectin from

Periplaneta americana agglutinates (effectively inhibited by galactose) human A-type red blood cells. This Periplaneta lectin can agglutinate both E. coli and Salmonella minnesota (Kubo et al., 1996). However, in B. mori, another LPS-binding protein recognizes rough types of E. coli and S. minnesota strains, but not the smooth types. This 86 indicates the lectin binds to the lipid A core of LPS rather than its oligosaccharide

complex. Two other lectins were reported in B. mori, βGRP (β1, 3-glucan recognition

protein) and PGRP (peptidoglycan recognition protein) which recognize the β1, 3-glucan

of yeast and the peptidoglycan of Gram-positive bacteria, respectively (Franc and White,

2000).

Four lectins from Blaberus discoidalis, BDL1, BDL2, BDL3 and GSL, have

specific binding affinities for mannose, N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D- galactosamine (GalNAc) and β1, 3-glucan. The lectins not only play roles in the phagocytosis of yeast, Gram-negative and Gram-positive bacteria, but interestingly, they also activate the ProPO cascade independently of their lectin activity (Franc and White,

2000). A newly discovered lectin of M. sexta, immulectin, was also shown to activate the ProPO cascade and bind to a broader range of microorganisms

Hemocytes of D. melanogaster synthesize cecropins after binding to a snail (Helix

pomatia) lectin, which is an activator of T-lymphocytes (Gillespie et al., 1997; Vilmos

and Kurucz, 1998). The ligand (receptor) was identified as a transmembrane protein

with an exterior mucin-like domain. In addition to the high levels of expression in

hemocytes, this protein, hemomucin, is deposited on the surface of eggs (Gillespie et al.,

1997).

The receptor, Croquemort (Crq), is a CD36-related molecule (Franc and White,

2000; Lavine and Strand, 2002). CD36 is a class B scavenger receptor and was the first

PRR described that can recognize and result in engulfing apoptotic cells in mammals.

87

3.1.2 Overview of entomopathogenic nematodes

3.1.2.1 Biology of Entomopathogenic nematodes

Nematodes, with 20,000 known species, are biologically diverse and versatile,

occupying an enormous range of habitats and displaying a fascinating array of lifestyles

(Ley, 2000). Nematodes, classified in the phylum Nemata, are structurally simple

organisms with approximately 1,000 somatic cells in the adults. Nematodes have no

appendages, segmentation and are composed of a tube within tube structure. The

alimentary canal extends from the mouth on the anterior end to the anus located near the

tail. A collagenous exoskeleton made by the epidermis is on the outside. Nematodes

possess digestive, nervous, excretory, and reproductive systems, but lack a discrete circulatory or respiratory system. Nematodes can be free-living or parasitic.

The small, free-living nematode Caenorhabditis elegans has been used for many

years as a model for analysis of complex biological problems. The complete, 97Mb, genome sequence of C. elegans has been determined by a consortium of researchers in the UK (Sanger Centre) and the USA (Washington University Genome Sequencing

Centre). The genome of C. elegans is organized as six chromosomes (five autosomes and a sex [X] chromosome) and encodes approximately 20,000 protein-coding genes and an additional 1000 RNA encoding genes. In C. elegans project, every single cell of C.

elegans is ascribed a function and tissue and there are over 2500 loci defined by

mutation. C. elegans project has helped researchers glean significant genetic

developmental and basic biological information. Work on model species of nematodes

can yield great insight into the general biology of related nematode species (Blaxter et al., 88 1998, 2000). The genome of C. briggsae is complete and the genome of Brugia malayi is being determined (Maizels, et al., 2001).

Parasitic nematodes are of great importance ecologically and in human terms

(Blaxter et al., 2000). The lymphatic and cutaneous filarial nematodes infect over 150 million people currently and cause significant losses in domestic animals (Blaxter et al.,

2000). Plant parasitic nematodes, such as root knot nematode, cause an estimated 80 billion dollars in crop damage annually (Blaxter et al., 1998, 2000). The complete genome of one human pathogen, filarial nematode, Brugia malayi, has been recently sequenced by the Filarial Genome Project. Researchers are now exploring the genes involved in evading and manipulating mammalian immune systems (Maizels, et al,

2001).

Extensive studies have been carried out on insect parasitic nematodes, entomopathogenic nematodes (EPN) from two families of rhabditid nematodes: the

Steinernematidae (Chitwood and Chitwood, 1937) and the Heterorhabditidae (Poinar,

1976; Burnell and Stock, 2000). The EPN, Heterorhabditis and Steinernema together with their symbiotic bacteria Photorhabdus and Xenorhabdus, respectively, are obligate and lethal parasites of insects. EPN can provide effective biological control of some important lepidopterans, dipterans and coleopteran pests (Burnell and Stock, 2000).

Although the two families share a similar life history, based on 18s rDNA sequences,

Blaxter et al. (1998, 2000) has found that heterorhabditids and steinernematids do not share a common ancestry.

The only free living stage of the EPN, the third stage dauer juvenile (DJ) or infective juvenile (IJ), seeks out and enters the hemocoel of an insect larva in the soil. 89 Steinernema gain entry through natural openings (mouth, anus, spiracles) of the insect

while Heterorhabditis can also invade through intersegmental membranes of the insect

(Cui et al., 1993; Burnell and Stock, 2000). After the nematode invades the insect, it

penetrates to the hemocoel. Before an extensive cellular immune response by the insect,

symbiotic bacteria must be released from the nematode’s alimentary tract to kill the

insect host and to digest host tissues to provide suitable nutrient conditions for the nematodes’ growth, development and reproduction (Wouts 1980, Poinar 1990; Burnell and Stock, 2000). In steinernematids, IJs are ensheathed and after invading the host, IJs will exsheath. The insect host dies rapidly, usually within 24 to 48 hours. Nematodes molt to the J4 stage and reach adulthood within 2 or 3 days. Eggs are produced and hatched in the females’ uterus. The second generation of juveniles feed on the contents of the uterus and then break out into the body cavity and feed within the mother’s body.

Once the resources of the female have been exhausted, the juveniles break out of the

nematode’s cuticle into the insect’s hemolymph. Then, juveniles continue to feed on the

nutrients digested by bacteria. The process continues for two to three generations until

nutrient status of the cadaver deteriorates and most of the juveniles differentiate into third

stage juveniles, the form that can survive in the environment. The insect cuticle then

ruptures and the third stage juveniles (Djs / IJs) emerge into the soil, where they can live

for several months in the absence of a suitable host (Wouts 1980, Poinar 1990).

Under optimal conditions, it takes 3 to 7 days for steinernematids and

heterorhabditids to complete one life cycle inside a host from egg to egg. Emergence of

infective juveniles from the host requires about 6 to 11 days for steinernematids and 12–

14 days for heterorhabditids (Kaya and Koppenhöfer 1999). Males and females are 90 produced in Steinernema species, but in Heterorhabditis species juveniles develop into a

hermaphroditic female that can produce eggs without mating.

3.1.2.2 Epicuticle and extracellular matrices of Entomopathogenic nematodes

Extracellular matrices (ECMs) are composed of complex mixtures of

glycoproteins and carbohydrates. Two types of ECMs have been identified in

Caenorhabditis elegans: the cuticle and basement membranes. Many of the same

molecules found in mammalian ECM (collagens, proteoglycans, laminins) have been

identified in C. elegans. Cuticular proteins are extensively cross-linked with disulfide

bonds and also with nonreducible tyrosine-derived cross-links. The cuticle is an

acellular, dynamic, biochemical compartment (Kramer, 1994).

The external plasma membrane of a nematode is the hypodermis (epidermis), but

the cuticle is enveloped by the membrane-like epicuticle. Lipids are the major

component of the epicuticle but no glycolipids are on the surface of C. elegans. The cuticle is connected to the hypodermis via hemidesmosomes. Filaments extend from body wall muscles through the basement membrane and connect to the hypodermis (Francis and Waterston 1985; Francis and Waterston 1991).

The cuticle has both surface specializations and internal layers that can differ at

different developmental stages. Loosely associated, a carbohydrate-rich surface coat

external to the epicuticle can be detected using specific fixation and staining methods

Primary Structure of Collagens

The basic unit of collagens is a polypeptide consisting of the repeating sequence

(glycine (Gly) - X - Y)n, where X is often proline (Pro) and Y is often hydroxyproline 91 (proline to which an -OH group is added after synthesis of the polypeptide). Chitin is

composed of beta-1, 4, N acetyl glucosamine.

Surface coat

Surface coat of nematodes is composed of carbohydrate rich layer and has a loose

association with the epicuticle. The surface coat or glycocalyx lies external to the

epicuticle. The surface coats of nematodes are polyanionic (probably due to sulfate or

phosphate groups) and contain carbohydrates and mucin-like proteins. The surface coat is dynamic in that its components appear to be synthesized continuously. Information

content of the surface is reduced, rendering it biochemically invisible to sensory or

adhesive products of predators and parasites. Active shedding of the coat by animal parasites is thought to be an adaptive defense against immune attack of the host (Blaxter

and Bird, 1997).

Basement membrane components

Basement membranes can be recognized as thin sheets of extracellular matrix

material closely associated with cell membranes. Type IV collagen is generally the most

abundant constituent of basement membranes and is found only in the basement

membranes. Proteoglycans consist of a protein core to which at least one

glycosaminoglycan chain is attached. Perlecan is a common component of basement membranes and has been shown to interact with other basement membrane molecules as

well as cell surface receptors. SPARC (also known as BM-40 or osteonectin) is an anti-

adhesive extracelluar matrix-associated glycoprotein that can modulate the interaction of

cells with the matrix. Laminins are a family of basement membrane molecules composed 92 of three disulfide-bonded subunits, αβγ. The α and β homologs are identified in C. elegans (Kramer, 1997).

3.1.2.3 Noncollagenous surface coat proteins

The major surface glycoprotein of Brugia malayi adults is an N-glycosylated glutathione peroxidase (GPX) homolog, which is made in the hypodermis and secreted through the cuticle. The filarial GPX is inactive against hydrogen peroxide but is active against lipid peroxides and is thus well placed to protect the epicuticle from peroxidative disruption. A second protein on the filarial surface is a secreted superoxide dismutase that is presumed to eliminate superoxide generated by the lipoperoxidase. A third protein is similar to (cystatin) cysteine proteinase inhibitors. It may be involved in cuticle maintenance or in abrogating the effects of proteases released by immune effector cells product (Maizels et al., 2001, 2004).

Glutathione S-transferases (GSTs) of Onchocerca volvulus are involved in xenobiotic detoxification and ligand binding / transport functions, which neutralise known cytotoxic products arising from reactive oxygen species (ROS) attack on cell membranes and in turn help the human filarial parasite to evade the hosts’ immune responses. The enzyme is suggested to be a secretory product (Maizels et al., 2001,

2004).

3.1.3 Overview of Steinernema glaseri, Heterorhabditis bacteriophora, host immune response and surface coat proteins

Originally, intensive researches in EPN and their symbionts were motivated by their biological control potential. Therefore, much of EPN research has focused on 93 applied aspects related to pest control (Burnell and Stock 2000). However, since they belong to the same family as C. elegans, EPN and their symbionts are increasingly being viewed as an exciting subject for basic research in biochemistry and molecular genetics.

Insect hosts defend against EPN infective juveniles in the hemocoel by a rapid cellular immune response that results in encapsulation and melanization. For EPNs, their symbiotic bacteria must be released in the host for EPNs’ to survival and reproduce. The release of symbiotic bacteria has to occur before the intensive immune responses block this process (Wang and Gaugler 1999).

Heterorhabditidae and Steinernematidae elicit different immune responses in different hosts. Steinernema glaseri enter Japanese beetle larvae, Popillia japonica, through the midgut and escape the immune response of P. Japonica larvae after penetrating into the larvae (Wang and Gaugler, 1998). Heterorhabditis bacteriophora induces a strong immune response after being injected into the hemocoel of P. japonica larvae; only 10% of nematodes escape from encapsulation after 48 hours (Wang et al.,

1995). The same injection experiment showed that only 24% of S. glaseri were melanized initially, and after 24 hours, all nematodes were free in the host hemocoel

(Wang et al., 1995). Symbiotic bacteria are released by EPNs to suppress the host immune system. In S. glaseri, the symbiont Xenorhabdus poinarii is released at 4 to 6 hours after entry into the hemocoel; whereas, the symbiont of H. bacteriophora

Photorhabdus luminescens can be detected within 30 minutes (Wang et al., 1994).

The surface coat proteins (SCPs) play a role in suppression of immune responses.

A 35% ethanol extract of S. glaseri can suppress the host immune system and protect both living and dead H. bacteriophora from encapsulation (Wang and Gaugler, 1998). 94 One protein SCP3a isolated from a nondenaturing gel was known to block out

encapsulation (Wang and Gaugler, 1998). Besides these initial studies, limited

information is available on mechanisms underlying the immune interactions between host

insects and EPNs.

The study of underlying mechanisms that permit EPNs to evade insect immune responses and how insects counteract EPNs will help us develop a better understanding of insect immunity. The study of EPN-host interaction may also help to discover the key

regulators of innate immunity and possibly lead to a better understanding of the

interaction between filial nematodes and their vector, the mosquito.

Moreover, EPNs are considered environmentally safe and are commercially used

as biological-control agents against insect pests in many Integrated Pest Management

projects. To understand the immune response between EPNs and their host insects will

make it possible to produce a genetically-modified biological control agent with higher

specificity and improved potency.

The insect hosts I studied varied from low to high levels of resistance. Low level

resistance refers to EPNs that can survive and produce well in the host whereas high level

resistance means the EPNs can not invade, (and / or) survive (and / or) reproduce in the

host. The insects in my study include larvae of the wax moth Galleria mellonella, larvae

of the Japanese beetle Popillia japonica, larvae of the Oriental beetle Exomala orientalis,

larvae of tobacco hornworm Manduca sexta, adults of house cricket Acheta domesticus

and larvae of northern masked chafer Cyclocephala borealis.

The two entomopathogenic nematodes studied were Heterorhabditis

bacteriophora, which is a commercialized biological control agent, and Steinernema 95 glaseri (FL strain and NC strain), whose SCPs have been studied by Wang et al. (1999).

The goal of my research was to understand the immune responses between two species of nematodes, H. bacteriophora and S. glaseri, and their insect hosts, as well as their relationship to host specificity. I hypothesized that the SCPs of EPNs induce or suppress the immune responses of their hosts. 96 Reference:

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Chapter 4

Infectivity of Entomopathogenic Nematodes

and Immune Responses of Their Insect Hosts 102 Abstract

Entomopathogenic nematodes (EPNs) are ecologically and economically important.

Two families of insect EPNs are good candidates for use as of biological-control agents,

but they have not significantly replaced pesticides. One limitation of nematodes is the immunity or partial immunity of certain target insects to infection. Infective juveniles

(IJ) of EPNs penetrate insect hosts and release symbiotic bacteria that kill the insects and serve as food resources for EPNs. EPNs have to overcome cellular immune response of insect hosts in order to release biosymbions.

I studied the immune responses of insect hosts to two species of nematodes

(Heterorhabditis bacteriophora and Steinernema glaseri). I found that these immune

responses were related to EPNs specificity. The insect hosts I tested were the wax worm

larvae (Galleria mellonella), oriental beetle larvae (Exomala orientalis), Japanese beetle

larvae (Popillia japonica), the tobacco hornworm (Manduca sexta), northern masked

chafer larvae (Cyclocephala borealis), and adults of the house cricket (Acheta

domesticus). I found that H. bacteriophora and S. glaseri infected wax worms and

reproduced well. Both H. bacteriophora and S. glaseri killed most larvae of Japanese

beetle, oriental beetle, and the tobacco hornworm. S. glaseri reproduced better than H.

bacteriophora in these insect hosts. Interestingly, S. glaseri NC strain had stronger

pathogenity compared with S. glaseri FL strain in the same hosts. Northern masked

chafer larvae and house crickets were resistant to both nematodes.

In injection assays, I found that in M. sexta, P. japonica and E. orientalis, high

percentages of H. bacteriophora were melanized while high percentages of S. glaseri 103 were moving freely. In M. sexta and P. japonica, higher percentages of S. glaseri FL

were encapsulated compared with S. glaseri NC. In the resistant host C. borealis, both H.

bacteriophora and S. glaseri were melanized. My results suggest that these nematodes elicit immune responses in hosts that correlate with their infectivity.

Using an in vitro assay, I also found that hemocytes from M. sexta recognized S.

glaseri at a low level during the first hour post nematode introduction, and after 24 hours,

H. bacteriophora escaped recognition of G. mellonella blood cells.

104 Introduction

Entomopathogenic nematodes (EPNs) are widely used biological control agents

targeting soil dwelling insect pests (Dunphy and Thurston, 1990; Gaugler et al., 1997).

Nematodes from two phylogenetically distant families, Heterorhabditidae and

Steinernematidae, share a similar life history (Kaya and Gaugler, 1993; Burnell and

Stock, 2000). Infective juveniles (IJs) of EPNs search for hosts and gain access to the

hemocoel of the host via body openings (Gaugler et al., 1996). EPNs then release their

symbiotic bacteria and the bacteria kill the insect host within 24 to 48 hours.

Steinernema glaseri, an EPN naturally associated with white grubs, releases its symbiotic

bacteria Xenorhabdus poinarii at 4-6 hours after entry into the insect host (Steiner, 1929;

Wang et al., 1995). Heterorhabditis bacteriophora releases its symbiotic bacteria

Photorhabdus luminescens 30 minutes after entry into the host hemocoel (Bowen et al.,

1998).

Insect hosts have evolved several strategies to defend against nematode infection. A

Japanese beetle larva exhibits evasive and aggressive behaviors when its cuticle contacts

EPNs (Gaugler et al., 1994). It is reported that scarab larvae have morphological defenses and chemical defenses (Forschler and Gardner, 1991; Wang et al., 1995).

Infectivity and viability of EPNs is also affected by environmental factors, such as soil depth, moisture and pressure (Koppenhofer et al., 1995; Fife et al., 2003). Four major factors – host-habitat finding, host finding, host acceptance and host suitability – determine EPNs’ host specificity (Gaugler et al., 1997). Host suitability is dependent on 105 evading or suppressing insect immune responses, thus surviving and reproducing (Wang et al., 1994, 1995; Peters and Ehlers, 1997; Peters et al., 1997; Hand and Ehlers, 1997).

Insect larvae defend themselves against bacterial or parasite infections with cellular and humoral immune responses (Hoffmann et al., 1999). Cellular immune responses include phagocytosis and encapsulation (Dunphy and Thurston, 1990; Gillespie et al.,

1997). Humoral immune responses include inducible antimicrobial peptides, cell adhesion molecules, lysozyme, lectins and the prophenoloxidase system (Lackie 1998;

Gillespie et al., 1997; Hoffmann et al., 1996, 1999; Johansson, 1999; Wilson and

Ratcliffe, 2000). One of the most important reactions of humoral immune responses is melanization, which is related to the production of reactive oxygen species that can kill invaders (Cox-Foster and Stehr, 1994, Nappi et al., 1995).

Symbiotic bacteria released by EPNs produce virulence factors that suppress host immune responses. These factors include high molecular weight toxin complexes, proteases, lipases and lipopolysaccharide (Forst and Nealson, 1996; Forst et al., 1997,

Owuama, 2001; Silva et al., 2002). P. luminescens suppresses the immune response of

Manduca sexta, and thus is able to reproduce within the caterpillar and kill the insects

(Silva et al., 2002). It is suggested that lipopolysaccharides on the surface of symbiotic bacteria trigger hemocyte damage and suppression of phenoloxidase activation (Dunphy and Webster, 1984, 1988, 1991). A single gene from Photorhabdus allows Esherichia coli to persist and kill an insect host (Daborn et al., 2002).

It is clear, however, that rapid host immune responses can kill EPNs and symbionts.

H. bacteriophora, S. glaseri and S. carpocapsae always are encapsulated and melanized in the adult house cricket Acheta domesticus before symbionts can be released (Wang et 106 al., 1994, 1995). P. japonica encapsulated and melanized H. bacteriophora and S. carpocapsae but not S. glaseri at 24 hours (Wang et al., 1994, 1995).

EPNs themselves have to evade or suppress host immune responses to ensure the

release of their symbionts (Brivio et al., 2002). In Galleria mellonella larvae, S.

carpocapsae are not recognized as being foreign due to cuticular lipid components and

are protected against encapsulation (Dunphy and Webster, 1987). Moreover, axenic IJs

of S. carpocapsae kill all wax worms and the hosts liquefy without any bacteria being

present (Han and Ehlers, 2000). In contrast, axenic H. bacteriophora IJs were unable to

kill G. mellonella (Han and Ehlers, 2000). In the Dipteran Tipula oleracea, 20 axenic S.

feltiae only caused 39% mortality of its hosts within 8 days (Ehlers, et al., 1997). S. feltiae is encapsulated (Peters and Ehlers, 1997, Peters et al., 1997), and its symbiotic bacteria were probably blocked by cellular defense mechanism in hemolymph (Ehlers, et al., 1997). In the same insect host, H. bacteriophora and H. megidis avoid encapsulation

(Peters et al., 1997). Different strains of H. bacteriophora have different immune responses in the same host, M. sexta. The H. bacteriophora Oswego strain is encapsulated in M. sexta starting from the head and tail and reactive oxygen molecules are produced by hemocytes (Unpublished personal communication at Dr. Cox-Foster’s lab, E. Troy, K. Miller, A. Kazi, S. Hayden and D. L. Cox-Foster).

Although H. bacteriophora is encapsulated in P. japonica initially, 10 % of the nematodes escaped encapsulation after 24 hrs by an unknown mechanism (Wang et al.,

1994, 1995). 24 % of S. glaseri were melanized initially in the hemocoel of P. japonica;

however, all nematodes were free after 24 hrs (Wang et al., 1995). Mammalian parasitic

nematodes use surface coat proteins (SCPs) and other surface components to counteract 107 host immunity (Dunphy and Webster, 1984, 1987, 1988; Maizels et al., 2001, 2004). It is

suggested that EPNs use a similar mechanism. It is reported that SCPs lyse hemocytes

and suppress melanization in P. japonica (Wang and Gaugler, 1999), and that the cuticle

of S. feltiae can immuno-suppress G. mellonella by down regulating the

prophenoloxidase pathway (Brivio et al, 2002). Besides these initial findings, limited

information is available about host specificity and its relationship to the immune

responses in insect hosts of EPNs.

The goal of this research is to determine the relationship between infectivity of EPNs

and the immune response of insect hosts to the EPNs. Moreover, I hypothesize that

differences in the SCPs of different species and strains of EPNs influence the induction of different immune responses in the insect hosts.

108 Materials and methods

Nematodes

Two species of entomopathogenic nematodes (EPNs) were tested: Steinernema

glaseri (NC and FL strains), and Heterorhabditis bacteriophora (HP88 strain). All EPNs

were cultured in last instar wax worms (Galleria mellonella (L.)) at room temperature.

Infective juveniles (IJs) of EPNs were harvested from white traps by filtration and cultured in distilled water bubbled with air for about 2-4 days at room temperature for all the experiments conducted. Nematodes were washed 3 times by MilliQ water before conducting all the experiments except the infectivity tests.

Insects

Last-instar Japanese beetles (Popillia japonica), oriental beetles (Exomala orientalis),

and northern masked chafers (Cyclocephala borealis) were field collected. The grubs

were stored at 8°C and fed on grass seeds and roots for one day before the experiments.

Last instar wax worms and adult house crickets (Acheta domesticus) were purchased

(Nature’s Way Inc.) and maintained at room temperature. Tobacco horn worm (Manduca

sexta) eggs were purchased (North Carolina State University Entomology Insectary,

Raleigh, NC) and the larvae were reared on an artificial diet (Yamamoto, 1969) at room

temperature.

Infectivity Experiment

300 IJs of EPNs were applied in 400 µl distilled water onto a dampened filter paper

inside of a 35 mm Petri dish. An insect was transferred into the Petri dish and examined

daily under a microscope. The numbers of insect hosts killed (out of n subjected to 109 testing) by EPNs were recorded. The relative reproductive capability (approximate

number of IJs produced) were observed and recorded qualitatively.

Recognition of EPNs by hemocytes of different hosts

The immune responses were studied in vitro (Lovallo and Cox-Foster, 1999). Insects

were rinsed thoroughly by sequentially flushing with Coverage PlusTM (Calgon Vestal

Laboratory, Merck and Co., Inc., St Louis, MO), distilled water and 70% ethanol for 5

minutes each before sacrifice (by bleeding through a severed foreleg). About 10 nematodes in 10 µl MilliQ water were combined with one or two drops of hemolymph of various insect hosts in 440μl Grace’s insect medium (Quality Biologicals, Gaithersburg,

MD, catalogue #117-048-100, with L-glutamine, but without lactalbumin hydrolysate,

TC yeastolate, or insect hemolymph) in coated 4-chambered coverglass wells (Lab-Tek,

Nunc. Naperville, IL). The tissue culture wells were coated with 0.2 % molecular-grade agarose in filter-sterilized MilliQ water. Cellular responses were recorded using an

inverted microscope (Axiovert 405 M, Zeiss, West Germany) equipped with a video

camera (Sony DXC-960MD 3CCD; Montvale, NJ) and linked to a computer (Macintosh

Quadra 800, Apple Computer Inc., Cupertino, CA) with NIH Image (version 1.59) video

imaging software.

Hemocyte recognition was recorded when blood cells attached to the nematodes.

Observations were carried out at 1 and 24 hours post-bleeding. H. bacteriophora and S.

glaseri NC strain were the nematodes tested against A. domesticus, M. sexta, P. japonica,

and G. mellonella.

110 Injection

Ten EPN IJs were delivered by a Drummond micro injector (positive displacement digital microdispenser 3-000-525, Drummond Scientific Company, Broomall, PA) equipped with a glass needle. The needle was pulled from a capillary tube (Drummond

Wiretrol 5-000-1050 50 µl Drummond Scientific Company, Broomall, PA) on a flame and then sharpened on a sharpening stone. For each insect host, 4 µl Ringers’ solution

(0.8 % sodium chloride, 0.02 % calcium chloride, 0.02 % potassium chloride, 0.02 % sodium bicarbonate in MilliQ water, pH 7.0) or MilliQ water with EPNs was injected into

Japanese beetle larvae and other hosts laterally to the base of foreleg. If the digestive system was damaged during injection, the host was not used. Insects were kept at room temperature in a Petri dish with a piece of moisture filter paper. H. bacteriophora, S. glaseri NC and FL strains, were the nematodes tested, versus E. orientalis, P. japonica,

M. sexta, and C. borealis.

Dissection to recover EPNs

Host insects were decapitated and a lateral cut was made around tip of the abdomen.

Then, the insect was carefully cut open ventrally and the digestive system was removed, taking care not to rupture it. Often, careful examination revealed EPNs trapped around the digestive system. Next, distilled water was flushed through the open hemocoel and examined for free moving IJs (these EPNs were classified as free-moving and therefore had not been recognized by the host immune system). Other tissues were then dissected and examined. All dark-colored and brown nematodes were classified as melanized, regardless of the number of hemocytes attached to them. Encapsulated nematodes had blood cells attached to them, but lacked melanization. Free-moving nematodes were 111 moving freely without blood cells attached. The number of melanized, encapsulated and

free-moving nematodes were recorded. In M. sexta, EPNs were recovered after 24 hours

because results of a pilot study indicated that melanization occurred at this time. In other

insects, IJs were recovered at 16 to18 hours after injection.

Immune responses of P. japonica to different EPNs

The insect host P. japonica and different species of EPNs, SgFL (S. glaseri FL

strain), SgNC (S. glaseri NC strain), Hb88 (H. bacteriophora HP88 strain), Sc (S.

scarabaei), and Sf (Steinernema feltiae) were used in this injection assay. The beetle larvae were injected and dissected as described above.

Immune responses of different hosts to different species/strains of EPNs

Different species/strains of EPNs, S. glaseri NC strain and FL strain, and H.

bacteriophora (HP88 strain), were injected into different insect hosts that included M.

sexta, E. orientalis, P. japonica, and C. borealis. The injection and recovery procedures

were as described above. The percentages of melanized, encapsulated and free moving

EPNs were recorded, statistically analyzed, and graphed.

Extraction of surface coat proteins (SCPs)

IJs were washed thoroughly by filtration before extraction of surface coat proteins

(SCPs). The IJs collected on Whatman filter paper (grade 4, 20-25 μm 1004-042,

Whatman Inc.) then were added into 150 ml of cold 35 % ethanol. The nematode/ethanol

solution was stirred at low speed at 4 °C for one hour, using a magnetic stir-bar. Then

the solution was filtered (how?) and was frozen at –80 °C. Next, the extract was

lyophilized and re-suspended in MilliQ water. The re-suspended solution was 112 centrifuged at 14,000 g, aliquotted and stored at –20 °C. The protein concentrations of

SCP solutions were determined by modified Bradford assay (Cox-Foster and Stehr, 1994).

Separation of SCPs from S. glaseri on 2D PAGE

The SCPs from S. glaseri NC strain, S. glaseri FL strain, and H. bacteriophora were

extracted as described above. The SCPs (4.6 µg) of each were loaded on a slab IEF gel,

pH 3.5 to 10, and run on a Multiphor apparatus for 1 hr (Cox and Willis, 1985, 1988).

The lanes for each sample were cut out and processed for 2D gel electrophoresis,

according to Cox and Willis, 1985. The sample lanes from the IEF gel were then

separated on a 2nd dimension 10 % SDS-PAGE. The procedures followed Cox and Willis

1985.

Effect of SCPs from S. glaseri NC strain on the immune response of M. sexta and E.

orientalis to H. bacteriophora

The injection technique was carried out as described above for oriental beetle larvae

and tobacco hornworms. For each host, 230 ng of SCPs from S. glaseri NC strain in 4 µl

MilliQ water was injected with 10 IJs of H. bacteriophora. Ringers’ solution was

injected with H. bacteriophora as control. E. orientalis was dissected at 8 hours post

injection. In M. sexta, EPNs were recovered after 24 hours because results of pilot study

indicated that melanization occurred at this time.

Statistical Analysis

The percentages of melanization, encapsulation and free-moving were calculated based upon the number of IJs injected and the number of IJs recovered, respectively.

When calculating percentages based on the number of EPNs injected, unrecovered 113 nematodes were assumed to be encapsulated and not visible. Standard residuals of the

data were investigated in order to determine the fit to the statistical model. For the

hemocyte recognition assay, percentage data was normalized using arcsine transformation prior to analysis. For examining immune responses of different EPNs in

Japanese beetle larvae and different species/strains of EPNs in different insect hosts, and

testing effect of SCPs in M. sexta and E. orientalis, percentage data was transformed

using arcsine square root. ANOVAs were performed for all assays. When two-way

ANOVA was used, an interaction term was included. If the results were significant,

Tukey pairwise comparisons were then performed. MINITAB 13.0 (Minitab Inc.) was

used for all statistical analyses. 114 Results

Do different species/strains of EPNs have different hosts?

Data of the infection assays were summarized in Table 4.1. Wax worms were susceptible hosts for both H. bacteriophora and S. glaseri; both EPN species infected wax worms and reproduced successfully (Table 4.1). Tobacco hornworms were a semi- susceptible host for both EPN species, which means the EPNs can infect third instar larvae of M. sexta but they did not reproduce well. Interestingly, the S. glaseri NC strain infected a higher percentage of hosts and reproduced better than the S. glaseri FL strain.

Oriental and Japanese beetles were semi-susceptible hosts for H. bacteriophora; in contrast, S. glaseri infected these hosts and reproduced in large numbers. Northern masked chafers and house crickets were resistant to both nematodes.

How do the blood cells of the host interact with the EPNs? Does this correspond to the host specificity?

The recognition of non-self is the first line of host defense against invaders in host hemocoel; therefore, I asked if the hemocytes of the hosts can recognize the EPNs. The hemocytes of G. mellonella, P. japonica, M. sexta and A. domesticus all recognized EPNs within minutes of exposure. However, for certain combinations, the recognition was intensive; for other combinations it was not. Even in the hemolymph from the same host, hemocytes reacted to the same species of EPN differently, suggesting that there were variations among IJs. Some IJs had a large amount of attached hemocytes while some were moving freely in the hemolymph (Fig. 4.1). 115

Table 4.1. Infectivity of EPNs against insect hosts.

EPNs G. mellonella M. sexta E. orientalis P. japonica* C. borealis A. domesticus 100% (20/20) 25% (18/73) 4% (1/23) 60% 0% (0/10) 0% (0/10) H. bacteriophora +++ + + + - - 100% (20/20) 75% (9/12) 0% (0/10) 0% (0/10) S. glaseri FL N. D. N. D. ++ ++ - - 100% (20/20) 100% (17/17) 86% (24/28) 80% 0% (0/10) 0% (0/10) S. glaseri NC +++ +++ +++ ++ - -

*Data from Wang et al., 1994;

N. D., Not determined;

Numbers in parenthesis are (number of infected hosts / number of host tested);

+ indicates EPNs reproduced successfully in the host;

- indicates EPNs did not reproduce very well in the host.

116

A M. sexta

IJ of H. IJ of S.

B P. japonica IJs of S. glaseri

IJ of H.

Fig 4.1 The degree of recognition by hemocytes from hosts to the IJs of EPNs after 60 mins of exposure. The hemolymph of insect host was mixed with Grace’s media. All images have the same magnification (100X) (Note: you may wish to insert a 100 micron scale bar into one of the photos). (A)

With hemocytes from larvae of M. sexta, IJs of H. bacteriophora were recognized at the head and tail by

hemocytes, while IJs of S. glaseri were free-moving. (B) For P. japonica, IJs of H. bacteriophora were

recognized by hemocytes, whereas, the responses to S. glaseri was varied, with some IJs being recognized

and others free-moving.

117

120 AaA A A A A a a 100 A a a a G. mellonella 80 a B P. japonica M. sexta 60 A. domestica b 40 cells of hosts of cells 20

0 1 hour 24 hour 1 hour 24 hour

percentage of EPNs recognized by blood blood by EPNs recognized of percentage H. bacteriophora S. glaseri NC

Fig. 4.2 Percentages of EPNs recognized by hemocytes from different hosts at 1 and 24 hours.

Bars represent mean ± SE. The letters indicates Tukey comparison results. The same letter indicates no statistical difference. Data were arranged and compared by time in H. bacteriophora and the S. glaseri

NC strain, respectively. Number of replicates ranged from 4 to 25.

(Note: You should use lower case letters on one half of the graph and all caps on the other side, because the two groups used separate mean comparison tests.) Y-axis label should probably read “EPNs recognized by blood (%)” 118 Data were analyzed and summarized from the in vitro recognition assay in Fig. 4.2.

In a three-way ANOVA, the three-way interaction of EPNs by hosts by time was

significant (F = 3.33; df = 3, 188; p < 0.05), which suggested that recognition at the 1 and

24 hours occurred in a EPN-host specific manner. The degree of recognition by the hosts

depended on the EPNs they encountered. Tukey pair-wise comparisons were carried out separately in the S. glaseri NC and H. bacteriophora groups (Fig. 4.2).

The percentages of two EPN species recognized by blood cells of different hosts

could explain the differences in infectivity of EPNs in the hosts to a limited extent. The

blood cells of wax worms, a susceptible host, recognized H. bacteriophora at 1 and 24

hours, but a significant percentage of the nematodes escaped from being recognized (p <

0.02). In the hemolymph of P. japonica, the recognition was high for H. bacteriophora

at both times (> 88 % recognized). S. glaseri was recognized at first by the blood cells of

the Japanese beetle (90 % recognized), but after 24 hours some S. glaseri escaped

recognition (75 % recognized) (not statistically significant for the two time points). For

H. bacteriophora, the blood cells of the resistant host M. sexta recognized the nematodes

strongly at both times (> 99 % recognized). For S. glaseri, M. sexta (which is a semi-

susceptible host) hemocytes recognized the nematodes weakly at 1 hour (28 %

recognized, p < 0.04) as compared with recognition by hemocytes from other hosts. The

recognition by M. sexta hemocytes was still low at 24 hours (64 % recognized), but the

difference was not statistically significant. In the resistant host A. domesticus, both

nematodes were recognized strongly at 1 and 24 hours (> 94 % recognized).

119 Do different species and strains of EPNs induce different immune responses in

Japanese beetles?

When analyzing the data, I assumed the numbers of EPNs not discovered in dissection were encapsulated (Fig. 4.3). This is a reasonable assumption because a similar pattern resulted when the data were graphed based on both the number of EPNs recovered and injected. (I don’t understand the last sentence.)

A one-way ANOVA indicated that species/strain effect was highly significant for percentages of EPNs melanized (F = 12.59; df = 4, 23; p < 0.001), encapsulated (F =

15.19; df = 4, 23; p < 0.001), and free-moving (F = 13.74; df = 4, 23; p < 0.001). This indicated that the species and strains of EPNs induced different immune responses in P. japonica larvae. The S. glaseri FL strain had the lowest percentage of melanization (12

%). S. glaseri NC strain were melanized less than H. bacteriophora (p < 0.01). H. bacteriophora were melanized the most (88 %) among all EPNs tested. Although the S. glaseri FL strain appeared to elicit the least immune response as indicated by melanization, it had the highest percentage of encapsulation (88 %) and almost no nematodes were free-moving in the host. The S. glaseri NC strain had the most free- moving nematodes (45 %; p < 0.02). The results indicated that the S. glaseri NC strain successfully suppressed the immune response in P. japonica larvae, and that H. bacteriophora induced the strongest immune responses.

In the other species of EPNs, a higher percentage of S. feltiae were melanized (67 %) rather than encapsulated (23 %) or free-moving (10 %). For S. scarabaei, most nematodes were encapsulated (53 %) while some were melanized (37 %) and free- moving (10 %). The percentages of free-moving nematodes for both S. feltiae and S. 120 scarabaei were lower than for the S. glaseri NC strain (p < 0.02) implying that neither

species of EPN could suppress the host immune response as well as the S. glaseri NC

strain.

How do hosts respond to different species and strains of EPNs?

For the percentages of melanized, encapsulated and free-moving EPNs in different

insect hosts, three two-way ANOVAs were carried out. Interaction between the EPNs and the hosts were included.

Based upon the numbers of EPNs injected, the interaction of EPNs by hosts was

significant for percentages of EPNs melanized (F = 6.65; df = 6, 107; p < 0.001),

encapsulated (F = 6.01; df = 6, 107; p < 0.001), and free-moving (F = 10.99; df = 6, 107;

p < 0.001). The interactions (EPNs by hosts) were all highly significant, which indicates

that each species of EPN induced specific immune responses in the different insect hosts.

The intensity of the immune responses was determined both by the species of EPNs and

by the species of insect hosts (Fig. 4.4).

121

Immune responses of different EPN species/strains JB 100 a C Melanized Encapsulated

n ac 80 Free-moving

up BC β

sed 60

a o bc bc

ge b 40 AB AB

enta A α α b α 20

perc α number of EPNs injected 0 Hb88 Sf SgFL SgNC Ss

Species and strains of EPNs

Fig. 4.3 Immune responses of P. japonica to different EPNs 16 to 18 hours after injection of 10 EPNs.

Percentages of melanized, encapsulated and free-moving nematodes (mean ± SE) were based on

observations following dissections of host insects 16 to18 hours post injection of EPNs. The nematodes not

recovered (< 50 % of the total number injected) were assumed to be encapsulated and not visible. The bars

of different colors (percentages of melanized, encapsulated and free-moving nematodes) were compared

separately. The letters above the bars (upper case, lower case, and Greek letter) indicate the results of

Tukey pairwise comparisons in each comparison group. The same letter indicates no statistical difference.

Number of replicates ranged from 3 to 6. SgFL, S. glaseri FL strain; SgNC, S. glaseri NC strain; Hb88,

H. bacteriophora HP88 strain; Sc, S. scarabaei; Sf, S. feltiae.

. 122

100 B Melanized and encapsulated B 80 BC BC

Percentage based upon number of EPNs injected DBC 60

AC AC 40 AC AC AD 20 A A

0 b 100 Encapsulated bc 80 bcd 60 ab ac ab a 40 ad ad a ad a 20

0 100 γ Free-moving 80 γ γ

60 βγ

40 βε αε αε 20 α α α α α 0 Hb88 SgFL SgNC Hb88 SgFL SgNC Hb88 SgFL SgNC Hb88 SgFL SgNC M. sexta E. orientalis P. japonica C. borealis (Note: please rotate the y-axis label 180 degrees)

Fig. 4.4 Immune responses of different hosts to different EPNs after 16 to 18 hours after injection of

10 EPNs. Percentages of melanized, encapsulated and free-moving nematodes (mean ± SE) were based on

observations following dissections of host insects 16 to18 hours post injection of EPNs (24 hours for M.

sexta)..The EPNs not recovered were assumed to be encapsulated and not visible. The bars of different

colors (percentages of melanized, encapsulated and free-moving nematodes) were compared separately.

The different letters above the bars (upper case, lower case, and Greek letter) indicate the results of Tukey

pairwise comparisons in each comparison group. The same letter indicates no statistical difference.

Number of replicates ranged from 3 to 16. Hb88, H. bacteriophora HP88 strain; SgFL, S. glaseri FL strain; SgNC, S. glaseri NC strain;; Sc, S. scarabaei; Sf, S. feltiae. 123 The host species responded differently to the various species/strains of EPNs. In all

four insect hosts, most of H. bacteriophora were melanized and encapsulated and very

few of the EPNs were free-moving. These results correspond well to the evidence that

H. bacteriophora could not infect and survive well in all the insect species tested. For the

S. glaseri FL strain, most nematodes were encapsulated in M. sexta and P. japonica. In

M. sexta, some of the S. glaseri FL strain could remained free (compared with this strain in P. japonica) which is consistent with the ability of S. glaseri FL strain to reproduce in

M. sexta. High percentages of S. glaseri NC strain were free-moving in M. sexta, E.

orientalis and P. japonica, which correlates with the S. glaseri NC strain being able to

infect and reproduce well in all three insect hosts. S. glaseri NC strain successfully

suppressed the immune responses in these susceptible hosts, whereas the S. glaseri FL

strain was less successful in these hosts. This indicates different strains within the same

species of EPN induced a different intensity of immune responses in the same host

species. Most of two EPN species were melanized in C. borealis, which may explain

why these species of EPNs were unable to reproduce in this insect. Overall, these results

suggest that the immune responses of the insects against EPNs corresponded to the

suitability of the host for EPN reproduction (Fig. 4.4).

Tobacco hornworm larvae, oriental beetle larvae and Japanese beetle larvae had a

similar pattern of immune responses to the S. glaseri NC strain, although, in Japanese

beetle larvae there was a lower percentage of free-moving S. glaseri NC strain. This

result was in agreement of the infectivity of the nematodes in these hosts.

124 8.0 7.0 6.1 5.1 4.2 3.2

A. S. glaseri 200KD —

an urea-IEF gel (pH 3.5-10) and then on a 2D PAGE (10% acrylamide PAGE (10% 2D a on then and 3.5-10) (pH gel urea-IEF an glaseri 97 KD —

Fig. 4.5SCPs from and and

H. bacteriophora 66 KD — NC strain

45 KD —

S. glaseri havedifferent patternsof protein spots onPAGE. 2D 21 KD — NC strain (A),

8.0 7.0 6.1 5.1 4.2 3.2 200KD — S. glaseri

B. S. glaseri 97 KD — FL strain (B), FL and

66 KD —

FL strain

SDS-PAGE). . The SCPs from two strains of 45 KD —

H. bacteriophora 21 KD —

8.0 7.0 6.1 5.1 4.2 3.2 (C) separated first on

C. 200KD — H. bacteriophora 97 KD —

66 KD — S.

45 KD —

21 KD — 125 Are SCPs from S. glaseri NC strain, S. glaseri FL strain, and H. bacteriophora the same?

Since the surface of the EPNs is involved in the immune responses, the different immune responses the EPNs encountered were possibly due to their SCPs. The SCPs from S. glaseri NC strain, S. glaseri FL strain, and H. bacteriophora were separated on

2D PAGE (Fig. 4.5). The SCPs from different strains and species of EPNs had different patterns on 2D SDS-PAGE. SCPs from S. glaseri NC strain had fewer high molecular weight acidic (pH 4.2-5.0) proteins as compared with SCPs from S. glaseri FL strain.

Moreover, SCPs from S. glaseri NC strain had more low molecular weight proteins (40-

50 KD) as compared with the latter. SCPs from H. bacteriophora had a different pattern as compared with the two strains of S. glaseri. The SCPs of H. bacteriophora were composed of some high molecular weight (200 KD) proteins but had fewer low molecular weight proteins (40-50 KD).

126

p 100 Melanized 80 Encapsulated Free-moving

60

40 Percentage

20

0 Control SCPs

Fig. 4.6 Surface coat proteins from S. glaseri NC strain failed to protect H. bacteriophora from host immune responses in M. sexta. 10 IJs of H. bacteriophora were injected with 4 μl Ringers’ solution or

with SCPs from S glaseri NC strain. Percentages of melanized, encapsulated and free-moving nematodes

(mean ± SE) were based upon observations following dissection at 24 hours post injection . The EPNs not

recovered were assumed to be encapsulated. The percentages of melanized, encapsulated and free-moving

H. bacteriophora were not different between coinjection with and without SCPs. Replicates ranged from 3

to 16. Control is injection of Ringers’ solution and SCPs is injection of SCPs from S glaseri NC strain in

MilliQ water. 360 ng of SCPs were injected.

127

100 a Melanized Encapsulated 80 Free-moving

60 β B

percentage 40

b 20 A α

0 Control SCPsSCPs

Fig. 4.7 Surface coat proteins from S. glaseri NC strain protected H. bacteriophora from host immune responses in E. orientalis larvae. 10 IJs of H. bacteriophora were injected with 4 μl Ringers’ solution or with SCPs from S glaseri NC strain. Percentages of melanized, encapsulated and free-moving nematodes (mean ± SE) were based upon observations following dissection at 8 hours post injection. The

EPNs not recovered were assumed to be encapsulated. The percentages of melanized, encapsulated and free-moving nematodes were compared separately. The different types of the letters above the bars (upper case, lower case, and Greek letter) indicate the results of Tukey pairwise comparisons in each comparison group. The same letter indicates no statistical difference. Control is injection of Ringers’ solution (n = 10) and injection of SCPs from S glaseri NC strain in MilliQ water (n = 12). 230 ng of SCPs were injected.

128 Can surface coat proteins (SCPs) from S. glaseri NC strain protect H. bacteriophora from being killed?

In E. orientalis and M. sexta, I have demonstrated that most H. bacteriophora were

melanized while a high percentage of S. glaseri NC strain was free moving inside of the

hosts. Since H. bacteriophora and S. glaseri NC strain had different SCPs, I

hypothesized that SCPs helped S. glaseri evade or suppress the immune responses of

insect hosts. Therefore, I extracted SCPs from S. glaseri NC strain and tested if these

SCPs could protect H. bacteriophora from being melanized and encapsulated in E.

orientalis and M. sexta.

Injection of S. glaseri NC strain SCPs with H. bacteriophora into M. sexta did not

suppress the melanization (F = 0.00; df = 1, 14; p = 0.973) and encapsulation (F = 0.03;

df = 1, 14; p = 0.856) of the nematodes (Fig. 4.6). There were no significant differences

in the percentages of free moving nematode between the injection of SCPs and injection

of Ringer’s solution with H. bacteriophora (F = 0.12; df = 1, 14; p = 0.733). One

explanation would be that the SCPs injected could have been degraded or lost function in

the hosts during 24 hour period. The other possibility is that S. glaseri evaded the

immune responses in M. sexta instead of suppressing the hosts’ immune system.

However, in E. orientalis larvae, the percentage of melanized H. bacteriophora when

coinjection of SCPs is significantly lower compared with injection of Ringers’ solution

with nematodes (F = 88.42; df = 1, 20; p < 0.001). The percentage of free-moving

nematodes is significant higher (F = 36.50; df = 1, 20; p < 0.001). This indicates that

SCPs from S. glaseri NC strain larvae protected H. bacteriophora from being melanized

(Fig. 4.7). 129 The differences between the immune responses of M. sexta and E. orientalis can be explained by several possible mechanisms. One is that S. glaseri interacts with M. sexta and E. orientalis differently. In M. sexta, S. glaseri may evade immune responses while in E. orientalis, S. glaseri inhibits the immune system. It is possible that M. sexta does not recognize the invader and that SCPs target specific insect host species. The other possible explanation is that SCPs from S. glaseri lost their immuno-suppressive function during 8 to 24 hours post injection. Thus, whichever hypothesis is correct, S. glaseri suppresses immune responses in E. orientalis but not in M. sexta.

130 Discussion

The present research demonstrates that different strains and species of EPNs have

different infectivity among insect groups. Among the four major factors that determine

EPNs’ host specificity (Gaugler et al., 1997), suppression of insect immune responses

play an important role in determining host suitability. Infectivity is correlated with immune responses that EPNs encounter in insect hosts. Initial recognition of non-self by hemocytes is a critical step in immune responses. However, recognition is only part of the story of EPN-host immunity interactions. The inconsistency between the degree of recognition of EPNs by host hemocytes and the intensity of immune responses EPNs encountered in hosts was probably caused by suppression of host immune responses by

EPNs and their symbiotic bacteria.

H. bacteriophora was melanized and encapsulated in P. japonica, in agreement with

previous research (Wang et al., 1994). However, Wang et al. (1994) showed that S.

glaseri could reproduce well in A. domesticus which conflicts with the results in this

paper. This difference may be attributable to different strains of S. glaseri used in the

experiments. Wang et al. (1994) showed that S. glaseri killed more P. japonica larvae

compared with H. bacteriophora which was consistent with reduced melanization and

encapsulation of S. glaseri compared with H. bacteriophora.

It is reported that S. feltiae cuticle plays a role in inactivation of the pro-

phenoloxidase pathway (Brivio et al., 2002). The present results indicate that different

strains and species of EPNs have different SCPs. Moreover, the results in the present

research indicate that S. glaseri can suppress the immune responses in E. orientalis larvae

with its SCPs. This supports previous reports that S. glaseri SCPs can suppress immune 131 responses and protect H. bacteriophora in P. japonica (Wang and Gaugler, 1998). This suppression protects nematodes, allowing their release of symbiotic bacteria. However, in M. sexta, SCPs from S. glaseri NC did not suppress the host immunity, therefore, S. glaseri probably evades rather than suppresses the immune responses in this insect host.

S. glaseri induced a strong melanization effect in the house cricket. These results suggest that S. glaseri interacts with different hosts in species-specific ways. 132 Acknowledgments

I appreciate intellectual contributions from Drs. Richard S. Cowles, Elizabeth Cowles and Randy Gaugler. I also thank Dr. Richard S. Cowles who collected and identified all oriental beetle larvae. The thank goes to Dr. Yi Wang who communicated with me on all kinds of issues. Last, I will express my thanks to my advisor Dr. Diana L. Cox-Foster who guided me through the experiments. Without her help on techniques and experimental designs, it would not be a good story.

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Chapter 5

Characterization of surface coat proteins from Steinernema glaseri that

suppress immune responses in Oriental beetle larvae 138 Abstract

Infective juveniles (IJ) of entomopathogenic nematodes (EPNs) penetrate insect hosts

and release symbiotic bacteria that kill the hosts. Insect hosts defend against EPNs by

rapid cellular immune responses that result in encapsulation and melanization which kills

EPNs. Nematodes have to overcome the innate immunity of the hosts to survive and

reproduce. Therefore, the release of symbiotic bacteria has to occur before the intensive

host immune responses occur.

Surface coat proteins (SCPs) of EPNs are suggested to play a role in the

suppression/evasion of host immune responses. I demonstrated that different species and

strains of EPNs have different surface coat protein profiles. I isolated and characterized

SCPs from Steinernema glaseri NC strain. These SCPs suppressed immune responses of

the Oriental beetle larva, a susceptible host for S. glaseri, thus protecting Heterorhabditis

bacteriophora from being killed in the same host, as it normally would. Here, I demonstrated that immuno-suppression was dose dependent. Also, multiple injections of the SCPs protected H. bacteriophora better in oriental beetle larvae. In a nondenatured state, two isolated proteins in the SCPs of S. glaseri each conveyed this immuno- suppressive effect. The two proteins were composed of smaller proteins when separated on two-dimensional PAGE. The sequences and characterization of these proteins were also investigated.

139 Introduction

Entomopathogenic nematodes (EPNs) in genera Steinernema and Heterorhabditis are important biocontrol agents for soil dwelling insects (Dunphy and Thurston, 1990; Kaya and Gaugler, 1993). Through convergent evolution, these two groups of EPNs share a similar life history and are closely related to two groups of vertebrate parasitic nematodes respectively (Blaxter et al., 2000; Burnell and Stock, 2000).

EPNs enter the hemocoel of the insect hosts via body openings and intersegmental membranes (Burnell and Stock, 2000). The symbiotic bacteria are released after the nematodes enter the insect hemocoel (Dunphy and Webster, 1998). Symbiotic bacteria produce proteases and toxins that inhibits encapsulation, melanization and the production of antimicrobial peptides (Dunphy and Thurston, 1990; Wang et al., 1994, 1995; Hatab et al, 1998; Gaugler et al., 1997)

Insects use morphological, behavioral and chemical mechanisms against EPNs

(Forschler and Gardner, 1991; Gaugler et al., 1994; Wang et al., 1995). One group of the most important insect defense mechanisms are humoral and cellular immune responses

(Hoffmann et al., 1996,1999; Gillespie et al, 1997). Encapsulation and melanization induced by EPNs result in killing of the nematodes (Wang et al., 1994, 1995; Peters and

Ehlers, 1997).

Steinernema glaseri, originally isolated from Japanese beetle larvae, is naturally associated with scarab larvae (Steiner, 1929). The symbiotic bacteria of S. glaseri,

Xenorhabdus poinarii (Akhurst) is released after 4-6 hour after nematode entry the host hemocoel (Akhurst and Dunphy 1993; Wang et al., 1995). Heterorhabditis bacteriophora is a commercialized biocontrol agent targeted to white grubs (Gaugler et 140 al., 2000). Its symbiotic bacteria Photorhabdus luminescens can be detected as soon as

30 minutes after nematodes entry the hemocoel (Bowen et al., 1998). Since bacteria act at a later stage, it has been suggested that short-term host parasite interaction is manipulated by the nematodes themselves (Brivio et al., 2002). In mammals, the parasitic nematodes have surface coat proteins (SCPs) that interacted with host immunity; these proteins include antioxidant enzymes (Glutathione peroxidase, superoxide dismutase, glutathione S-transferase and peroxiredoxin) and serine protease inhibitors

(serpins) (Maizels et al., 2001, 2004).

There is remarkable conservation in the biochemical and basic structure pattern of

cuticle among nematode groups, which includes free living Caenorhabditis elegans,

mammalian parasitic nematodes and EPNs (Burglin et al., 1998). The genome projects of

C. elegans and a filarial parasite Brugia malayi have demonstrated great homology

between these two species (Burglin et al., 1998). However, together with other projects it

also has revealed that 35% ETSs of B. malayi have no homologues in C. elegans or other

species (Burglin et al., 1998). Most considerable differences between the C. elegans and

parasitic nematodes are species and stage specific surface proteins (Burglin et al., 1998).

All nematodes share a surface coat of glycocalyx composed of a few glycoproteins.

Mammalian parasitic nematodes secret some glycoproteins conveying the immuno-

evasive effect (Maizels et al, 1993; Haslam et al., 1999). There are two main strategies to

avoid recognition by the host immune system. One is immune evasion and the other is

immune suppression. Both strategies always involve surface of the parasites (Dunphy

and Webster, 1984, 1987, 1988; Ratcliffe et al, 1985; Politz and Philipp, 1992). 141 H. bacteriophora is encapsulated in Japanese beetle larvae Popillia japonica, however 10 % of the nematodes escape the encapsulation after 24 hrs (Wang et al., 1994,

1995). The mechanism of this evasion is still unknown. In comparison only 24% of S. glaseri is melanized initially in P. japonica; and after 24 hours, all nematodes are free in the host hemocoel (Wang et al., 1995). The SCPs of S. glaseri are separated as five bands on nondenaturing PAGE. Two of them each convey the immuno-suppression effect in P. japonica (Wang and Gaugler, 1999). The two SCPs lyse the hemocytes and suppress melanization in the P. japonica (Wang and Gaugler, 1999). It also has been demonstrated that cuticle of an EPN Steinernema feltiae is involved in immune suppression of wax moth Galleria mellonella (Brivio et al, 2002). Besides theses initial studies, there is dearth of the information on the proteins that involved in suppression or evasion of host immune response in insects.

SCPs from S. glaseri NC strain protect H. bacteriophora from immune responses in

E. orientalis larvae (unpublished, Chapter 4 of this thesis) but not in M. sexta. In this research, the goal is to study the function of SCPs from S. glaseri NC strain in detail and to separate the components that have immuno suppressive function. This research will help us have a better understanding of the mechanism SCPs used to suppress the host immune responses. The sequences of these SCPs will help to tackle the puzzle of the evolution of the SCPs of parasitic nematodes.

142 Materials and methods

Nematodes

Two species of entomopathogenic nematodes (EPNs) were tested: Steinernema

glaseri (NC strain and FL strain), and Heterorhabditis bacteriophora (HP88 strain). All

EPNs were cultured in last instar wax worms Galleria mellonella (L.) (purchased from

Nature’s Way Inc.) at room temperature. Infective juveniles (IJs) of EPNs were

harvested from white trap by filtration and cultured in distilled water bubbled with air for

about 2-4 days at room temperature for all the experiments conducted. Nematodes were

washed 3 times by MilliQ water before conducting all the experiments.

Insects

Last-instar Oriental beetles Exomala orientalis were field collected. The grubs were stored at 8°C incubator and fed on grass seeds and roots for one day before the experiments. G. mellonella were purchased and maintained at room temperature.

Extraction of surface coat proteins (SCPs)

IJs were washed thoroughly by filtration before extraction of surface coat proteins

(SCPs). The IJs collected on Whatman filter paper (0.25μm D) then were added into 150

ml of cold 35 % ethanol. The nematode/ethanol solution was stirred at low speed at 4 °C

for one hour, using a stir-bar. Then the solution was filtered and was frozen at -80 °C.

Next, the extract was lyophilized and re-suspended in MilliQ water. The re-suspended

solution was centrifuged at 14,000 g, aliquotted and stored at –20 °C. The protein

concentration of SCPs solution was determined by modified Bradford assay (Cox-Foster and Stehr, 1994).

143 Injection

10 IJs of H. bacteriophora were delivered by a Drummond micro injector (positive

displacement digital microdispenser 3-000-525, Drummond Scientific Company,

Broomall, PA) equipped with a glass needle. The needle was pulled from a capillary tube

(Drummond Wiretrol 5-000-1050 50 µl Drummond Scientific Company, Broomall, PA) on a flame and then sharpened on a sharpening stone. For each insect host, either 4 µl

Ringers’ solution (0.8 % sodium chloride, 0.02 % calcium chloride, 0.02 % potassium chloride, 0.02 % sodium bicarbonate in MilliQ water, pH 7.0), IEF buffer (2 mM PMSF,

36 mM Tris-HCL (pH 8.4) in MilliQ H2O), MilliQ water or Bovine Serum Albumin

(BSA) solution (BSA in MilliQ water, served as a protein control) with H. bacteriophora

was injected into Oriental beetle larvae in the following experiments. Injection site was

laterally to the base of foreleg. If the digestive system was damaged during injection, the

host was not used. Insects were kept at room temperature in a Petri dish with a piece of

moisture filter paper. IJs were recovered at 8 hours after injection. H. bacteriophora, S.

glaseri NC strain and E. orientalis were used in the experiments.

Test effect of SCPs from S. glaseri NC strain in E. orientalis

In this experiment, several SCPs extraction methods were compared (Table 5.1). The

concentrations of SCPs solution were determined by modified Bradford assay (Cox and

Willies, 1985).

The SCPs were either extracted by cold 35 % ethanol, or by cold MilliQ water (at 4

°C or –20 °C; Table 5.1). The extraction was either on ice or at 4 °C for half hour or for

one hour. The lyophilized SCPs were resuspended either in Ringers’ solution or IEF

buffer. 144 The storage water of S. glaseri was added to acetone at a 5:95 v:v ratio. The mixture

was stored overnight at –20 °C. The proteins in storage water then precipitated, and the

mixture were centrifuged under 4 °C at 3000 g for 1 hour. The precipitates then were

collected and rinsed twice with cold acetone. Then the proteins were resuspended in

Ringers’ solution, and marked as extraction number 3.

The desheathment method was followed Wang and Gaugler, 1999. In brief, IJs were

desheathed using 0.5 % sodium hypochlorite at room temperature for 15 min, nematodes

then were rinsed by centrifugation in distilled water 3 times. Nematodes were stored in 2

liters of distilled water with bubbled air for 2 days to redevelop surface coat. After 2

distilled water rinses, nematodes were put in 35 %, precooled ethanol (at –20 °C) for 30 min and extracted on ice.

All SCPs were coinjected with H. bacteriophora into Oriental beetle larvae as

described as ‘injection’ part. IJs were recovered at either 8 hours or 16 to 18 hours post

injection.

Percentages of melanized H. bacteriophora were calculated. To analyze the data,

GLM model was used and Dunnett comparisons to control were carried out.

Dosage effect and exposure time effect of SCPs

E. orientalis were used for both assays. To test the dosage effect of SCPs from S.

glaseri NC strain, 0, 50, 100, 230 and 940 ng of SCPs from S. glaseri NC strain were

injected with 10 IJs of H. bacteriophora. Insects were dissected at 8 hour post injection.

To test the effect of time of exposure of SCPs on the immune response of the host,

each of the E. orientalis larvae was injected twice. For a group of five insects, at the 1st

hour time, two of the insects were injected with Ringers, and other three received 145 injection of SCPs. At the 8th hour, one of the first two insects received another Ringers’ injection. The two of the last 3 insects were injected Ringers’ and SCPs respectively. All insects were dissected 24 hours after the first injection (Fig. 5.3).

Electroelution of the SCPs from S. glaseri NC strain

Nondenaturing gel molecular markers (Nondenatured protein molecular weight marker kit, MW-ND-137, Sigma Inc.) and SCPs from S. glaseri NC were separated on 8

% nondenaturing PAGE. The outer lanes were stained with Coomassie Brilliant Blue

(0.1 % Coomassie Brilliant Blue R250, 50 % methanol, 16 % glacial acetic acid), and inner lanes were cut out according to the bands showed on the outer lanes. The gel pieces then were electroeluted overnight in a micro-Electroeluter (Centrilutor 57005, Amicon

Inc.). The electroelution buffer is 1:1 ratio of dilution gel running buffer. Each electroelutes were collected and concentrated by microconcentrators (Centricon YM-30

4208 30KD cutoff, Amicon Inc.) at 3000 g and 4 °C. The 30 KD cutoff concentrators were used to eliminate small molecules and salts. Concentrators with fractions of SCPs then was filled up with MilliQ water and concentrated again. Next, each fraction was aliquotted and stored at –20 °C. To test the effect of electroeluted fractions, each of the fractions were coinjected with H. bacteriophora into the Oriental beetle larvae. Total

SCPs from S. glaseri NC and BSA solution were injected as control groups. The insects were dissected at 8 hour post injection.

Dissection to recover EPNs

Host insects were decapitated and a lateral cut was made around tip of the abdomen.

Then, the insect was carefully cut open ventrally and the digestive system was dissected out. Care was taken so that the digestive system did not rupture and it was examined 146 carefully. Often, EPNs were trapped around the digestive system. Next, distilled water

was flushed through the open hemocoel and examined for free moving IJs (these EPNs were classified as free-moving and not recognized by the host immune system). Other tissues were then dissected and examined. All dark-colored and brown nematodes were classified as melanized, regardless of the number of hemocytes attaching to them.

Encapsulated nematodes had blood cells attaching to them, but lacked melanization.

Free-moving nematodes were moving freely without blood cells attached. The number of melanized, encapsulated and free-moving nematodes were recorded.

Running nondenaturing PAGE, SDS-PAGE and 2D PAGE

For SDS-PAGE, the sample was boiled with SDS sample buffer for 5 minutes.

SDS-PAGE broad range protein molecular weight markers (SDS-PAGE Molecular weight standards, broad range 161-0317, Bio-Rad) were applied on SDS-PAGE to

calculate the molecular weight of the proteins. Nondenaturing PAGE was made with the

same recipe as SDS-PAGE but without SDS. On native PAGE, Native PAGE molecular

weight standards were used and BSA was also applied. BSA consistently separated into

several bands that were served as markers. 8 % or 10 % PAGE were used to separate the

SCPs. For 2D PAGE, the first dimension slab IEF gel was run with or without urea, and pH ranged from 3.5 to 10.5. On SDS PAGE, broad range molecular weight marker was used to calculate the protein weight. All gels were stained by coomassie blue or silver staining. All procedures followed Cox and Willies, 1985. Glycosylation was investigated by GelCode® Glycoprotein Staining Kit (Pierce 24562).

147 Effect of SCPa and SCPb on hemocytes of oriental beetle larvae

The effect of SCPs on hemocytes of Oriental beetle larvae was studied in vitro

(Lovallo and Cox-Foster, 1999). Insects were rinsed thoroughly by sequentially flushing

coverage plus, distilled water and 70 % ethanol for 5 minutes for each solution before

sacrifice. One or two drops of hemolymph of insect hosts added to 250 µl Grace’s insect

medium (Quality Biologicals, Gaithersburg, MD, catalogue #117-048-100, with L- glutamine, without lactalbumin hydrolysate, TC yeastolate, and insect hemolymph) in a

coated 8-chambered coverglass wells (Lab-Tek, Nunc. Naperville, IL). The tissue culture

wells were coated with 0.2 % molecular-grade agarose in filter-sterilized MilliQ water.

Cellular responses were recoded using an inverted microscope (Axiovert 405M, Zeiss,

West Germany) equipped with a video camera (Sony DXC-960MD 3CCD; Montvale,

NJ) and linked to a computer (Macintosh Quadra 800, Apple Computer Inc., Cupertino,

CA) with NIH Image (version 1.59) video imaging software.

Observation was carried out at 1st, 3rd, 5th and 24th hour after initial introduction of the

hemocytes.

Statistical Analysis

The percentages of melanization, encapsulation and free-moving were calculated based upon the numbers of IJs injected. When calculating percentages based on the number of EPNs injected, unrecovered nematodes were assumed to be encapsulated and not visible. Residuals of the data were investigated to determine model fits. Percentages data were arcsine square root transformed for all the experiments. ANOVAs were performed for each assay. If the results were significant, Tukey pairwise comparisons or 148 Dunnett comparisons were then performed. MINITAB 13.0 (Minitab Inc.) was used for all statistical analysis.

149 Results

Can SCPs from S. glaseri protect H. bacteriophora from being melanized in E. orientalis?

Different methods of exaction were described in Table 5.1. The amount of SCPs

injected, the buffer SCPs in was also included in the table. Group 0 served as a control.

There were no differences between recovering EPNs at 8 or 16-18 hours post injection (F

= 0.33, df = 1, 89; p = 0.569); and protein concentration was not a significant covariate (F

= 1.01; df = 1, 89; p = 0.317). Therefore, the data were pooled for further analysis.

There was no significant differences between IEF buffer and Ringers solution among

injection groups (F = 3.22; df = 1, 91; p=0.076). Concentration of SCPs was not a

significant covariate which indicated including concentration in the analysis did not

explain part of the effect observed. Therefore, the results from different time groups and

different buffer groups were pooled to perform statistical analysis.

The results indicated that SCPs from S. glaseri NC strain suppressed melanization

in E. orientalis thus protected H. bacteriophora (Fig. 5.1). However, SCPs with different

extraction methods had different degrees of suppression.

The percentages of melanized H. bacteriophora were greatly reduced by

coinjection of SCPs from S. glaseri NC strain ((F = 13.97, df = 8, 92; p < 0.000),

regardless of extraction methods used. The proteins precipitated from storage water

(group 3) also suppressed melanization significantly (Dunnet test, t = -3.007, p = 0.0258).

The SCPs extracted at 4 °C and without desheathment had the lowest percentages of

melanization (12 ± 7 %). The difference was not due to different amount of SCPs

injected. Since injections of (extraction 6 and 7) less amount of protein had lower 150 percentages of melanization compared to injections larger amount of proteins (extraction

2 and 8) (Table 5.1 and Fig. 5.1).

Is there a dosage effect of SCPs from S. glaseri NC strain on the hosts’ immune

responses?

Injection of various amounts of SCPs with H. bacteriophora into E. orientalis

indicated a significant dosage response. The percentages of melanized (F = 29.48; df = 4,

19; p<0.001) and free-moving (F = 13.73; df = 4, 19; p<0.001) EPNs were significantly

different. When injected with 0 ng SCPs, more than 80 % of H. bacteriophora were

melanized. With injections of 50 ng and 100 ng of SCPs, less than 40 % of H.

bacteriophora were melanized. The SCPs also increased the percentage of free moving

nematodes(Fig. 5.2). When injected 230 ng and 940 ng SCPs, very few H. bacteriophora

were melanized in the insect host and about half of the nematodes were moving freely.

The results indicated that with increasing of the amount of SCPs injected, more

nematodes were protected from being melanized and more nematodes were free moving

inside the host.

What the effect of exposure time of SCPs on host immune responses?

There were significant differences of melanized (F = 13.73; df = 4, 10; p < 0.001) and

free-moving EPNs (F = 9.63; df = 4, 10; p < 0.002) between the treatment groups. Both

Ringers’-no injection and Ringers’-Ringers’ injection did not suppress melanization in E. orientalis. Most H. bacteriophora were melanized and almost no nematodes were free moving in the host. However, SCPs- no injection and SCPs-Ringers’ injection significantly inhibited melanization in the host (t = 3.7, p < 0.026) and about 30 % of the 151 nematodes were free moving. When SCPs were injected twice, the melanization is

significantly inhibited (t = 4.5, p < 0.01) and more than 50 % of the nematodes were

moving freely within the host (Fig. 5.3). After initial injection of SCPs, another injection

of SCPs increased the percentage of free moving nematodes. The results suggested that

the SCPs effect decreased after 8 hours post injection, and this could be caused by

degradation of the SCPs.

What SCPs have the immuno-suppressive effect in E. orientalis?

The SCPs from S. glaseri NC strain were separated on both nondenaturing PAGE and

SDS-PAGE (Fig. 5.4). There were many protein bands on Native PAGE. The same sample was run SDS-PAGE, the resulting gel had more protein bands. This suggested that some larger molecular weight proteins under native state were composed of several smaller proteins (Fig. 5.4). The Glycosylation kit did not detect significant glycoproteins on the gel. A limited number of minor bands reacted with the periodic acid Schiff reagent (PAS). It is possible that the sensitivity of the kit (μg grade) was not great enough to detect all forms of glycosylation (Cox and Willies, 1985).

The SCPs from S. glaseri NC strain were separated by nondenaturing PAGE and

major bands were electroeluted and concentrated (Fig 5.4). Each fraction was tested

against E. orientalis. Two fractions of the SCPs, SCPa and SCPb both protected H.

bacteriophora from melanization and increased percentages of free moving nematodes

(Fig. 5.5). Each of two SCPs had the same immuno-suppressive effect as the total extract of SCPs. Other proteins electroeluted from the total SCPs did not suppress the immune

responses in the host. Similarly, the Bovine Serum Albumin (BSA) did not inhibit the

host immune response with more than 80 % of the nematodes being melanized and less 152 than 10 % of nematodes were free moving (Fig. 5.5). The results indicated that the

suppression of host immune responses was caused by injection of specific SCPs, but not injection of a certain amount of proteins. A small amount of SCPa (0.12μg) and SCPb

(0.3μg) had the same effect as ten times amount (1.2 μg) of total SCPs.

SCPa and SCPb were separated on nondenaturing PAGE (Fig 5.6a) and SDS-PAGE

(Fig. 5.6b). The estimated molecular weight for SCPa was 150 KD and for SCPb was

100 KD (Fig. 5.6a). Given that a nondenaturing PAGE separates proteins based on

molecular weight, charge and conformation, the estimated molecular weight of the SCPs

was not as accurate as SDS-PAGE.

When SCPa and SCPb were denatured and separated on SDS-PAGE, both of the

proteins were composed of smaller proteins (Fig. 5.6b). One of major protein was about

38KD. SCPa and SCPb were then loaded on IEF PAGE and then 2nd dimension SDS-

PAGE. The results indicated that both of the SCPs were composed of three major

proteins (Fig 5.7a, 5.7b). The pH of the proteins ranged from 3.5 to 4.2 and molecular weight were from 36 KD to 56 KD. The two proteins had the same pI at 4.2. Two proteins about 38 KD with different pI (Fig 5.7a, 5.7b) probably were the two proteins of

38KD on SDS-PAGE (Fig 5.6b). There were also minor proteins stained on 2nd

dimension SDS-PAGE. It was also possible that those proteins were the ones that

conveyed immuno-suppressive function.

What is the effect of SCPs on hemocytes of the hosts?

When hemocytes of E. orientalis exposed to SCPa and SCPb, the hemocytes started

degrading at the 3 hour time. Degradation of the hemocytes was more significant at 5

hour time. At 24 hour time, large amount of hemocytes degraded compared to the 153 hemocytes unexposed to the SCPs (Fig. 5.8a, 5.8b). These results suggested that SCPs from S. glaseri NC strain probably lysed the hemocytes and thus suppressed immune responses in E. orientalis larvae. 154

Table 5.1 Description methods of SCPs extractions used in Fig. 5.1.

Amount Extraction methods (S. glaseri NC strain) Batch Abbreviation Buffer injected

(µg) T Extraction buffer length sheath Other

Ringers or 0 H2O IEF precooled – 20 °C 1 NCDS Ringer’s 1.49 on ice 0.5 hr desheathment desheathment 35 % Ethanol precooled – 20 °C 2 NCWS Ringer’s 1.32 on ice 0.5 hr with sheath 35 % Ethanol storage water 3 NCSW Ringer’s 0.50 – 20 °C acetone overnight with sheath precipitated precooled – 20 °C 4 NCWS Ringer’s 0.59 on ice 0.5 hr with sheath 35 % Ethanol

5 NCWSH2O Ringer’s 0.91 on ice water 0.5 hr with sheath * after 4

6 NCWSH2O IEF 0.94 at 4 °C water 1 hr with sheath 7 NCWS IEF 0.74 at 4 °C 35 % ethanol 1 hr with sheath

8 NCWSH2O IEF 1.24 at 4 °C water 1 hr with sheath * after 7

After 4 and after 7 indicate the extractions were performed after extraction 4 and extraction 7 respectively.

155

Fig. 5.1 Comparison of effect of suppression of melanization by SCPs from S. glaseri NC strain among different extraction methods.

10 IJs of H. bacteriophora were injected with 4 μl Ringers’ solution, IEF buffer or with various amounts of

SCPs from S glaseri NC strain (Table. 5.1). SCPs extraction methods were described in Table 5.1. EPNs

were recovered at 8 hours or 16-18 hours post injection. Percentages of melanized nematodes graphed

based upon EPNs injected. The EPNs not recovered were assumed to be encapsulated. Bars represent

Means ± SE. The letters above the bars indicate the Dunnett comparisons to control. * indicates statistical

difference (p < 0.05) from control (0 group); and ** indicates the difference from control is very significant

(p < 0.01). Replicates ranged from 3 to 27. Control (0 group) was injection of Ringers’ solution or IEF

buffer and group 1 to 8 were injection of SCPs from S glaseri NC strain. ANCOVA indicated protein

concentration was not a significant factor (p > 0.05). IEF buffer had no significant difference from Ringers

solution (p > 0.05). Blank bar (0) is control. Doted bars (1, 2, 4 and 7) indicate SCPs were extracted with

35 % ethanol. Grey bars (1, 2, 4 and 5) indicate extractions were on ice and black bars (6, 7 and 8) indicate

extractions were at 4 °C. Striped bar (3) indicates SCPs were participated with acetone.

156

Relation of Melanization to Concentration of SCPs A Melanized 100 Free-moving 80 c bc 60 BC b BC 40 ab (mean+SD)

20 a C C category in percentage 0 0 50 100 230 940 Amount of protein injected (ng)

Fig. 5.2 Dosage effect of SCPs from S. glaseri NC strain on immune responses in E. orientalis larvae.

10 IJs of H. bacteriophora were injected with 4μl Ringers’ solution or with various amounts of SCPs from

S glaseri NC strain. EPNs were recovered after 8 hours post injection. Percentages of melanized, encapsulated and free-moving nematodes graphed based upon the number of EPNs injected. The EPNs not recovered were assumed to be encapsulated. Bars represent Means ± SE. The letters above the bars indicate the Tukey pairwise comparison results. The same letter indicates no statistical difference.

Replicates ranged from 9 to 27. Control was injection of Ringers’ solution and SCPs was injection of SCPs from S glaseri NC strain in MilliQ water. 157

Relation of free-moving IJs to time of SCP injection A A 100 Melanized Free-moving 80 b

60 ab ab 40 B

Percentage Percentage B B 20 ac c 0 R- N R- R P- N P- R P- P Protein injected at 1 hour and at 8 hour

Fig. 5.3 Effect of exposure time of SCPs from S. glaseri NC strain on immune responses in E. orientalis larvae. 10 IJs of H. bacteriophora were injected with 4μl Ringers’ solution or with 230 ng of

SCPs from S glaseri NC strain. Two injections were carried out for each grubs. One at 1st hour post injection and the second is 8 hours later. EPNs were recovered at 24 hours after first injection. Injection at the 1st hour was indicated by letter in front of ‘-‘, and injection at the 8th hour was indicated by letter after ‘-

‘. R indicates injection of Ringers’ solution; N indicates no injection carried out; P indicates injection of

SCPs. For example, ‘P-R’ indicates injection of SCPs at the 1st hour and injection of Ringer’s solution at

8th hour. Percentages of melanized, encapsulated and free-moving nematodes graphed based upon the number of EPNs injected. The EPNs not recovered were assumed to be encapsulated. Bars represent

Means ± SE. The letters above the bars indicate the Tukey pairwise comparison results. The same letter indicates no statistical difference. There were 3 replicates for each group. Experimental design was as following chart.

st At 1 hour injected SCPs or Ringers’ solution Ringers’ Ringers’ SCP SCP SCP

At 8th hour injected SCP, Ringers’ solution or did nothing Ringers’-No Ringers-Ringer SCP-No SCP-Ringer SCP-SCP 158 A B

200KD — BSA 3rd band — SCPa SCPb 116KD — nd BSA 2 band — 97 KD —

BSA 1st band — 66 KD —

45 KD —

21 KD —

Fig. 5.4 Separation of SCPs from S. glaseri NC strain on 8% Nondenaturing PAGE and 8% SDS

PAGE. 20 μg of SCPs from S. glaseri NC strain were separated by 8 % nondenaturing PAGE (A) and 8%

SDS-PAGE (B). BSA solution was applied on native PAGE as a relative standard together with other

native PAGE standards. Each bands (marked with arrow) on the native PAGE were electroeluted and

concentrated, washed in microconcentrators. 8 % SDS PAGE shows that SCPs from S. glaseri NC strain

were composed of smaller proteins.

159

Test electro-eluted proteins b 100 Melanized Free-moving b 80

60 A A a A 40 a percentage a 20 B B

0 SCPa SCPb Other SCPs BSA Isolated SCPs

Fig. 5.5 Test electroeluted fractions of SCPs from S. glaseri NC strain on immune responses of E.

orientalis larvae. SCPs from S. glaseri NC strain were separated by 8 % nondenaturing PAGE and each

band on the gel were electroeluted then concentrated as described in text. SCPa, SCPb and Other Isolated

SCPs were fraction electroeluted from 8% nondenaturing PAGE. 0.12 μg protein from SCPa, 0.30 μg protein from SCPb were injected with H. bacteriophora into E. orientalis. ‘Other isolated SCPs’ indicates

0.08 to 1.20 μg proteins electroeluted from fractions other than SCPa and SCPb. These proteins were tested separately and data were pooled. ‘SCPs’ indicates 1.20 μg total SCPs injected with EPNs. BSA indicates injection of 0.60 μg Bovine Serum Albumin with EPNs. 10 IJs of H. bacteriophora were injected with 4μl Ringers’ solution, BSA, total SCPs, or fractions electroeluted from total SCPs. EPNs were recovered at 8 hours after injection. Percentages of melanized, encapsulated and free-moving nematodes graphed based upon EPNs injected. The EPNs not recovered were assumed to be encapsulated. Bars represent Means ± SE. The letters above the bars indicate the Tukey pairwise comparison results. The same letter indicates no statistical difference. Replicates ranged from 4 to 36. 160

A

Molecular weight Native PAGE

150KD —

3rd band of BSA — 100KD —

BSA high band —

B

Molecular weight SDS PAGE 56 KD —

46 KD —

38 KD —

Fig. 5.6. Separation of SCPa and SCPb on 8 % Nondenaturing PAGE and SDS PAGE. 6 μg SCPa

and SCPb isolated from S. glaseri NC strain were separated by 8 % nondenaturing PAGE and SDS PAGE.

Molecular weight on Nondenaturing PAGE was estimated based on Nondenaturing PAGE markers which

were both estimation of molecular weight and conformation of the native proteins. SDS PAGE indicated

that both SCPs were composed of smaller proteins. BSA high band indicated the higher band of BSA

nondenaturing PAGE standard. 3rd band BSA indicated the 3rd band of BSA from the lowest band. 161 4.2 3.5

| | A 8.9 8.0 7.0 6.1 5.1 4.2 3.2 2.3

200KD —

116KD — 97 KD — Molecular weight

66 KD — — 54 KD 45 KD — — 39 KD — 36 KD

21 KD —

B 8.9 8.0 7.0 6.1 5.1 4.2 3.2 2.3

200KD —

116KD — 97 KD —

66 KD — — 56 KD

45 KD — — 40 KD

21 KD —

Fig. 5.7 Separation of SCPa and SCPb on 2D PAGE. 3 μg SCPa and SCPb (proteins isolated from native PAGE, Fig. 5.4) from S. glaseri NC strain were separated by first dimension of IEF PAGE (pH 3.5-

9.5), then 2nd dimension 8 % SDS PAGE. 2D PAGE show that SCPa and SCPb were composed of the three major proteins. These proteins ranged from pH 3.5 to 4.2. 162 B With SCPs A Without SCPs Grace’ media in vitro 24 hours post introduction of the SCPa and SCPb. Hemocyte degradation occurred at 3 hours post introduction of the SCPa and SC from Oriental beetle larvae by severing the foreleg. Pictures were taken via an inverted microscope at X320 by NIH image. Ima

Fig.5.8.Effect of SCPa andSCPb on hemocytes of 1 hour 1 hour

. B ,

3

μ g SCPa and SCPb from

3 hour 3 hour

S. glaseri

E. orientalis NC strain was added into the mixture. One or two drops of hemolymph were collected

larvae.

SCPs A, no wereadded into 250 5 hour 5 hour

μ l mixturel of hemocytesand

24 hour 24 hour ges showed 1, 3, 5 and

Pb.

163 Discussion

The cuticle of S. feltiae is reported to play a role in inactivation of the pro-

Phenoloxidase pathway (Brivio et al., 2002). The present research demonstrated that

SCPs from S. glaseri NC strain suppressed the immune responses in E. orientalis larvae.

Suppression of immune responses in oriental beetle larvae protected H. bacteriophora from being melanized. However, it is reported that S. glaseri induced a strong melanization effect in the house cricket (Wang et al., 1994). These results suggest that S. glaseri interacts with the host immune system differently. There was a dosage effect of the SCPs. Because nematodes have small surface area, proteins on the surface could be concentrated compared to the proteins released.

The results indicated that SCPa and SCPb each conveyed the immuno-suppression

function, which supports the results reported by Wang and Gaugler (Wang and Gaugler,

1998). In the present research, total SCPs separated into more than five bands on nondenaturing PAGE and without molecular markers on the nondenaturing PAGE images shown in previous research, my results could not confirm the same proteins have been found (Wang and Gaugler, 1998). It is possible that the differences of SCPs pattern on nondenaturing PAGE is caused by changes of SCPs of the S. glaseri strain. The S. glaseri NC strain has been maintained in wax worms in lab condition without interacting with P. japonica, its natural host, for 7 years.

The suppression of melanization effect by SCPs from S. glaseri in this research was

about 20 % to 30 % among assays, which is higher than the less 20 % reported (Wang and Gaugler, 1998). The amount of SCPs injected was 5 ng in previous research (Wang

and Gaugler, 1998), which has the similar inhibition effect of 100-230 ng in our dosage 164 effect assays. The present research supported that the SCPs lyse the hemocytes of the insect hosts (Wang and Gaugler, 1998), and this lysis occurred at 3 hour after exposure to

SCPa and SCPb.

Interestingly, both SCPa and SCPb had the same 3 smaller protein components. Two of the proteins had the same pI. It is possible that these proteins have to work together to convey the immuno-suppressive function.

The future research of sequencing the component proteins of SCPa and SCPb will help us understand the evolution of SCPs among nematodes. A study of mechanisms that the SCPs use to interact with host immune responses will result in a better understanding of the native immunity.

165 Acknowledgments

I appreciate intellectual contributions from Drs. Richard S. Cowles, Elizabeth Cowles and

Randy Gaugler. I also thank Dr. Richard S. Cowles who collected and identified all Oriental beetle larvae. The thank goes to Dr. Yi Wang who communicated with me on all kinds of issues.

I also like to express my appreciation to Dr. A. Daniel Jones who contributed valuable opinions in this paper.

Last, I will express my thanks to my advisor Dr. Diana L. Cox-Foster who guided me through the experiments. Without her help on techniques and experimental designs, it would not be a good story.

166

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Identification of surface coat proteins from Steinernema glaseri 170 Abstract

Entomopathogenic nematodes (EPNs) infect soil-dwelling insects. Infective juvenile

(IJ) of EPNs release their symbiotic bacteria after entry into the host hemocoel. The

bacteria suppress the host immune responses and kill the insect. Insect hosts defend

against EPNs by rapid immune responses which include encapsulation and melanization.

The nematodes can suppress the host immune responses before they release the symbiotic bacteria. Surface coat proteins (SCPs) of IJs are suggested to play a role in suppression/evasion of host immune responses. Different species and strains EPNs have different SCPs. SCPs from Steinernema glaseri NC strain could protect Heterorhabditis bacteriophora from being melanized and encapsulated in oriental beetle larvae. SCPs of

S. glaseri NC were isolated and separated on PAGE. SCPs were identified using

LC/MS/MS and MALDI-TOF and one of the SCPs was enolase that is also secreted by other parasites. The other proteins had no match against the database of Brugia malayi

and Caenorhabditis elegans, which suggests that these SCPs are EPN specific proteins. 171 Introduction

Entomopathogenic nematodes (EPNs) in genera Steinernema and Heterorhabditis are parasites that infect soil dwelling insects (Dunphy and Thurston, 1990; Kaya and Gaugler,

1993). These two groups of EPNs share a similar life history (Blaxter et al., 2000;

Burnell and Stock, 2000). Infective juvenile (IJ) of EPNs is the only free moving stage.

They enter the hemocoel of the insect hosts via insect body openings and intersegmental membranes (Burnell and Stock, 2000). The symbiotic bacteria are released after nematodes entry the insect hemocoel (Dunphy and Webster, 1998). Symbiotic bacteria produce proteases and toxins that inhibit host immune responses (Dunphy and Thurston,

1990; Wang et al., 1994, 1995; Gaugler et al., 1997). Rapid encapsulation and melanization induced by EPNs will block release of symbionts and thus kill the nematodes (Wang et al., 1994, 1995; Peters and Ehlers, 1997).

The symbiotic bacteria of Steinernema glaseri are released after 4-6 hour after nematode entry the host hemocoel (Akhurst and Dunphy 1993; Wang et al., 1995).

During this time, S. glaseri has to evade or suppress host immune response.

Mammalian parasitic nematodes evade or suppress host immune responses by surface coat proteins (SCPs) or excretory factors. These manipulations are carried out by mainly groups of enzymes such as glutathione peroxidase, glutathione S-transferase, superoxide dismutases, cysteine protease inhibitors, serine protease inhibitors (Maizels et al., 2001,

2004).

It is reported that only 24 % of S. glaseri were melanized initially in Japanese beetle larvae Popillia. japonica. It has been demonstrated that SCPs from S. glaseri NC strain 172 protected H. bacteriophora from immune responses in oriental beetle larvae Exomala orientalis larvae (unpublished, chapter 5) but not in tobacco hornmoth larvae M. sexta.

However, what SCPs conveyed immuno-suppressive function and the mechanism of this immuno-suppression are still question marks. The SCPs of S. glaseri separated as five bands on nondenaturing PAGE, and two of them can convey the immuno- suppression effect in P. japonica (Wang and Gaugler, 1999). The two SCPs lyse hemocytes and suppress melanization in the P. japonica (Wang and Gaugler, 1999). The information had limited its usefulness because the nondenaturing PAGE did not have molecular marker with the SCPs. Besides these initial findings, there is a dearth of information related to EPN SCPs. 173 Materials and methods

Nematodes

Steinernema glaseri NC strain and Heterorhabditis bacteriophora (HP88 strain) were studied. All EPNs were cultured in last instar of the wax worm Galleria mellonella (L.) at room temperature. Wax worms were purchased from Nature’s way Inc. Infective juveniles (IJs) of EPNs were harvested from white trap by filtration and cultured in distilled water bubbled with air for about 2 to 4 days at room temperature for all the experiments conducted.

Extract surface coat proteins (SCPs)

For S. glaseri, desheathment step was carried out before extracting the SCPs. The

method followed Wang and Gaugler (1999). In brief, 15 g of IJs of S. glaseri were

desheathed using 0.5 % sodium hypochlorite at room temperature for 15 min, nematodes

then were rinsed by centrifugation in distilled water 3 times. Nematodes were stored in

water with bubbled air for 2 days to redevelop surface coat.

H. bacteriophora and S. glaseri were rinsed 2 times with distilled water. Then IJs

were put in 35 %, precooled ethanol (at –20 °C) for 30 min and extracted on ice with

mild stirring.

Then the mixture was filtered through a 0.22 μm filter apparatus (Falcon® Sterile

Bottle Top Filters, 357111, by Becton-Dickinson®). Then the filtrates were collected

and froze at –80 °C. The extracts then were lyophilized and resuspended in MilliQ water.

The resuspended solution was aliquotted and stored at –20 °C. The modified Bradford 174 assay was used to determine protein concentration of SCPs solution (Cox-Foster and

Stehr, 1994).

Running nondenaturing PAGE and SDS-PAGE

Nondenaturing polyacrylamide gel electrophoresis (PAGE) was made with the same

recipe as SDS-PAGE but without SDS. For SDS-PAGE, the sample was boiled with

SDS sample buffer for 5 minutes. Native PAGE molecular weight standards markers

(Nondenatured protein molecular weight marker kit, MW-ND-137, Sigma Inc.) were

used. 8 % or 10 % PAGE were used to separate the SCPs. After running the 1st dimension nondenaturing PAGE, gel strips were cut out and lay on top of 2nd dimension

SDS-PAGE. All gels were stained by coomassie blue or silver staining. The procedures

followed the red book ‘current protocols in molecular biology’.

Protein identification

LC/MS/MS (ThermoFinnigan ProteomeX Workstation, 2-D LC/MS/MS, ESI, ion

trap, components of the ProteomeX workstation are LCQ DecaXP plus Mass

Spectrometer, 2 Dimensional ‘SurveyorMS’ Liquid Chromatography system, computer,

Data system with Software (Xcalibur and Bioworks)) was used to identify the SCPs of

interest.

SEQUEST, a database-searching algorithm was used for analysis of peptide tandem

mass (MacCoss et al., 2002) following LC/MS/MS. SEQUEST using a cross-correlation

(Xcorr) function scoring to match tandem mass spectra to model spectra derived from

peptide sequences. Xcorr above 2 are usually indicating a good correlation. However,

because Xcorr values are dependent on the quality of the tandem mass spectrum, they are

usually higher for larger peptides and lower for smaller peptides (MacCoss et al., 2002). 175 DelCn (∆Cn), the difference between the first and second-rank sequences was used to normalized SEQUEST scores to assess a match. A value of above 0.1 is an acceptable match. However, ∆Cn is larger when the searching is against a smaller database

(MacCoss et al., 2002). Sp is a preliminary score and a value greater than 200 is considered reasonable.

Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass

spectrometry (MS) technique was also used to identify the SCPs.

176 Results

How do the SCPs compare between H. bacteriophora and S. glaseri?

The SCPs of S. glaseri and H. bacteriophora were separated by 8 % non-denaturing

PAGE. Then the second dimension 10 % SDS-PAGE was followed to get a better resolution of the proteins.

The non-denaturing PAGE separated SCPs without denaturing them, based on both

SCPs molecular weight and conformation. SDS-PAGE, on other hand, separated proteins based on molecular weight because the SCPs will be denatured and all special conformation will be destroyed.

The results (Fig 6.1) indicated that different species of EPNs had different SCPs.

SCPs from H. bacteriophora had large amount of high molecular weight components

(around 116 KD). SCPs from S. glaseri were composed of many smaller proteins ranged from 45 KD to 66 KD.

What proteins are in SCPs of S. glaseri?

On the first dimension nondenaturing gel, SCPs from S. glaseri were separated into many bands (Fig. 6.2). The circled proteins were de novo sequenced by LC/MS/MS and analyzed by SQUEST (Fig. 6.2, Fig. 6.3). Sample 1, about 45 KD on nondenaturing gel, was identified as enolase. The result was acceptable because delta Cn was 60.28 with 6 peptides hits for the match against enolase from Caenorhabditis elegans. For other circle proteins (Fig. 6.2, Fig.6.3), there were no known protein matches. Also, the matched peptides had lower confidence because their delta Cn values, Xcorr values and peptides hits were low. Five major spots on SDS PAGE were identified using MALDI-TOF MS 177 technique (Fig. 6.4). Spot S1, about 66 KD, was identified as enolase with a match score of 776. This was a confirmation that one of SCPs was enolase. Again, there were no known matches for other SCPs.

On 2nd dimension SDS-PAGE (Fig. 6.3, Fig. 6.4), protein about 200 KD on nondenaturing PAGE had moved to 20 KD region. This indicated that the larger proteins on native PAGE probably were composed of smaller proteins.

178

A) SCPs of H. bacteriophora B) SCPs of S. glaseri

200 200 116 116 97 97

66 66

45 45

31 31

14 14

Fig. 6.1 Second dimension separation on 10 % SDS PAGE gel of surface coat proteins of and H. bacteriophora (6.1A) and S. glaseri (6.1B). Proteins were extracted from IJs by 35 % ethanol and first separated on 8 % nondenaturing gel. On the first dimension PAGE, 20 μg SCPs from S. glaseri NC strain and 6.3 μg SCPs from H. bacteriophora were loaded. SDS-PAGE Molecular weight standards (Bio-Rad SDS-PAGE Molecular weight standards, broad range 161-0317) were used to determine the molecular weight on SDS-PAGE.

179

200 KD —

116 KD — 97 KD —

6 3 2 66 KD —

45 KD — 1 31 KD —

14 KD —

Fig. 6.2 The first dimension separation of surface coat proteins from S. glaseri on 10 % nondenaturing PAGE gel. 46 μg of SCPs were loaded. The SCPs spots circled were characterized by

Tibor Pechan at Mississippi State University. Spot 1 was enolase. The bands on right side of the gel are

10 μg nondenaturing PAGE molecular weight markers (SIGMA nondenatured protein molecular weight marker kit, MW-ND-137). From left to right, they were Urease, Bovine Serum Albumin (66 KD),

Albumin, Chicken Egg (45 D), Carbonic anhydrase (29 KD), and α-Lactalbumin, (14 D) respectively.

180

200 116 97 66 45 31 14 | | | | | | |

200 KD —

D 116 KD —

97 KD — C B 66 KD —

E 45 KD —

31 KD — A

21 KD —

Fig. 6.3. The 2nd dimension separation of surface coat proteins from S. glaseri on 10 % nondenaturing PAGE gel. The first dimension was 8 % native PAGE. Second dimension separation of

SCPs of S. glaseri separated on 10 % SDS PAGE with Coomassie Blue stain. The SCPs spots circled were characterized by Tibor Pechan at Mississippi State University.

181 Table 6.1 Sequences of SCPs from S. glaseri NC strain

A Referencea Score Accession Peptides Scan(s) Sequence MH+ ChargeXC Delta Cn Sp (Hits) Ions gi|3874824|emb|CAA86316.1| glutamate and serine rich; #1 cDNA EST EMBL:D28017 14.11 3874824.0 2 (1 0 0 1 0) 202 - 206 R.TVLKLYHR.L 1030.25 2 2.27 0.14 878.5 11/14 208 - 210 R.TVLKLYHR.L 1030.25 1 1.61 0.08 170.9 4/7 gi|3878527|emb|CAA88867.1| similar to DNA #2 topoisomerase II; cDNA EST EMBL: 10.12 3878527.0 1 (1 0 0 0 0) 223 - 226 K.IYDEILVNAADNK.Q 1478.63 2 2.44 0.01 224.0 5/12 #3 gi|3876589|emb|CAB04227.1| 10.12 3876589.0 1 (1 0 0 0 0) 223 - 226 K.IYDEILVNAADNK.Q 1478.63 2 2.44 0.01 224.0 5/12 #4 gi|3877971|emb|CAA91994.1| 10.10 3877971.0 1 (1 0 0 0 0) 186 - 190 K.FAINLKKVDLGK.A 1346.64 2 2.08 0.01 208.3 6/11 gi|3877196|emb|CAA93773.1| similar to Zinc finger, #5 C3HC4 type (RING finger) 8.12 3877196.0 1 (0 1 0 0 0) 223 - 226 K.KFTKNDLENELK.Y 1479.66 2 2.42 0.03 406.7 13/22 B gi|39581367|emb|CAE69264.1| Hypothetical protein #1 CBG15316 [Caenorhabditis 20.25 39581367.0 2 (2 0 0 0 0) 219 - 222 K.GILAADESTGSMEK.R 1409.55 2 4.96 0.46 1868.7 11/13 295 - 298 K.KPWALTFSYGR.A 1326.53 3 3.05 0.34 1063.4 11/20 gi|5052015|gb|AAD38403.1| fructose 1,6 bisphosphate #2 aldolase [Onchocerca v 20.15 5052015.0 2 (2 0 0 0 0) 246 - 251 K.KGILPGIK.V 826.06 2 2.14 0.27 407.8 6/7 295 - 298 K.KPWALTFSYGR.A 1326.53 3 3.05 0.34 1063.4 11/20 C gi|39585560|emb|CAE65320.1| Hypothetical protein #1 CBG10243 [Caenorhabditis 10.10 39585560.0 1 (1 0 0 0 0) 186 - 190 R.GRGGPQLRSVHR.V 1320.49 2 2.05 0.12 136.5 5/11 1 gi|17536383|ref|NP_495900.1| enolase (46.6 kD) (2J223) #1 [Caenorhabditis ele 60.28 17536383.0 6 (6 0 0 0 0) 222 - 226 K.YNQLLR.I 806.93 2 2.02 0.09 408.3 4/5 227 - 232 K.YNQLLR.I 806.93 1 1.75 0.00 156.1 3/5 234 - 238 K.IAPALIAK.G 797.02 1 2.02 0.42 360.4 9/14 247 - 251 R.ANGWGVMVSHR.S 1214.38 2 3.87 0.29 769.5 3/4 272 - 279 R.YGLDATAVGDEGGFAPNIQDNK.E 2253.37 2 5.64 0.55 1242.5 13/21 316 - 319 K.VVLPVPAFNVINGGSHAGNK.L 1991.28 2 5.34 0.54 1133.5 12/19 gi|32440997|gb|AAP81756.1| enolase [Onchocerca #2 volvulus] [MASS=47152] 40.28 32440997.0 4 (4 0 0 0 0) 222 - 226 K.YNQILR.I 806.93 2 2.02 0.00 408.3 4/5 227 - 232 K.YNQILR.I 806.93 1 1.75 0.00 156.1 3/5 243 - 246 K.AC#NC#LLLK.V 992.54 2 2.52 0.18 365.7 11/14 272 - 279 R.YGLDATAVGDEGGFAPNIQDNK.E 2253.37 2 5.64 0.55 1242.5 13/21 gi|32563855|ref|NP_871916.1| enolase and Enolase (36.4 #3 kD) (2J223) [Caenor 40.19 32563855.0 4 (4 0 0 0 0) 222 - 226 K.YNQLLR.I 806.93 2 2.02 0.09 408.3 4/5 227 - 232 K.YNQLLR.I 806.93 1 1.75 0.00 156.1 3/5 234 - 238 K.IAPALIAK.G 797.02 1 2.02 0.42 360.4 9/14 247 - 251 R.ANGWGVMVSHR.S 1214.38 2 3.87 0.29 769.5 3/4 182 gi|17568987|ref|NP_508842.1| actin (act-4) #4 [Caenorhabditis elegans 20.19 17568987.0 2 (2 0 0 0 0) 168 - 172 K.DSYVGDEAQSK.R 1199.21 2 3.70 0.48 1032.3 9/10 270 - 274 R.VAPEEHPVLLTEAPLNPK.A 1955.24 3 2.62 0.31 561.9 6/17 gi|17557190|ref|NP_505818.1| actin (41.8 kD) (act-2) #5 [Caenorhabditis elegans 20.19 17557190.0 2 (2 0 0 0 0) 168 - 172 K.DSYVGDEAQSK.R 1199.21 2 3.70 0.48 1032.3 9/10 270 - 274 R.VAPEEHPVLLTEAPLNPK.A 1955.24 3 2.62 0.31 561.9 6/17 gi|3182894|sp|P90689|ACT_BRUMA actin [Brugia #6 malayi] 20.19 3182894.0 2 (2 0 0 0 0) 168 - 172 K.DSYVGDEAQSK.R 1199.21 2 3.70 0.48 1032.3 9/10 270 - 274 R.VAPEEHPVLLTEAPLNPK.A 1955.24 3 2.62 0.31 561.9 6/17 gi|477248|pir||A48449 Actin-1A - nematode #7 (Onchocerca volvulus) 20.19 477248.0 2 (2 0 0 0 0) 168 - 172 K.DSYVGDEAQSK.R 1199.21 2 3.70 0.48 1032.3 9/10 270 - 274 R.VAPEEHPVLLTEAPLNPK.A 1955.24 3 2.62 0.31 561.9 6/17 2 gi|25152769|ref|NP_500112.2| nuclear Hormone #1 Receptor (41.6 kD) (nhr-87) [ 10.22 25152769.0 2 (0 1 0 0 1) 232 - 235 K.M*LDDMELM*YIMNKK.D 1840.20 2 2.46 0.09 221.0 7/13 232 - 235 K.M*LDDM*ELMYIMNKK.D 1840.20 2 2.67 0.07 204.4 1/2 gi|17508537|ref|NP_493078.1| putative membrane #2 protein (1M779) [Caenorhabd 10.14 17508537.0 1 (1 0 0 0 0) 271 - 274 K.FAWTKAIDVDM*ATVVSEK.D 2044.32 3 2.82 0.05 1072.0 25/68 3 gi|17560094|ref|NP_506093.1| SyNaptoBrevin related, #1 synaptic vesicle-assoc 10.11 17560094.0 1 (1 0 0 0 0) 203 - 206 R.M*SANNEANK.D 1011.05 2 2.25 0.05 477.8 13/16 6 gi|39595875|emb|CAE67378.1| Hypothetical protein #1 CBG12856 [Caenorhabditis 10.14 39595875.0 1 (1 0 0 0 0) 258 - 264 K.SNESKQAAPPVITTNEEPK.T 2041.21 3 2.87 0.04 1101.4 29/72 gi|17565362|ref|NP_507202.1| predicted CDS, #2 serpentine Receptor, class W ( 10.13 17565362.0 1 (1 0 0 0 0) 204 K.VSSVGSSASNDR.S 1166.18 3 2.62 0.40 928.3 5/11 gi|17532265|ref|NP_494204.1| cyclin-like F-box family #3 member (2C458) [Caen 10.11 17532265.0 1 (1 0 0 0 0) 306 K.DTGSKLVIFDDGAQNSLNQDSTK.I 2454.59 2 2.26 0.13 146.4 1/4 gi|17508537|ref|NP_493078.1| putative membrane #4 protein (1M779) [Caenorhabd 6.19 17508537.0 1 (0 0 1 0 0) 258 - 264 K.FAWTKAIDVDM*ATVVSEK.D 2044.32 3 2.71 0.07 736.3 23/68 “D” Sample: “E” Sample:

1. NHYS 1. QGFLWK 6. APVQTAAQDSSYE 2. AYNAGRYCE QGFLDAK 7. APFLPASFFK AYNAGASYCE 2. AAAR 8. HVFWSNWLSQK 3. CGAAVACWDFSK 3. WTMRRD 9. EFWTVLQGTEK 4. APANG 4. GSAMDVSL EFTWVLQGTEK 5. AQFLCAGMVA 10. FEPMMK

183

2 1 9 6 4 3 1 0 1 7 6 5 1 4 0 6

200 KD —

116 KD —

97 KD — S2 S1 S3 66 KD —

45 KD — S4 31 KD — S5

21 KD —

Fig. 6.4 The 2nd dimension separation of surface coat proteins from S. glaseri on 10 % SDS-

PAGE gel. The first dimension was 8 % native PAGE. 46 μg Spots marked were loaded on the first dimension PAGE. SDS-PAGE was silver stained and spots digested by trypsin and sequenced by MALDI-

TOF mass spectrometry, the proteins were identified by mass matches. I am grateful to Dr. A. Daniel

Jones who carried out the analysis at Department of Chemistry at Penn State University. Spot S1 was identified as enolase with a match score of 776.

184 Table 6.2 Protein Masses of SCPs from S. glaseri NC strain

#S1 #S2 #S3 #S4 #S5 568.11 547.26 825.05 568.04 614.07 550.10 841.02 613.09 550.01 650.03 561.28 850.00 629.96 568.02 656.04 568.1 857.00 649.95 587.03 666 587.13 1325.66 655.97 613.96 669.42 614.05 1353.64 665.94 649.92 672.02 625.35 1515.7 671.94 665.21 675.31 650.02 1533.76 825.00 669.31 754.35 656.04 1560.62 840.97 824.95 781.36 669.4 1654.79 849.94 840.93 806.44 825.07 1663.88 856.94 1029.39 825.09 841.05 1776.86 860.96 1322.48 841.05 1308.72 1779.78 865.91 1434.53 842.49 1837.8 2077.07 876.94 1459.78 1196.56 1965.91 2268.14 881.18 1836.86 1213.58 1966.86 2296.18 1030.02 1951.56 1214.58 1983.87 2298.09 1325.56 2704.70 1447.71 1984.82 1654.71 1515.78 2041.97 2076.92 1569.82 2179.14 2268.00 1778.94 2899.41 1882.88 2973.46 1990.96 2987.54 2252.13 2988.54 3015.49 3146.81 3864.27 3991.74

#S1 Score: 6/24 matches (25%). Accession #: Q27527 Species: CAEEL Protein Name: Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase) MS-Digest Index #: 40655 MW: 46617 Da pI: 5.6

#S2 Score: #/28(%) Accession #: P52709 Species: CAEEL Protein Name: Threonyl-tRNA synthetase, cytoplasmic (Threonine--tRNA ligase) (ThrRS) MS-Digest Index #: 62547 Protein MW: 84418 Da pI: 7.1

185 Discussion

Enolase is an enzyme involved in basic energy metabolism. In glycolytic pathway, it

converts 2-phosphoglycerate (PGA) to phosphoenolpyruvate, and vice versa. The

forward reaction occurs in glycolysis, the reverse reaction occurs in gluconeogenesis. In

vertebrates the enzyme exists in three isoforms: alpha, beta and gamma. Enolase from

parasites can bind plasmin and plasminogen of the hosts and in turn help the parasites

penetrate the tissues of the hosts.

In human opportunistic fungal pathogen, Candida albicans, enolase is a plasmin(ogen)-binding protein and helped the parasites cross an in vitro blood-brain barrier system (Jong et al., 2003).. In human respiratory tract pathogenic bacteria,

Streptococcus pneumoniae, enolase is also shown to present abundantly in the cell wall of parasites and is a key component in parasite-human interaction (Bergmann et al., 2001;

Jong et al., 2003). Enolase is a secretory/excretory product of Haemonchus contortus, a parasitic nematode of small ruminants (Yatsuda et al., 2003). Moreover, enolase (Jolodar et al., 2003) is involved in the interaction between humans and their filarial parasite,

Onchocerca volvulus. Two species of spiroplasma, an insect pathogenic gram-positive

eubacteria, secrete enolase (personal communication with Dr. Saskia Hogenhout at Ohio

State University ). Therefore, enolase from SCPs of S. glaseri could have the same

function, i.e. bind the plasmin(ogen) of the host insects and help the nematode penetrate the tissue of the host.

S. glaseri and H. bacteriophora all have complicate SCPs. For S. glaseri, many of its

peptides tandem mass of SCPs had no match with that of the known proteins. This

indicates the SCPs of S. glaseri are EPN specific. To obtain the knowledge of these 186 SCPs will help us understand the evolution of SCPs in nematodes. And the further study of their functions probably will enlighten the immune interaction between the parasitic nematodes and their hosts.

187 Acknowledgements

I appreciate Dr. Tibor Pechan at Mississippi State University who helped me sequenced the proteins. I wish to thank Dr. Elizabeth Cowles who cooperated with me and also sequenced enolase. I also want to thank Dr. A. Daniel Jones of Chemistry

Department at Penn State University who helped sequenced proteins and gave me lots of advices. 188

References:

Bergmann, S., Rohde, M., Chhatwal, G. S., and Hammerschmidt, S. (2001) alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol. 40(6), 1273-87.

Blaxter, M. L., Dorris, M. and De Ley, P. (2000) Patterns and processes in the evolution of animal parasitic nematodes. Nematology 2, 43-55.

Burnell, A. M. and Stock, S. Patricia. (2000) Heterorhabditis, Steinernema and their bacterial symbionts - lethal pathogens of insects. Nematology 2(1), 31-42.

Cox-Foster, D. L. and Stehr, J. E. (1994). Induction and localization of FAD-glucose dehydrogenase (GLD). during encapsulation of abiotic implants in Manduca sexta larvae. J. Insect Physiol. 40(3), 235-249.

Dunphy, G. B. and Thurston, G. S. (1990). Insect immunity. In: Entomopathogenic nematodes in biological control. Editors, Gaugler, R. Kaya, H. K. CRC Press Inc, Boca Raton, FL. pgs. 301-323.

Dunphy, G. B. and Webster, J. M. (1988). Virulence mechanism of Heterorhabditis heliotidis and its bacterial associate, Xenarhabdus luminescens, in nonimmune larvae of the greater wax moth, Galleria mellonella. Int’l. J. Parasitol. 18, 729–737.

Frederick, M., Ausubel, Roger, Brent, Robert, E. Kingston, David, D. Moore, J. G. Seidman, and Kevin, Struhl. Current Protocols in Molecular Biology, ISBN: 0-471-50338-X; published by John Wiley & Sons Inc.

Jolodar , A., Fischer , P., Bergmann, S., Buttner, D. W., Hammerschmidt, S., and Brattig, N. W.. (2003) Molecular cloning of an alpha-enolase from the human filarial parasite Onchocerca volvulus that binds human plasminogen. Biochim Biophys Acta. 1627(2-3), 111-20.

Jong, A. Y., Chen S. H., Stins, M. F., Kim, K. S., Tuan, T. L., and Huang, S. H. (2003) Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells. J Med Microbiol. 52(Pt 8), 615-22.

Kaya, H. and Gaugler, R. (1993). Entomopathogenic nematodes. Ann. Rev. Entomol. 38, 181-206. 189 MacCoss, M. J., Wu, C. C., Yates, J. R., III. (2002) Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal. Chem. 74, 5593-5599.

Maizels, R. M., Blaxter, M. L., and Scott, A. L. (2001). Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity. Parasite Immunology 23, 327-344.

Maizels, R. M., Balic, A., Gomez-Escobar, N., Nair, M., Taylor, M., and Allen, J. E. (2004) Helminth parasites – masters of regulation. Immunological Reviews 201, 89-116.

Peters, A. and Ehlers, R.-U. (1997). Encapsulation of the entomopathogenic nematode Steinernema feltiae in Tipula oleracea. J. Invertebrate Pathology 69, 218-222.

Yatsuda, A. P., Krijgsveld, J., Cornelissen, A. W., Heck, A. J., and de Vries, E. (2003) Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition. J Biol Chem. 278(19), 16941- 51.

Wang, Y. and Gaugler, R. (1999). Steinernema glaseri surface coat protein suppresses the immune response of Japanese beetle larvae. Biol. Contr. 14, 45-50.

Wang, Y., Campbell, J., and Gaugler, R. (1995). Infection of entomopathogenic nematodes Steinernema glaseri and Heterorhabditis bacteriophora against Popillia japonica larvae. J. Invertebr. Pathol. 66, 178-84.

Wang, Y., Gaugler, R., and Cui, L. (1994). Variations in immune response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema spp. J. Nematol. 26, 11-8.

190 Summary

Juvenile hormone (JH), produced by the corpora allata (CA), regulates molting

and reproduction in cockroaches. JH is produced only after dorsal closure, a conspicuous event in embryogenesis. I characterized embryo development time and dorsal closure time in thirteen cockroach species. I found that dorsal closure occurred at a consistent

percentage of embryo development time across most cockroach species. This consistency was related to the reproductive biology of the cockroaches. The viviparous cockroach Diploptera punctata completed dorsal closure at 20.8 % of developmental

time and Blattella germanica finished its dorsal closure at 38.5 %. Theses two species of

cockroach have different reproductive biology compared to other cockroaches. Other

oviparous and ovoviviparous cockroaches all completed dorsal closure at similar

percentage of the embryo development time.

Embryonic CA produce both JH and its immediate unepoxidized precursor methyl

farnesoate (MF) in N. cinerea. Using a radiochemical assay, I found that cockroach

embryos produced and released both JH and MF in many species across all three

reproductive modes. These cockroaches included Periplaneta americana, Eurycotis

floridana, Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, and Diploptera punctata. I also found that the conversion of MF into JH by epoxidase was a rate-limiting step, the last step of biosynthesis of JH, was species dependent.

In the future, using more sensitive techniques to study the JH/MF production in

the cockroach embryo will be very informative. Moreover, to study the JH/MF titer in

191

the whole embryo body together with production and release by CA will

enlighten the understanding the role of MF in cockroach embryos. A real time PCR

study of epoxidase among cockroach species will be very helpful in understanding the

regulation of conversion of MF into JH.

Entomopathogenic nematodes (EPNs) are ecologically and economically important.

Two families EPNs are good candidates for biological-control agents target to soil

dwelling insects. Infective juveniles (IJs) of EPNs penetrate insect hosts and release

symbiotic bacteria that kill the insect hosts and serve as food resources for EPNs. Insect

hosts defend against EPNs by a rapid cellular immune response resulting in encapsulation

and melanization that kills EPNs. They have to overcome insect innate immunity to release their symbiotic bacteria before intensive host immune responses occur.

I studied the immune responses between two species of nematodes, Heterorhabditis

bacteriophora and Steinernema glaseri and their insect hosts, and the relationship to host specificity. I also hypothesized that EPNs induced or suppressed the immune responses

in their host because of their surface coat proteins (SCPs).

I found that H. bacteriophora and S. glaseri infected wax worms and reproduced

well. Both H. bacteriophora and S. glaseri killed most Japanese beetle larvae, Oriental

beetle larvae, and tobacco horn worms. S. glaseri reproduced better than H.

bacteriophora in these insects. S. glaseri NC strain was more successful compare to the

S. glaseri FL strain in the same hosts. Northern masked chafer larvae and house crickets

were resistant hosts to both nematodes.

192

I found that in Manduca sexta, Popillia japonica and Exomala orientalis,

most of H. bacteriophora were melanized while most of S. glaseri were moving freely.

In M. sexta and P. japonica, higher percentages of S. glaseri FL strain were encapsulated

compared to S. glaseri NC strain. In resistant host Cyclocephala borealis, both H.

bacteriophora and S. glaseri were melanized. These results suggested EPNs elicited immune responses in hosts that correlate with EPNs’ infectivity.

Using an in vitro assay, I found that hemocytes from M. sexta recognized S. glaseri at

a low percentage during the first hour post nematode introduction, and after 24 hours, H.

bacteriophora escaped recognition of wax worm hemocytes.

Surface coat proteins of EPNs are suggested to play a role in suppression/evasion of

host immune responses. I demonstrated that different species and strains EPNs had different SCPs. I isolated and characterized SCPs from S. glaseri NC strain. These SCPs suppressed immune responses in the Oriental beetle larvae, a susceptible host for S. glaseri, thus protecting H. bacteriophora from being killed in the same host, as it normally would be. The immuno-suppression was dose dependent. Also, multiple injections of the SPCs protected H. bacteriophora better in the Oriental beetle larvae. In the nondenatured state, two isolated proteins in the SCPs of S. glaseri each conveyed this immuno-suppressive effect. The two SCPs were composed of smaller proteins when separated on two dimensional PAGE. Hemocytes of Oriental beetle larvae started degrading after exposure to the proteins for 3 hours.

We sequenced some of the SCPs from S. glaseri NC strain and found one of the SCPs

was enolase which was also secreted by other parasites.

193

The future study of sequences of these SCPs and SCPs from other stains/species of EPNs will help us understanding evolution of SCPs of nematodes. This information will also be the promising step in constructing a genetic modified biological control agent that are more successful against Oriental beetle larvae. The further study of mechanism of these SCPs suppression of the immune response will also cast light on understanding of interaction of the parasitic nematodes with their hosts.

194

Appendix A

Protocols

Surface coat proteins extraction protocol

Yi Wang’s protocol, from Yi Wang et al, 1999 (15g Ijs was desheathed using 0.5% sodium hypochlorite at room temperature for 15 min, nematodes then were rinsed by centrifugation in distilled water 3 times, stored in 2 liters of distilled water with bubbled air. nematodes were allowed 2 days to redevelop Surface coat. After 2 distilled water rinses, nematodes were put in 35%, 50 and 100% ethanol for 30 min. the resulting extracts were lyophilized and stored at –20C .)

Old protocol modified by Xinyi Li and Chris Brey from Yi Wang et al, 1998.

• On the 1st day, wash IJs 3X with MQ H2O. After the 3rd wash let the IJs stay on the filter papers in a large Petri dish • Wash the cylinder with soap and water and rinse about 5 X with MQ water • (Regenerate new SCPs). Fill up each of the washed cylinders with the dilute detergent, 0.05- 0.1% solution of 10X hyamine (I use 0.06% sodium hypochlorite), and dump the IJs back into the cylinder. Let the IJs bubble for 15 minutes. After bubbling, wash them 3X and let them bubble at least for 2 days in MQ water. 0.06% sodium hypochlorite sodium hypochlorite (6%) 10ml:1000ml H2O v/v • On the 3rd day,: You need to have the following supplies ready 1) 200 ml flask with a stir bar (autoclaved) 2) Erlmeyer flask that will be used to collect the extract (autoclaved) 3) Stir plate, glass bottom dish, ice, 200 ml 35% ethanol ( -20C for >1 hour; Chris used 200ml for from 4 -12g nematodes). 4) Set up an ice bath on the stir plate (no heat!) Next, place the sterile 200 ml flask with cold 35% ethanol (200 ml) on the stir plate (always keep this covered before adding your samples). 5) Filter the IJs onto a regular filter paper. You should be able to get all of them on one or two papers. Next dump them with a sterile forceps into the 35% ethanol and let them stir for 30-60 minutes (This is the stage were the SCPs are extracted from the nematodes cuticle) 6) After 30 minutes filter the 35% ethanol with IJs through filter paper. Collect the suppernant, the product, into the flask. Next aliquot at least 40 ml into sterile 50 ml polypropylene tubes. Take the tubes to the -80 freezer and let them stay over night. 7) The next day place the tubes into the lyophilizer. After the alcohol and water evaporates you are left with yellowish white flakes. Place the tubes in the -80 or –20C. 8) Suspend the proteins. Re-suspend the lyophilized protein with IEF sample buffer with 20X protein inhibitors.

195

New! Protocol of extracting Surface Coat Proteins --- modified by Xinyi Li. • Set up inoculation about 1000 wax worms, you will be able to get 15-20 g nematodes in the end. • Infective Juveniles (IJs) of S. glaseri are harvested from white trap daily by filtering 2 or 3 times with distilled water on 0.25 μm filter paper on erlmeyer flask. • Nematodes are kept in distilled water with bubbling air for 2-4 days before exaction. Keep the air just enough bubble but not stir the water too much. • Make 35% ethanol and keep it at 4 C. • Filter IJs 3 times before protein extraction. • Dump IJs into a 200 ml beaker with 150 ml cold ethanol. Stir the solution in 4C cold room for 1 - 2 hours. Keep stirring at speed as low as possible. You can add 0.1- 1mM EDTA and 0.1-1mM PMSF to the 35% ethanol at this step if you wants add protein inhibitor. • Filter the 35% ethanol with IJs through filter paper. Collect up to 40 ml the solution into each sterile 50 ml polypropylene tubes. Take the tubes to the -80 freezer and let them stay over night. • The leftover can be extracted by MilliQ water at 4 for 1- hours too. Then filter the water through filter paper and collect solution into tubes also. • When solution freezes in the freezer, place the tubes into the lyophilizer. After the alcohol and water evaporates you are left with yellowish white flakes. Store the tubes at the -80 or –20C for future. • Re-suspend the lyophilized proteins with MilliQ water or IEF sample buffer (with 20X protein inhibitors) on ice. Aliquot the solution into eppendorf tubes. • Centrifuge eppendorf tubes at the highest speed at 4C and aliquots the supernatant into small tubes. Add 0.1-1mM EDTA and 0.1-1mM PMSF to the 35% ethanol.

EtOH 35% 200ml EtOH 35% = 70 ml 100% EtOH+ 130 ml MQ H2O EDTA 1mM 0.4ml 0.5M EDTA : 200ml 35% EtOH PMSF 1mM 1.0ml 0.2M PMSF : 200ml 35% EtOH

196

IEF sample buffer: Native gels PMSF 200 mM in MeOH 40 µl 3.6M Tris-HCL (pH8.4) 40 µl

MQ H2O 4.0 ml Denaturing gels PMSF 200 mM in MeOH 40 µl 3.6M Tris-HCL (pH8.4) 40 µl ß-mercaptoethanol 40 µl 10M Ultrapure urea (Kept in freezer in aliquots) 4.0 ml ***PMSF (phenyl methyl sulfonyl fluoride ) (200 mM in MeOH) --mix at ratio of 0.0348 mg/µl of MeOH --disregard the methanol volume in final concentrations (it evaps off quickly)

197

Bradford Assay for Protein Quantification with Spectramax 1. Turn spectramax on; power switch in on back, upper right corner 2. Open Bradford protocol; External Part 1: Softmax Pro: DCF lab protocols: Bradford Std Crv Helpful Hints for working with Softmax pro Parts of program are called sections, you may only work in one section at a time and you must first make that section active. To make a section active, click anywhere in the section. When active the tool bar at the top of the section will be highlighted. To delete a section: make the section active, then under edit menu choose "delete section" To have a section appear on the computer screen but not print on the final report: Make the section active, under section menu choose section settings . When the dialog box appears inactivate "include in report" To make scrolling through the program faster, you may open and close sections; to do this click on the arrow to the left of the section name To print one section, make that section active, under file menu choose "print section" 3. First section is called overview, use this to type in any information or experimental parameters you wish to record. 4. Second section is Plate#1, this plate contains data for the standard curve. On the right side of the plate the date standard curve was made is printed. DO NOT CHANGE SETTINGS ON PLATE 1 OR YOU WILL LOSE THE STANDARD CURVE DATA! Go directly to plate 2 5. Third section is Plate#2, this will be your data plate for each experiment. Make this section active then select "settings" from the section tool bar. Parameters are already set for Bradford assays. To indicate which wells the spectramax will read select "strips", highlight the wells you will be using, click OK *after you have data you can NOT change the settings without deleting your data **parameters are: Mode= Endpoint Three wavelengths; L1=590; L2=595; L3=465

198 Automix 300 sec before first reading Autocalibrate on No pre-read 6. Return to plate section and click "template"; this is where you tell the program where samples are located. Highlight wells for blanking, under "group" choose "blank" Highlight all wells with a given sample, under "group" choose "Unknown", when finished click OK *you need to include at least 1 blank, this well will contain water instead of sample and its absorbance will be subtracted from sample absorbances **for each sample you must separately assign unknown, if you assign them all at once Softmax will think they are replicates and will average everything together ***any unassigned wells in the strips to be read will be read but not analyzed ****you may print the template to help when loading wells, click "print" 7. To tell the program how to analyze the data, click "reduction" In the dialog box which appears, under "wavelength combination", select "custom", the formula L2-L3 should appear to the right Subtract plate blank should be selected, click "ok" 8. You are now ready to prepare the plate: To each well add: 10 µl 1M NaOH (made fresh) sample (total volume must = 14 µl) 200 µl Bradford reagent

*total sample volume must = 14 µl, for molting fluid load 1 µl fluid and 13 µl H2O *you must use 200 µl Bradford and 14 µl sample. If you change these volumes, you change the absorbance, which is effected by path length (i.e. the depth of sample in the well). To use different volumes you must create a new standard curve 9. To read the plate: Be sure "Plate #2" is active. Insert the plate and click "read". *NEVER try to read a plate when Plate #1 is active; this will delete the std curve 10. When spectramax is finished go to the section titled "unknown" your data is displayed in a table

199 *Values are Abs595 - Abs465; Result is your protein concentration in µg 11. If you wish to calculate protein concentration in µg/µl you need to add a column; To do this: click on the result column to highlight it, click the left-most button in the unknown toolbar For "name", type the name of the new column in "Formula", type "Result/volume", click "OK" *Remember that the number you enter for volume is not necessarily 14, it is the volume of your sample only, for example for molting fluid the volume is 1 µl **this program can only handle equal volumes, if you volumes are not equal for all samples, export result to a spreadsheet (e.g. Excel) and calculate µg/µl there

Bradford Reagent Solution A 50 mg Coomassie Brilliant Blue G-250 bring to 25 mls with 95% EtOH Solution B 50mls 85% Phosphoric Acid 25 mls Soln A

425 mls MQ H2O Stir 3-4 hrs or overnight Filter through filter paper and store in brown bottle.

Vincent R. and Nadeau D. (1983) A micromethod for the quantitation of cellular proteins in percoll with the coomassie blue dye-binding assay. Analyt. Biochem. 135, 355-362.

200

BCA method for protein concentration determination BCA solution A: 1 g sodium Bicinchoninate (BCA)

2 g Na2CO3.H2O, 0.16 g sodium tartrate 0.4 g NaOH

0.95 g NaHCO3

Solid sodium bicarbonate (NaHCO3) or NaOH to adjust the pH to 11.25.

100 ml MilliQ H2O. BCA solution B:

4% copper sulfate hydrated (CuSO4.5H2O) Working reagent: 1. Mix 50 volumes of BCA solution A with 1 volume of BCA solution B. 2. Prepare a set of BSA standards in the range of 20-2000 g/ml. 3. Place 100 μl of each standard (or water blank!) into each tube 4. Add 3 ml of BCA working reagent and mix well 5. Incubate the tubes for 37oC for 30 minutes 6. Cool to room temperature and read at A560. Set up a standard curve. In alkaline conditions, divalent copper (Cu(II); cupric) binds to the peptide nitrogen of proteins where the ion is reduced to the monovalent form (Cu (I); cuprous). This reaction is the basis for the Biuret, Lowry and BCA reactions. Two molecules of BCA chelate to a cuprous ion, resulting in an intense purple color with an absorbance maximum at 560 nm. Microtiter Assay Using BCA

Use 10 μl of sample (or 5 μl of sample plus 5 μl of H2O ) Add 200 μl of working BCA reagent Incubate microtiter plate at 37-50oC. Read at A560 using a microtiter plate reader.

201 Bleeding Honey Bee Adults and Hemocyte GLD Activity Staining

When making GLD stain, you may also try MTT instead of NBT, with the same concentration. Sometimes, MTT may work better but the reaction is faster (In step 5, 10 minutes may be enough).

1. In the sterile hood, add 400 µl Grace’s medium in to each chamber of the Lab-Tek® Chambered Coverglass System (#1 Borosilicate coverglass). Cover the chambers with lids, and keep the chambers in a sterile Petri dish. 2. Wash bees in a beaker filled with Coverage Plus® solution (1/250, v/v) for 5

minutes, wash the bees with 500 ml sterile MQ H2O in aliquots, and wash the bees with 70% alcohol for 30 seconds. The Buchmer funnel should be used to remove above liquids. Keep the bees in a sterile Petri dish. If the bees start to move put the Petri dish on ice. 3. Use a pair of sterile forceps to pick a surface-sterilized bee from step 2. Hold the bee with a hand on its thorax and abdomen. Cut off one half of an antenna with a pair of 70% EtOH-sterilized dissecting scissors. Invert and gently squeeze the bee to allow hemolymph drops to fall into (or to touch onto, but touch the drop only, not the cuticle) the Grace’s medium in the chambers prepared in step 1. Record the number of drops of hemolymph in each chamber. 4. Let the hemocytes to settle on the bottom for 30 minutes. Meanwhile, make GLD stain solution from stock solutions* as follows. • 920 µl Grace’s medium • 500 µl NBT (Nitro BT, Kodak) stock solution, 10 mg/ml • 80 µl PMS (phenazine methosulfate) stock solution, 10 mg/ml • 1000 µl 0.6 M glucose 5. In the dark or very dim light, add 400 µl GLD stain solution into each chamber, and leave the chambers in the dark for 30 minutes. Remove the stain solution. 6. Add 400 µl Grace’s medium, and let it sit for 5 minutes. Repeat this step once.

202

* Make the stock solutions: 5 ml NBT solution:

50 mg NBT + MQ H2O Æ 5 ml. Cover the solution tube with two layers of foil and store it in refrigerator. It should be good for 1 month. 5 ml PMS solution:

50 mg PMS + MQ H2O Æ 5 ml. Cover the solution tube with two layers of foil and store it in refrigerator. It should be good for 1 month. 5 ml 0.6 M glucose:

weight 0.55732 g β-D-glucose + MQ H2O Æ 5 ml. Make 1000 µl aliquots with 1.7 ml centrifuge tubes, and store them in freezer. If the solution becomes turbid, get rid of it.

203 Silver Staining Protocol Gel fix solution

100% 50 % methanol, 12 % acetic acid, 38% H2O

250 ml 125 methanol, 30 acetic acid, 95 H2O 300 ml 150 methanol, 36 acetic acid, 114 H2O

Sodium thiosulfate (Na2S2O3.5H2O) solution

0.2 g Na2S2O3.5H2O / 1 L H2O 100mg/500ml 50mg/250ml

Silver nitrate (AgNO3) solution

2 g AgNO3 / L H2O 0.75 ml/ L of 37 % formaldehyde stock solution 500 mg / 250ml 187.5 µl/250ml of 37 % formaldehyde Developing solution

Na2CO3, 30 g/ L; 0.5 ml/ L of 37 % formaldehyde stock solution; 4 mg /

L Na2S2O3.5H2O) = 20 ml sodium thiosulfate solution (0.2g Na2S2O3.5H2O / L) 7.5 g/250ml 125 µl/250ml 1 mg/250ml = 5ml 0.2g/L 6 g/200ml 100 µl/200ml 0.8 mg/200ml = 4ml 0.2g/L Ethanol solutions 50 % or 30 % Methanol 50 %

1. Submerge the separating gel in a large volume of gel fix solution. The gel can be stored in this solution for several days with no effect on the quality of the staining. 2. Wash the gel in 50 % ethanol three times for 20 minutes each. Gel that is < 1 mm thick should be kept in 30 % ethanol for the final 20 minutes wash to prevent gel deformation. 3. Submerge the gel in the solution of sodium thiosulfate for 1 minute 4. Rinse the gel with water three times for 20 seconds each. 5. Submerge the gel for 20 minutes in the silver nitrate solution. 6. Rinse the gel with water twice for 20 seconds each. 7. Incubate the gel at room temperature for 10 minutes in developing solution. 8. Stop the reaction by rinsing the gel in 6% acetic acid for 6 minutes. 9. Wash the gel in 50 % methanol for at least 20 minutes. 10. Store the gel at cold room in 50 % methanol.

204 Table 2.1 Solutions for preparing resolving gels for Tris-glycine SDS- polyacrylamide Gel Electrophoresis Components volumes (ml) per gel mold volume of Solution components 5 ml 10 ml 15 ml 20 ml 25 ml 30ml 35 ml 40 ml 50 ml 6% H2O 2.7 5.3 8.0 10.6 13.3 15.9 18.6 21.2 26.5 30% acrylamide mix 1 2 3 4 5 6 7 8 10 1.5 M Tris (pH 8.8) 1.3 2.6 3.9 5.2 6.5 7.8 9.1 10.4 13 10% SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 TEMED 0.004 0.008 0.012 0.016 0.02 0.024 0.028 0.032 0.04 8% H2O 2.3 4.6 7.0 9.3 11.6 13.9 16.2 18.6 23.2 30% acrylamide mix 1.3 2.7 4.0 5.3 6.7 8.0 9.3 10.6 13.3 1.5 M Tris (pH 8.8) 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.5 10% SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 TEMED 0.003 0.006 0.009 0.012 0.015 0.018 0.021 0.024 0.03 10 H2O 2.0 4.0 5.9 7.9 9.9 11.9 13.9 15.8 19.8 % 30% acrylamide mix 1.7 3.3 5.0 6.7 8.4 10.0 11.7 13.4 16.7 1.5 M Tris (pH 8.8) 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.5 10% SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.02 12 H2O 1.7 3.3 5.0 6.6 8.3 9.9 11.6 13.2 16.5 % 30% acrylamide mix 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 20.0 1.5 M Tris (pH 8.8) 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.5 10% SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.02 15 H2O 1.2 2.3 3.5 4.6 5.8 6.9 8.1 9.2 11.5 % 30% acrylamide mix 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 25.0 1.5 M Tris (pH 8.8) 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.5 10% SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.02

Table 2.3 Solutions for preparing stacking gels for Tris-glycine SDS-Polyacrylamide Gel Electrophoresis Component volumes (ml) per gel mold volume of Solution components 1 ml 2 ml 3 ml 4 ml 5 ml 6 ml 8 ml 10 ml 15 ml H2O 0.61 1.22 1.83 2.44 3.05 3.66 4.88 6.1 9.15 30% acrylamide mix 0.13 0.26 0.39 0.52 0.65 0.78 1.04 1.3 1.95 0.5M Tris (pH 6.8) 0.25 0.5 0.75 1 1.25 1.5 2 2.5 3.75 10% SDS 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 0.15 10% ammonium persulfate 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 0.15 TEMED 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01 0.015

205 Solutions for SDS-Polyacrylamide Gel Electrophoresis 1.5 M Tris (pH ~8.8) 4X conc. resolving gel buffer recipes – Weigh and pour Adjust pH to ~8.8

90.75g Tris 91 g Tris base in 300 ml MillQ H2O 8.8ml con HCl adjust to pH 8.8 with 1 N HCl

424ml MillQ H2O add H2O to 500ml final volume

0.5 M Tris (pH ~6.8) 4X conc. stacking gel buffer Weigh and pour Adjust pH to ~6.8

7.8g Tris HCl 6.05 g Tris base 40 ml MillQ H2O 0.3g Tris adjust to pH 6.8 with 1 N HCl

96ml MillQ H2O add H2O to 100ml final volume

30% acrylamide / 0.8% bisacrylamide (2nd dimension) 30 g acrylamide 0.8 g N,N’-methylene-bisacrylamide

100 ml MilliQ H2O Store at 4C in the dark, discard after 30 days

30% acrylamide / 1.8% bisacrylamide (1st dimension) 30 g acrylamide 1.8 g N,N’-methylene-bisacrylamide

100 ml MilliQ H2O. Store at 4C in the dark, discard after 30 days

Samples buffer 2x 4x 6x Final volume 10 ml 10 ml 10 ml

Distilled H2O 5.3 ml 0.9 ml 0 ml 0.5 M Tris-Hcl (4x pH 6.8) 2.5 ml 5 ml 3.4 ml Glycerol 2 ml 4 ml 6 ml SDS 0.4 g 0.8 g 1.2 g 2-mercaptoethanol / DTT 0.2 ml / 0.31 g 0.1 ml / 0.15g 0.6 ml / 0.93 g Bromophenol blue 0.1 mg 0.2 g 0.3 mg

206

Running Buffer 5x Running buffer 10x Running buffer 15 g Tris 30 g Tris 72 g glycine 144 g glycine

1000 ml MillQ H2O 1000 ml MillQ H2O (5 g SDS) (10 g SDS) For large proteins, more HCl (actually, NaCl) will give better stacking. If you're interested in small proteins, less HCl will release more proteins from the "stack".

Table Gel Loading Form Gel # ____ 8% / 10% _Urea _SDS Date ___/___05 Voltage____ Ampere____ Watt____ Time ____hrs (start_____end_____)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample Sample# Amount (µl) Conc (µg/µl) Comments: ______Stain method: __Commasie Blue / __Silver Stain / __PAS / ___

IEF gel solutions Weigh and pour Without urea With urea Acrylamide 2.6 g 2.6 g Bisacrylamide 0.2 g 0.2 g Ultrapure urea 0 g 18.72 g PH 4-6 gel pH4-6 ampholytes 2.40 ml 2.40 ml PH 3.5-10 ampholytes 0.24 ml 0.24 ml PH 3.5-10 gel PH 3.5-10 ampholytes 2.64 ml 2.64 ml

MillQ H2O 47.2 ml 32.8 ml Ammonium persulfate 16 mg 16 mg

Trichloracetic acid TCA staining Coomasie blue R-250 0.04 g 0.2 g 0.4 g

MilliQ H2O 40% 40 ml 200 ml 400 ml Methanol 50% 50 ml 250 ml 500 ml

207 100% TCA 10% 10 ml 50 ml 100 ml Total 100% 100 ml 500 ml 1000 ml Destain MeOH 10% 20 ml 350 ml Acetic acid 7.5% 15 ml 262 ml

MillQ H2O 82.5% 165 ml 2888 ml Total 100% 200 ml 3500 ml

(Trichloracetic acid) TCA water 100% TCA 0.4ml 1ml 2 ml

MillQ H2O 100ml 250ml 500 ml

Serva Blue W stain Serva blue 0.04 % 0.06 g 0.08 g 0.2 g

MillQ H2O 80 % 120 ml 160 ml 400 ml 100% Tricholoracetic acid (TCA) 20 % 30 ml 40 ml 100 ml Total 100 % 150ml 200 ml 500 ml *Mix serva blue W and water first then add TCA

TEMED Water 5 drops TEMED in 1000 ml water

SDS Sample buffer SDS 10 ml 15 ml 40 ml 20% SDS Glycerol 8.5 ml 12.75 ml 34 ml 100% glycerol 0.5 M Tris-Hcl, pH=6.8 12.5 ml 18.75 ml 50 ml Mercaptoethanol / DTT 5 ml / 0.309g 7.5 ml / 0.463g 20 ml / 1.234 g

MillQ H2O 64ml / 69ml 96 / 103.5 ml 256 ml / 276 ml Total 100ml 150ml 400 ml *DTT dithiothreitol

208 1.% Agarose sample buffer SDS Sample buffer 10 ml 15 ml 40 ml Sigma Type V agarose 0.1g 0.15 g 0.4 g Bromophenol Blue-Agarose SDS Sample buffer 10 ml 15 ml 40 ml Sigma Type V agarose 0.1g 0.15 g 0.4 g Bromophenol blue trace

Equilibration buffer SDS 2.1 g 5.25g 8.4 g Glycerol 10 ml 25 ml 40 ml Tris base 1.5 g 3.75 g 6 g dithiothreitol (DTT) 0.133 g 0.333g 0.532 g Total 100 ml 250 ml 400 ml Adjust pH to 6.8 with 6M HCl

Coomassie Blue staining solution Methanol 50% 75 ml 100 ml 250 ml Coomassie Brilliant Blue R-250 0.05% 0.075 g 0.1 g 0.25 g Acetic acid 10% 15 ml 20 ml 50 ml

H2O 40% 60 ml 80 ml 200 ml Total 100% 150 ml 200 ml 500 ml

Destaining solution Methanol 5% 7.5 ml 10 ml 25 ml Acetic acid 7% 10.5 ml 14 ml 35 ml

H2O 88% 132 ml 176 ml 440 ml Total 100% 150 ml 200 ml 500 ml

209 Fixing solution Methanol 50% 75 ml 100 ml 250 ml Acetic acid 10% 15 ml 20 ml 50 ml

H2O 40% 60 ml 80 ml 200 ml Total 100% 150 ml 200 ml 500 ml JH MF Protocol Incubation media: (2 Ci/mmol L-[methyl-3H]-mehtoinine ) 1.25 mM methoinine in L15B. Pre-incubation and incubation: Pre-incubation CA about 45 to 60 minutes in 400 µl hot L15B media then transferred CA to 20 µl fresh radiolabeled medium in 6 × 25 mm borosilicate glass culture tubes, where CA will be incubated for 6 hr at 27˚C with shaking on a variable plane mixer. Vertex-centrifuge and storage: The culture tubes are vertexed well (15-20seconds) when isooctane is transferred to the tube with culture media. And then, each tube is centrifuged at 4000 g for 5 minutes. Isooctane will be the upper layer in the aliquots. The isooctane aliquots are pooled and stored in a conical-bottomed tube filled up with nitrogen at –80C. The synthesis of JH and MF of cockroach embryonic CA, broods 6-7 pairs of CA of cockroach embryos are dissected out from each brood of cockroaches. After pre-incubation for 45 to 60 minutes, CA are incubated separately for 6 hours as described above, then transfer 100µl isooctane into the tube and then vertex it well. Then, the culture tube is centrifuged at 4000 g for 5 minutes. The isooctane aliquots are pooled and stored in a conical-bottomed tube. Then, store the tube under –80C with nitrogen filled up the tube. The release of JH and MF of cockroach embryonic CA, broods 6-7 pairs of CA of cockroach embryos are dissected out from each of cockroach broods After preincubation-incubation described as above, pippet 100µl (cold) L15B media into the tube. Vertex and then centrifuge it. Then, I collect 100 µl culture media (without CA which is at the bottom of the tube) from the upper party of the media carefully, and transfer it into a 6 × 50 mm borosilicate glass culture tube. Next, transfer 250µl isooctane to each tube. After vertex and centrifugation, the isooctane will be collected and pooled, stored as described above. Add 100µl isooctane to the leftovers of 6 × 25 mm culture tubes, which probably have CA at their bottom. Vertex well and centrifuge, then the isooctane is collected and I mark this as synthesis-release, or synthesis-after-release. Farnesol experiment, split glands: After pre-incubation one pair of CA is split into two corporal allatum, and cultured with and without farnesol for 6 hours in hot media in 6 × 25 mm borosilicate glass culture tubes on the shaker. To mix farnesol into the culture media, just add 10µl 2mM farnesol (in methanol) into 6 × 25 mm borosilicate glass culture tubes and evaporate it under nitrogen stream (Mark the tube with SF). Then transfer 20 µl hot media into the tube and incubated one corporal allatum in this media, and the other corporal allatum in the tube with hot media but no farnesol (Mark it as S) on the shaker for 6 hours. After incubation, add 100µl cold media into the tube, vertex and centrifuge very quick. Transfer 80µl media to 6 × 50 mm tube (mark it as R indicating release, the one with Farnesol, which is marked as RF) by pipeting the surface of media, and then add 200 µl isooctane to the tube. Add 100 µl isooctane into the remaining 40 µl media (6 × 25 mm tube) which should have a corporal allatum at the bottom. Vertex all the tubes thoroughly and transfer 90% of the isooctane (90 and 180 µl isooctane respectively), into conical-bottomed vials marked corresponding with the culture tubes. a pair of CA preincubation Split 2tubes S/SF (20 µl hot media without/with Farnesol) Incubation Transfer 80µl into tubes marked R/RF 40µl remains in the tubes S/SF centrifuge Vertex, Add 100 µl into each tubes transfer isooctane transfer 200 µl isooctane into R/RF transfer 100µl isooctane into S/SF vertex pipet 180µl and 90µl from R/RF and S/SF respectively Notes: So we have 4 tubes, or say 4 extracts from one pair of CA in the farnesol experiment. R, S, RF, SF.

210 Appendix B Table B.1 Electro elution results fractions hrs Hb M E F M_I E_I F_I E_A conc dilut amt A_M #53-1 8 10 0.068 #53-2 8 10 8 0 0 0.8 0 0 0.2 0.059 1 to 2 0.079 0.80 #53-3 8 10 9 0 1 0.9 0 0.1 0 0.096 1 to 2 0.128 0.90 #53-4 8 10 1 0 6 0.1 0 0.6 0.3 0.092 1 to 2 0.123 0.24 #53-4 8 10 7 0 1 0.7 0 0.1 0.2 0.092 1 to 2 0.123 #53-4 8 10 0 1 4 0 0.1 0.4 0.6 0.092 1 to 2 0.123 #53-4 8 10 2 2 6 0.2 0.2 0.6 0.2 0.092 1 to 2 0.123 #53-4 8 10 2 2 4 0.2 0.2 0.4 0.4 0.092 1 to 2 0.123 #53-5 8 10 2 0 1 0.2 0 0.1 0.7 0.228 1 to 2 0.304 0.10 #53-5 8 10 2 3 5 0.2 0.3 0.5 0.3 0.228 1 to 2 0.304 #53-5 8 10 0 2 4 0 0.2 0.4 0.6 0.228 1 to 2 0.304 #53-5 8 10 0 2 5 0 0.2 0.5 0.5 0.228 1 to 2 0.304 #53-6 8 10 6 0 4 0.6 0 0.4 0 0.691 1 to 2 0.921 0.53 #53-6 8 10 4 2 4 0.4 0.2 0.4 0.2 0.691 1 to 2 0.921 #53-6 8 10 6 1 2 0.6 0.1 0.2 0.2 0.691 1 to 2 0.921 #47 E1 16 10 8 0 0 0.8 0 0 0.2 0.412 3 to 1 1.236 0.80 #47 E2 16 10 10 0 0 1 0 0 0 1.203 1 to 1 2.406 0.95 #47 E2 16 10 9 0 0 0.9 0 0 0.1 1.203 1 to 1 2.406 #47 E3 16 10 6 0 1 0.6 0 0.1 0.3 0.561 1 to 1 1.122 0.60 #47 E3 16 10 7 0 0 0.7 0 0 0.3 0.561 1 to 1 1.122 #47 E3 8 10 2 1 3 0.2 0.1 0.3 0.5 0.561 1 to 1 1.122 #47 E3 8 10 9 0 0 0.9 0 0 0.1 0.561 1 to 1 1.122 #47 E3 8 10 6 0 0 0.6 0 0 0.4 0.561 1 to 1 1.122 #47 E4 16 10 8 0 0 0.8 0 0 0.2 0.596 1 to 1 1.192 0.80 #47 E5 16 10 1 6 1 0.1 0.6 0.1 0.8 0.538 1 to 1 1.076 0.55 #47 E5 16 10 10 0 0 1 0 0 0 0.538 1 to 1 1.076 #47 E6 16 10 10 0 0 1 0 0 0 1.467 1 to 1 2.934 0.40 #47 E6 16 10 4 2 1 0.4 0.2 0.1 0.5 1.467 1 to 1 2.934 #47 E6 8 10 2 4 2 0.2 0.4 0.2 0.6 1.467 1 to 1 2.934 #47 E6 8 10 0 2 3 0 0.2 0.3 0.7 1.467 1 to 1 2.934 #47 E7 16 10 9 0 1 0.9 0 0.1 0 0.263 1 to 1 0.526 0.95 #47 E7 16 10 10 0 0 1 0 0 0 0.263 1 to 1 0.526 #48 E7 8 10 1 1 4 0.1 0.1 0.4 0.5 0.855 1 to 2 1.140 0.05 #48 E7 8 10 0 3 4 0 0.3 0.4 0.6 0.855 1 to 2 1.140 #48 E8 8 10 6 0 1 0.6 0 0.1 0.3 0.818 1 to 2 1.091 0.60 #48 E9 16 10 10 0 0 1 0 0 0 1 to 2 0.000 0.93 #48 E9 16 10 8 0 0 0.8 0 0 0.2 1 to 2 0.000 #48 E9 8 10 10 0 0 1 0 0 0 1 to 2 0.000 #49 E1 8 10 1 0 1 0.1 0 0.1 0.8 0.898 1 to 2 1.197 0.15 #49 E1 8 10 2 0 5 0.2 0 0.5 0.3 0.898 1 to 2 1.197 #49 E2 8 10 8 0 0 0.8 0 0 0.2 0.818 1 to 2 1.091 0.85 #49 E2 8 10 9 0 1 0.9 0 0.1 0 0.818 1 to 2 1.091 #49 E3 8 10 9 1 1 0.9 0.1 0.1 0 0.73 #49 E3 8 10 3 0 1 0.3 0 0.1 0.6 #49 E3 8 10 10 0 0 1 0 0 0 #49 E3 16 10 9 0 1 0.9 0 0.1 0 0.55 #49 E3 16 10 2 0 1 0.2 0 0.1 0.7 #49 E4 16 10 9 1 0 0.9 0.1 0 0.1 0.85 #49 E4 16 10 8 1 0 0.8 0.1 0 0.2 #49 E5 16 10 7 0 0 0.7 0 0 0.3 0.85 #49 E5 16 10 10 0 0 1 0 0 0 #49 E6 16 10 5 0 1 0.5 0 0.1 0.4 0.63 #49 E6 16 10 8 0 1 0.8 0 0.1 0.1 #49 E6 16 10 6 1 0 0.6 0.1 0 0.4 protein43 8 10 2 4 2 0.2 0.4 0.2 1.510 0.30 protein43 8 10 4 2 2 0.4 0.2 0.2 1.510 BSA 8 10 8 0 1 0.8 0 0.1 0.590 0.9 BSA 8 10 10 0 0 1 0 0 0.590 BSA 8 10 10 0 0 1 0 0 0.590 BSA 8 10 9 0 1 0.9 0 0.1 0.590 BSA 8 10 10 0 0 1 0 0 0.590 BSA 8 10 9 0 0 0.9 0 0 0.590 BSA 8 10 7 1 0 0.7 0.1 0 0.590

211 Fractions, the number follows the # sign are the gel number. The number following are the protein band numbers which is indicated on the gel image. Hrs: Hours post injection Hb: Number of H. bacteriophora (HP88 strain) injected M, number of melanized H. bacteriophora E, number of encapsulated H. bacteriophora F, number of free-moving H. bacteriophora M_I, percentage of melanized H. bacteriophora based upon the number of nematodes injected E_I, percentage of encapsulated H. bacteriophora based upon the number of nematodes injected F_I, percentage of free-moving H. bacteriophora based upon the number of nematodes injected E_A, percentage of encapsulated H. bacteriophora based upon the number of nematodes injected with assumption that lost nematodes were encapsulated Concentration, protein concentration Dilut, protein dilution Amtt, the amount of protein injected A_M, average of percentage of melanized H. bacteriophora based upon the number of nematodes injected. Protein43 is total extracts of SCPs of SgNC. BSA serves are a negative control.

Descriptive Statistics: melan_inj by Proteins Proteins N Mean StDev SE Mean #47-E6 4 0.400 0.432 0.216 #48-E7 2 0.0500 0.0707 0.0500 #49-E1 2 0.1500 0.0707 0.0500 #53-4 5 0.240 0.270 0.121 #53-5 4 0.1000 0.1155 0.0577 BSA 7 0.9000 0.1155 0.0436 others 36 0.7361 0.2497 0.0416 SCP 5 0.2600 0.0894 0.0400

Explanation of the results The table is part of electro elution result. The numbers with bold size and blue background are the fractions that have activity. I will figure out the accurate molecular weight when I am back. The protein which I believe has activity is between 100K to 200K, and the protein is located between the 2nd and 3rd bands of BSA. The proteins electro eluted from #53 (gel number 53) are separated on the last image on native gel. Bands #4 and #5 have the strongest activity.

212

Gel #47 10%

aL,CA, 200 1 6 116

97 2 7

66 3 c 4

45 5

31 14

Gel #48 10%

200 7

116 8 97

66 9

45

31

14

213

Gel #49 10%

200

1 116

97 2

3 66 4

5

45 6

31 14

BSA BS #53-123 456 200

116

97

66

214

Test electro-eluted proteins 1.2 b 1.0 b 0.8

0.6 a 0.4 a a a a 0.2 percentage of melanization of percentage 0.0 #48-E7 #49-E1 #53-4 #53-5 others SCP BSA proteins on the gel

Fig B.1. Immune response by oriental beetle larvae to H. bacteriophora with injection of proteins electroeluted from native gels. Others are other protein bands on the gel pooled together. Bovine serum albumin (BSA) and surface coat proteins (SCP) are served as negative and positive control respectively. Bars represent mean ± SE. Letters above the column indicate results of Tukey pairwise comparisons. The same letter indicates no significant difference.

215 Appendix C

Statistical analysis

Chapter 2 Embryogenesis, Eggs and nymphs

General Linear Model: SqrtGest, sqrtNym, sqrtEgg versus species

Factor Type Levels Values species fixed 13 Blaberus discoidalis Blatta orientalis Blattella germanica Byrsotria fumigata Diploptera punctata Eurycotis floridana Nauphoeta cinerea Panchlora nivea Periplaneta americana Pheotalia palluda Rhyparobia maderae Schultesia landyriformis Supella longipalpa

Analysis of Variance for SqrtGest, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species 12 400.148 400.148 33.346 2775.91 0.000 Error 397 4.769 4.769 0.012 Total 409 404.917

Analysis of Variance for sqrtNym, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species 12 644.130 644.130 53.678 93.98 0.000 Error 397 226.749 226.749 0.571 Total 409 870.880 Analysis of Variance for sqrtEgg, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species 12 805.072 805.072 67.089 366.16 0.000 Error 397 72.739 72.739 0.183 Total 409 877.812

Kruskal-Wallis Test: SqrtGest versus species 503 cases were used 192 cases contained missing values Kruskal-Wallis Test on SqrtGest species N Median Ave Rank Z Blaberus 35 7.810 421.5 7.15 Blatta o 28 7.000 292.3 1.51 Blattell 31 4.583 16.0 -9.33 Byrsotri 34 7.583 397.6 6.05 Diplopte 30 8.185 485.7 9.08 Eurycoti 28 7.141 312.6 2.27 Nauphoet 33 6.325 219.0 -1.35 Panchlor 42 5.745 96.2 -7.25 Periplan 37 6.557 254.8 0.12 Pheotali 83 6.000 152.1 -6.85 Rhyparob 34 8.000 449.0 8.18 Schultes 47 5.745 76.2 -8.71 Supella 41 7.211 349.0 4.46 Overall 503 252.0

216 H = 486.59 DF = 12 P = 0.000 H = 487.36 DF = 12 P = 0.000 (adjusted for ties)

Kruskal-Wallis Test: sqrtNym versus species 412 cases were used 283 cases contained missing values Kruskal-Wallis Test on sqrtNym species N Median Ave Rank Z Blaberus 31 6.000 301.5 4.62 Blatta o 28 3.936 117.6 -4.09 Blattell 31 6.325 349.4 6.95 Byrsotri 24 3.535 80.5 -5.34 Diplopte 30 3.162 45.8 -7.68 Eurycoti 28 3.606 107.7 -4.55 Nauphoet 27 5.657 301.4 4.28 Panchlor 36 7.582 359.7 8.08 Periplan 37 4.000 124.9 -4.37 Pheotali 56 5.385 258.9 3.54 Rhyparob 24 5.384 250.0 1.85 Schultes 19 4.796 226.7 0.76 Supella 41 3.873 116.3 -5.11 Overall 412 206.5

H = 313.00 DF = 12 P = 0.000 H = 313.63 DF = 12 P = 0.000 (adjusted for ties)

Kruskal-Wallis Test: sqrtEgg versus species 662 cases were used 33 cases contained missing values Kruskal-Wallis Test on sqrtEgg species N Median Ave Rank Z Blaberus 58 5.958 477.1 6.07 Blatta o 62 3.873 127.1 -8.84 Blattell 32 6.782 593.9 7.96 Byrsotri 30 4.899 299.9 -0.93 Diplopte 42 3.464 42.4 -10.12 Eurycoti 56 4.243 209.7 -4.98 Nauphoet 57 6.083 496.0 6.79 Panchlor 49 8.485 636.1 11.59 Periplan 56 3.873 144.9 -7.63 Pheotali 54 5.657 408.6 3.09 Rhyparob 56 5.745 444.9 4.64 Schultes 54 5.000 341.2 0.39 Supella 56 4.000 168.1 -6.68 Overall 662 331.5

H = 573.44 DF = 12 P = 0.000 H = 574.30 DF = 12 P = 0.000 (adjusted for ties)

General Linear Model: SqrtGest, sqrtNym, sqrtEgg versus Type, species

Factor Type Levels Values species(Type) fixed 13 Blatta orientalis Blattella germanica Eurycotis floridana Periplaneta americana Supella longipalpa Blaberus discoidalis Byrsotria fumigata Nauphoeta cinerea Panchlora nivea Pheotalia palluda Rhyparobia maderae Schultesia landyriformis Diploptera punctata

217 Type fixed 3 oviparity ovoviviparity viviparity

Analysis of Variance for SqrtGest, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species(Type) 10 326.812 326.879 32.688 2721.15 0.000 Type 2 73.337 73.337 36.668 3052.50 0.000 Error 397 4.769 4.769 0.012 Total 409 404.917

Analysis of Variance for sqrtNym, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species(Type) 10 457.798 408.693 40.869 71.56 0.000 Type 2 186.332 186.332 93.166 163.12 0.000 Error 397 226.749 226.749 0.571 Total 409 870.880

Analysis of Variance for sqrtEgg, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species(Type) 10 546.147 490.898 49.090 267.93 0.000 Type 2 258.926 258.926 129.463 706.59 0.000 Error 397 72.739 72.739 0.183 Total 409 877.812

Descriptive Statistics: nymphs, eggs, Gestation time by Type Variable Type N N* Mean Median TrMean nymphs oviparit 165 97 18.921 16.000 18.309 ovovivip 217 174 31.52 31.00 30.60 vivipari 30 12 9.600 10.000 9.769 eggs oviparit 262 0 19.218 16.000 18.127 ovovivip 358 33 36.503 33.000 35.109 vivipari 42 0 11.786 12.000 11.947 Gestatio oviparit 165 97 43.600 49.000 44.289 ovovivip 308 83 44.016 37.000 43.514 vivipari 30 12 67.300 67.000 67.269

Variable Type StDev SE Mean Minimum Maximum Q1 nymphs oviparit 10.684 0.832 1.000 47.000 14.000 ovovivip 15.52 1.05 1.00 105.00 23.00 vivipari 1.673 0.306 4.000 12.000 9.000 eggs oviparit 10.524 0.650 6.000 50.000 14.000 ovovivip 15.728 0.831 10.000 90.000 27.000 vivipari 1.335 0.206 5.000 13.000 11.000 Gestatio oviparit 11.534 0.898 20.000 56.000 43.000 ovovivip 12.290 0.700 30.000 68.000 35.000 vivipari 1.236 0.226 65.000 70.000 66.000

Tukey Simultaneous Tests Response Variable SqrtGest All Pairwise Comparisons among Levels of species(Type)

Type = oviparit species = Blatta o subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Blattell -2.445 0.02857 -85.55 0.0000 oviparit Eurycoti 0.101 0.02929 3.45 0.0322 oviparit Periplan -0.420 0.02745 -15.31 0.0000 oviparit Supella 0.257 0.02687 9.58 0.0000

218 ovovivip Blaberus 0.792 0.02857 27.72 0.0000 ovovivip Byrsotri 0.583 0.03049 19.11 0.0000 ovovivip Nauphoet -0.643 0.02956 -21.76 0.0000 ovovivip Panchlor -1.175 0.02762 -42.54 0.0000 ovovivip Pheotali -1.023 0.02552 -40.08 0.0000 ovovivip Rhyparob 0.949 0.03049 31.12 0.0000 ovovivip Schultes -1.263 0.03258 -38.78 0.0000 vivipari Diplopte 1.201 0.02880 41.70 0.0000

Type = oviparit species = Blattell subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Eurycoti 2.546 0.02857 89.09 0.0000 oviparit Periplan 2.024 0.02669 75.85 0.0000 oviparit Supella 2.702 0.02609 103.58 0.0000 ovovivip Blaberus 3.237 0.02784 116.27 0.0000 ovovivip Byrsotri 3.027 0.02980 101.58 0.0000 ovovivip Nauphoet 1.801 0.02885 62.43 0.0000 ovovivip Panchlor 1.270 0.02685 47.28 0.0000 ovovivip Pheotali 1.422 0.02470 57.56 0.0000 ovovivip Rhyparob 3.393 0.02980 113.88 0.0000 ovovivip Schultes 1.181 0.03193 37.00 0.0000 vivipari Diplopte 3.646 0.02807 129.88 0.0000

Type = oviparit species = Eurycoti subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Periplan -0.521 0.02745 -18.99 0.0000 oviparit Supella 0.156 0.02687 5.82 0.0000 ovovivip Blaberus 0.691 0.02857 24.19 0.0000 ovovivip Byrsotri 0.481 0.03049 15.79 0.0000 ovovivip Nauphoet -0.744 0.02956 -25.18 0.0000 ovovivip Panchlor -1.276 0.02762 -46.20 0.0000 ovovivip Pheotali -1.124 0.02552 -44.04 0.0000 ovovivip Rhyparob 0.848 0.03049 27.81 0.0000 ovovivip Schultes -1.364 0.03258 -41.88 0.0000 vivipari Diplopte 1.100 0.02880 38.19 0.0000

Type = oviparit species = Periplan subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Supella 0.6778 0.02485 27.27 0.0000 ovovivip Blaberus 1.2126 0.02669 45.44 0.0000 ovovivip Byrsotri 1.0029 0.02873 34.91 0.0000 ovovivip Nauphoet -0.2230 0.02774 -8.04 0.0000 ovovivip Panchlor -0.7545 0.02566 -29.41 0.0000 ovovivip Pheotali -0.6026 0.02339 -25.76 0.0000 ovovivip Rhyparob 1.3692 0.02873 47.66 0.0000 ovovivip Schultes -0.8428 0.03093 -27.24 0.0000 vivipari Diplopte 1.6214 0.02693 60.22 0.0000

Type = oviparit species = Supella subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Blaberus 0.535 0.02609 20.50 0.0000 ovovivip Byrsotri 0.325 0.02817 11.54 0.0000 ovovivip Nauphoet -0.901 0.02716 -33.16 0.0000

219 ovovivip Panchlor -1.432 0.02503 -57.22 0.0000 ovovivip Pheotali -1.280 0.02270 -56.40 0.0000 ovovivip Rhyparob 0.691 0.02817 24.54 0.0000 ovovivip Schultes -1.521 0.03042 -49.99 0.0000 vivipari Diplopte 0.944 0.02633 35.83 0.0000

Type = ovovivip species = Blaberus subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Byrsotri -0.210 0.02980 -7.04 0.0000 ovovivip Nauphoet -1.436 0.02885 -49.76 0.0000 ovovivip Panchlor -1.967 0.02685 -73.25 0.0000 ovovivip Pheotali -1.815 0.02470 -73.50 0.0000 ovovivip Rhyparob 0.157 0.02980 5.26 0.0000 ovovivip Schultes -2.055 0.03193 -64.36 0.0000 vivipari Diplopte 0.409 0.02807 14.56 0.0000

Type = ovovivip species = Byrsotri subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Nauphoet -1.226 0.03075 -39.87 0.0000 ovovivip Panchlor -1.757 0.02888 -60.85 0.0000 ovovivip Pheotali -1.606 0.02689 -59.71 0.0000 ovovivip Rhyparob 0.366 0.03164 11.58 0.0000 ovovivip Schultes -1.846 0.03366 -54.84 0.0000 vivipari Diplopte 0.619 0.03002 20.61 0.0000

Type = ovovivip species = Nauphoet subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Panchlor -0.5315 0.02790 -19.05 0.0000 ovovivip Pheotali -0.3796 0.02583 -14.70 0.0000 ovovivip Rhyparob 1.5922 0.03075 51.78 0.0000 ovovivip Schultes -0.6198 0.03282 -18.88 0.0000 vivipari Diplopte 1.8444 0.02907 63.44 0.0000

Type = ovovivip species = Panchlor subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Pheotali 0.15192 0.02358 6.442 0.0000 ovovivip Rhyparob 2.12376 0.02888 73.531 0.0000 ovovivip Schultes -0.08822 0.03108 -2.839 0.1845 vivipari Diplopte 2.37597 0.02709 87.693 0.0000

Type = ovovivip species = Pheotali subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Rhyparob 1.9718 0.02689 73.335 0.0000 ovovivip Schultes -0.2401 0.02924 -8.214 0.0000 vivipari Diplopte 2.2241 0.02496 89.114 0.0000

Type = ovovivip species = Rhyparob subtracted from:

Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value

220 ovovivip Schultes -2.212 0.03366 -65.72 0.0000 vivipari Diplopte 0.252 0.03002 8.40 0.0000

Type = ovovivip species = Schultes subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value vivipari Diplopte 2.464 0.03213 76.68 0.0000

Tukey Simultaneous Tests Response Variable sqrtNym All Pairwise Comparisons among Levels of species(Type)

Type = oviparit species = Blatta o subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Blattell 2.5278 0.1970 12.829 0.0000 oviparit Eurycoti -0.0849 0.2020 -0.420 1.0000 oviparit Periplan 0.1355 0.1893 0.716 1.0000 oviparit Supella -0.0771 0.1853 -0.416 1.0000 ovovivip Blaberus 1.9814 0.1970 10.056 0.0000 ovovivip Byrsotri -0.5007 0.2102 -2.382 0.4571 ovovivip Nauphoet 1.9910 0.2038 9.767 0.0000 ovovivip Panchlor 3.4944 0.1904 18.350 0.0000 ovovivip Pheotali 1.5436 0.1760 8.770 0.0000 ovovivip Rhyparob 1.4474 0.2102 6.885 0.0000 ovovivip Schultes 1.1151 0.2246 4.964 0.0001 vivipari Diplopte -0.6417 0.1986 -3.232 0.0641

Type = oviparit species = Blattell subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Eurycoti -2.613 0.1970 -13.26 0.0000 oviparit Periplan -2.392 0.1840 -13.00 0.0000 oviparit Supella -2.605 0.1799 -14.48 0.0000 ovovivip Blaberus -0.546 0.1920 -2.85 0.1811 ovovivip Byrsotri -3.028 0.2055 -14.74 0.0000 ovovivip Nauphoet -0.537 0.1989 -2.70 0.2534 ovovivip Panchlor 0.967 0.1852 5.22 0.0000 ovovivip Pheotali -0.984 0.1703 -5.78 0.0000 ovovivip Rhyparob -1.080 0.2055 -5.26 0.0000 ovovivip Schultes -1.413 0.2202 -6.42 0.0000 vivipari Diplopte -3.170 0.1936 -16.38 0.0000

Type = oviparit species = Eurycoti subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Periplan 0.2203 0.1893 1.164 0.9948 oviparit Supella 0.0078 0.1853 0.042 1.0000 ovovivip Blaberus 2.0663 0.1970 10.487 0.0000 ovovivip Byrsotri -0.4158 0.2102 -1.978 0.7478 ovovivip Nauphoet 2.0759 0.2038 10.184 0.0000 ovovivip Panchlor 3.5793 0.1904 18.796 0.0000 ovovivip Pheotali 1.6285 0.1760 9.253 0.0000 ovovivip Rhyparob 1.5323 0.2102 7.289 0.0000 ovovivip Schultes 1.2000 0.2246 5.342 0.0000 vivipari Diplopte -0.5569 0.1986 -2.804 0.2001

221 Type = oviparit species = Periplan subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Supella -0.2125 0.1714 -1.240 0.9908 ovovivip Blaberus 1.8460 0.1840 10.032 0.0000 ovovivip Byrsotri -0.6361 0.1981 -3.211 0.0680 ovovivip Nauphoet 1.8556 0.1913 9.701 0.0000 ovovivip Panchlor 3.3589 0.1769 18.985 0.0000 ovovivip Pheotali 1.4081 0.1613 8.731 0.0000 ovovivip Rhyparob 1.3119 0.1981 6.623 0.0000 ovovivip Schultes 0.9797 0.2133 4.593 0.0003 vivipari Diplopte -0.7772 0.1857 -4.186 0.0020

Type = oviparit species = Supella subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Blaberus 2.0585 0.1799 11.444 0.0000 ovovivip Byrsotri -0.4236 0.1942 -2.181 0.6054 ovovivip Nauphoet 2.0681 0.1873 11.041 0.0000 ovovivip Panchlor 3.5714 0.1726 20.690 0.0000 ovovivip Pheotali 1.6207 0.1565 10.352 0.0000 ovovivip Rhyparob 1.5244 0.1942 7.848 0.0000 ovovivip Schultes 1.1922 0.2097 5.684 0.0000 vivipari Diplopte -0.5647 0.1816 -3.110 0.0912

Type = ovovivip species = Blaberus subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Byrsotri -2.482 0.2055 -12.08 0.0000 ovovivip Nauphoet 0.010 0.1989 0.05 1.0000 ovovivip Panchlor 1.513 0.1852 8.17 0.0000 ovovivip Pheotali -0.438 0.1703 -2.57 0.3280 ovovivip Rhyparob -0.534 0.2055 -2.60 0.3106 ovovivip Schultes -0.866 0.2202 -3.93 0.0056 vivipari Diplopte -2.623 0.1936 -13.55 0.0000

Type = ovovivip species = Byrsotri subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Nauphoet 2.4917 0.2120 11.7523 0.0000 ovovivip Panchlor 3.9951 0.1992 20.0598 0.0000 ovovivip Pheotali 2.0443 0.1854 11.0259 0.0000 ovovivip Rhyparob 1.9480 0.2182 8.9292 0.0000 ovovivip Schultes 1.6158 0.2321 6.9624 0.0000 vivipari Diplopte -0.1411 0.2070 -0.6816 1.0000

Type = ovovivip species = Nauphoet subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Panchlor 1.503 0.1924 7.81 0.0000 ovovivip Pheotali -0.447 0.1781 -2.51 0.3663 ovovivip Rhyparob -0.544 0.2120 -2.56 0.3323 ovovivip Schultes -0.876 0.2263 -3.87 0.0072 vivipari Diplopte -2.633 0.2005 -13.13 0.0000

Type = ovovivip

222 species = Panchlor subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Pheotali -1.951 0.1626 -12.00 0.0000 ovovivip Rhyparob -2.047 0.1992 -10.28 0.0000 ovovivip Schultes -2.379 0.2143 -11.10 0.0000 vivipari Diplopte -4.136 0.1868 -22.14 0.0000

Type = ovovivip species = Pheotali subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Rhyparob -0.096 0.1854 -0.52 1.0000 ovovivip Schultes -0.428 0.2016 -2.13 0.6459 vivipari Diplopte -2.185 0.1721 -12.70 0.0000

Type = ovovivip species = Rhyparob subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Schultes -0.332 0.2321 -1.43 0.9696 vivipari Diplopte -2.089 0.2070 -10.09 0.0000

Type = ovovivip species = Schultes subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value vivipari Diplopte -1.757 0.2216 -7.929 0.0000

Tukey Simultaneous Tests Response Variable sqrtEgg All Pairwise Comparisons among Levels of species(Type) Type = oviparit species = Blatta o subtracted from:

Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Blattell 2.9807 0.11160 26.709 0.0000 oviparit Eurycoti 0.3459 0.11440 3.024 0.1155 oviparit Periplan -0.0191 0.10722 -0.178 1.0000 oviparit Supella 0.1116 0.10494 1.063 0.9978 ovovivip Blaberus 1.9678 0.11160 17.633 0.0000 ovovivip Byrsotri 0.9895 0.11907 8.310 0.0000 ovovivip Nauphoet 2.1790 0.11545 18.873 0.0000 ovovivip Panchlor 4.6030 0.10786 42.677 0.0000 ovovivip Pheotali 1.6563 0.09968 16.616 0.0000 ovovivip Rhyparob 1.7871 0.11907 15.009 0.0000 ovovivip Schultes 1.3097 0.12723 10.294 0.0000 vivipari Diplopte -0.3547 0.11248 -3.154 0.0805 Type = oviparit species = Blattell subtracted from:

Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Eurycoti -2.635 0.11160 -23.61 0.0000 oviparit Periplan -3.000 0.10422 -28.78 0.0000 oviparit Supella -2.869 0.10188 -28.16 0.0000 ovovivip Blaberus -1.013 0.10872 -9.32 0.0000 ovovivip Byrsotri -1.991 0.11638 -17.11 0.0000 ovovivip Nauphoet -0.802 0.11268 -7.11 0.0000 ovovivip Panchlor 1.622 0.10488 15.47 0.0000

223 ovovivip Pheotali -1.324 0.09645 -13.73 0.0000 ovovivip Rhyparob -1.194 0.11638 -10.26 0.0000 ovovivip Schultes -1.671 0.12471 -13.40 0.0000 vivipari Diplopte -3.335 0.10963 -30.43 0.0000 Type = oviparit species = Eurycoti subtracted from:

Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Periplan -0.3650 0.10722 -3.404 0.0374 oviparit Supella -0.2343 0.10494 -2.233 0.5667 ovovivip Blaberus 1.6219 0.11160 14.533 0.0000 ovovivip Byrsotri 0.6436 0.11907 5.405 0.0000 ovovivip Nauphoet 1.8330 0.11545 15.877 0.0000 ovovivip Panchlor 4.2571 0.10786 39.469 0.0000 ovovivip Pheotali 1.3104 0.09968 13.145 0.0000 ovovivip Rhyparob 1.4412 0.11907 12.104 0.0000 ovovivip Schultes 0.9637 0.12723 7.575 0.0000 vivipari Diplopte -0.7006 0.11248 -6.229 0.0000

Type = oviparit species = Periplan subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value oviparit Supella 0.1307 0.09706 1.346 0.9815 ovovivip Blaberus 1.9869 0.10422 19.064 0.0000 ovovivip Byrsotri 1.0086 0.11219 8.990 0.0000 ovovivip Nauphoet 2.1980 0.10834 20.288 0.0000 ovovivip Panchlor 4.6221 0.10021 46.125 0.0000 ovovivip Pheotali 1.6754 0.09135 18.340 0.0000 ovovivip Rhyparob 1.8062 0.11219 16.100 0.0000 ovovivip Schultes 1.3287 0.12081 10.999 0.0000 vivipari Diplopte -0.3356 0.10516 -3.192 0.0721

Type = oviparit species = Supella subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Blaberus 1.8562 0.10188 18.220 0.0000 ovovivip Byrsotri 0.8779 0.11001 7.980 0.0000 ovovivip Nauphoet 2.0674 0.10609 19.487 0.0000 ovovivip Panchlor 4.4914 0.09777 45.940 0.0000 ovovivip Pheotali 1.5447 0.08867 17.421 0.0000 ovovivip Rhyparob 1.6756 0.11001 15.230 0.0000 ovovivip Schultes 1.1981 0.11879 10.085 0.0000 vivipari Diplopte -0.4663 0.10284 -4.534 0.0004

Type = ovovivip species = Blaberus subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Byrsotri -0.978 0.11638 -8.41 0.0000 ovovivip Nauphoet 0.211 0.11268 1.87 0.8107 ovovivip Panchlor 2.635 0.10488 25.13 0.0000 ovovivip Pheotali -0.311 0.09645 -3.23 0.0645 ovovivip Rhyparob -0.181 0.11638 -1.55 0.9440 ovovivip Schultes -0.658 0.12471 -5.28 0.0000 vivipari Diplopte -2.322 0.10963 -21.19 0.0000

Type = ovovivip species = Byrsotri subtracted from:

224 Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Nauphoet 1.189 0.1201 9.91 0.0000 ovovivip Panchlor 3.613 0.1128 32.03 0.0000 ovovivip Pheotali 0.667 0.1050 6.35 0.0000 ovovivip Rhyparob 0.798 0.1236 6.46 0.0000 ovovivip Schultes 0.320 0.1314 2.44 0.4184 vivipari Diplopte -1.344 0.1172 -11.47 0.0000

Type = ovovivip species = Nauphoet subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Panchlor 2.424 0.1090 22.24 0.0000 ovovivip Pheotali -0.523 0.1009 -5.18 0.0000 ovovivip Rhyparob -0.392 0.1201 -3.26 0.0583 ovovivip Schultes -0.869 0.1282 -6.78 0.0000 vivipari Diplopte -2.534 0.1135 -22.31 0.0000

Type = ovovivip species = Panchlor subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Pheotali -2.947 0.09210 -31.99 0.0000 ovovivip Rhyparob -2.816 0.11280 -24.96 0.0000 ovovivip Schultes -3.293 0.12138 -27.13 0.0000 vivipari Diplopte -4.958 0.10582 -46.85 0.0000

Type = ovovivip species = Pheotali subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Rhyparob 0.131 0.10501 1.25 0.9904 ovovivip Schultes -0.347 0.11418 -3.04 0.1118 vivipari Diplopte -2.011 0.09747 -20.63 0.0000

Type = ovovivip species = Rhyparob subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value ovovivip Schultes -0.477 0.1314 -3.63 0.0172 vivipari Diplopte -2.142 0.1172 -18.27 0.0000

Type = ovovivip species = Schultes subtracted from: Level Difference SE of Adjusted (Type )species of Means Difference T-Value P-Value vivipari Diplopte -1.664 0.1255 -13.26 0.0000

Regression Analysis: nymphs/brood versus eggs/brood The regression equation is nymphs/brood = 1.95 + 0.781 eggs/brood

Predictor Coef SE Coef T P Constant 1.950 2.014 0.97 0.354 eggs/bro 0.78105 0.06143 12.71 0.000

S = 3.507 R-Sq = 93.6% R-Sq(adj) = 93.0%

Analysis of Variance Source DF SS MS F P

225 Regression 1 1988.2 1988.2 161.64 0.000 Residual Error 11 135.3 12.3 Total 12 2123.5

Unusual Observations Obs eggs/bro nymphs/b Fit SE Fit Residual St Resid 7 23.4 11.292 20.201 1.027 -8.909 -2.66R 12 71.2 54.694 57.596 2.788 -2.902 -1.36 X

Regression Analysis: DC day versus gestation time/day The regression equation is DC day = 6.86 + 0.326 gestation time/day Predictor Coef SE Coef T P Constant 6.855 5.517 1.24 0.240 gestatio 0.3265 0.1133 2.88 0.015

S = 5.413 R-Sq = 43.0% R-Sq(adj) = 37.8%

Analysis of Variance Source DF SS MS F P Regression 1 243.36 243.36 8.30 0.015 Residual Error 11 322.33 29.30 Total 12 565.69

Regression Analysis: DC day versus gestation time/day The regression equation is DC day = 2.88 + 0.450 gestation time/day

Predictor Coef SE Coef T P Constant 2.884 1.805 1.60 0.145 gestatio 0.44956 0.03722 12.08 0.000

S = 1.280 R-Sq = 94.2% R-Sq(adj) = 93.5%

Analysis of Variance

Source DF SS MS F P Regression 1 238.89 238.89 145.85 0.000 Residual Error 9 14.74 1.64 Total 10 253.64

JH release and synthesis comparisons among all cockroach species

226

normality test of JH normality test of LOGJH

.999 .999 .99 .99 .95 .95 .80 .80 .50 .50 .20

Probability .20 Probability .05 .05 .01 .01 .001 .001

0 50000 100000 2345 DPM logJH Average: 10164.6 Anderson-Darling Normality Test Average: 3.56992 Anderson-Darling Normality Test StDev: 17963.8 A-Squared: 13.225 StDev: 0.655061 A-Squared: 0.241 N: 94 P-Value: 0.000 N: 92 P-Value: 0.768

Normal Probability Plot of the Residuals Histogram of the Residuals (response is logJH) (response is logJH)

3 20 2

e 1 r

0 10 mal Sco r

-1 Frequency No

-2

0 -3 -3 -2 -1 0 1 2 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Standardized Residual Standardized Residual

General Linear Model: logJH versus rel-syn, type, species Factor Type Levels Values species(type) fixed 8 Blatta o Eurycoti Periplan Blaberu Byrsortr Nauphoet Rhyparob Diplopte rel-syn fixed 2 release synthesi type fixed 3 oviparity ovoviviparity vivparity

Analysis of Variance for logJH, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P species(type) 5 17.6553 17.0800 3.4160 23.65 0.000 rel-syn 1 1.3603 0.8657 0.8657 5.99 0.017 type 2 8.2331 7.9501 3.9751 27.52 0.000 rel-syn*type 2 0.0568 0.0252 0.0126 0.09 0.917 rel-syn*species(type) 5 0.7657 0.7657 0.1531 1.06 0.389 Error 76 10.9774 10.9774 0.1444 Total 91 39.0486

Tukey Simultaneous Tests Response Variable logJH All Pairwise Comparisons among Levels of rel-syn*species(type) type = oviparit rel-syn = release species = Blatta o subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value

227 oviparit release Eurycoti 0.2221 0.2776 0.800 1.0000 oviparit release Periplan -0.1192 0.2776 -0.430 1.0000 oviparit synthesi Blatta o -0.0532 0.2301 -0.231 1.0000 oviparit synthesi Eurycoti 0.5740 0.2301 2.494 0.4867 oviparit synthesi Periplan 0.2608 0.2301 1.133 0.9988 ovovivip release Blaberu 0.8813 0.2549 3.457 0.0635 ovovivip release Byrsortr 0.1441 0.2404 0.599 1.0000 ovovivip release Nauphoet -0.5586 0.2167 -2.578 0.4287 ovovivip release Rhyparob 0.8706 0.2301 3.783 0.0250 ovovivip synthesi Blaberu 0.9225 0.2301 4.008 0.0124 ovovivip synthesi Byrsortr 0.3190 0.2167 1.472 0.9825 ovovivip synthesi Nauphoet -0.0163 0.2082 -0.078 1.0000 ovovivip synthesi Rhyparob 1.1146 0.2120 5.258 0.0002 vivparit release Diplopte 1.1689 0.2776 4.212 0.0064 vivparit synthesi Diplopte 1.5310 0.2549 6.005 0.0001 type = oviparit rel-syn = release species = Eurycoti subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit release Periplan -0.3414 0.3103 -1.100 0.9991 oviparit synthesi Blatta o -0.2753 0.2687 -1.024 0.9996 oviparit synthesi Eurycoti 0.3519 0.2687 1.309 0.9944 oviparit synthesi Periplan 0.0386 0.2687 0.144 1.0000 ovovivip release Blaberu 0.6591 0.2903 2.271 0.6454 ovovivip release Byrsortr -0.0780 0.2776 -0.281 1.0000 ovovivip release Nauphoet -0.7808 0.2573 -3.034 0.1799 ovovivip release Rhyparob 0.6485 0.2687 2.413 0.5441 ovovivip synthesi Blaberu 0.7003 0.2687 2.606 0.4102 ovovivip synthesi Byrsortr 0.0969 0.2573 0.377 1.0000 ovovivip synthesi Nauphoet -0.2384 0.2502 -0.953 0.9998 ovovivip synthesi Rhyparob 0.8925 0.2534 3.522 0.0530 vivparit release Diplopte 0.9468 0.3103 3.051 0.1734 vivparit synthesi Diplopte 1.3089 0.2903 4.509 0.0023 type = oviparit rel-syn = release species = Periplan subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Blatta o 0.0660 0.2687 0.246 1.0000 oviparit synthesi Eurycoti 0.6932 0.2687 2.580 0.4279 oviparit synthesi Periplan 0.3800 0.2687 1.414 0.9880 ovovivip release Blaberu 1.0005 0.2903 3.447 0.0652 ovovivip release Byrsortr 0.2633 0.2776 0.949 0.9999 ovovivip release Nauphoet -0.4394 0.2573 -1.708 0.9383 ovovivip release Rhyparob 0.9899 0.2687 3.683 0.0336 ovovivip synthesi Blaberu 1.0417 0.2687 3.876 0.0188 ovovivip synthesi Byrsortr 0.4382 0.2573 1.703 0.9396 ovovivip synthesi Nauphoet 0.1030 0.2502 0.412 1.0000 ovovivip synthesi Rhyparob 1.2338 0.2534 4.870 0.0007 vivparit release Diplopte 1.2881 0.3103 4.151 0.0078 vivparit synthesi Diplopte 1.6502 0.2903 5.685 0.0001 type = oviparit rel-syn = synthesi species = Blatta o subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Eurycoti 0.6272 0.2194 2.858 0.2604 oviparit synthesi Periplan 0.3139 0.2194 1.431 0.9866 ovovivip release Blaberu 0.9345 0.2453 3.809 0.0231 ovovivip release Byrsortr 0.1973 0.2301 0.857 1.0000

228 ovovivip release Nauphoet -0.5055 0.2053 -2.463 0.5090 ovovivip release Rhyparob 0.9238 0.2194 4.210 0.0064 ovovivip synthesi Blaberu 0.9757 0.2194 4.446 0.0029 ovovivip synthesi Byrsortr 0.3722 0.2053 1.813 0.9038 ovovivip synthesi Nauphoet 0.0369 0.1963 0.188 1.0000 ovovivip synthesi Rhyparob 1.1678 0.2003 5.830 0.0001 vivparit release Diplopte 1.2221 0.2687 4.548 0.0020 vivparit synthesi Diplopte 1.5842 0.2453 6.457 0.0000 type = oviparit rel-syn = synthesi species = Eurycoti subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Periplan -0.313 0.2194 -1.428 0.9869 ovovivip release Blaberu 0.307 0.2453 1.252 0.9965 ovovivip release Byrsortr -0.430 0.2301 -1.868 0.8818 ovovivip release Nauphoet -1.133 0.2053 -5.518 0.0001 ovovivip release Rhyparob 0.297 0.2194 1.352 0.9923 ovovivip synthesi Blaberu 0.348 0.2194 1.588 0.9658 ovovivip synthesi Byrsortr -0.255 0.2053 -1.242 0.9967 ovovivip synthesi Nauphoet -0.590 0.1963 -3.008 0.1909 ovovivip synthesi Rhyparob 0.541 0.2003 2.699 0.3509 vivparit release Diplopte 0.595 0.2687 2.214 0.6849 vivparit synthesi Diplopte 0.957 0.2453 3.901 0.0174 type = oviparit rel-syn = synthesi species = Periplan subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Blaberu 0.6205 0.2453 2.529 0.4622 ovovivip release Byrsortr -0.1167 0.2301 -0.507 1.0000 ovovivip release Nauphoet -0.8194 0.2053 -3.992 0.0131 ovovivip release Rhyparob 0.6099 0.2194 2.779 0.3032 ovovivip synthesi Blaberu 0.6617 0.2194 3.016 0.1875 ovovivip synthesi Byrsortr 0.0582 0.2053 0.284 1.0000 ovovivip synthesi Nauphoet -0.2770 0.1963 -1.412 0.9882 ovovivip synthesi Rhyparob 0.8538 0.2003 4.263 0.0054 vivparit release Diplopte 0.9082 0.2687 3.379 0.0780 vivparit synthesi Diplopte 1.2702 0.2453 5.178 0.0002 type = ovovivip rel-syn = release species = Blaberu subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Byrsortr -0.737 0.2549 -2.891 0.2437 ovovivip release Nauphoet -1.440 0.2327 -6.187 0.0001 ovovivip release Rhyparob -0.011 0.2453 -0.043 1.0000 ovovivip synthesi Blaberu 0.041 0.2453 0.168 1.0000 ovovivip synthesi Byrsortr -0.562 0.2327 -2.416 0.5422 ovovivip synthesi Nauphoet -0.898 0.2248 -3.992 0.0131 ovovivip synthesi Rhyparob 0.233 0.2284 1.022 0.9996 vivparit release Diplopte 0.288 0.2903 0.991 0.9998 vivparit synthesi Diplopte 0.650 0.2687 2.418 0.5409 type = ovovivip rel-syn = release species = Byrsortr subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Nauphoet -0.7027 0.2167 -3.243 0.1102 ovovivip release Rhyparob 0.7265 0.2301 3.157 0.1358 ovovivip synthesi Blaberu 0.7784 0.2301 3.382 0.0774

229 ovovivip synthesi Byrsortr 0.1749 0.2167 0.807 1.0000 ovovivip synthesi Nauphoet -0.1604 0.2082 -0.770 1.0000 ovovivip synthesi Rhyparob 0.9705 0.2120 4.578 0.0018 vivparit release Diplopte 1.0248 0.2776 3.692 0.0327 vivparit synthesi Diplopte 1.3869 0.2549 5.440 0.0001 type = ovovivip rel-syn = release species = Nauphoet subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Rhyparob 1.4293 0.2053 6.964 0.0000 ovovivip synthesi Blaberu 1.4811 0.2053 7.216 0.0000 ovovivip synthesi Byrsortr 0.8776 0.1900 4.619 0.0016 ovovivip synthesi Nauphoet 0.5424 0.1803 3.009 0.1904 ovovivip synthesi Rhyparob 1.6732 0.1847 9.061 0.0000 vivparit release Diplopte 1.7276 0.2573 6.714 0.0000 vivparit synthesi Diplopte 2.0896 0.2327 8.979 0.0000 type = ovovivip rel-syn = release species = Rhyparob subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Blaberu 0.0518 0.2194 0.236 1.0000 ovovivip synthesi Byrsortr -0.5516 0.2053 -2.688 0.3579 ovovivip synthesi Nauphoet -0.8869 0.1963 -4.519 0.0023 ovovivip synthesi Rhyparob 0.2440 0.2003 1.218 0.9974 vivparit release Diplopte 0.2983 0.2687 1.110 0.9991 vivparit synthesi Diplopte 0.6603 0.2453 2.692 0.3553 type = ovovivip rel-syn = synthesi species = Blaberu subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Byrsortr -0.6035 0.2053 -2.940 0.2205 ovovivip synthesi Nauphoet -0.9387 0.1963 -4.783 0.0009 ovovivip synthesi Rhyparob 0.1921 0.2003 0.959 0.9998 vivparit release Diplopte 0.2465 0.2687 0.917 0.9999 vivparit synthesi Diplopte 0.6085 0.2453 2.480 0.4963 type = ovovivip rel-syn = synthesi species = Byrsortr subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Nauphoet -0.3353 0.1803 -1.860 0.8854 ovovivip synthesi Rhyparob 0.7956 0.1847 4.308 0.0046 vivparit release Diplopte 0.8499 0.2573 3.303 0.0949 vivparit synthesi Diplopte 1.2120 0.2327 5.208 0.0002 type = ovovivip rel-syn = synthesi species = Nauphoet subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Rhyparob 1.131 0.1746 6.476 0.0000 vivparit release Diplopte 1.185 0.2502 4.737 0.0010 vivparit synthesi Diplopte 1.547 0.2248 6.881 0.0000 type = ovovivip rel-syn = synthesi species = Rhyparob subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value vivparit release Diplopte 0.05431 0.2534 0.2144 1.0000

230 vivparit synthesi Diplopte 0.41637 0.2284 1.8231 0.9001 type = vivparit rel-syn = release species = Diplopte subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value vivparit synthesi Diplopte 0.3621 0.2903 1.247 0.9966

MF release and synthesis comparisons among all cockroach species normality test of sqrtsqrtMF normality test of MF

.999 .999 .99 .99 .95 .95 .80 .80 .50 .50 obability

r .20

.20 P Probability .05 .05 .01 .01 .001 .001

0 5000 10000 15000 2 7 12 sqrtmf DPM Average: 1415.33 Anderson-Darling Normality Test Average: 4.45948 Anderson-Darling Normality Test StDev: 2857.80 A-Squared: 14.834 StDev: 2.54234 A-Squared: 1.369 N: 94 P-Value: 0.000 N: 94 P-Value: 0.001

Normal Probability Plot of the Residuals Histogram of the Residuals (response is sqrtsqrt) (response is sqrtsqrt)

3 30

2

1 20

0 Frequency -1 10 Normal Score

-2

0 -3 -4 -3 -2 -1 0 1 2 3 4 -4 -3 -2 -1 0 1 2 3 4 Standardized Residual Standardized Residual

General Linear Model: sqsqmf versus rel-syn, type, species Factor Type Levels Values rel-syn fixed 2 release synthesi species(type) fixed 8 Blatta o Eurycoti Periplan Blaberu Byrsortr Nauphoet Rhyparob Diplopte type fixed 3 oviparity ovoviviparity viviparity

Analysis of Variance for sqsqmf, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P rel-syn 1 82.471 5.584 5.584 1.49 0.227 species(type) 5 253.988 208.384 41.677 11.08 0.000 type 2 15.552 8.773 4.387 1.17 0.317 rel-syn*species(type) 5 59.516 51.308 10.262 2.73 0.025 rel-syn*type 2 35.989 35.989 17.995 4.79 0.011 Error 78 293.305 293.305 3.760 Total 93 740.822

231

Tukey Simultaneous Tests Response Variable sqsqmf All Pairwise Comparisons among Levels of rel-syn*species(type) type = oviparit rel-syn = release species = Blatta o subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit release Eurycoti -0.380 1.416 -0.268 1.0000 oviparit release Periplan 1.330 1.416 0.939 0.9999 oviparit synthesi Blatta o -1.098 1.174 -0.935 0.9999 oviparit synthesi Eurycoti 2.457 1.174 2.093 0.7638 oviparit synthesi Periplan 1.928 1.174 1.642 0.9551 ovovivip release Blaberu -2.718 1.301 -2.090 0.7656 ovovivip release Byrsortr -0.871 1.226 -0.710 1.0000 ovovivip release Nauphoet 1.007 1.105 0.911 0.9999 ovovivip release Rhyparob 2.441 1.174 2.079 0.7721 ovovivip synthesi Blaberu 0.710 1.174 0.604 1.0000 ovovivip synthesi Byrsortr 0.850 1.105 0.769 1.0000 ovovivip synthesi Nauphoet 5.762 1.032 5.582 0.0001 ovovivip synthesi Rhyparob 3.516 1.082 3.251 0.1076 vivipari release Diplopte 1.288 1.416 0.909 0.9999 vivipari synthesi Diplopte -0.107 1.301 -0.082 1.0000 type = oviparit rel-syn = release species = Eurycoti subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit release Periplan 1.709 1.583 1.080 0.9993 oviparit synthesi Blatta o -0.718 1.371 -0.524 1.0000 oviparit synthesi Eurycoti 2.837 1.371 2.069 0.7782 oviparit synthesi Periplan 2.307 1.371 1.683 0.9452 ovovivip release Blaberu -2.339 1.481 -1.579 0.9675 ovovivip release Byrsortr -0.491 1.416 -0.347 1.0000 ovovivip release Nauphoet 1.387 1.313 1.057 0.9995 ovovivip release Rhyparob 2.821 1.371 2.057 0.7851 ovovivip synthesi Blaberu 1.089 1.371 0.794 1.0000 ovovivip synthesi Byrsortr 1.230 1.313 0.937 0.9999 ovovivip synthesi Nauphoet 6.141 1.252 4.906 0.0006 ovovivip synthesi Rhyparob 3.896 1.293 3.014 0.1877 vivipari release Diplopte 1.667 1.583 1.053 0.9995 vivipari synthesi Diplopte 0.273 1.481 0.184 1.0000 type = oviparit rel-syn = release species = Periplan subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Blatta o -2.428 1.371 -1.770 0.9192 oviparit synthesi Eurycoti 1.128 1.371 0.822 1.0000 oviparit synthesi Periplan 0.598 1.371 0.436 1.0000 ovovivip release Blaberu -4.048 1.481 -2.733 0.3296 ovovivip release Byrsortr -2.201 1.416 -1.554 0.9717 ovovivip release Nauphoet -0.322 1.313 -0.246 1.0000 ovovivip release Rhyparob 1.112 1.371 0.811 1.0000 ovovivip synthesi Blaberu -0.620 1.371 -0.452 1.0000 ovovivip synthesi Byrsortr -0.479 1.313 -0.365 1.0000 ovovivip synthesi Nauphoet 4.432 1.252 3.541 0.0500

232 ovovivip synthesi Rhyparob 2.187 1.293 1.692 0.9429 vivipari release Diplopte -0.042 1.583 -0.027 1.0000 vivipari synthesi Diplopte -1.436 1.481 -0.970 0.9998 type = oviparit rel-syn = synthesi species = Blatta o subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Eurycoti 3.555 1.1196 3.176 0.1294 oviparit synthesi Periplan 3.026 1.1196 2.703 0.3482 ovovivip release Blaberu -1.620 1.2517 -1.295 0.9950 ovovivip release Byrsortr 0.227 1.1742 0.193 1.0000 ovovivip release Nauphoet 2.105 1.0473 2.010 0.8120 ovovivip release Rhyparob 3.539 1.1196 3.161 0.1338 ovovivip synthesi Blaberu 1.808 1.1196 1.615 0.9608 ovovivip synthesi Byrsortr 1.948 1.0473 1.860 0.8853 ovovivip synthesi Nauphoet 6.860 0.9696 7.075 0.0000 ovovivip synthesi Rhyparob 4.615 1.0220 4.515 0.0022 vivipari release Diplopte 2.386 1.3712 1.740 0.9291 vivipari synthesi Diplopte 0.991 1.2517 0.792 1.0000 type = oviparit rel-syn = synthesi species = Eurycoti subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value oviparit synthesi Periplan -0.530 1.1196 -0.473 1.0000 ovovivip release Blaberu -5.176 1.2517 -4.135 0.0081 ovovivip release Byrsortr -3.328 1.1742 -2.834 0.2724 ovovivip release Nauphoet -1.450 1.0473 -1.384 0.9903 ovovivip release Rhyparob -0.016 1.1196 -0.014 1.0000 ovovivip synthesi Blaberu -1.748 1.1196 -1.561 0.9706 ovovivip synthesi Byrsortr -1.607 1.0473 -1.534 0.9747 ovovivip synthesi Nauphoet 3.305 0.9696 3.408 0.0718 ovovivip synthesi Rhyparob 1.059 1.0220 1.036 0.9996 vivipari release Diplopte -1.170 1.3712 -0.853 1.0000 vivipari synthesi Diplopte -2.564 1.2517 -2.048 0.7904 type = oviparit rel-syn = synthesi species = Periplan subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Blaberu -4.646 1.2517 -3.712 0.0306 ovovivip release Byrsortr -2.799 1.1742 -2.383 0.5653 ovovivip release Nauphoet -0.920 1.0473 -0.879 0.9999 ovovivip release Rhyparob 0.514 1.1196 0.459 1.0000 ovovivip synthesi Blaberu -1.218 1.1196 -1.088 0.9993 ovovivip synthesi Byrsortr -1.077 1.0473 -1.029 0.9996 ovovivip synthesi Nauphoet 3.834 0.9696 3.954 0.0145 ovovivip synthesi Rhyparob 1.589 1.0220 1.555 0.9716 vivipari release Diplopte -0.640 1.3712 -0.467 1.0000 vivipari synthesi Diplopte -2.034 1.2517 -1.625 0.9586 type = ovovivip rel-syn = release species = Blaberu subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Byrsortr 1.847 1.301 1.420 0.9876

233 ovovivip release Nauphoet 3.726 1.187 3.137 0.1417 ovovivip release Rhyparob 5.160 1.252 4.122 0.0085 ovovivip synthesi Blaberu 3.428 1.252 2.739 0.3263 ovovivip synthesi Byrsortr 3.569 1.187 3.005 0.1912 ovovivip synthesi Nauphoet 8.480 1.120 7.575 0.0000 ovovivip synthesi Rhyparob 6.235 1.165 5.351 0.0001 vivipari release Diplopte 4.006 1.481 2.705 0.3468 vivipari synthesi Diplopte 2.612 1.371 1.905 0.8658 type = ovovivip rel-syn = release species = Byrsortr subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Nauphoet 1.8783 1.105 1.6991 0.9409 ovovivip release Rhyparob 3.3124 1.174 2.8209 0.2797 ovovivip synthesi Blaberu 1.5807 1.174 1.3461 0.9926 ovovivip synthesi Byrsortr 1.7213 1.105 1.5571 0.9712 ovovivip synthesi Nauphoet 6.6328 1.032 6.4259 0.0000 ovovivip synthesi Rhyparob 4.3875 1.082 4.0564 0.0105 vivipari release Diplopte 2.1586 1.416 1.5243 0.9761 vivipari synthesi Diplopte 0.7642 1.301 0.5875 1.0000 type = ovovivip rel-syn = release species = Nauphoet subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip release Rhyparob 1.434 1.0473 1.3693 0.9913 ovovivip synthesi Blaberu -0.298 1.0473 -0.2842 1.0000 ovovivip synthesi Byrsortr -0.157 0.9696 -0.1619 1.0000 ovovivip synthesi Nauphoet 4.754 0.8851 5.3717 0.0001 ovovivip synthesi Rhyparob 2.509 0.9423 2.6629 0.3729 vivipari release Diplopte 0.280 1.3128 0.2135 1.0000 vivipari synthesi Diplopte -1.114 1.1875 -0.9382 0.9999 type = ovovivip rel-syn = release species = Rhyparob subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Blaberu -1.732 1.1196 -1.547 0.9728 ovovivip synthesi Byrsortr -1.591 1.0473 -1.519 0.9768 ovovivip synthesi Nauphoet 3.320 0.9696 3.425 0.0687 ovovivip synthesi Rhyparob 1.075 1.0220 1.052 0.9995 vivipari release Diplopte -1.154 1.3712 -0.841 1.0000 vivipari synthesi Diplopte -2.548 1.2517 -2.036 0.7977 type = ovovivip rel-syn = synthesi species = Blaberu subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Byrsortr 0.1407 1.0473 0.1343 1.0000 ovovivip synthesi Nauphoet 5.0522 0.9696 5.2107 0.0002 ovovivip synthesi Rhyparob 2.8068 1.0220 2.7463 0.3218 vivipari release Diplopte 0.5779 1.3712 0.4215 1.0000 vivipari synthesi Diplopte -0.8165 1.2517 -0.6523 1.0000 type = ovovivip rel-syn = synthesi

234 species = Byrsortr subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Nauphoet 4.9115 0.8851 5.5491 0.0001 ovovivip synthesi Rhyparob 2.6661 0.9423 2.8295 0.2751 vivipari release Diplopte 0.4373 1.3128 0.3331 1.0000 vivipari synthesi Diplopte -0.9572 1.1875 -0.8060 1.0000 type = ovovivip rel-syn = synthesi species = Nauphoet subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value ovovivip synthesi Rhyparob -2.245 0.8551 -2.626 0.3968 vivipari release Diplopte -4.474 1.2517 -3.574 0.0455 vivipari synthesi Diplopte -5.869 1.1196 -5.242 0.0002 type = ovovivip rel-syn = synthesi species = Rhyparob subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value vivipari release Diplopte -2.229 1.293 -1.724 0.9338 vivipari synthesi Diplopte -3.623 1.165 -3.109 0.1513 type = vivipari rel-syn = release species = Diplopte subtracted from: Level Difference SE of Adjusted (type )rel-syn *species of Means Difference T-Value P-Value vivipari synthesi Diplopte -1.394 1.481 -0.9415 0.9999

Contrast of JH synthesis and release (Ovoviviparity vs Oviparity) Parameter Estimate Error t Value Pr > |t| Ovoviviparity vs Oviparity -0.31223088 0.09105974 -3.43 0.0010 proc glm; class species relsyn; model logJH = species relsyn species*relsyn; contrast 'Ovoviviparity vs Oviparity' species -0.25 0.333 -0.25 0 0.333 - 0.25 0.333 -0.25; /*Blaberu Blatta_o Byrsortr Diplopte Eurycoti Nauphoet Periplan Rhyparob */ estimate 'Ovoviviparity vs Oviparity' species -3 4 -3 0 4 -3 4 -3/ divisor=12; run;

Contrast of MF synthesis and release (Ovoviviparity vs Oviparity)

Parameter Estimate Error t Value Pr > |t| Ovoviviparity vs Oviparity -0.71382837 0.40488531 -1.76 0.0818 contrast 'Ovoviviparity vs Oviparity' species -0.25 0.333 -0.25 0 0.333 -0.25 0.333 -0.25; /*Blaberu Blatta_o Byrsortr Diplopte Eurycoti Nauphoet Periplan Rhyparob */ estimate 'Ovoviviparity vs Oviparity' species -3 4 -3 0 4 -3 4 -3/ divisor=12; run;

Split Gland experiment

235

normality test of log(JH) after farnesol Normal test of JH after farnesol .999 .99 .95 .999 .99 .80 .95 .50 .80 .20 .50 Probability

.05 Probability .20 .01 .05 .01 .001 .001

-3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 log(JH) JH Average: -1.70155 Anderson-Darling Normality Test Average: 0.0485592 Anderson-Darling Normality Test StDev: 0.623643 A-Squared: 0.494 StDev: 0.0934645 A-Squared: 24.262 N: 159 P-Value: 0.213 N: 164 P-Value: 0.000

Normal Probability Plot of the Residuals (response is log(JH)) Histogram of the Residuals (response is log(JH)) 3

25 2

20

e 1 r

15 0 mal Sco r 10 -1 No Frequency

-2 5

-3 0

-1 0 1 -1 0 1 Residual Residual

General Linear Model: log(JH) versus species, Treat, gland Factor Type Levels Values species fixed 4 Blaberus discoidalis Byrsotria fumigata Diploptera punctata Rhyparobea maderea gland(species) fixed 41 34 35 36 37 38 39 40 41 28 29 30 31 32 33 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Treat fixed 4 R RF S SF

Analysis of Variance for log(JH), using Adjusted SS for Tests Source Model DF Reduced DF Seq SS species 3 3 7.66441 gland(species) 37 37 14.61280 Treat 3 3 20.73975 species*Treat 9 9 1.67106 Treat*gland(species) 111 105+ 16.76283 Error -5 1 0.00025 Total 158 158 61.45110 + Rank deficiency due to empty cells or collinearity. No storage of results or further analysis will be done.

General Linear Model: log(JH) versus species, Treat, gland

Factor Type Levels Values

236 species fixed 4 Blaberus discoidalis Byrsotria fumigata Diploptera punctata Rhyparobea maderea gland(species) fixed 41 34 35 36 37 38 39 40 41 28 29 30 31 32 33 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Treat fixed 4 R RF S SF

Analysis of Variance for log(JH), using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P species 3 7.6644 7.5842 2.5281 15.99 0.000 gland(species) 37 14.6128 14.9591 0.4043 2.56 0.000 Treat 3 20.7397 18.9102 6.3034 39.86 0.000 species*Treat 9 1.6711 1.6711 0.1857 1.17 0.319 Error 106 16.7631 16.7631 0.1581 Total 158 61.4511

Tukey Simultaneous Tests Response Variable log(JH) All Pairwise Comparisons among Levels of species species = Blaberus subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Byrsotri -0.2756 0.10941 -2.519 0.0627 Diplopte 0.4061 0.09239 4.395 0.0002 Rhyparob 0.1144 0.08739 1.309 0.5592 species = Byrsotri subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Diplopte 0.6817 0.10307 6.614 0.0000 Rhyparob 0.3901 0.09861 3.955 0.0008 species = Diplopte subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Rhyparob -0.2917 0.07931 -3.678 0.0021

Tukey Simultaneous Tests Response Variable log(JH) All Pairwise Comparisons among Levels of Treat

Treat = R subtracted from: Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value RF 0.3684 0.09380 3.927 0.0009 S -0.5792 0.09443 -6.134 0.0000 SF -0.3998 0.09672 -4.133 0.0004

Treat = RF subtracted from: Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value S -0.9476 0.09443 -10.04 0.0000 SF -0.7681 0.09672 -7.94 0.0000

Treat = S subtracted from: Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value SF 0.1795 0.09739 1.843 0.2592

237 Tukey Simultaneous Tests Response Variable log(JH) All Pairwise Comparisons among Levels of species*Treat species = Blaberus Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus RF 0.258 0.1988 1.295 0.9953 Blaberus S -0.659 0.1988 -3.312 0.0867 Blaberus SF -0.456 0.1988 -2.295 0.6280 Byrsotri R -0.309 0.2148 -1.437 0.9868 Byrsotri RF -0.011 0.2148 -0.052 1.0000 Byrsotri S -1.012 0.2148 -4.714 0.0008 Byrsotri SF -0.628 0.2306 -2.723 0.3304 Diplopte R 0.376 0.1848 2.037 0.7986 Diplopte RF 0.868 0.1848 4.696 0.0009 Diplopte S -0.179 0.1848 -0.970 0.9998 Diplopte SF -0.298 0.1848 -1.613 0.9625 Rhyparob R -0.070 0.1722 -0.406 1.0000 Rhyparob RF 0.357 0.1722 2.075 0.7764 Rhyparob S -0.469 0.1776 -2.638 0.3845 Rhyparob SF -0.219 0.1776 -1.231 0.9973 species = Blaberus Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus S -0.916 0.1988 -4.608 0.0012 Blaberus SF -0.714 0.1988 -3.591 0.0395 Byrsotri R -0.566 0.2148 -2.636 0.3861 Byrsotri RF -0.269 0.2148 -1.251 0.9968 Byrsotri S -1.270 0.2148 -5.913 0.0001 Byrsotri SF -0.885 0.2306 -3.841 0.0182 Diplopte R 0.119 0.1848 0.644 1.0000 Diplopte RF 0.610 0.1848 3.302 0.0890 Diplopte S -0.437 0.1848 -2.364 0.5780 Diplopte SF -0.556 0.1848 -3.007 0.1844 Rhyparob R -0.327 0.1722 -1.902 0.8693 Rhyparob RF 0.100 0.1722 0.579 1.0000 Rhyparob S -0.726 0.1776 -4.088 0.0080 Rhyparob SF -0.476 0.1776 -2.681 0.3566 species = Blaberus Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus SF 0.2022 0.1988 1.017 0.9997 Byrsotri R 0.3501 0.2148 1.630 0.9590 Byrsotri RF 0.6475 0.2148 3.015 0.1809 Byrsotri S -0.3538 0.2148 -1.648 0.9552 Byrsotri SF 0.0307 0.2306 0.133 1.0000 Diplopte R 1.0351 0.1848 5.602 0.0001 Diplopte RF 1.5264 0.1848 8.260 0.0000 Diplopte S 0.4793 0.1848 2.594 0.4141 Diplopte SF 0.3606 0.1848 1.951 0.8453 Rhyparob R 0.5887 0.1722 3.419 0.0648 Rhyparob RF 1.0159 0.1722 5.900 0.0001 Rhyparob S 0.1901 0.1776 1.070 0.9994 Rhyparob SF 0.4400 0.1776 2.477 0.4958

238 species = Blaberus Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri R 0.1478 0.2148 0.688 1.0000 Byrsotri RF 0.4453 0.2148 2.073 0.7773 Byrsotri S -0.5561 0.2148 -2.589 0.4173 Byrsotri SF -0.1715 0.2306 -0.744 1.0000 Diplopte R 0.8329 0.1848 4.507 0.0018 Diplopte RF 1.3241 0.1848 7.166 0.0000 Diplopte S 0.2771 0.1848 1.499 0.9803 Diplopte SF 0.1583 0.1848 0.857 1.0000 Rhyparob R 0.3865 0.1722 2.244 0.6642 Rhyparob RF 0.8137 0.1722 4.725 0.0008 Rhyparob S -0.0121 0.1776 -0.068 1.0000 Rhyparob SF 0.2377 0.1776 1.339 0.9934 species = Byrsotri Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri RF 0.2974 0.2296 1.295 0.9953 Byrsotri S -0.7039 0.2296 -3.066 0.1608 Byrsotri SF -0.3193 0.2444 -1.307 0.9949 Diplopte R 0.6850 0.2018 3.394 0.0694 Diplopte RF 1.1763 0.2018 5.828 0.0001 Diplopte S 0.1292 0.2018 0.640 1.0000 Diplopte SF 0.0105 0.2018 0.052 1.0000 Rhyparob R 0.2386 0.1904 1.254 0.9967 Rhyparob RF 0.6658 0.1904 3.498 0.0519 Rhyparob S -0.1600 0.1953 -0.819 1.0000 Rhyparob SF 0.0899 0.1953 0.460 1.0000 species = Byrsotri Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri S -1.001 0.2296 -4.361 0.0030 Byrsotri SF -0.617 0.2444 -2.523 0.4631 Diplopte R 0.388 0.2018 1.920 0.8606 Diplopte RF 0.879 0.2018 4.354 0.0031 Diplopte S -0.168 0.2018 -0.833 1.0000 Diplopte SF -0.287 0.2018 -1.422 0.9881 Rhyparob R -0.059 0.1904 -0.309 1.0000 Rhyparob RF 0.368 0.1904 1.935 0.8534 Rhyparob S -0.457 0.1953 -2.343 0.5938 Rhyparob SF -0.208 0.1953 -1.063 0.9995 species = Byrsotri Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri SF 0.3846 0.2444 1.573 0.9697 Diplopte R 1.3889 0.2018 6.882 0.0000 Diplopte RF 1.8802 0.2018 9.316 0.0000 Diplopte S 0.8332 0.2018 4.128 0.0069 Diplopte SF 0.7144 0.2018 3.540 0.0459 Rhyparob R 0.9426 0.1904 4.951 0.0003 Rhyparob RF 1.3697 0.1904 7.195 0.0000 Rhyparob S 0.5439 0.1953 2.786 0.2937 Rhyparob SF 0.7938 0.1953 4.065 0.0086

239 species = Byrsotri Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte R 1.0044 0.2185 4.5957 0.0013 Diplopte RF 1.4956 0.2185 6.8436 0.0000 Diplopte S 0.4486 0.2185 2.0526 0.7897 Diplopte SF 0.3299 0.2185 1.5093 0.9791 Rhyparob R 0.5580 0.2080 2.6825 0.3559 Rhyparob RF 0.9852 0.2080 4.7361 0.0008 Rhyparob S 0.1594 0.2125 0.7500 1.0000 Rhyparob SF 0.4092 0.2125 1.9258 0.8580 species = Diplopte Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte RF 0.4913 0.1696 2.897 0.2343 Diplopte S -0.5558 0.1696 -3.278 0.0950 Diplopte SF -0.6745 0.1696 -3.978 0.0116 Rhyparob R -0.4464 0.1558 -2.866 0.2501 Rhyparob RF -0.0192 0.1558 -0.123 1.0000 Rhyparob S -0.8450 0.1617 -5.226 0.0001 Rhyparob SF -0.5951 0.1617 -3.680 0.0301 species = Diplopte Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte S -1.047 0.1696 -6.175 0.0000 Diplopte SF -1.166 0.1696 -6.875 0.0000 Rhyparob R -0.938 0.1558 -6.020 0.0001 Rhyparob RF -0.510 0.1558 -3.277 0.0951 Rhyparob S -1.336 0.1617 -8.264 0.0000 Rhyparob SF -1.086 0.1617 -6.719 0.0000 species = Diplopte Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte SF -0.1187 0.1696 -0.700 1.0000 Rhyparob R 0.1094 0.1558 0.702 1.0000 Rhyparob RF 0.5366 0.1558 3.445 0.0602 Rhyparob S -0.2892 0.1617 -1.789 0.9150 Rhyparob SF -0.0393 0.1617 -0.243 1.0000 species = Diplopte Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob R 0.2281 0.1558 1.465 0.9841 Rhyparob RF 0.6553 0.1558 4.207 0.0053 Rhyparob S -0.1705 0.1617 -1.054 0.9995 Rhyparob SF 0.0794 0.1617 0.491 1.0000 species = Rhyparob Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob RF 0.4272 0.1406 3.038 0.1715

240 Rhyparob S -0.3986 0.1472 -2.709 0.3394 Rhyparob SF -0.1488 0.1472 -1.011 0.9997 species = Rhyparob Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob S -0.8258 0.1472 -5.612 0.0001 Rhyparob SF -0.5759 0.1472 -3.914 0.0143 species = Rhyparob Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob SF 0.2499 0.1540 1.622 0.9606

General Linear Model: sqrtsqrtmf versus species, Treat, gland

normality test of SQRTSQRT(MF) after farnesol normality test of MF after farnesol

.999

.999 .99 .99 .95 .95 .80 .80 .50 .50 .20 Probability

Probability .20 .05 .05 .01 .01 .001 .001

0.00 0.05 0.10 0.0 0.1 0.2 0.3 0.4 0.5 0.6 MF sqrtsqrtmf Average: 0.0033574 Anderson-Darling Normality Test Average: 0.105034 Anderson-Darling Normality Test StDev: 0.0142268 A-Squared: 45.455 StDev: 0.125717 A-Squared: 10.728 N: 164 P-Value: 0.000 N: 164 P-Value: 0.000

Normal Probability Plot of the Residuals Histogram of the Residuals (response is sqrtsqrt) (response is sqrtsqrt)

3

30 2

e 1 r 20 0 mal Sco r

-1 Frequency No 10

-2

0 -3 -0.2 -0.1 0.0 0.1 0.2 -0.2 -0.1 0.0 0.1 0.2 Residual Residual

Factor Type Levels Values gland(species) fixed 41 34 35 36 37 38 39 40 41 28 29 30 31 32 33 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 species fixed 4 Blaberus discoidalis Byrsotria fumigata Diploptera punctata Rhyparobea maderea Treat fixed 4 R RF S SF

241 Analysis of Variance for sqrtsqrt, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P gland(species) 37 0.779744 0.779744 0.021074 3.00 0.000 species 3 0.371712 0.371712 0.123904 17.63 0.000 Treat 3 0.369159 0.181307 0.060436 8.60 0.000 species*Treat 9 0.275651 0.275651 0.030628 4.36 0.000 Error 111 0.779923 0.779923 0.007026 Total 163 2.576190

Tukey Simultaneous Tests Response Variable sqrtsqrt All Pairwise Comparisons among Levels of species species = Blaberus subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Byrsotri 0.04932 0.02263 2.1789 0.1355 Diplopte 0.01764 0.01947 0.9056 0.8019 Rhyparob 0.11267 0.01815 6.2083 0.0000 species = Byrsotri subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Diplopte -0.03168 0.02127 -1.490 0.4473 Rhyparob 0.06335 0.02006 3.157 0.0109 species = Diplopte subtracted from: Level Difference SE of Adjusted species of Means Difference T-Value P-Value Rhyparob 0.09503 0.01642 5.789 0.0000

Tukey Simultaneous Tests Response Variable sqrtsqrt All Pairwise Comparisons among Levels of Treat

Treat = R subtracted from: Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value RF 0.007691 0.01977 0.3890 0.9799 S 0.020039 0.01977 1.0135 0.7419 SF 0.089568 0.01977 4.5302 0.0001

Treat = RF subtracted from: Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value S 0.01235 0.01977 0.6245 0.9240 SF 0.08188 0.01977 4.1412 0.0004

Treat = S subtracted from:

Level Difference SE of Adjusted Treat of Means Difference T-Value P-Value SF 0.06953 0.01977 3.517 0.0035

Tukey Simultaneous Tests Response Variable sqrtsqrt All Pairwise Comparisons among Levels of species*Treat species = Blaberus Treat = R subtracted from:

242 Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus RF 0.020571 0.04191 0.4908 1.0000 Blaberus S 0.024606 0.04191 0.5871 1.0000 Blaberus SF -0.008134 0.04191 -0.1941 1.0000 Byrsotri R 0.093828 0.04527 2.0727 0.7779 Byrsotri RF 0.022171 0.04527 0.4897 1.0000 Byrsotri S 0.020606 0.04527 0.4552 1.0000 Byrsotri SF 0.097713 0.04527 2.1585 0.7232 Diplopte R -0.008913 0.03895 -0.2288 1.0000 Diplopte RF -0.003938 0.03895 -0.1011 1.0000 Diplopte S 0.012964 0.03895 0.3328 1.0000 Diplopte SF 0.107472 0.03895 2.7593 0.3083 Rhyparob R 0.014453 0.03630 0.3982 1.0000 Rhyparob RF 0.091329 0.03630 2.5162 0.4678 Rhyparob S 0.121348 0.03630 3.3432 0.0791 Rhyparob SF 0.260590 0.03630 7.1795 0.0000 species = Blaberus Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus S 0.00403 0.04191 0.0963 1.0000 Blaberus SF -0.02870 0.04191 -0.6849 1.0000 Byrsotri R 0.07326 0.04527 1.6182 0.9616 Byrsotri RF 0.00160 0.04527 0.0353 1.0000 Byrsotri S 0.00004 0.04527 0.0008 1.0000 Byrsotri SF 0.07714 0.04527 1.7041 0.9415 Diplopte R -0.02948 0.03895 -0.7570 1.0000 Diplopte RF -0.02451 0.03895 -0.6292 1.0000 Diplopte S -0.00761 0.03895 -0.1953 1.0000 Diplopte SF 0.08690 0.03895 2.2311 0.6735 Rhyparob R -0.00612 0.03630 -0.1685 1.0000 Rhyparob RF 0.07076 0.03630 1.9495 0.8466 Rhyparob S 0.10078 0.03630 2.7765 0.2982 Rhyparob SF 0.24002 0.03630 6.6127 0.0000 species = Blaberus Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Blaberus SF -0.03274 0.04191 -0.7811 1.0000 Byrsotri R 0.06922 0.04527 1.5291 0.9766 Byrsotri RF -0.00243 0.04527 -0.0538 1.0000 Byrsotri S -0.00400 0.04527 -0.0883 1.0000 Byrsotri SF 0.07311 0.04527 1.6149 0.9623 Diplopte R -0.03352 0.03895 -0.8606 1.0000 Diplopte RF -0.02854 0.03895 -0.7328 1.0000 Diplopte S -0.01164 0.03895 -0.2989 1.0000 Diplopte SF 0.08287 0.03895 2.1275 0.7435 Rhyparob R -0.01015 0.03630 -0.2797 1.0000 Rhyparob RF 0.06672 0.03630 1.8383 0.8967 Rhyparob S 0.09674 0.03630 2.6653 0.3663 Rhyparob SF 0.23598 0.03630 6.5016 0.0000 species = Blaberus Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri R 0.101962 0.04527 2.25232 0.6586 Byrsotri RF 0.030304 0.04527 0.66942 1.0000

243 Byrsotri S 0.028740 0.04527 0.63486 1.0000 Byrsotri SF 0.105847 0.04527 2.33814 0.5969 Diplopte R -0.000780 0.03895 -0.02002 1.0000 Diplopte RF 0.004196 0.03895 0.10773 1.0000 Diplopte S 0.021098 0.03895 0.54167 1.0000 Diplopte SF 0.115605 0.03895 2.96810 0.2002 Rhyparob R 0.022587 0.03630 0.62228 1.0000 Rhyparob RF 0.099463 0.03630 2.74028 0.3196 Rhyparob S 0.129481 0.03630 3.56732 0.0418 Rhyparob SF 0.268723 0.03630 7.40355 0.0000 species = Byrsotri Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri RF -0.0717 0.04840 -1.481 0.9826 Byrsotri S -0.0732 0.04840 -1.513 0.9787 Byrsotri SF 0.0039 0.04840 0.080 1.0000 Diplopte R -0.1027 0.04254 -2.415 0.5408 Diplopte RF -0.0978 0.04254 -2.298 0.6259 Diplopte S -0.0809 0.04254 -1.901 0.8700 Diplopte SF 0.0136 0.04254 0.321 1.0000 Rhyparob R -0.0794 0.04013 -1.978 0.8318 Rhyparob RF -0.0025 0.04013 -0.062 1.0000 Rhyparob S 0.0275 0.04013 0.686 1.0000 Rhyparob SF 0.1668 0.04013 4.156 0.0061 species = Byrsotri Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri S -0.00156 0.04840 -0.0323 1.0000 Byrsotri SF 0.07554 0.04840 1.5609 0.9719 Diplopte R -0.03108 0.04254 -0.7307 1.0000 Diplopte RF -0.02611 0.04254 -0.6137 1.0000 Diplopte S -0.00921 0.04254 -0.2164 1.0000 Diplopte SF 0.08530 0.04254 2.0051 0.8172 Rhyparob R -0.00772 0.04013 -0.1923 1.0000 Rhyparob RF 0.06916 0.04013 1.7235 0.9360 Rhyparob S 0.09918 0.04013 2.4716 0.4998 Rhyparob SF 0.23842 0.04013 5.9416 0.0001 species = Byrsotri Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Byrsotri SF 0.07711 0.04840 1.5933 0.9664 Diplopte R -0.02952 0.04254 -0.6939 1.0000 Diplopte RF -0.02454 0.04254 -0.5769 1.0000 Diplopte S -0.00764 0.04254 -0.1796 1.0000 Diplopte SF 0.08687 0.04254 2.0419 0.7963 Rhyparob R -0.00615 0.04013 -0.1533 1.0000 Rhyparob RF 0.07072 0.04013 1.7625 0.9241 Rhyparob S 0.10074 0.04013 2.5105 0.4718 Rhyparob SF 0.23998 0.04013 5.9805 0.0001 species = Byrsotri Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte R -0.1066 0.04254 -2.506 0.4748

244 Diplopte RF -0.1017 0.04254 -2.389 0.5595 Diplopte S -0.0847 0.04254 -1.992 0.8243 Diplopte SF 0.0098 0.04254 0.229 1.0000 Rhyparob R -0.0833 0.04013 -2.075 0.7766 Rhyparob RF -0.0064 0.04013 -0.159 1.0000 Rhyparob S 0.0236 0.04013 0.589 1.0000 Rhyparob SF 0.1629 0.04013 4.059 0.0086 species = Diplopte Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte RF 0.004976 0.03574 0.1392 1.0000 Diplopte S 0.021878 0.03574 0.6121 1.0000 Diplopte SF 0.116385 0.03574 3.2562 0.0997 Rhyparob R 0.023367 0.03283 0.7117 1.0000 Rhyparob RF 0.100242 0.03283 3.0532 0.1649 Rhyparob S 0.130261 0.03283 3.9676 0.0117 Rhyparob SF 0.269503 0.03283 8.2087 0.0000 species = Diplopte Treat = RF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte S 0.01690 0.03574 0.4729 1.0000 Diplopte SF 0.11141 0.03574 3.1170 0.1416 Rhyparob R 0.01839 0.03283 0.5602 1.0000 Rhyparob RF 0.09527 0.03283 2.9017 0.2313 Rhyparob S 0.12529 0.03283 3.8160 0.0193 Rhyparob SF 0.26453 0.03283 8.0571 0.0000 species = Diplopte Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Diplopte SF 0.094508 0.03574 2.64414 0.3801 Rhyparob R 0.001489 0.03283 0.04535 1.0000 Rhyparob RF 0.078365 0.03283 2.38688 0.5613 Rhyparob S 0.108383 0.03283 3.30121 0.0886 Rhyparob SF 0.247625 0.03283 7.54232 0.0000 species = Diplopte Treat = SF subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob R -0.09302 0.03283 -2.833 0.2666 Rhyparob RF -0.01614 0.03283 -0.492 1.0000 Rhyparob S 0.01388 0.03283 0.423 1.0000 Rhyparob SF 0.15312 0.03283 4.664 0.0010 species = Rhyparob Treat = R subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob RF 0.07688 0.02964 2.594 0.4136 Rhyparob S 0.10689 0.02964 3.607 0.0371 Rhyparob SF 0.24614 0.02964 8.305 0.0000 species = Rhyparob Treat = RF subtracted from: Level Difference SE of Adjusted

245 species *Treat of Means Difference T-Value P-Value Rhyparob S 0.03002 0.02964 1.013 0.9997 Rhyparob SF 0.16926 0.02964 5.711 0.0001 species = Rhyparob Treat = S subtracted from: Level Difference SE of Adjusted species *Treat of Means Difference T-Value P-Value Rhyparob SF 0.1392 0.02964 4.698 0.0008 Residual Histogram for sqrtsqrt Normplot of Residuals for sqrtsqrt

Chapter 4

Blood cell recognition of EPNs at 1 hour and 24 hour

General Linear Model: arcsine versus EPN, host, time Factor Type Levels Values EPN fixed 2 Hb88 SgNC host fixed 4 Ad Gm Ms Pj time fixed 2 1h 24h

Analysis of Variance for arcsine, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPN 1 1.8117 1.9839 1.9839 10.09 0.002 host 3 5.5925 4.5014 1.5005 7.63 0.000 EPN*host 3 8.0805 7.9056 2.6352 13.40 0.000 time 1 0.0651 0.0025 0.0025 0.01 0.910 EPN*time 1 0.8770 0.3134 0.3134 1.59 0.208 host*time 3 2.3162 2.1375 0.7125 3.62 0.014 EPN*host*time 3 1.9659 1.9659 0.6553 3.33 0.021 Error 188 36.9770 36.9770 0.1967 Total 203 57.6859

246 Normal Probability Plot of recognition Normal Probability Plot of acrsine

.999 .999 .99 .99 .95 .95 .80 .80 .50 .50 .20 .20 Probability .05 Probability .05 .01 .01 .001 .001

0.0 0.5 1.0 0.0 0.5 1.0 1.5 recognition arcsine(recognition) Average: 0.773760 Anderson-Darling Normality Test Average: 1.08936 Anderson-Darling Normality Test StDev: 0.306812 A-Squared: 20.069 StDev: 0.533073 A-Squared: 14.374 N: 204 P-Value: 0.000 N: 204 P-Value: 0.000

Normal Probability Plot of the Residuals Histogram of the Residuals (response is arcsine) (response is arcsine)

3

20 2

1

0 10

-1 Frequency Normal Score

-2

0 -3 -2 -1 0 1 2 -3 -2 -1 0 1 2 Standardized Residual Standardized Residual

For H. bacteriophora Tukey Simultaneous Tests Response Variable arcsineh All Pairwise Comparisons among Levels of hosthb*timehb hosthb = Ad timehb = 1h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Ad 24h 0.1827 0.2925 0.625 0.9984 Gm 1h -0.0836 0.2299 -0.364 1.0000 Gm 24h -0.6001 0.2299 -2.610 0.1680 Ms 1h 0.2668 0.2486 1.073 0.9605 Ms 24h 0.3090 0.2486 1.243 0.9162 Pj 1h 0.0324 0.2365 0.137 1.0000 Pj 24h -0.0033 0.2365 -0.014 1.0000 hosthb = Ad timehb = 24h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Gm 1h -0.2663 0.2299 -1.159 0.9410 Gm 24h -0.7828 0.2299 -3.405 0.0223 Ms 1h 0.0841 0.2486 0.338 1.0000 Ms 24h 0.1263 0.2486 0.508 0.9996 Pj 1h -0.1503 0.2365 -0.636 0.9982 Pj 24h -0.1860 0.2365 -0.787 0.9934 hosthb = Gm timehb = 1h subtracted from:

247 Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Gm 24h -0.5164 0.1419 -3.640 0.0110 Ms 1h 0.3504 0.1705 2.055 0.4528 Ms 24h 0.3927 0.1705 2.303 0.3054 Pj 1h 0.1160 0.1524 0.761 0.9946 Pj 24h 0.0803 0.1524 0.527 0.9995 hosthb = Gm timehb = 24h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Ms 1h 0.8669 0.1705 5.084 0.0001 Ms 24h 0.9091 0.1705 5.331 0.0000 Pj 1h 0.6324 0.1524 4.150 0.0021 Pj 24h 0.5967 0.1524 3.916 0.0046 hosthb = Ms timehb = 1h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Ms 24h 0.0423 0.1950 0.217 1.0000 Pj 1h -0.2344 0.1794 -1.307 0.8936 Pj 24h -0.2701 0.1794 -1.506 0.8020 hosthb = Ms timehb = 24h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Pj 1h -0.2767 0.1794 -1.542 0.7820 Pj 24h -0.3124 0.1794 -1.742 0.6604 hosthb = Pj timehb = 1h subtracted from: Level Difference SE of Adjusted hosthb*timehb of Means Difference T-Value P-Value Pj 24h -0.03571 0.1622 -0.2201 1.000

For S. galseri Tukey Simultaneous Tests Response Variable arcsines All Pairwise Comparisons among Levels of Hostsg*timesg

Hostsg = A. domes timesg = 1h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value A. domes 24h -0.018 0.2477 -0.072 1.0000 G. mello 1h -0.527 0.1982 -2.661 0.1461 G. mello 24h -0.436 0.2049 -2.126 0.4053 M. sexta 1h -1.165 0.2173 -5.362 0.0000 M. sexta 24h -0.578 0.2204 -2.623 0.1590 P. Japon 1h -0.159 0.2049 -0.776 0.9940 P. Japon 24h -0.429 0.2100 -2.041 0.4596

Hostsg = A. domes timesg = 24h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value G. mello 1h -0.510 0.1982 -2.571 0.1779

248 G. mello 24h -0.418 0.2049 -2.039 0.4610 M. sexta 1h -1.147 0.2173 -5.280 0.0000 M. sexta 24h -0.560 0.2204 -2.542 0.1892 P. Japon 1h -0.141 0.2049 -0.689 0.9971 P. Japon 24h -0.411 0.2100 -1.956 0.5159

Hostsg = G. mello timesg = 1h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value G. mello 24h 0.0918 0.1411 0.651 0.9980 M. sexta 1h -0.6378 0.1585 -4.024 0.0026 M. sexta 24h -0.0508 0.1628 -0.312 1.0000 P. Japon 1h 0.3684 0.1411 2.612 0.1630 P. Japon 24h 0.0987 0.1484 0.665 0.9977

Hostsg = G. mello timesg = 24h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value M. sexta 1h -0.7296 0.1668 -4.373 0.0007 M. sexta 24h -0.1426 0.1709 -0.834 0.9907 P. Japon 1h 0.2766 0.1504 1.840 0.5948 P. Japon 24h 0.0069 0.1573 0.044 1.0000

Hostsg = M. sexta timesg = 1h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value M. sexta 24h 0.5870 0.1855 3.164 0.0408 P. Japon 1h 1.0062 0.1668 6.031 0.0000 P. Japon 24h 0.7365 0.1731 4.256 0.0011

Hostsg = M. sexta timesg = 24h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value P. Japon 1h 0.4192 0.1709 2.4528 0.2272 P. Japon 24h 0.1495 0.1770 0.8445 0.9900

Hostsg = P. Japon timesg = 1h subtracted from: Level Difference SE of Adjusted Hostsg *timesg of Means Difference T-Value P-Value P. Japon 24h -0.2697 0.1573 -1.715 0.6775

249 Interaction Plot - LS Means for arcsine 8 C 8 N h b g h 4 d m s j H S 1 2 A G M P EPN 1.5 SgNC 1.0

Hb88 0.5 time 1.5 24h 1.0

1h 0.5 host 1.5 Pj Ms 1.0 Gm Ad 0.5

Immune responses of different species/strains of EPNs in Japanese beetle

General Linear Model: asnsrMel, asnsrEnc, asnsrFree versus EPNs Factor Type Levels Values EPNs fixed 5 Hb88 Sf SgFL SgNC Ss

Analysis of Variance for asnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 4 3.84939 3.84939 0.96235 12.59 0.000 Error 19 1.45199 1.45199 0.07642 Total 23 5.30138 Unusual Observations for asnsrMel Obs asnsrMel Fit SE Fit Residual St Resid 3 0.57964 1.04720 0.15960 -0.46756 -2.07R 5 1.57080 1.04720 0.15960 0.52360 2.32R

Analysis of Variance for asnsrEnc, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 4 4.7713 4.7713 1.1928 15.19 0.000 Error 19 1.4918 1.4918 0.0785 Total 23 6.2631

Analysis of Variance for asnsrFre, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 4 1.96505 1.96505 0.49126 13.74 0.000 Error 19 0.67938 0.67938 0.03576 Total 23 2.64444

Unusual Observations for asnsrFre Obs asnsrFre Fit SE Fit Residual St Resid 3 0.579640 0.193213 0.109174 0.386426 2.50R 21 0.463648 0.077275 0.077198 0.386373 2.24R

Tukey Simultaneous Tests Response Variable asnsrMel All Pairwise Comparisons among Levels of EPNs

EPNs = Hb88 subtracted from:

250 Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value Sf -0.238 0.1955 -1.218 0.7412 SgFL -1.075 0.1596 -6.733 0.0000 SgNC -0.656 0.1596 -4.108 0.0048 Ss -0.642 0.1955 -3.285 0.0282

EPNs = Sf subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgFL -0.8365 0.1955 -4.279 0.0033 SgNC -0.4176 0.1955 -2.136 0.2460 Ss -0.4041 0.2257 -1.790 0.4074

EPNs = SgFL subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgNC 0.4189 0.1596 2.625 0.1053 Ss 0.4324 0.1955 2.212 0.2176

EPNs = SgNC subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value Ss 0.01349 0.1955 0.06903 1.000

Tukey Simultaneous Tests Response Variable asnsrEnc All Pairwise Comparisons among Levels of EPNs

EPNs = Hb88 subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value Sf 0.2133 0.1981 1.076 0.8162 SgFL 1.1519 0.1618 7.120 0.0000 SgNC 0.2064 0.1618 1.276 0.7084 Ss 0.6108 0.1981 3.083 0.0429

EPNs = Sf subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgFL 0.938617 0.1981 4.73725 0.0012 SgNC -0.006870 0.1981 -0.03467 1.0000 Ss 0.397505 0.2288 1.73744 0.4362

EPNs = SgFL subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgNC -0.9455 0.1618 -5.844 0.0001 Ss -0.5411 0.1981 -2.731 0.0861

EPNs = SgNC subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value Ss 0.4044 0.1981 2.041 0.2852

Tukey Simultaneous Tests Response Variable asnsrFre All Pairwise Comparisons among Levels of EPNs

EPNs = Hb88 subtracted from: Level Difference SE of Adjusted

251 EPNs of Means Difference T-Value P-Value Sf 0.11594 0.1337 0.8671 0.9054 SgFL -0.07727 0.1092 -0.7078 0.9522 SgNC 0.65632 0.1092 6.0116 0.0001 Ss 0.18452 0.1337 1.3800 0.6470

EPNs = Sf subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgFL -0.1932 0.1337 -1.445 0.6079 SgNC 0.5404 0.1337 4.041 0.0056 Ss 0.0686 0.1544 0.444 0.9913

EPNs = SgFL subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value SgNC 0.7336 0.1092 6.719 0.0000 Ss 0.2618 0.1337 1.958 0.3227

EPNs = SgNC subtracted from: Level Difference SE of Adjusted EPNs of Means Difference T-Value P-Value Ss -0.4718 0.1337 -3.528 0.0169

Immune responses of different species/strains of EPNs in four insect hosts

General Linear Model: asnsrMel, asnsrEnc, ... versus EPNs, beetle larvae

Factor Type Levels Values EPNs fixed 3 Hb88 SgFL SgNC beetle l fixed 4 JB MS NMC OB

Analysis of Variance for asnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 2 4.8881 4.0889 2.0444 23.98 0.000 beetle l 3 4.2024 2.3911 0.7970 9.35 0.000 EPNs*beetle l 6 3.3998 3.3998 0.5666 6.65 0.000 Error 107 9.1233 9.1233 0.0853 Total 118 21.6136

Analysis of Variance for asnsrEnc, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 2 0.54763 0.54210 0.27105 3.15 0.047 beetle l 3 3.91839 1.53655 0.51218 5.95 0.001 EPNs*beetle l 6 3.10097 3.10097 0.51683 6.01 0.000 Error 107 9.20346 9.20346 0.08601 Total 118 16.77046

Analysis of Variance for asnsrFre, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P EPNs 2 5.58718 5.25983 2.62992 67.24 0.000 beetle l 3 2.61222 3.34395 1.11465 28.50 0.000 EPNs*beetle l 6 2.57909 2.57909 0.42985 10.99 0.000 Error 107 4.18495 4.18495 0.03911 Total 118 14.96344

Tukey Simultaneous Tests Response Variable asnsrMel All Pairwise Comparisons among Levels of beetle l*EPNs

252 beetle l = JB EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgFL -0.877 0.1686 -5.200 0.0001 JB SgNC -0.567 0.1625 -3.491 0.0324 MS Hb88 -0.707 0.1398 -5.057 0.0001 MS SgFL -0.696 0.1625 -4.283 0.0023 MS SgNC -1.076 0.2065 -5.209 0.0001 NMC Hb88 -0.298 0.1686 -1.771 0.8300 NMC SgFL -0.334 0.1625 -2.054 0.6560 NMC SgNC -0.410 0.1508 -2.720 0.2310 OB Hb88 -0.109 0.1288 -0.846 0.9994 OB SgFL -0.745 0.1625 -4.585 0.0008 OB SgNC -0.996 0.1577 -6.319 0.0000 beetle l = JB EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgNC 0.3096 0.1625 1.9055 0.7532 MS Hb88 0.1698 0.1398 1.2147 0.9867 MS SgFL 0.1810 0.1625 1.1139 0.9934 MS SgNC -0.1989 0.2065 -0.9634 0.9981 NMC Hb88 0.5782 0.1686 3.4298 0.0388 NMC SgFL 0.5431 0.1625 3.3430 0.0498 NMC SgNC 0.4665 0.1508 3.0938 0.0975 OB Hb88 0.7678 0.1288 5.9632 0.0000 OB SgFL 0.1318 0.1625 0.8114 0.9996 OB SgNC -0.1198 0.1577 -0.7596 0.9998 beetle l = JB EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS Hb88 -0.1398 0.1323 -1.056 0.9958 MS SgFL -0.1286 0.1561 -0.824 0.9996 MS SgNC -0.5085 0.2015 -2.523 0.3373 NMC Hb88 0.2687 0.1625 1.654 0.8845 NMC SgFL 0.2335 0.1561 1.496 0.9386 NMC SgNC 0.1570 0.1439 1.091 0.9945 OB Hb88 0.4583 0.1206 3.799 0.0123 OB SgFL -0.1777 0.1561 -1.139 0.9921 OB SgNC -0.4293 0.1511 -2.841 0.1783 beetle l = MS EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgFL 0.0112 0.13232 0.084 1.0000 MS SgNC -0.3687 0.18371 -2.007 0.6877 NMC Hb88 0.4084 0.13978 2.922 0.1483 NMC SgFL 0.3733 0.13232 2.821 0.1864 NMC SgNC 0.2967 0.11771 2.521 0.3389 OB Hb88 0.5980 0.08774 6.816 0.0000 OB SgFL -0.0380 0.13232 -0.287 1.0000 OB SgNC -0.2896 0.12644 -2.290 0.4899 beetle l = MS EPNs = SgFL subtracted from:

253 Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgNC -0.3799 0.2015 -1.885 0.7656 NMC Hb88 0.3973 0.1625 2.445 0.3858 NMC SgFL 0.3621 0.1561 2.320 0.4692 NMC SgNC 0.2856 0.1439 1.984 0.7027 OB Hb88 0.5869 0.1206 4.865 0.0003 OB SgFL -0.0491 0.1561 -0.315 1.0000 OB SgNC -0.3007 0.1511 -1.990 0.6990 beetle l = MS EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC Hb88 0.77713 0.2065 3.7638 0.0139 NMC SgFL 0.74200 0.2015 3.6824 0.0180 NMC SgNC 0.66542 0.1922 3.4618 0.0353 OB Hb88 0.96673 0.1755 5.5094 0.0000 OB SgFL 0.33073 0.2015 1.6413 0.8895 OB SgNC 0.07912 0.1977 0.4002 1.0000 beetle l = NMC EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgFL -0.0351 0.1625 -0.216 1.0000 NMC SgNC -0.1117 0.1508 -0.741 0.9998 OB Hb88 0.1896 0.1288 1.473 0.9448 OB SgFL -0.4464 0.1625 -2.748 0.2182 OB SgNC -0.6980 0.1577 -4.426 0.0014 beetle l = NMC EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgNC -0.0766 0.1439 -0.532 1.0000 OB Hb88 0.2247 0.1206 1.863 0.7787 OB SgFL -0.4113 0.1561 -2.635 0.2741 OB SgNC -0.6629 0.1511 -4.386 0.0016 beetle l = NMC EPNs = SgNC subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB Hb88 0.3013 0.1044 2.887 0.1609 OB SgFL -0.3347 0.1439 -2.326 0.4653 OB SgNC -0.5863 0.1385 -4.233 0.0028 beetle l = OB EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB SgFL -0.6360 0.1206 -5.273 0.0001 OB SgNC -0.8876 0.1141 -7.777 0.0000 beetle l = OB EPNs = SgFL subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB SgNC -0.2516 0.1511 -1.665 0.8798

254

Tukey Simultaneous Tests Response Variable asnsrEnc All Pairwise Comparisons among Levels of beetle l*EPNs beetle l = JB EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgFL 0.9137 0.1693 5.3960 0.0001 JB SgNC 0.2750 0.1632 1.6855 0.8708 MS Hb88 0.7535 0.1404 5.3669 0.0001 MS SgFL 0.5895 0.1632 3.6131 0.0223 MS SgNC 0.3216 0.2074 1.5507 0.9223 NMC Hb88 0.3659 0.1693 2.1610 0.5809 NMC SgFL 0.3049 0.1632 1.8688 0.7754 NMC SgNC 0.3979 0.1514 2.6271 0.2783 OB Hb88 0.1463 0.1293 1.1316 0.9925 OB SgFL 0.1420 0.1632 0.8703 0.9993 OB SgNC 0.2763 0.1584 1.7445 0.8432 beetle l = JB EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgNC -0.6387 0.1632 -3.914 0.0084 MS Hb88 -0.1602 0.1404 -1.141 0.9920 MS SgFL -0.3241 0.1632 -1.987 0.7013 MS SgNC -0.5921 0.2074 -2.855 0.1728 NMC Hb88 -0.5478 0.1693 -3.235 0.0672 NMC SgFL -0.6087 0.1632 -3.731 0.0154 NMC SgNC -0.5158 0.1514 -3.406 0.0416 OB Hb88 -0.7673 0.1293 -5.933 0.0000 OB SgFL -0.7717 0.1632 -4.729 0.0004 OB SgNC -0.6374 0.1584 -4.024 0.0058 beetle l = JB EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS Hb88 0.4785 0.1329 3.600 0.0233 MS SgFL 0.3145 0.1568 2.006 0.6882 MS SgNC 0.0466 0.2024 0.230 1.0000 NMC Hb88 0.0909 0.1632 0.557 1.0000 NMC SgFL 0.0299 0.1568 0.191 1.0000 NMC SgNC 0.1228 0.1445 0.850 0.9994 OB Hb88 -0.1287 0.1211 -1.062 0.9956 OB SgFL -0.1330 0.1568 -0.848 0.9994 OB SgNC 0.0013 0.1518 0.009 1.0000 beetle l = MS EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgFL -0.1639 0.13290 -1.234 0.9850 MS SgNC -0.4319 0.18452 -2.341 0.4552 NMC Hb88 -0.3876 0.14040 -2.761 0.2124 NMC SgFL -0.4486 0.13290 -3.375 0.0455 NMC SgNC -0.3556 0.11823 -3.008 0.1207 OB Hb88 -0.6072 0.08812 -6.890 0.0000 OB SgFL -0.6115 0.13290 -4.601 0.0007

255 OB SgNC -0.4772 0.12699 -3.757 0.0141 beetle l = MS EPNs = SgFL subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgNC -0.2679 0.2024 -1.324 0.9741 NMC Hb88 -0.2236 0.1632 -1.371 0.9666 NMC SgFL -0.2846 0.1568 -1.816 0.8059 NMC SgNC -0.1917 0.1445 -1.326 0.9738 OB Hb88 -0.4432 0.1211 -3.658 0.0194 OB SgFL -0.4475 0.1568 -2.855 0.1729 OB SgNC -0.3132 0.1518 -2.064 0.6492 beetle l = MS EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC Hb88 0.0443 0.2074 0.2137 1.0000 NMC SgFL -0.0167 0.2024 -0.0823 1.0000 NMC SgNC 0.0763 0.1931 0.3951 1.0000 OB Hb88 -0.1753 0.1762 -0.9944 0.9975 OB SgFL -0.1796 0.2024 -0.8874 0.9991 OB SgNC -0.0453 0.1986 -0.2281 1.0000 beetle l = NMC EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgFL -0.0610 0.1632 -0.374 1.0000 NMC SgNC 0.0319 0.1514 0.211 1.0000 OB Hb88 -0.2196 0.1293 -1.698 0.8653 OB SgFL -0.2239 0.1632 -1.372 0.9663 OB SgNC -0.0896 0.1584 -0.566 1.0000 beetle l = NMC EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgNC 0.0929 0.1445 0.643 1.0000 OB Hb88 -0.1586 0.1211 -1.309 0.9762 OB SgFL -0.1629 0.1568 -1.039 0.9964 OB SgNC -0.0286 0.1518 -0.189 1.0000 beetle l = NMC EPNs = SgNC subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB Hb88 -0.2515 0.1048 -2.399 0.4158 OB SgFL -0.2559 0.1445 -1.770 0.8301 OB SgNC -0.1216 0.1391 -0.874 0.9992 beetle l = OB EPNs = Hb88 subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB SgFL -0.004331 0.1211 -0.03575 1.0000 OB SgNC 0.129974 0.1146 1.13382 0.9924

256 beetle l = OB EPNs = SgFL subtracted from:

Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB SgNC 0.1343 0.1518 0.8848 0.9991

Tukey Simultaneous Tests Response Variable asnsrFre All Pairwise Comparisons among Levels of beetle l*EPNs beetle l = JB EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgFL -0.07479 0.11418 -0.6550 1.0000 JB SgNC 0.56965 0.11003 5.1773 0.0001 MS Hb88 0.01905 0.09467 0.2013 1.0000 MS SgFL 0.33755 0.11003 3.0679 0.1041 MS SgNC 0.82587 0.13984 5.9058 0.0000 NMC Hb88 -0.07479 0.11418 -0.6550 1.0000 NMC SgFL 0.16388 0.11003 1.4894 0.9404 NMC SgNC 0.13203 0.10213 1.2928 0.9784 OB Hb88 0.01088 0.08721 0.1247 1.0000 OB SgFL 0.78054 0.11003 7.0941 0.0000 OB SgNC 0.82705 0.10681 7.7435 0.0000 beetle l = JB EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value JB SgNC 0.644436 0.11003 5.85706 0.0000 MS Hb88 0.093842 0.09467 0.99121 0.9976 MS SgFL 0.412338 0.11003 3.74760 0.0146 MS SgNC 0.900662 0.13984 6.44056 0.0000 NMC Hb88 0.000000 0.11418 0.00000 1.0000 NMC SgFL 0.238667 0.11003 2.16916 0.5752 NMC SgNC 0.206815 0.10213 2.02509 0.6755 OB Hb88 0.085665 0.08721 0.98232 0.9978 OB SgFL 0.855332 0.11003 7.77382 0.0000 OB SgNC 0.901839 0.10681 8.44369 0.0000 beetle l = JB EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS Hb88 -0.5506 0.08962 -6.144 0.0000 MS SgFL -0.2321 0.10571 -2.196 0.5565 MS SgNC 0.2562 0.13647 1.877 0.7702 NMC Hb88 -0.6444 0.11003 -5.857 0.0000 NMC SgFL -0.4058 0.10571 -3.838 0.0108 NMC SgNC -0.4376 0.09746 -4.490 0.0011 OB Hb88 -0.5588 0.08169 -6.840 0.0000 OB SgFL 0.2109 0.10571 1.995 0.6957 OB SgNC 0.2574 0.10235 2.515 0.3425 beetle l = MS EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgFL 0.31850 0.08962 3.5538 0.0268

257 MS SgNC 0.80682 0.12443 6.4844 0.0000 NMC Hb88 -0.09384 0.09467 -0.9912 0.9976 NMC SgFL 0.14483 0.08962 1.6160 0.8995 NMC SgNC 0.11297 0.07972 1.4171 0.9577 OB Hb88 -0.00818 0.05942 -0.1376 1.0000 OB SgFL 0.76149 0.08962 8.4968 0.0000 OB SgNC 0.80800 0.08564 9.4353 0.0000 beetle l = MS EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value MS SgNC 0.4883 0.13647 3.578 0.0249 NMC Hb88 -0.4123 0.11003 -3.748 0.0146 NMC SgFL -0.1737 0.10571 -1.643 0.8889 NMC SgNC -0.2055 0.09746 -2.109 0.6178 OB Hb88 -0.3267 0.08169 -3.999 0.0063 OB SgFL 0.4430 0.10571 4.191 0.0032 OB SgNC 0.4895 0.10235 4.782 0.0004 beetle l = MS EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC Hb88 -0.9007 0.1398 -6.441 0.0000 NMC SgFL -0.6620 0.1365 -4.851 0.0003 NMC SgNC -0.6938 0.1302 -5.330 0.0001 OB Hb88 -0.8150 0.1188 -6.858 0.0000 OB SgFL -0.0453 0.1365 -0.332 1.0000 OB SgNC 0.0012 0.1339 0.009 1.0000 beetle l = NMC EPNs = Hb88 subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgFL 0.23867 0.11003 2.1692 0.5752 NMC SgNC 0.20682 0.10213 2.0251 0.6755 OB Hb88 0.08566 0.08721 0.9823 0.9978 OB SgFL 0.85533 0.11003 7.7738 0.0000 OB SgNC 0.90184 0.10681 8.4437 0.0000 beetle l = NMC EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value NMC SgNC -0.0319 0.09746 -0.327 1.0000 OB Hb88 -0.1530 0.08169 -1.873 0.7730 OB SgFL 0.6167 0.10571 5.834 0.0000 OB SgNC 0.6632 0.10235 6.479 0.0000 beetle l = NMC EPNs = SgNC subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB Hb88 -0.1212 0.07069 -1.714 0.8580 OB SgFL 0.6485 0.09746 6.654 0.0000 OB SgNC 0.6950 0.09381 7.409 0.0000 beetle l = OB EPNs = Hb88 subtracted from: Level Difference SE of Adjusted

258 beetle l*EPNs of Means Difference T-Value P-Value OB SgFL 0.7697 0.08169 9.421 0.0000 OB SgNC 0.8162 0.07730 10.558 0.0000 beetle l = OB EPNs = SgFL subtracted from: Level Difference SE of Adjusted beetle l*EPNs of Means Difference T-Value P-Value OB SgNC 0.04651 0.1024 0.4544 1.000

SCPs vs Control and time effect (8 hours vs 16-18 hours) in oriental beetle larvae

Time effect (8 vs 16-18 hours; control vs SCPs extracts #30 #31)

General Linear Model: acnsrMel, acnsrEnc, ... versus hours after , protein batc Factor Type Levels Values hours af fixed 2 16-18 8 protein fixed 3 0 30 31

Analysis of Variance for acnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P hours af 1 0.4458 0.0038 0.0038 0.07 0.791 protein 2 8.4440 8.3142 4.1571 76.57 0.000 hours af*protein 2 0.9636 0.9636 0.4818 8.87 0.001 Error 50 2.7145 2.7145 0.0543 Total 55 12.5679

Analysis of Variance for acnsrEnc, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P hours af 1 0.22340 0.26967 0.26967 5.30 0.025 protein 2 1.99140 1.89829 0.94915 18.66 0.000 hours af*protein 2 0.26699 0.26699 0.13350 2.62 0.082 Error 50 2.54299 2.54299 0.05086 Total 55 5.02477

Analysis of Variance for acnsrFre, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P hours af 1 1.3517 0.1532 0.1532 3.65 0.062 protein 2 4.0782 4.0927 2.0463 48.76 0.000 hours af*protein 2 0.2132 0.2132 0.1066 2.54 0.089 Error 50 2.0985 2.0985 0.0420 Total 55 7.7417

Control vs SCPs (protein extract #30) 8 hours in Oriental beetle larvae

General Linear Model: acnsrMel, acnsrEnc, acnsrFree versus protein batch Factor Type Levels Values protein fixed 2 0 30

Analysis of Variance for acnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 6.2023 6.2023 6.2023 88.42 0.000 Error 20 1.4030 1.4030 0.0701 Total 21 7.6053

Analysis of Variance for acnsrEnc, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 1.4797 1.4797 1.4797 18.48 0.000

259 Error 20 1.6017 1.6017 0.0801 Total 21 3.0814

Analysis of Variance for acnsrFre, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 2.3526 2.3526 2.3526 36.50 0.000 Error 20 1.2889 1.2889 0.0644 Total 21 3.6415

Effect of SCPs in Manduca sexta

General Linear Model: melan_inject, encap_assume, . versus protein desc Factor Type Levels Values protein fixed 2 0 NCWS

Analysis of Variance for melan_in, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.00006 0.00006 0.00006 0.00 0.973 Error 14 0.75744 0.75744 0.05410 Total 15 0.75750

Analysis of Variance for encap_as, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.00160 0.00160 0.00160 0.03 0.856 Error 14 0.65590 0.65590 0.04685 Total 15 0.65750

Unusual Observations for encap_as Obs encap_as Fit SE Fit Residual St Resid 4 0.20000 0.69231 0.06003 -0.49231 -2.37R R denotes an observation with a large standardized residual.

Analysis of Variance for free_inj, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.001026 0.001026 0.001026 0.12 0.733 Error 14 0.118974 0.118974 0.008498 Total 15 0.120000

General Linear Model: melan_recove, encap_recove, ... versus protein desc

Factor Type Levels Values protein fixed 2 0 NCWS

Analysis of Variance for melan_re, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.0067 0.0067 0.0067 0.04 0.849 Error 14 2.5032 2.5032 0.1788 Total 15 2.5100

Analysis of Variance for encap_re, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.05207 0.05207 0.05207 0.64 0.436 Error 14 1.13231 1.13231 0.08088 Total 15 1.18438

Analysis of Variance for free_rec, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 0.09625 0.09625 0.09625 0.98 0.340 Error 14 1.38139 1.38139 0.09867

260 Total 15 1.47764

Chapter 5

SCPs extraction method comparison

Use protein concentration as a covariate

General Linear Model: melan_injection versus protein batch Factor Type Levels Values protein fixed 9 0 17 18 23 26 29 30 31 32

Analysis of Variance for melan_in, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P protein 1 2.30133 0.01172 0.01172 0.25 0.617 protein 8 3.51919 3.51919 0.43990 9.47 0.000 Error 91 4.22887 4.22887 0.04647 Total 100 10.04939

Term Coef SE Coef T P Constant 0.3342 0.1103 3.03 0.003 protein 0.2589 0.5157 0.50 0.617

Means for Covariates Covariate Mean StDev protein 0.1524 0.1228

Least Squares Means for melan_in protein Mean SE Mean 0 0.8357 0.08885 17 0.3375 0.12681 18 0.4042 0.14356 23 0.5373 0.06971 26 0.3507 0.13049 29 0.3499 0.05393 30 0.1856 0.06041 31 0.1279 0.07275 32 0.2342 0.13501

Test effect of dissection time (8 hour vs 16-18 hour)

General Linear Model: asnsrMel versus protein batc, buffer, hours after Factor Type Levels Values protein fixed 9 0 17 18 23 26 29 30 31 32 buffer fixed 2 IEF Ringer hours af fixed 2 16-18 8

Analysis of Variance for asnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 1 3.34839 0.08347 0.08347 1.01 0.317 protein 8 6.25571 5.42413 0.67802 8.23 0.000 buffer 1 0.38117 0.39267 0.39267 4.76 0.032 hours af 1 0.02692 0.02692 0.02692 0.33 0.569 Error 89 7.33637 7.33637 0.08243 Total 100 17.34856

261 Effect of different SCPs extraction methods

General Linear Model: asnsrMel versus protein batch

Factor Type Levels Values protein fixed 9 0 17 18 23 26 29 30 31 32

Analysis of Variance for asnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 8 9.5161 9.5161 1.1895 13.97 0.000 Error 92 7.8325 7.8325 0.0851 Total 100 17.3486

Dunnett Simultaneous Tests Response Variable asnsrMel Comparisons with Control Level protein = 0 subtracted from:

Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 17 -0.5069 0.13169 -3.849 0.0017 18 -0.4133 0.13169 -3.138 0.0175 23 -0.3247 0.10801 -3.007 0.0258 26 -0.5271 0.17757 -2.968 0.0288 29 -0.5740 0.09206 -6.236 0.0000 30 -0.7541 0.08608 -8.761 0.0000 31 -0.8735 0.11231 -7.778 0.0000 32 -0.6356 0.15632 -4.066 0.0008

Least Squares Means for melan_in protein Mean SE Mean 0 0.7963 0.04132 17 0.3833 0.08765 18 0.4611 0.08765 23 0.5300 0.06789 26 0.3704 0.12395 29 0.3490 0.05367 30 0.2039 0.04801 31 0.1222 0.07156 32 0.2750 0.10735

Histogram of the Residuals Normal Probability Plot of the Residuals (response is asnsrMel) (response is asnsrMel)

3 25

2 20 e r 15 1

0

10 mal Sco r Frequency No 5 -1

0 -2

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 -2 -1 0 1 2 3 Standardized Residual Standardized Residual

Test IEF buffer and Ringers solution

262

General Linear Model: melan_injection versus protein batch, buffer Factor Type Levels Values protein fixed 9 0 17 18 23 26 29 30 31 32 buffer fixed 2 IEF Ringer

Analysis of Variance for melan_in, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 8 5.80881 4.71926 0.58991 13.11 0.000 buffer 1 0.14487 0.14487 0.14487 3.22 0.076 Error 91 4.09571 4.09571 0.04501 Total 100 10.04939

Test Time effect of SCPs in oriental beetle larvae

General Linear Model: acsnsrMel, acsnsrFree versus treatment Factor Type Levels Values treatmen fixed 5 pn pp pr rn rr

Analysis of Variance for acsnsrMe, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P treatmen 4 4.6682 4.6682 1.1671 13.73 0.000 Error 10 0.8502 0.8502 0.0850 Total 14 5.5184

Analysis of Variance for acsnsrFr, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P treatmen 4 1.71283 1.71283 0.42821 9.63 0.002 Error 10 0.44448 0.44448 0.04445 Total 14 2.15732

Tukey Simultaneous Tests Response Variable acsnsrMe All Pairwise Comparisons among Levels of treatmen treatmen = pn subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value pp -0.1896 0.2381 -0.7963 0.9259 pr -0.2282 0.2381 -0.9587 0.8673 rn 0.8815 0.2381 3.7027 0.0264 rr 1.0808 0.2381 4.5395 0.0074 treatmen = pp subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value pr -0.03866 0.2381 -0.1624 0.9998 rn 1.07109 0.2381 4.4989 0.0079 rr 1.27033 0.2381 5.3358 0.0024 treatmen = pr subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value rn 1.110 0.2381 4.661 0.0062 rr 1.309 0.2381 5.498 0.0019 treatmen = rn subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value rr 0.1992 0.2381 0.8369 0.9130

263

Tukey 95.0% Simultaneous Confidence Intervals Tukey Simultaneous Tests Response Variable acsnsrFr All Pairwise Comparisons among Levels of treatmen treatmen = pn subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value pp 0.2968 0.1721 1.724 0.4623 pr 0.0488 0.1721 0.283 0.9983 rn -0.4825 0.1721 -2.803 0.1059 rr -0.5958 0.1721 -3.461 0.0384 treatmen = pp subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value pr -0.2481 0.1721 -1.441 0.6179 rn -0.7794 0.1721 -4.528 0.0076 rr -0.8926 0.1721 -5.186 0.0029 treatmen = pr subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value rn -0.5313 0.1721 -3.086 0.0686 rr -0.6446 0.1721 -3.745 0.0248 treatmen = rn subtracted from: Level Difference SE of Adjusted treatmen of Means Difference T-Value P-Value rr -0.1133 0.1721 -0.6581 0.9611

Test dosage effect of SCPs

Factor Type Levels Values protein fixed 5 0 50 100 200 900

Analysis of Variance for acnsrMel, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 4 7.5591 7.5591 1.8898 29.48 0.000 Error 19 1.2181 1.2181 0.0641 Total 23 8.7773

Analysis of Variance for acnsrFre, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P protein 4 2.03561 2.03561 0.50890 13.73 0.000 Error 19 0.70437 0.70437 0.03707 Total 23 2.73999

Tukey Simultaneous Tests Response Variable acnsrMel All Pairwise Comparisons among Levels of protein protein = 0 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 50 -0.742 0.1387 -5.347 0.0003 100 -1.108 0.1667 -6.648 0.0000 200 -1.370 0.1667 -8.219 0.0000

264 900 -1.263 0.1667 -7.576 0.0000 protein = 50 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 100 -0.3665 0.1849 -1.982 0.3115 200 -0.6283 0.1849 -3.398 0.0223 900 -0.5211 0.1849 -2.818 0.0728 protein = 100 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 200 -0.2618 0.2067 -1.266 0.7139 900 -0.1545 0.2067 -0.748 0.9423 protein = 200 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 900 0.1073 0.2067 0.5188 0.9844

Tukey Simultaneous Tests Response Variable acnsrFre All Pairwise Comparisons among Levels of protein protein = 0 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 50 0.4456 0.1055 4.225 0.0037 100 0.2039 0.1267 1.609 0.5098 200 0.6487 0.1267 5.118 0.0005 900 0.7611 0.1267 6.005 0.0001 protein = 50 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 100 -0.2416 0.1406 -1.718 0.4468 200 0.2032 0.1406 1.445 0.6080 900 0.3155 0.1406 2.244 0.2065 protein = 100 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 200 0.4448 0.1572 2.829 0.0712 900 0.5572 0.1572 3.544 0.0163 protein = 200 subtracted from: Level Difference SE of Adjusted protein of Means Difference T-Value P-Value 900 0.1124 0.1572 0.7147 0.9505

Least Squares Means .. melan_in .. .. free-inj .. protein Mean SE Mean Mean SE Mean 0 0.920000 0.05137 0.030000 0.04296 50 0.360000 0.07264 0.270909 0.06075 100 0.166667 0.09378 0.133333 0.07843 200 -0.000000 0.09378 0.466667 0.07843 900 0.033333 0.09378 0.566667 0.07843

265 Test Electroeluted proteins

General Linear Model: acsnsrMel, acsnsrFree versus Proteins Factor Type Levels Values Proteins fixed 5 #53-4 #53-5 BSA others SCP

Analysis of Variance for acsnsrMe, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Proteins 4 5.9412 5.9412 1.4853 15.14 0.000 Error 52 5.1020 5.1020 0.0981 Total 56 11.0432

Analysis of Variance for acsnsrFr, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Proteins 4 2.52705 2.52705 0.63176 14.52 0.000 Error 52 2.26319 2.26319 0.04352 Total 56 4.79024

Tukey Simultaneous Tests Response Variable acsnsrMe All Pairwise Comparisons among Levels of Proteins

Proteins = #53-4 subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value #53-5 -0.2162 0.2101 -1.029 0.8408 BSA 0.8818 0.1834 4.808 0.0001 others 0.6446 0.1495 4.312 0.0007 SCP 0.0830 0.1981 0.419 0.9934

Proteins = #53-5 subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value BSA 1.0980 0.1963 5.593 0.0000 others 0.8608 0.1651 5.214 0.0000 SCP 0.2992 0.2101 1.424 0.6152

Proteins = BSA subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value others -0.2372 0.1294 -1.833 0.3662 SCP -0.7988 0.1834 -4.355 0.0006

Proteins = others subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value SCP -0.5616 0.1495 -3.757 0.0038

Tukey Simultaneous Tests Response Variable acsnsrFr All Pairwise Comparisons among Levels of Proteins

Proteins = #53-4 subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value #53-5 -0.0484 0.13995 -0.346 0.9968 BSA -0.6007 0.12216 -4.918 0.0001 others -0.5184 0.09957 -5.207 0.0000 SCP -0.0981 0.13194 -0.744 0.9451

Proteins = #53-5 subtracted from:

266 Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value BSA -0.5524 0.1308 -4.224 0.0009 others -0.4700 0.1100 -4.275 0.0008 SCP -0.0498 0.1399 -0.356 0.9965

Proteins = BSA subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value others 0.08234 0.08618 0.9555 0.8735 SCP 0.50260 0.12216 4.1144 0.0013

Proteins = others subtracted from: Level Difference SE of Adjusted Proteins of Means Difference T-Value P-Value SCP 0.4203 0.09957 4.221 0.0009

VITA

I was born in a beautiful city of Urumqi in Xinjiang province in northwest of China. During the 12-year elementary and middle school education in my hometown, I have been honored by many awards. The most important event for my life is that I got the highest score in college entrance examination in the province, which is regarded as the most competitive exam in one’s life. I have been admitted to the Fudan University in Shanghai, one of the top five universities in China. I have received my Bachelor’s Degree in Department of Biology. During 4-years education, I have received many awards; one of them is first rank of the people’s scholarship that honors the highest GPA in the department. I continued my academic life in the same university. I got my Master’s degree in Zoology, during these three years, I published two articles and also managed to obtain more awards. In 2000, I came the peaceful town of state college and continued my education in Department of Entomology at Penn State University. In this friendly department, I have been learning new techniques and scientific reasoning. In my Ph.D. life, I participated many academic activities. I have presented my research many times in each annual meeting of Entomological Society of American, Pennsylvania Pest Management Association, Environmental Chemical Student Symposium, graduate exhibition and Agricultural student research expo. Besides communicating with researchers and other students, I have accumulated experience and exchanged ideas by both oral and poster presentation. I also worked as a teaching assistant for three semesters in the department, which is a good education to me. In five years, I also work hard in statistics and earned a Master of Applied Statistics from Department of Statistics. Based on my research, I am preparing three manuscripts and I hope they can be published in near future. When I think about this five-year time, I really appreciate the people in this department, the faculties, students and working staff. I know when I am old and think of my life, the time I spent at Penn State will always be a treasure.