Molecular Mechanisms Underlying Cockroach Host Manipulation by a Parasitoid Wasp

Thesis submitted in partial fulfillment

of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

Maayan Kaiser Paltin

Submitted to the Senate of Ben-Gurion University

of the Negev

June 2018

Beer-Sheva

Molecular Mechanisms Underlying Cockroach Host Manipulation by a Parasitoid Wasp

Thesis submitted in partial fulfillment

of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

Maayan Kaiser Paltin

Submitted to the Senate of Ben-Gurion University

of the Negev

Approved by the advisor

Approved by the Dean of the Kreitman School of Advanced Graduate Studies

June 2018

Beer-Sheva

This work was carried out under the supervision of Prof. Frederic Libersat

In the Department of Life Sciences

Faculty of Natural Sciences

Research-Student's Affidavit when Submitting the Doctoral Thesis for Judgment

I, Maayan Kaiser Paltin, whose signature appears below, hereby declare that:

___ I have written this Thesis by myself, except for the help and guidance offered by my Thesis Advisors.

___ The scientific materials included in this Thesis are products of my own research, culled from the period during which I was a research student.

___ This Thesis incorporates research materials produced in cooperation with others, excluding the technical help commonly received during experimental work. Therefore, I am attaching another affidavit stating the contributions made by myself and the other participants in this research, which has been approved by them and submitted with their approval.

Date: ______31/12/18 Student's name: ______Maayan Kaiser Paltin Signature:______

Affidavit stating the contributions to present work:

The venom proteomics which is described at chapter 2, was performed in collaboration with Prof. Michael Adams and Dr. Ryan Arvidson from the University of California, Riverside. Mass spectrometry analysis was based on the transcriptomes and differential expression analysis of the venom apparatus, which were done there (described at method section, chapter 2). In chapter 3, in collaboration with Prof. Michael Adams, I used the transcriptome of the cockroach cerebral ganglia as a database for mass spectrometry analysis.

Acknowledgments

First, I would like to express my utmost gratitude to my advisor and mentor, Prof. Frederic Libersat, for giving me the opportunity to work in his lab, for having the patient when it was needed, for teaching me the basics of being a scientist, and for his wise advices and help in all matters.

I would like to thanks some people who led me to the decision of pursuing science. First and most important are my parents, Rene and Leonardo Kaiser, who I have so much to thank for, but especially on always insisting on the importance of expanding own knowledge. My parents inhered me the love for the written word, such an important thing a parent can give to his child. This introduced me to Daglas Adams and Richard Dawkins who inspired me to pursuit biology. I thank those great persons for leading my way to where I needed to be.

Thanks to my other and better half, my favored person in the whole universe, Daniel. I could not have done a thing without him, as support and inspiration.

Thanks to my brothers Noam and Ariel. Noam, the best person I know, for teaching me so much, and Ariel, for always taking care of me. Thanks to my parents at law, Veronika and Uri Paltin, for being the amazing people they are, for their precious and wise advices, for their kindness and help in anything I could possibly need.

I would like to thank Gustavo Glusman, who made the lab more like a home, for his kindness and help. My gratitude goes also to Ram Gal, who mentored me at the beginning of my way and Stav Emanuel, for the companionship and moral support.

I would like to thank many more from our life sciences department: Prof. Raz Zarivach and Nitzan Kutnowski for their help with the venom affinity chromatography, Prof. Noam Zilberberg and Prof. Uri Abdu for their wise advices and comments and to all members of Prof. Ofer Yifrach and Prof. Noam Zilberberg labs, for their kind help.

Special thanks to Prof. Michael Adams for the collaboration, helpful comments and editing part of this thesis and to Dr. Ryan Arvidson for his collaboration on the venomics of the wasp and for his work on the transcriptomic of the cockroach cerebral ganglia.

To Lia

Table of Contents List of figures and tables ...... 1 Abstract ...... 4 General Introduction ...... 6 1.The role of the cerebral ganglia in the venom-induced behavioral manipulation of cockroaches stung by the parasitoid Jewel Wasp ...... 10 1.1 Abstract ...... 10 1.2 Introduction ...... 10 1.3 Material and Methods ...... 13 1.4 Results ...... 16 1.5 Discussion ...... 22 2. Parasitoid Jewel Wasp Mounts Multi-Pronged Neurochemical Attack to Hijack a Host Brain ...... 25 2.1 Abstract ...... 25 2.2 Introduction ...... 25 2.3 Material and Methods ...... 27 2.4 Results ...... 31 1.5 Discussion ...... 39 3. Molecular cross-talk in a unique parasitic manipulation strategy ...... 46 3.1 Abstract ...... 46 3.2 Introduction ...... 46 3.3 Methods ...... 48 3.3 Results ...... 51 3.4 Discussion ...... 71 4. General Discussion and Future Research Directions ...... 80 References ...... 88 Appendix 1 ...... 99 Appendix 2 ...... 100 Appendix 3 ...... 101 Appendix 4 ...... 103

List of figures and tables Figure 1: Life cycle of the Jewel Wasp...... 7 Figure 1.1: The venom is injected directly into the cockroach cerebral ganglia...... 11 Figure 1.2 Brain exposure for the surgical procedures ...... 16 Figure 1.3: A procaine injection to the CX decreases spontaneous walking...... 17 Figure 1.4: Representative images for postmortem verification of injection site. .... 18 Figure 1.5: A procaine injection to the MBs increases spontaneous walking...... 19 Figure 1.6. A venom injection to the CX decreases spontaneous walking...... 20 Figure 1.7: Stung crushed CirC cockroaches show decreased spontaneous walking...... 21 Figure 2.1: Proteins/peptides in the venom are necessary for the behavioral manipulation...... 31 Figure 2.2: Morphological analysis of A. compressa venom apparatus...... 32 Figure 2.3: Domains of venom proteins...... 33 Figure 2.4: Proteomic analysis of A. compressa venom...... 34 Figure 2.5: Partial list of A. compressa venom proteins with expression and abundance values...... 37 Figure 2.6: Bioinformatic analysis of the venom proteome ...... 38 Figure 2.7: Comparative genomic analysis of A. compressa venom proteins...... 39 Figure.2.8: Schematic representation of venom proteins that could be localized at the synspase...... 45 Figure 3.1: Venom affinity column revealed multiple bands in a wide range of molecular weights...... 52 Figure 3.2: The venom targets database is enriched with proteins that are associated with synaptic processes...... 54 Figure 3.3: The venom targets database is enriched with proteins that are associated with synaptic processes...... 55 Figure 3.4: Venom targets in brain show enriched terms that that are associated with cytoskeleton organization and synapse assembly...... 56 Figure 3.5: Venom targets in SEG show enriched terms that are associated with cytoskeleton organization and synapse assembly...... 57 Figure 3.6: The percentage of differentially expressed (DE) proteins in the cerebral ganglia of stung cockroaches, at the different time points after the wasps sting. .... 58

Figure 3.7: Heatmap of differentially expressed proteins in CX, 24 hours after the sting...... 61 Figure 3.8: Changes that are associated with the long term effect of the venom. .... 62 Figure 3.9: Heatmap of differentially expressed proteins in brain, 24 hours after the sting...... 63 Figure 3.10: Heatmap of differentially expressed proteins in SEG, 24 hours after the sting...... 64 Figure 3.11: Changes that are associated with the short term effect of the venom. . 64 Figure 3.12: Changes that are associated with recovery from venom effect...... 67 Figure 3.13: The highest count of enriched Go terms is found 24 hours after the sting...... 67 Figure 3.14: Functional enrichment analysis of the differentially expressed proteins reveals common and unique processes for the different time point...... 69 Figure 3.15: Recovered cockroaches are immune to the wasp sting...... 70 Figure 3.16: Representation of the venom’s molecular targets at the synapse...... 73 Figure 3.17: Representation of differentially expressed proteins in the CX of stung cockroaches...... 77 Figure 4: An integrated model for the molecular mechanisms underlying the venom- induced behavioral manipulation...... 81

Table1. Summary of the mass spectrometry identification of venom targets...... 53 Table 2. Summary of the number of identified differentially expressed proteins at different time points after the wasp’s sting ...... 58

List of abbreviations

SEG: sub-esophageal ganglion

CirC: circumesophageal connectives

CX: central complex

MBs: mushroom bodies

MudPIT: multiple dimension Protein Identification Technology

MS: mass spectrometry

ORFs: open reading frames

VG: venom gland

VS: venom sac

OBCAM: Opioid-binding protein/cell adhesion molecule

Ig: immunoglobulin

CAM: cell adhesion molecules

NHS: N-hydroxysuccinimide

BLAST: Basic Local Alignment Search Tool

GO: Gene Ontology

DAVID: The Database for Annotation, Visualization and Integrated Discovery

GEF: Guanine nucleotide exchange factor

GAP: GTPase-activating protein

PICK1: Protein interacting with C kinase 1

Abstract The Jewel Wasp (Ampulex compressa) stings cockroaches and injects venom into their cerebral ganglia, namely, the subeosophageal ganglion (SEG) and supraeosophageal ganglion (brain). Most of the venom in the brain of cockroaches is primarily concentrated in and around the central complex (CX), a region in the cockroach brain which is important for the initiation and maintenance of walking. In order to provide a fresh food supply for its offspring, the wasp venom induces a long-lasting sedentary condition in the cockroach, termed “hypokinesia”. In that respect, the Jewel Wasp sting is unusual compared to the vast majority of parasitoid wasps, whose stings completely paralyze the host. However, viewed more broadly in the world of host-parasitoid interactions, many types of behavioral alterations are induced in the host for benefit of the parasitoid. Neuro-parasitology, an emerging branch of science, deals with parasites that can control the nervous system of the host. The ability of parasites to alter the behavior of their hosts has recently generated a great interest in both scientists and non- scientists. Yet very little is known about the precise molecular mechanisms underlying such behavioral manipulations.

In the work described here, I show that venom injection to either the SEG or the CX of the brain is, alone, sufficient to decrease walking, at least for the short term.

Next, I show that the proteins in the venom are necessary for the behavioral change in the cockroach and that the venom contains a complex mixture of many proteins, enzymes and peptides. Notable among those are proteases, phospholipases, ampulexins, tachykinins, along with a large number of novel components. This suggests that the wasp uses the venom to mount a multi-pronged neurochemical attack on the cockroach cerebral ganglia.

In addition, since behavioral changes induced by the sting are long lasting and reversible, I hypothesized that long-term effects of the venom must be mediated by up or down regulation of cerebral ganglia proteins. My results show that the venom binds to synaptic proteins and therefore likely interferes with synaptic processes. I show that numerous proteins are differentially expressed in cerebral ganglia of stung cockroaches, many of which are involved in signal transduction pathways such as the Rho GTPase pathway, which is implicated in synaptic plasticity. Altogether, my data suggest that venom could affect many pathways, causing changes in synaptic efficacy in cockroach

4 cerebral ganglia to induce behavioral changes that benefit the wasp. My findings establish the groundwork for suggesting and testing mechanisms which underlie the behavioral manipulation.

Key words: Wasp; Cockroach; Neuroethology; Parasitoid; Neuro-parasitology; Venom; Central complex; Synaptic efficacy

General Introduction Neuro-parasitology is a field of biology that deals with how parasite manipulate the nervous system of their host to their benefit. Viruses, macroscopic worms and insects (notably, parasitic wasps) have evolved this ability to manipulate behavior. Most of the known manipulated hosts are insects, which is not surprising considering that most animals are insects1-3. For example, in some of the fascinating parasitic behavioral manipulations, some parasite can induce a suicidal behavior in their host. Crickets can be infected by the Nematomorph hairworm through feeding, which then develops inside the host and changes its responses to water. Instead of avoiding water, as crickets usually do, they in fact, seek or are attracted to water. This water seeking behavior is a suicidal behavior for the cricket: the infected insects jump into an aquatic environment to allow for emergence of the adult worm, while the cricket cannot survive in the aquatic environment4, 5. In another fascinating example, a parasitic wasp manipulates the behavior of caterpillar and uses it as a bodyguard for its offspring. First, the wasp stings and injects eggs into the caterpillar. The caterpillar quickly recovers from the attack and resumes feeding. The wasp larvae mature by feeding on the host, and after two weeks, up to 80 fully grown larvae emerge from the host prior to pupation. One or two larvae remain within the caterpillar while their siblings perforate the caterpillar body and begin to pupate. After emergence of the larval wasps to pupate, the remaining larvae take control of the caterpillar behavior, causing the host to snap its upper body back and forth violently, deterring predators and protecting their pupating siblings. This bodyguard behavior of the wasp offspring results in a reduction in mortality of the parasitic wasp pupae6. Many other examples are known in nature, however, the best understood example is wasp-cockroach association. The parasitoid Jewel wasp (Ampulex compressa) hunts and stings cockroaches (Periplaneta americana) to use them as a live fresh food supply for its offspring (Fig.1). The wasp first stings the cockroach in thorax and induces a transient paralysis of the front legs (2-3 minutes). The temporally paralyzed legs allow the wasp to execute a second precise sting into the cockroach head. This time, the Jewel wasp venom does not simply paralyze the cockroach. Instead, the venom causes specific changes in the cockroach behavior 7. After the cockroach is stung, it grooms continuously for 30 minutes and then it enters a long-term hypokinetic state, during which it does not voluntarily engage in spontaneous locomotion and escapes neither wind nor tactile stimuli. Such a behavioral manipulation allows the wasp to walk the

6 cockroach to a burrow, where it affixes its egg. The larva that hatches from the egg feeds on the cockroach hemolymph at first and then enters the cockroach abdomen to devour the cockroach’s internal organs 8,9. Eventually, the larva pupates and a fully grown wasp emerges from the body of the dead cockroach.

Figure 1: Life cycle of the Jewel Wasp. An adult wasp stings a cockroach into the head (A) to manipulate its behavior. The wasp then leads the cockroach (B) into a nest and lays an egg on its cuticle. The hatching larva (C) feeds on the cockroach, pupates inside its abdomen and emerges roughly 30 days later (D).

During the venom induced Hypokinesia, the wasp grabs the cockroach at the base of the antenna and walks the cockroach to a burrow, resembling an obedient dog pulled on a leash. This shows that the cockroach is able to walk and in effect, cockroaches can swim (walk on water) if submerged in water and fly in a wind tunnel. The venom appears to modulate descending signals from cerebral ganglia, resulting in suppression of host escape behavior and decreased spontaneous walking, without affecting other

7 behaviors. More specifically, the wasp’s sting affects the spontaneous initiation and the maintenance of walking 10-12. This specificity of behavioral modification is particularly interesting and unique among host-parasitoid interactions13, 14. Remarkably, the hypokinetic state is reversible: if egg deposition is prevented following the sting, the escape response of a stung cockroach returns to normal within days. It has been shown that the wasp injects its venom directly into the cockroach cerebral ganglia, namely, the subeosophageal ganglion (SEG) and the supraeosophageal ganglion (Brain) 15. The SEG and Brain, which are connected to each other by the circumesophageal connectives (CirC), are considered as ‘higher- order’ neuronal centers which modulate different aspects of locomotion 16-19. Since the venom is injected directly into the cerebral ganglia, the behavioral change observed in stung cockroaches must be the result of a pharmacological manipulation of neuronal circuits in these ganglia. However, prior to this study, the contribution of venom injection into each of those ganglia to the observed behavioral manipulation was still unclear. I will focus on in this subject at the first chapter, which has been published in Kaiser and Libersat, 201520. Mostly of what we know about this system involves the short term effect of the venom: the transient paralysis of the front legs and the grooming phase. The short-term effect of the venom seems to be mediated by neuro-pharmacological mechanisms. The transient paralysis of the front legs seems to be mediated by gamma-aminobutyric acid (GABA) and the grooming phase, which its purpose is unclear, is induced by dopamine. In order to understand the mechanisms underlying the long term effect of the venom, we first need to identify the peptides/proteins composition of the venom. Prior to the present study, the only known relevant components of the venom were GABA (and its receptor agonists taurine and beta-alanine) and Dopamine21, 22. However, today, nucleotide sequencing and mass spectrometry technologies have greatly facilitated protein discovery in non-model systems and allowed us to identify to venom protein components. The study of the venom protein composition is described at the second chapter, which is based on the submitted paper: "Parasitoid Jewel Wasp Mounts Multi- Pronged Neurochemical Attack to Hijack a Host Brain" (Arvidson, Kaiser, et al.)23. The same technological advances that helped us to investigate venom proteome, were used to investigate the venom proteins targets in the cockroach cerebral ganglia and changes in proteins expression in the hypokinetic cockroaches. This work, described at the third chapter, serves as a large database, and provides the first clues in understating 8 the long term effect of the venom in the cockroach cerebral ganglia. In the general discussion, I will combine all this data and propose a model for the long term behavioral manipulation.

1. The role of the cerebral ganglia in the venom-induced behavioral manipulation of cockroaches stung by the parasitoid Jewel Wasp

This chapter is based on Kaiser and Libersat, 2015 20.

1.1 Abstract The Jewel Wasp stings cockroaches and injects venom into their cerebral ganglia, namely, the subeosophageal ganglion (SEG) and supraeosophageal ganglion (brain). The venom induces a long-term hypokinetic state, during which the stung cockroach shows little or no spontaneous walking.

It was shown that venom injection to the SEG reduces neuronal activity thereby suggesting a similar effect of venom injection in the Brain. Paradoxically, Brain-ablated cockroaches show increased spontaneous walking in comparison to control. Yet, most of the venom in the brain of cockroaches is primarily concentrated in and around the central complex (CX). Thus, the venom could chiefly decrease activity in the CX to contribute to the hypokinetic state. My first aim was to resolve this discrepancy by using a combination of behavioral and neuro-pharmacological tools. My results show that the CX is necessary for the initiation of spontaneous walking and that focal injection of procaine to the CX is sufficient to induce the decrease in spontaneous walking. Furthermore, it was shown that artificial venom injection to the SEG decreases walking. Hence, my second aim was to test the interactions between the Brain and SEG in the venom-induced behavioral manipulation. I show that, in the absence of the inhibitory control of the brain on walking initiation, injection of venom in the SEG alone by the wasp is sufficient to induce the hypokinetic state. To summarize, in chapter I show that venom injection to either the SEG or the CX of the Brain is, by itself, sufficient to decrease walking, at least for the short term.

1.2 Introduction Behavioral and electrophysiological studies have demonstrated that the venom effect on locomotion is limited to walking and escape behaviors 10. Other behaviors, such as flying or righting, remain unaffected and the walking pattern generator, which locally controls the spatiotemporal motor pattern of leg movements during walking, remains

10 functional in stung cockroaches. More specifically, the wasp’s sting affects the spontaneous initiation and the maintenance of walking 10, 11 It has been shown that the wasp injects its venom directly into the cockroach cerebral ganglia (Fig. 1.1), namely, the subeosophageal ganglion (SEG) and the supraeosophageal ganglion (Brain) 15. The SEG and brain, which are connected to each other by the circumesophageal connectives (CirC), are considered as ‘higher-order’ neuronal centers which modulate different aspects of locomotion 16-19. Since the venom is injected directly into the cerebral ganglia, the behavioral change observed in stung cockroaches must be the result of a pharmacological manipulation of neuronal circuits in these ganglia. However, the contribution of venom injection into each of those ganglia to the observed behavioral manipulation is still unclear.

Figure 1.1: The venom is injected directly into the cockroach cerebral ganglia, namely, the SEG and the Brain. (A) Autoradiographs of the Brain and SEG of a cockroach stung by a radio-labelled wasp. Black staining indicates the presence of venom 15. (B) Schematic representation of the cerebral ganglia. Different colors marks different regions/neuropils as described in the legend.

Based on experiments performed on the SEG, venom injected into the brain, almost certainly decreases neuronal activity there12. Paradoxically, brain-ablated cockroaches show increased spontaneous walking in comparison to control cockroaches 24-26 indicating that the brain has a general inhibitory role in the control of walking. While

11 the entire brain has a general inhibitory role, it is still possible that the wasp targets sub- regions in the brain. Most of the venom injected by the wasp into the brain is predominantly concentrated in its central part of the brain 15, especially in and around the central complex (CX; also know previously as CC 27). Thus, the venom could affect primarily this region to contribute to the hypokinetic state, leaving other regions unaffected. In fact, the CX is a target of choice for the wasp’s venom, as many studies suggest that the CX is important for the initiation and maintenance of walking 17, 28-32. In addition, studies carried out on the fruit fly suggest that the CX has a permissive role on the control of walking, whereas the mushroom bodies (MBs), a bilateral region in the brain, have a suppressive effect on walking 33. I suggest that the venom injected into the brain could affect mainly the CX to decrease spontaneous walking. To test this hypothesis and resolve the aforementioned paradox, I first performed a series of experiments in which I injected compounds into discrete regions of the brain and evaluated the resulting behavioral deficits.

The SEG, anatomically, is the second cerebral ganglion, located below and connected to the brain through the circumesophageal connectives (CirC). A previous study has shown that injection of procaine (a reversible voltage-dependent sodium and potassium channels blocker) into the honey bee brain 34, or into the SEG of cockroaches 12 reduces neuronal activity in these ganglia. In the latter, such an injection results in a decrease in spontaneous walking. Likewise, venom injections to the SEG also decrease neuronal activity and, consequently, decrease spontaneous locomotion 12. Thus, this study and others show that the SEG exerts a net tonic permissive effect in the control of initiation and maintenance of walking 19, 25, 35. Hence, the second aim of the present study was to examine the contribution of each cerebral ganglion to the manipulation of walking. To this aim, I performed a series of experiments involving lesions and natural venom injection by wasps into the cerebral ganglia.

My results show that the CX is predominantly permissive for the initiation of spontaneous walking and that its role in the control of spontaneous walking is antagonistic to that of the MBs. I show that a focal injection of venom or procaine to the CX is sufficient to induce the decrease in spontaneous walking. However, I also show that when the wasp stings a cockroach with a disabled brain but a normally 12 functioning SEG, the cockroach shows decreased spontaneous locomotion. Thus, venom in each of the two cerebral ganglia is sufficient to induce a decrease in spontaneous walking, as seen in stung hypokinetic cockroaches.

1.3 Material and Methods Animals Ampulex compressa Fabricius (Hymenoptera: Ampulicidae) wasps and Periplaneta americana cockroaches were reared in crowded colonies under laboratory conditions of 40–60% humidity, 30°C and a 12L:12D cycle. All animals were supplied with water and food (cat chow for cockroaches and honey for wasps) ad libitum. To obtain stung cockroaches, a single cockroach was introduced to a wasp and the stinging duration was measured to ensure normal stinging behavior 7.

Venom milking Wasps were immobilized by chilling on ice for 5 min and were confined in a small, conical, plastic tube open at both ends. A modified syringe plunger was fit to one end of the tube and was used to provoke the wasp to sting a small piece of Parafilm held in front of the other end. Venom droplets were collected from the distal side of the Parafilm with a nanovolumetric injector (NVI-570A/V, Medical Systems, Greenvale, NY). From each wasp, approximately 10 nl of crude venom were collected to a glass micropipette containing 10 nl saline.

Pharmacology Procaine was freshly prepared and dissolved to a concentration of 500 mg/ml in vehicle containing cockroach saline (composition in mM: NaCl 214, KCl 3.1, CaCl2 9, Sucrose 50, HEPES 5, pH 7.236) and 0.1% Janus Green dye. Venom was freshly milked and dissolved 1:1 in cockroach saline containing 0.1% Janus Green dye. For injections into both CX and SEG, venom was freshly milked and kept in ice until and between injections.

Injections and surgical procedures General Prior to the procedures, cockroaches were anesthetized with carbon dioxide and immobilized dorsal side up with modeling clay on a wax platform. A staple-shaped pin

13 was softly pressed against the neck to regulate hemolymph flow to the head during the procedure. A U-shaped incision was then performed to open a small flap between the ocelli. Then, I either injected various compounds in specific regions of the brain or performed a surgical crush of the CirC. After the procedure, the cuticular incisions were sealed with wax. Procedures: 1. Micro-injection: A nanovolumetric injector (NVI-570A/V, Medical Systems, Greenvale, NY, USA). was used to deliver solutions directly into the CX or MB, or SEG. Using physical landmarks (Fig 1.2, arrow mark), a glass needle was inserted to the specific location and the solution was injected. For two groups of cockroaches, I used procaine, a reversible voltage-dependent sodium and potassium channels blocker, which eliminate neuronal activity in the injected area as shown by Devaud 34 and by Gal and Libersat 12. 'Procaine-CX' cockroaches were injected with 10 nl of procaine into the CX; 'Procaine-MB' cockroaches were injected with 20 nl of procaine (bilateral injection) into the MB; and 'Venom-CX' cockroaches were injected with 20 nl of venom diluted in saline into the CX. Control cockroaches were injected with saline containing 0.1% Janus Green dye (10-20 nl) to the CX or MBs. The effect of procaine injection on neuronal activity is known to be restricted to the injection site 37. 2. Surgical crush of the CirC: Two groups of cockroaches were prepared as follows. For the first group, the CirC were gently crushed at first (carefully to not cut the connectives and to avoid damage to surrounding tissue) and afterward the cockroach was introduced to a wasp for a sting (‘crushed CirC - stung’). For the second group, the CirC were crushed after the cockroach was stung by a wasp (‘Stung - crushed CirC’). The experimental timeline is explained in ‘behavioral assays’.

Behavioral assays 1. Micro-injections: Behavioral assays were performed on freely-moving cockroaches in an open-field arena (radius = 30 cm). Spontaneous walking was quantified with a stopwatch in continuous 10 minute bins. Baseline walking duration was measured prior to the nano-injections procedure. After the procedure, the cockroach was given 10 minutes to recover. Then, spontaneous walking was again recorded continuously for one hour. 14

2. Surgical crush of the CirC: Freely-moving 'Brain-ablated' cockroaches in an open-field arena have a tendency to collide with the arena wall and often get turned on their back. This could affect the spontaneous walking measurements. Since 'Crushed CirC' cockroaches were expected to behave similar to 'Brain- ablated' cockroaches, behavioral assays were performed on tethered cockroaches walking on an oiled glass plate. In such a fixed position, cockroaches are able to move their legs in short bouts of stationary walking or running. The walking and escape movements are similar to those of free ranging animals 38. Spontaneous walking was measured in 10 minute bins in three time points. For both groups, the cockroaches were first tethered and let to acclimate for 20 minutes. Afterwards, baseline walking duration was measured. Following the baseline measurement, the protocol differed for both groups: for ‘Stung - crushed CirC’ cockroaches, the second time point (70 min) was an hour after the cockroach was stung by a wasp. After the second time point, the CirC were crushed (see Injections and surgical procedures) and the third time point (140 min) was an hour afterwards. For ‘Crushed CirC - stung’ cockroaches, the second time point (70 min) was an hour after the CirC were crushed. After the second time point, the cockroach was stung by a wasp and the third time point (140 min) was an hour afterwards.

Statistical analysis For the different nano-injections into the CX and MB treatments, the data passed equal variance (P = 0.207, Brown-Forsythe method) and normal distribution (P = 0.378, Shapiro-Wilk method) tests. I used a Two-way Repeated Measures ANOVA with Treatment as the between-subject factor and Time as the within-subject factor, to compare spontaneous walking between different treatment groups (n=22, n=8, n=8 and n=8 cockroaches in the Control, 'Procaine-CX', 'Procaine-MB' and 'Venom-CX' groups, respectively). All pairwise multiple comparison procedures were tested using Student- Newman-Keuls Method. For ‘Crushed CirC - stung’ and ‘Stung - crushed CirC’ cockroaches, the data passed equal variance (P = 0.230, Brown-Forsythe method) and normal distribution (P = 0.268, Shapiro-Wilk method) tests. I used a Two Way Repeated Measures ANOVA with a Student-Newman-Keuls post-hoc test to identify differences in time and treatment between the groups (n=8 for each group). 15

Postmortem verification of the injection site After the behavioral assay, the cockroach head was removed and fixed overnight in formalin. Then, the brain was removed from the head, embedded in 6% agarose and sliced (60 µm) with a vibratome. The location of the Janus Green tracer was used to verify the exact injection site.

Figure 1.2 Brain exposure for the surgical procedures. Red arrow shows the CX injections point, marked by the intersection of the main surface tracheae. Blue arrows show the injection point for MB injections.

1.4 Results A procaine injection to the CX decreases spontaneous walking First, to determine the role that the CX plays in the control of spontaneous walking, I focally injected procaine to the CX to reversibly decrease neuronal activity specifically in this region 12, 34, 37. Compared to the baseline walking duration, cockroaches that were injected with procaine to the CX walked significantly less, if at all, for 30 minutes after the injections (p<0.05; Fig. 1.2). After this time period, the walking duration returned to baseline, as expected given the reversible nature of procaine. In addition, as compared to Control cockroaches, the walking duration was significantly lower in the ‘Procaine-CX’ cockroaches, especially 20 and 30 min after injections (p=0.013 and

16 p=0.009, respectively; Fig. 1.2). Postmortem verification of the injection site confirmed that the injections were focused in the central body of the CX (Fig. 1.3). These results show that procaine injections to the CX have an inhibitory effect on walking.

Figure 1.3: A procaine injection to the CX decreases spontaneous walking. Spontaneous walking duration measured in seconds, in 10 minute bins, for baseline walking and for a continuous hour after nano-injection treatments. Data points represent mean ± s.e.m. Data points with same capital letter are not significantly different, whereas data points with different letters are significantly different (p<0.05). Asterisks represent significant difference between treated and control groups (*=p<0.05). n=22 and n=8 cockroaches in Control and 'Procaine-CX' groups, respectively.

Figure 1.4: Representative images for postmortem verification of injection site. In the center, a schematic drawing showing the general anatomy of the brain 39. Darker spots in the drawing indicate the injections site as shown in the representative images (bottom and top). Bottom photograph shows the injection site in the central body of the Central Complex and top photograph shows the injections sites in the Calyces of the Mushroom Bodies. CX: central complex, Ca.: Calyces of the Mushroom bodies. The all area of the mushroom bodies is marked in yellow.

A procaine injection to the MBs increases spontaneous walking I then tested the effect of a bilateral procaine injection into the MBs, which are also known to be involved in walking. Compared to baseline walking duration, cockroaches that were injected with procaine to the MBs walked significantly more for 30 minutes after the injections (p<0.005; Fig. 1.4). Likewise, compared to Control cockroaches, the walking duration was significantly higher in the ‘Procaine-MBs’ cockroaches for 30 min after the injection (p<0.001; Fig. 1.4). As expected, the walking duration returned to baseline 30 min following the injections. Postmortem verification of the injection site confirmed the location of the injections to the MBs (Calyces region, Fig. 1.3). These results show that the procaine injections to MBs have an opposite effect to procaine

18 injections to CX, and that these two regions in the brain appear to have antagonistic roles in the control of initiation of walking.

Figure 1.5: A procaine injection to the MBs increases spontaneous walking. Spontaneous walking duration measured in seconds, in 10 minute bins, for baseline walking and for a continuous hour after nano-injection treatments. Data points represent mean ± s.e.m. Data points with same capital letter are not significantly different, whereas data points with different letters are significantly different (p<0.05). Asterisks represent significant difference between treated and control groups (***=p<0.001). n=22 and n=8 cockroaches in Control and 'Procaine-MBs' groups, respectively.

A venom injection to the CX decreases spontaneous walking Most of the venom injected to the brain is concentrated in or around the CX. This means that the wasp could target specifically the CX to decrease spontaneous walking in the cockroach. If true, then venom injection to the CX should have a comparable effect to procaine injections to the CX, and should decrease spontaneous walking. To test my hypothesis, I focally injected freshly milked venom to CX. Compared to the baseline walking duration, cockroaches injected with venom to the CX walked significantly less for the entire duration of the behavioral test (p<0.05; Fig. 1.5). Likewise, compared to Control cockroaches, the walking duration was lower in the ‘Venom-CX’ injected cockroaches throughout the entire duration of behavioral testing

(p<0.05, 20-60 min; Fig. 1.5). Postmortem verification of the injection site confirmed the location of the injections to the central body in the CX (not shown). These results show that venom injection to the CX alone is sufficient to decrease spontaneous walking.

Figure 1.6. A venom injection to the CX decreases spontaneous walking. Spontaneous walking duration measured in seconds, in 10 minute bins, for baseline walking and for a continuous hour after nano-injection treatments. Data points represent mean ± s.e.m. Data points with same capital letter are not significantly different, whereas data points with different letters are significantly different (p<0.05). Asterisks represent significant difference between treated and control groups (*=p<0.05, **=p<0.01). n=22 and n=8 cockroaches in Control and 'Venom-CX' groups, respectively.

Stung crushed CirC cockroaches show decreased spontaneous walking. To examine the contribution of each cerebral ganglion to the drastic effect of the sting on walking, I performed another series of experiments. Although it has been shown that venom injection to SEG is, in itself, sufficient to decrease spontaneous walking, it is not known whether a natural direct injection by the wasp to cockroaches with only SEG will be sufficient to decrease spontaneous walking. If this was the case, the effect of venom in the SEG will need to overcome the increased walking observed previously in brain ablated cockroaches 24.

To investigate this possibility, I measured spontaneous walking in two groups: ‘Crushed CirC - stung’ cockroaches and ‘Stung - crushed CirC’ cockroaches. In the first group, the CirC were crushed before the cockroach was stung whereas in the second group, the CirC were crushed after the cockroach was stung. By crushing the CirC I removed descending inhibitory control of the brain on walking (Gal and Libersat 2006). I expected that, if venom in the SEG is sufficient to decrease spontaneous walking, then crushing the CirC before or after the sting would not change the effect of the sting and the cockroach will be hypokinetic in either one of the treatments. As expected, for ‘Crushed CirC - stung’, there was a significant increase in walking duration after surgery (70min, p<0.001; Fig. 1.6) compared to baseline. Following the sting, the walking duration dramatically decreased (140 min, p<0.001; Fig. 1.6), similar to when a wasp stung a normal cockroach (‘Stung - crushed CirC’, 70 min, p<0.001 compare to baseline; Fig. 1.6). Crushing the CirC after the sting (‘Stung - crushed CirC’ group) did not affect the walking duration of stung cockroaches as they remained hypokinetic (140 min; Fig. 1.6). These results show that venom injection to the SEG alone is sufficient to induce a decrease in spontaneous walking and to overcome the increased walking of brain-ablated cockroaches.

Figure 1.7: Stung crushed CirC cockroaches show decreased spontaneous walking. Spontaneous walking duration measured in seconds, in 10 minute bins in 3 time points: baseline (before treatment), 70 min (an hour after the first treatment) and 140 min (an hour the after second treatment). For ‘Crushed CirC stung’ cockroaches, the first treatment was crushing the CirC and the second one was stinging by a wasp. For ‘Stung Crushed CirC’ cockroaches, the first treatment was stinging by a wasp and the second one was crushing the CirC. Data points represent mean + s.e.m. Data points with same capital letter are not significantly different, whereas data points with different letters

21 are significantly different (p<0.05). Asterisks represents significant difference between treatments (***=p<0.001). n=8 cockroaches in each treatment.

1.5 Discussion The parasitoid Jewel Wasp stings cockroaches and injects venom directly into their cerebral ganglia, namely, into the SEG and the Brain 15. While venom or procaine injection to the SEG alone decreases spontaneous walking, venom or procaine injection to the entire brain paradoxically increases spontaneous walking 12. Yet, the wasp does not inject venom into the entire brain but, rather, mostly into a discrete region of the brain known as the CX 15. Hence, the goal of the present study was to resolve this paradox by using behavioral, surgical and neuro-pharmacological methods.

I show here that procaine focally injected to the CX dramatically decreased spontaneous walking in cockroach; in fact, most cockroaches did not show any spontaneous walking (Fig. 1.2). This clearly indicates that the CX is permissive for the initiation of spontaneous walking. My result is consistent with work carried out on Drosophila melanogaster that shows that flies with genetically manipulated neurons or structural mutations of the CX demonstrate shorter activity duration, lower walking speed, decreased levels of locomotory activity and changes in walking time intervals 18, 28, 30, 40-43. It is also in good agreement with experiments showing that current injection in the CX can elicit walking in crickets 32 and cockroaches 29. While most CX units show an increase in their firing rate preceding the initiation of locomotion some show a decrease indicating that the CX is predominantly permissive on walking 44. In a recently published study, injection of procaine into the CX of Blaberus discoidalis has been performed, to investigate its role on optomotor behavior (Kathman, et al. 2014). They found deficits in the optomotor behavior after procaine injection but no significant change in general activity (i.e. walking). I can only speculate as to what could account for such a difference. First, it is possible that size matters as Blaberus CX is much larger then Periplaneta CX. A more convincing argument would be a simple concentration effect. In the present study, I used high concentration solution of procaine (50% versus 20% and 10% in Kathman’s study) and a larger volume of diluted procaine (10 nl in the present study versus 2 nl in Kathman’s study). Hence, I expect that in the Blaberus study, the effect is limited to a smaller portion of the CX whereas in the present study, the effect results from a more complete inhibition of the entire CX.

While the CX is at least necessary for spontaneous walking, the entire brain in general has a suppressive role in the control of walking 24. The cockroach’s head ganglia play a major role in initiating and maintaining the walking pattern generated in the thoracic ganglia 16, 24. It was shown previously that the brain inhibits walking, whereas the SEG exerts the opposite effect 12, 24. This suggests the existence of other regions within the brain that exert inhibitory control on walking initiation. As already suggested by studies on Drosophila 33, I hypothesized that this observed suppressive effect of brain removal is a result of the inhibitory influence of the MBs on walking. My results showed that the procaine injections to the MBs have the opposite effect on walking to that of injections to the CX. A procaine injection to the MBs reversibly increases spontaneous walking. My results are in agreement with other studies that suggest that these two major 'higher' brain centers of the brain have an antagonistic role in the control of spontaneous walking 18, 32, 33, 45, 46.

As it was shown before that the venom decreases neuronal activity similarly to procaine 12, I focally injected venom to the CX to mimic the wasp’s venom injection in this discrete region. I show that an accurate venom injection to CX alone is sufficient to decrease spontaneous walking. Thus, the Jewel Wasp's venom injection to brain must be fairly accurate in order to impact only the CX, without affecting the MBs, to achieve the desired behavioral manipulation.

Next, in the present study, I also addressed the role of the venom in each ganglion in the venom-induced hypokinesia. Since it not possible to coerce the wasp to sting in the SEG alone and prevent it from stinging in the brain, I used the following approaches. I surgically crushed the CirC of normal cockroaches or of cockroaches that were already stung by a wasp, thereby removing the inhibitory input from the brain. Those 'Crushes CirC' cockroaches tend to be more active due to the removal of inhibition. When these cockroaches were stung by the wasp, their spontaneous walking significantly decreased comparably to the effect of the sting in both ganglia of normal cockroaches. Crushing the CirC after the sting did not change the spontaneous walking of the cockroaches as they remained hypokinetic. These experiments show that the venom injection into the SEG is sufficient to decrease spontaneous walking, and that the venom’s effect in the SEG is sufficient to ‘override’ the effect of the ablation of the Brain. Thus, the Jewel Wasp injects venom to both cerebral ganglia, while venom injection to either one would be sufficient to cause a decrease in spontaneous walking in the cockroach, at least for 23 the short term. It is only possible to speculate about the adaptive significance of such a double venom injection, where one alone would be sufficient to induce hypokinesia. It is possible that the venom injection to both ganglia is a kind of ‘insurance policy’, in which the wasp maximizes the odds that the cockroach will remain hypokinetic. Indeed, if the wasp were to fail to sting properly in the brain, it would still achieve its goal. Similar ‘insurance’ mechanisms can be found in other venomous organisms, such as the cone snail that produces different types of conotoxins, each of which should, in principle, be sufficient for blocking the neuro-muscular junction ensuring paralysis of the prey 47. Another, yet not exclusive, alternative is that the injection in the central portion of the brain is involved in other aspects of the manipulation. For instance, an important aspect of the manipulation is the modification of the cockroach’s metabolism 9. Venom in the brain could slow down the metabolism in the stung cockroach by affecting, directly or indirectly, neuro-peptidergic modulation in the CX 48, 49. Another possibility is that the venom in the brain is involved in the stereotypic grooming behavior of stung cockroaches. Indeed, injection of dopamine, which is present in the venom 22, of dopamine agonists 50, or of venom (data not shown) in the central portion of the brain induces intense grooming 22.

To summarize, in the present study, I was able to demonstrate the role of different regions in the brain in the control of spontaneous walking by using the wasp–cockroach association as a model. I have shown that the venom injection to each of the cerebral ganglia is, by itself, sufficient to decrease spontaneous walking, as observed in stung cockroaches. My work also provides further insights into the interplay between the brain and the SEG of insects and their role in the processes of motivation and decision to engage in a behavioral act.

2. Parasitoid Jewel Wasp Mounts Multi-Pronged Neurochemical Attack to Hijack a Host Brain

This chapter is based on the published paper: "Parasitoid Jewel Wasp Mounts Multi- Pronged Neurochemical Attack to Hijack a Host Brain" (Arvidson, Kaiser, et al.23).

2.1 Abstract In this chapter I show that proteins in the venom are necessary for the behavioral change in the cockroach. Second, to understand biochemical bases underlying venom-induced hypokinesia, we used proteomics based on transcriptomics analyses to construct a comprehensive “venome”. The venome contains a complex mixture of enzymes, neurotransmitters and peptides, including M13 family metalloproteases, phospholipases, adenosine deaminase, ampulexins, tachykinins, corazonin, and eclosion hormone, along with a large number of novel components. Also included are members of the Toll signaling pathway, including Persephone, Snake, Easter and Spätzle. Our findings deepen mechanistic understanding of the host-parasitoid interaction and in particular neuromodulatory mechanisms that alter host behavior for the benefit of opportunistic parasites and parasitoids.

2.2 Introduction Venom-induced hypokinesia raises an interesting biological question: How can such a biochemical cocktail causes such a long-lasting, specific, and yet reversible effects on behavior? To address this question, we generated a comprehensive A. compressa venome to relate the biochemical composition of the venom to hypokinesia induction. A pilot analysis of the wasp's venom showed it consists of a cocktail of proteins/peptides and non-peptides components, similar to other known venoms 21, 51, 52. This analysis revealed biogenic amines, such as dopamine (or a dopamine like compound) in venom and the presence of GABA (and its receptor agonists taurine and beta-alanine). In addition, it was shown that the venom contains a protein component with pore forming activity in artificial lipid bilayers52. Recently, novel α-helical, amphipathic venom peptides were identified and termed ampulexins. However, their function, target or contribution to the behavioral manipulation is still unknown53. Venomous animals extend throughout the animal kingdom, producing a variety of toxin proteins and peptides, mostly for defense or prey capture. The venoms of scorpions, snakes, spiders, cone snails and other venomous animals have been and are the subject

25 of countless of studies, demonstrating an exquisite form of natural selection in developing a lethal cocktail of protein and peptides47, 54-56.The study of Hymenopterans venom has been especially focused on social Hymenoptera such as bees, ants and social wasps14, 57-59. In contrast to other venomous animals, parasitoid wasps use venom for reproduction and completion of their parasitic life cycle. The venom of parasitoid wasps has evolved, not to simply paralyze or kill the prey, but to specifically affect host metabolism, developmental arrest, immune suppression and in some cases to affect behavior. One limitation for studying of the venom of parasitoid wasps, is their relativity small size, which leads to a limited amount of venom that could be collected at a reasonable time. However, in the recent years advances in nucleotide sequencing and mass spectrometry technologies have greatly facilitated protein discovery in non- model systems, allowing the use of relatively low amounts of proteins and thus, advancing the field of venomics58, 60. The parasitoid wasp, Nasonia vitripennis, was the first parasitoid with a sequenced genome, which has become an efficient genetic model61-63. N. vitripennis feeds and lays eggs on Diptera pupae and eventually leads to arrested or delayed development in the envenomated hosts. Using Mass spectrometry techniques 79 proteins were identified in N. vitripennis venom, comprising the most complete ectoparasitoid venom data 61, 63. Accumulating studies have shown the parasitoid wasp venom contain a complex mixture of proteins, polydnaviruses (PDVs), virus-like particles (VLPs), microRNAs, small molecules, and ovarian fluids. However, there are still a limited number of parasitoid venom analyses currently available. So far, venom proteins of 17 parasitoid wasp species have been examined64, although comprehensive proteomic analysis has been undertaken in only seven species61, 65-70. In this study, transcriptomes and differential expression analysis of the venom apparatus were generated de novo, using Illumina short read sequencing and the Trinity pipeline71, 72. This analysis serves two purposes: 1) Expression profiles of each glandular tissue reveal its specialization within the venom apparatus, and the location where each venom component is expressed, and 2) Protein coding sequences extracted from the transcriptome assembly serve as a custom database for mass spectrometry- based proteomics. The proteomics approach, coined MudPIT (Multiple dimension Protein Identification Technology), has been used to profile complex proteomes, including venoms73-75.

While the biochemical basis of venom-induced hypokinesia remains obscure, the venom proteome elucidated here has generated new hypotheses for functional analysis of the means by which A. compressa manipulates host behavior to its own advantage.

2.3 Material and Methods Animals A. compressa and P. americana were reared as described in chapter 1.

Venom and protease injections

Venom was collected as mentioned in chapter 1. The milked venom was diluted 1:1 with PBS alone or with PBS containing proteinase K (300µg/ml) and kept for one hour at room temperature. Behavioral observations for baseline responses were recorded before treatment. Then, using a Nano injector I injected 30 nl of either venom alone or venom with protease into the CX of the cockroach brain (n=4 for each group). Following the injection, the cockroach was given 10 minutes to recover and the behavioral effects were observed and quantified.

Behavioral assays

Behavioral assays were performed on freely-moving cockroaches in an open-field arena (radius = 30 cm). Spontaneous walking duration was quantified with a stopwatch for a continuous 10-minutes period before and after treatment (after 10 min recovery). The escape response distance was measured in response to a tactile stimulus after the spontaneous walking observations. Cockroaches were placed at the center of the arena and were given a tactile stimulus by a gentle touch of a brush. This test was repeated 3 times in interval of one minute between each stimulus and the escape distance was measured and averaged for each cockroach.

Statistical analysis

For both groups of cockroaches after venom injection and venom with protease injection, the comparison was done for after and before treatment, for both spontaneous walking and escape response. For comparing spontaneous walking after venom or venom/protease injection, the data passed normality test (Shapiro-Wilk, P = 0.939 and P=0.369) and paired t-test was used to test for significant different before and after the treatment. For comparing escape responses after venom or venom/protease injection,

Wilcoxon signed-rank test was used since the data for both groups did not pass the normality test.

Venom sac contraction In order to test if the venom sac or venom gland is contractile, venom sacs and glands were dissected out of the wasp abdomen and immersed in saline (130 mM NaCl, 5 mM KCl, 4 mM MgCl2, 5 mM CaCl2, 15 mM HEPES, 25 mM glucose, 160 mM sucrose, pH 7.276) containing high concentration of potassium (150mM KCl) or calcium ionophore, ionomycin (50 µM). The contraction of the venom sac was observed under a binocular.

RNA extraction and Sequencing Venom sacs and venom glands were dissected from nine wasps and pooled into two biological replicates of each tissue type. RNA was extracted from each tissue using the Trizol method (Invitrogen), and quality was assessed on an Agilent 2100 Bioanalyzer. Sequencing libraries were generated and multiplexed using the Illumina TruSeq RNA Library Preparation Kit, according to manufacturer’s instructions. All four libraries were combined and sequenced on the Illumina HiSeq 2000 platform in the Institute for Integrative Genome Biology at UC Riverside (IIGB). Sequencing data from each sample were combined and assembled using the Trinity software suite with the CuffFly and extended lock options, and a k-mer overlap of 2, to minimize spurious isoforms. RSEM and Deseq2 plugins for Trinity were used to quantify transcripts and calculate differential expression between tissue types, respectively77, 78. The Transdecoder plugin for Trinity was used to extract putative ORFs with a minimum length of 30 amino acids72. The ORF database (896984 sequences) generated with Transdecoder was used for MudPIT. All computational analyses were performed on the IIGB Linux Cluster.

Sodium dodecyl sulfate -Polyacrylamide gel electrophoresis (SDS-PAGE) Proteins were separated by TRIS-Tricine SDS-PAGE on a 16.5% gel (BioRad) with 20 µg protein in each lane at a constant 50 volts and stained with AcquaStain Protein Gel Stain (Bulldog Bio). Precision Plus Protein Dual Xtra Prestained Protein Standards were used as a reference (BioRad). Mass spectrometry sample preparation

Venom was milked from adult female A. compressa as described previously19. In brief: CO2 anesthetized wasps were placed into a modified P1000 tip with the abdomen protruding from the tip, covered with parafilm, and allowed to recover. Wasps were aggravated to sting through the parafilm and venom drops were absorbed into 5 µl of deionized water, frozen on dry ice, and stored at -80°C until processed. For analysis by mass spectrometry, 1000 sting equivalents of SepPak-purified milked venom protein were split into two samples, one of which was subjected to standard trypsin digestion before analysis, while the other was analyzed without protease treatment. For assays involving identification of mature signaling peptides, ~100 sting equivalents were analyzed without protease treatment per sample.

MudPIT Nano-UPLC-MS/MS analysis and protein identification A total of four trypsinized samples and three samples without protease treatment were analyzed. All samples were desalted using C18 Zip Tip (Harvard) or C18 SepPak (Waters) cartridges, dried and resuspended in 0.1% formic acid. Two trypsinized samples and two samples without protease treatment were analyzed at the Smoler Proteomics Center at the Technion Israel Institute of Technology via reverse-phase liquid chromatography on 0.075 X 180-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material and analyzed on a Q-Exactive plus mass spectrometer (Thermo). Two trypsinized samples and one sample without protease treatment were analyzed at the Institute of Integrative Genome Biology at the University of California, Riverside as described previously79, 80. Peptides were separated using two-dimensional nanoAcquity UPLC (Waters) and analyzed with an Orbitrap Fusion mass spectrometer (Thermo Fisher). All raw MS data were processed with Proteome Discoverer version 1.4 (Thermo Fisher, San Jose, CA) to generate .mgf files that were used in Mascot searches (version 2.5) against a custom ORF database. All searches were performed with the following settings: peptide mass tolerance: ± 10 ppm, fragment mass tolerance: ± 0.3 Da, Variable modifications: Acetyl (N-term), Amidated (C-term), Formyl (N-term), Gln-> pyro-Glu (N-term Q), Glu-> pyro-Glu (N-term E), Oxidation (M), with 1 max missed trypsin cleavages for trypsinized samples. Spectra were accepted for the venom samples if the MASCOT score of the identified protein was greater than the MASCOT score that corresponds to a false discovery rate (FDR) of 1% against a reversed-decoy database. The mass spectrometry proteomics data have been deposited with the

ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD00634081. Relative protein abundance was calculated using an exponentially modified protein abundance index (emPAI) – as protein concentration is proportional to the logarithm of the protein abundance index (ratio of observed to observable peptides). For relative abundance estimation of processed neuropeptides, protein spectral counts (PSM) were used.

Protein annotation ORFs identified as venom proteins via MudPIT were assessed for predicted secretory signals by SignalP 4.1. Molecular mass and isoelectric points for ORFs were calculated by ExPASy Compute pI/Mw tool (web.expasy.org/compute_pi/) with secretory signals removed from those sequences for which they were predicted. ORFs were searched against NCBI-nr, Uniprot and PfamA databases using standalone BLAST (Basic Local Alignment Search Tool) 2.2.30+, hmmscan or phmmer (Hmmer 3.0) where indicated.

Comparative genomic analysis Protein sequences from genomes of Nasonia vitripennis, Solenopsis invicta, Pogonomyrmex barbatus, Linepithema humile, Harpegnathos saltator, Acromyrmex echinatoir, Cardiocondyla obsucior, Atta cephalotes, Bombus impatiens, Apis mellifera, were obtained from the Hymenoptera Genome Database; Drosophila melanogaster sequences from Flybase, Tribolium castaneum sequences from iBeetle- Base; Orussus abietinus, Loxosceles reclusa, Latrodectus Hesperus, Centruroides exilicauda, Strigamia maritima, from Baylor College of Medicine Human Genome Sequencing Center; Mus musculus and Ophiophagus Hannah sequences from NCBI. Each genome protein set was interrogated with A. compressa venom ORFs using phmmer (Hmmer 3.0), with an expect cutoff of 10-5. Species key: Jewel wasp, N. vitripennis66; Wood Wasp, O. abietinus82; Fire Ant, S. invicta83; Harvester Ant, P. barbatus 84; Argentine Ant, L. humile 85; Jumping Ant, H. saltator86; Leaf-Cutter Ant, A. cephalotes87; Tramp Ant, C. obscurior88; Leaf-Cutter Ant, A. echinatior89; Bumble Bee, B. impatiens90; Honey Bee, A. mellifera91, 92; Fruit Fly, D. melanogaster93; Flour Beetle, T. castaneum94; Brown Recluse Spider, L. reclusa82; Black Widow Spider, L. hesperus82; Bark Scorpion, C. exilicauda82; Centipede, S. maritima82; Mouse, M. musculus95, 96; King Cobra, O. Hannah97.

2.4 Results Proteins or peptides in the venom are necessary for the behavioral manipulation

To determine whether the long lasting effect of the venom is mediated by the protein components of the venom, venom mixed with protease was injected into the Central Complex of the cockroach brain, and then behavioral observations were used to determine whether the cockroaches show the stereotypic hypokinetic behavior. In contrast to venom injection, injection of venom mixed with protease into the Central complex of cockroaches does not decrease the escape distance or spontaneous walking (Fig. 2.1). These results suggest that proteins or peptide in the venom are necessary for the behavioral manipulation.

Figure 2.1: Proteins/peptides in the venom are necessary for the behavioral manipulation. Injection of venom mixed with protease into the Central complex of cockroaches does not decrease the escape distance or spontaneous walking , in contrast to the effect of venom injections(*=p<0.05). n=4 for each group, Data points represent the means + SD.

The venom sac is contractile

The venom apparatus is composed of two components: venom gland and venom sac (Fig. 2.2A). The venom gland is bifurcated, highly branched, and larger than that of

31 most Hymenoptera with respect to body size. It is distinct and separate from the bulbous, glandular venom sac (also previously referred to as “venom reservoir”98), situated between the left and right major branches of the venom gland. Ducts emanating from venom gland and venom sac converge at the ductus venatus, or venom duct, which projects into the stinger.

The venom sac (VS) of Ampulex is distinctive among hymenopterans in that it is separate from the venom gland, connecting independently to the ductus venatus (DV) (Fig. 2.2A). While the VS serves as a storage reservoir, we find that it also makes distinctive contributions to the venom cocktail (see below). Because the venom sac is enveloped by musculature, we assessed its ability to contract by exposure to the calcium ionophore, ionomycin or high potassium; both evoke a robust contractile response (Fig. 2.2A, before treatment, Fig. 2.2B after treatment). No contractions of the venom gland were observed under the same conditions. The possibility of VS contraction allowed us to increase to amount of venom collected, in a relatively short time.

Figure 2.2: Morphological analysis of A. compressa venom apparatus. A. The venom apparatus of A. compressa is composed of two distinct glandular organs, the long, highly branched tubular venom gland (VG), and the bulbous venom sac (VS). Both glands are connected together and to the stinger via the ductus venatus (DV). B. The venom sac is contractile, here imaged after exposure to the calcium ionophore, ionomycin.

Venom proteomics reveals a rich mixture of proteins and peptides

The venom proteome was generated by combining MuDPIT results from seven milked venom sample preparations. The combined results, filtered using a p < 0.01 significance threshold, a minimum of 2 unique peptides per hit, and replicated at least twice, totaled 267 identified proteins. Phmmer searches against NCBI-nr and Uniprot databases returned 57 results with homology to characterized proteins, 132 results to putative/uncharacterized proteins, and 78 with no homology to any proteins in any

Figure 2.3: Domains of venom proteins. The top 10% identified domains by HMMscan of the venom proteins. 120 domains were identified in 211 venom proteins. database. Hmmscan identified 120 domain hits from the PfamA database, among the identified domains are the M13 peptidase family, phospholipase A2, and immunoglobulin domain (Fig. 2.3). In total 154 proteins out of 267 proteins had no homology in any of the databases, or homology to putative/uncharacterized proteins. The venom proteome contains hundreds of proteins, many with multiple isoforms, paralogs, or representatives of the same enzyme family, indicating a high degree of functional redundancy in venom components. For example, the M13 peptidase family is well represented in the venom with 31 separate domain hits in the PfamA database (Fig. 2.3). One isoform of endothelin-converting enzyme 1 stands out in both transcript counts and protein abundance, suggesting that the dominant M13 action is from this enzyme (Fig. 2.4A). Hyaluronidase is an exception to this pattern, as it is a highly-

33 represented venom protein in both protein abundance and expression in both VG and VS, with only one isoform.

Figure 2.4: Proteomic analysis of A. compressa venom. A. 3D ‘gel’ analysis of the venom proteome, based on theoretical values of isoelectric point (pI) and molecular weight (MW) of venom proteins plotted against exponentially modified Protein Abundance Index (emPAI). The pI and MW were extrapolated from entire coding sequences identified by MudPIT. The M13 family peptidases (orange) and phospholipase A2 (blue) fall into similar mass ranges, but different pI values. The most abundant venom proteins are highlighted, notably endothelin-converting enzyme, hyaluronidase, and ampulexin 1. All other proteins identified in PfamA and SWISS- PROT databases by Hmmscan and BLAST are in black (Annotated). Uncharacterized proteins (dark gray) are differentiated from novel proteins (light gray), in that uncharacterized proteins were found represented in the Uniport database as “putative” or “uncharacterized”, whereas proteins classified as “novel” did not return any significant hits (E-value < 10-5) from Uniprot or PfamA databases. B. Protein extracts from milked venom, venom gland and venom sac, separated by tris-tricine SDS-PAGE.

A. compressa venom is composed of proteins ranging from below 2 kDa to over 100 kDa (Figure 2.4B). The VG proteome is enriched in large molecular weight proteins (> 15 kDa), while the VS is enriched in low molecular weight (< 12 kDa) peptides. The SDS-PAGE banding pattern of the VS protein extract bears a striking similarity to that

34 of milked venom in the molecular size range below 25 kDa. On the other hand, the milked venom banding profile above 25 kDa resembles that of the VG, although correspondence between the two is not as pronounced. Milked venom therefore contains a combination of VG- and VS-specific proteins, with the VG contributing to the larger protein fraction of the venom and the VS contributing the smaller peptide components. This trend is reflected in the RNAseq counts as well, with VG read counts higher for larger molecular weight proteins, in particular the M13 peptidase family member neprilysin. In contrast, VS protein read counts are much lower overall than the VG, except for low molecular weight peptide toxins, in particular the ampulexins. Ampulexin 1 is the second-most highly expressed protein in the VS, but with much lower expression in the VG. There are several secreted, novel peptides with molecular mass between 2.4 and 10.3 kDa in the venom proteome that have higher transcript counts in the VG, though much lower peptide abundance (emPAI)99 than VS peptides. Comparing protein abundance in either the VG or VS to respective transcript levels reveals that some venom proteins more highly expressed in the VG are more abundant in the VS, supporting the idea that the VS serves as a reservoir for proteins synthesized in the VG.

The ampulexin family of peptides is well represented in both the venom apparatus transcriptome and venom proteome (Figure 2.5). The majority species of these peptides, as determined by spectral count, corresponds to the major peptide in the venom.

Some venom components appear to target nucleotides and nucleotide/nucleoside monomers. Such as Poly(U)-specific endoribonuclease Adenosine deaminase (Figure 2.5).

The majority of enzymes in A. compressa venom are predicted to be proteases, a common component of animal venoms100, 101. These proteases fall into multiple families, including serine-, cysteine- and zinc-containing metalloproteases (Figure 2.4, 2.5, 2.6).

Second to proteases are carbohydrate targeting enzymes (CTE) including hyaluronidase, trehalase, carbohydrate sulfotransferase, glucose dehydrogenase, and N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase (Figure 2.6). These enzymes could target extracellular proteoglycan domains in the cockroach brain. Extracellular

35 polysaccharides are involved in many critical functions in the central nervous system, including axonal growth and synapse integrity102, 103.

Remarkably, the venom contains members of the Toll signaling pathway, including the Toll receptor ligand Spätzle along with upstream serine proteases responsible for its activation, including Persephone, Easter, Snake, and Gastrulation defective (Gd) (Figure 2.5). While Persephone occurs in the upstream signaling pathway initiated by fungal and gram-positive virulence factors, Easter, Snake, and Gd are activated via signaling associated with dorso-ventral patterning during embryonic development.

The venom contains a number of predicted integral membrane proteins, such as the vesicular glutamate transporter, sortilin-related receptor, and renin-like receptor. These venom components may intercalate into membranes of host cells or could be translocated into target cells. The venom also contains conserved hymenopteran venom proteins icarapin and venom allergen 3, whose functions are currently unknown (Figure 2.5).

Raw sequencing data was submitted to NCBI under BioProject PRJNA356979.

Figure 2.5: Partial list of A. compressa venom proteins with expression and abundance values. Select venom proteins are grouped categorically with venom gland and venom sac expression values in blue and protein abundance in milked venom proteomics in red. A brief description of function from the Uniprot database is provided at right.

Figure 2.6: Bioinformatic analysis of the venom proteome. Hierarchical distribution of GO terms associated with identified venom proteins. The venom proteome contains representatives of many protein activities, but proteases are a dominant fraction. The pie chart summarizes and represents venom proteins with enzymatic activity by broad enzymatic classification. Protease activity is the dominant activity, representing more than half of venom GO terms. Proteases in the venom include serine, cysteine, and metalloproteases. A significant fraction of enzyme activity target carbohydrates or glycans as substrates and are referred to as carbohydrate-targeting-enzymes (CTE). Lipases include almost exclusively phospholipase A2 isoforms, which may not have lipase activity.

Comparative genomics revels that about half of the identified A. compressa venom proteins are shared with other venomous animals

To assess how the composition of A. compressa venom compares to that of other animal venoms, we made comparisons to genomes of sixteen venomous species. Three non- venomous animal genomes are included as controls. Approximately 50% of 201 identified A. compressa venom proteins are shared with other venomous animals, with the highest proportion of positive hits coming from ants and bees (115-122) (Fig. 2.7). The proportion of positive hits is lowest in non-venomous species, including mouse (24), fruit fly, and flour beetle (51 and 75 respectively). Surprisingly, the number of

38 positive hits is relatively low in the jewel wasp Nasonia vitripennis (78) compared to other Hymenoptera and other venomous animals.

Figure 2.7: Comparative genomic analysis of A. compressa venom proteins. Homologs for Ampulex venom proteins were searched against the respective genome using phmmer. Rows are venom proteins by theoretical molecular mass. Columns indicate whether at least one homolog was identified with an E-value cutoff < 10-5. The total number of positive hits is summed at the bottom of the column. Common protein families across species are identified at right. Scientific names for each species are given in Methods.

1.5 Discussion Hypokinesia induced by A. compressa in its envenomated cockroach host is remarkable for its specificity, duration, and reversibility. To understand the biochemical basis of this unique behavioral alteration, we undertook a comprehensive analysis of venom

39 composition. Our iterative study began with characterization of the venom apparatus on the transcriptomic level to build a database of transcripts and relative abundance. By combining transcriptomics with proteomics of milked venom, we identified venom- specific transcript open reading frames (ORFs), which greatly facilitate comprehensive identification and characterization, as the entire ORF can be assayed against global annotated databases instead of peptide fragments. Furthermore, this approach facilitates discovery of novel venom proteins, since peptide fragments would remain unidentified with proteomics alone. While our transcriptomic analysis has yielded quantitative information regarding transcript number, it does not guarantee direct correlation with translated protein levels. Conversely, proteomics alone often does not provide complete sequence information, given that sequences of non-conserved peptides may not be present in existing hymenopteran genome databases. Combining transcriptome data with the proteomics data has allowed us to generate a comprehensive inventory of venom components and to profile the gene expression pattern of the venom apparatus.

Transcriptomics of both VG and VS has revealed the compartmentalization and tissue specific expression of identified venom components. VG and VS differ greatly in expression levels of certain venom transcripts, though each have at least some level of expression for the great majority of venom proteins, suggesting that each tissue type shares a common ancestry. Therefore, transcriptomics reveals an interesting functional morphology of the venom apparatus as two related, yet distinct glandular structures that jointly contribute to overall venom composition. In other words, neither structure alone accounts for the entire protein repertoire of the venom, precluding the notion that the venom sac serves strictly as a passive venom reservoir. Indeed, the ampulexins, among the most abundant venom components, are products of the VS, confirming its functional role as a venom gland-independent contributor to the venom. It is therefore reasonable to infer that the venom is secreted from the venom gland into the VS, where it is supplemented with additional proteins and peptides and maintained under acidic conditions. Our findings confirm that the muscle-bound VS is innervated and contracts in response to calcium entry. This structure is thus ready to expel venom under the appropriate cue, presumably induced by mechanoreceptors on the stinger shaft104.

Only 65% of A. compressa venom proteins are predicted to have a secretory signal, which might be counter-intuitive assuming all venom proteins are to be secreted. However, this trend is consistent with other hymenoptera-venom proteins uploaded in 40

NCBI-nr data base, as 56% are predicted to have secretory signals, while 45% of all venom proteins in NCBI-nr contain secretory signals. Proteomics analysis of black widow venom found that 67% of venom gland specific transcripts have secretory signals75. In contrast only 2% of 1000 randomly selected A. compressa ORFs contained secretory signals, while 10% of all hymenopteran proteins from NCBI-nr contain secretory signals, demonstrating enrichment of secretory signals in venom-specific genes. The mechanism by which the non-signal bearing proteins are secreted into the venom is unknown. Mass spectrometry based proteomics can facilitate understanding of signal cleavage in small peptides.

We have identified a diverse range of proteases in the venom that could have a number of different functions, including the following: 1) processing of venom protein precursors into active form, including zymogens and propeptide precursors, leading to 2) disruption of host synaptic signaling, and 3) activation of the host immune response. The large representation of M13 proteases, especially members of the neprilysin and endothelin-converting enzyme families is particularly noteworthy. Indeed, Hmmscan (Hidden Markov Models profile scan) of the venom against SWISS-PROT and PfamA databases shows that more than 10% of venom proteins contain M13 protease domains. These proteases are reported to be anchored on the extra-cellular surface of expressing cells, where they deactivate neurotransmitter signaling peptides. Alternatively, such proteases could be involved in processing peptides from precursors. M13 proteases are the most well represented proteins in the venom, as measured by either peptide spectral counts or RNA expression level in the venom gland. Yet only four of the proteins with M13 domains are predicted to have secretory signals; the only highly expressed member predicted to have a secretory signal is neprilysin-2, an enzyme known to inactivate neuropeptides105 (Fig 2.8).

One of our more striking findings is presence in the venom of the Toll receptor ligand Spätzle, along with upstream serine proteases that process it into active form, including Easter, Persephone, Snake, and gastrulation defective (Gd)106. Activation of the Toll pathway could trigger expression of antimicrobial peptides that would protect the stung host from infection. Serine proteases could have other functions; for example, serine protease Bi-VSP in bee venom activates the phenoloxidase cascade, but also targets fibrinogen, affecting blood clotting in mammals107.

Besides proteases that activate zymogens and propeptide convertases, the venom contains enzymes involved in post-translational modification of neuropeptides, including amidation and pyroglutamyl capping. Venom neuromodulators precursors of tachykinin and corazonin, prominent in the venom cocktail, have canonical dibasic cleavages sites, serving as potential substrates for venom dibasic endopeptidases such as endothelin-converting enzyme and furin. Additionally, furin targets the motif R/K- X-R/K-R/K just C-terminal to each tachykinin sequence in its precursor, leaving C- terminal basic residues on the cleavage product to become substrates for carboxypeptidase D. This in turn exposes C-terminal glycine to alpha amidation by peptidylglycine alpha-amidating monooxygenase. Fully processed corazonin has N- terminal pyroglutamate, which forms spontaneously from N-terminal glutamate or glutamine residues, but is also catalyzed by glutaminyl-peptide cyclotransferase. Each of these enzyme activities are found in the venom proteome.

Other major enzyme components in the venom are phospholipase A2-like proteins, which are ubiquitous in venoms. In honeybees, it has cytolytic activity, especially in the presence of melittin, although we reported previously that A. compressa venom is not lytic53. Some phospholipase A2 enzymes in venom are small (~16 kDa), and in some snake venoms their toxicity is attributed to agonism of secretory phospholipase A2 receptors, rather than their catalysis of lipids108, 109.

Hyaluronidase, present at relatively high spectral count and expression level in A. compressa venom, is also found in other venoms and is thought to target the extracellular matrix110, 111. Hyaluronan, a major component of the extra-cellular matrix, is important in maintaining synapse connectivity112, 113. Disrupting synaptic integrity in the SEG or central complex of the brain could disrupt integration of sensory data and impair motor function. This disruption could also be reversible, as is the effect of the venom. Phospholipase A2 and hyaluronidase have been characterized as venom spreading factors through “loosening” of the extracellular space to allow penetration deeper into the tissue114-117. It is interesting to consider what the effect of “loosening” cellular connectivity of a brain, without killing the cells, would have on synaptic transmission (Fig. 2.8). A. compressa venom also contains isoforms of a cysteine-rich secretory protein known as Venom Allergen 3. Homologous proteins were found to block cyclic nucleotide-gated ion channels in snake venom.

Another interesting finding in the venom is proteins that contain an Immunoglobulin domain. Similar proteins were identified in N. vitripennis61. Surprisingly one of those was identified as Nuerotrimin/ Opioid-binding protein/cell adhesion molecule (OBCAM). This protein is member of the IgLON family of immunoglobulin (Ig) domain-containing glycosylphosphatidylinositol (GPI)-anchor cell adhesion molecules (CAMs). OBCAM was shown to bind opioids in the presence of acidic lipids and it is probably involved in cell contact118. The GPI-anchor of this CAM could incorporate into the membrane and could possibly interfere with opioids signaling in the CX or/and SEG119. This is in agreement to previous study that suggested that the venom of A. compressa might contribute to the manipulation of cockroach behavior by affecting the opioid system 120. Among the Ig domain containing proteins in the venom, one was found to have similarity to Roundabout, the receptor for SLIT. Another cell adhesion molecule in the venom was identified as Plexin. Members of the Plexin family, are known as Semaphorin receptors121. All those proteins can incorporate into the membrane, as described in figure 2.8, and affect neurite response to growth repulsive signal like SLIT or Semaphorin. Their incorporation into the membrane at the synapse of the CX or SEG could increase the sensitivity to repulsive signal and cause a neurite retraction. Even a slight neurite retraction at synapse could affect synaptic efficacy and therefore reduce neuronal activity in the CX and SEG, which will cause as described earlier, to decreased spontaneous walking (Fig. 2.8).

Comparison of A. compressa venom proteins to other venomous animals highlights those functions that are conserved in envenomation and those that may be unique to A. compressa. A significant portion of A. compressa venom proteins have some homology to other venomous animals, such as the king cobra, black widow, brown recluse spiders, bark scorpion and centipede.

This is perhaps surprising considering its unique target location, the cockroach central nervous system, and the specific behavioral modification caused by the venom. These results suggest that conservation of certain venom proteins, in particular the protease and lipase families, extends beyond the hymenoptera clade to include venomous animals in general. On the other hand, almost half of identified A. compressa venom proteins remain uncharacterized or are novel. A. compressa proteins in common with other venomous animals are generally confined to specific protein families. The M13 protease family is represented in all genomes examined; it is preserved in venomous 43 animals, with a more limited representation in the non-venomous animals and N. vitripennis. The serpin family and cysteine-rich secretory family of proteins are present in all animals examined. The phospholipase A2 family, a ubiquitously identified venom component, has good representation in all animals examined except mouse, and to a lesser extent in N. vitripennis and the non-venomous insects.

The large molecular weight fraction of A. compressa venom contains proteins that tend to be homologous to those in other venomous animals, whereas the small molecular weight fraction peptides are likely to be novel. Included in the more conserved venom set are known common venom allergens such as the phospholipase A2, icarapin, and venom acid phosphatases. The specialized ability of animal venoms to block or modify ion channel gating in the target nervous system, can often be conferred by small peptides122-125. So far, A. compressa venom peptides have not shown this type of activity, though its small molecule fraction activates GABAA receptors in the cockroach central nervous system21. A. compressa venom contains several novel small peptide toxins whose role in hypokinesia is yet to be determined.

The transcriptomic/proteomic analysis presented here provides a comprehensive survey of proteins in the venom cocktail of A. compressa. While inferences can be made from this list about which proteins might be sufficient to induce hypokinesia, further hypothesis testing is required before concluding which proteins are necessary. The nature and sensitivity of this approach is to detect as many proteins as possible, many of which may be “innocent bystanders” in the induction of hypokinesia. The dilemma then is how to identify proteins as being true venom components, i.e. are necessary in the pathology induced by the venom, from what may be proteins found in the venom fluid with no role in the venom action that confers fitness to the wasp, or apparent negligible effect on the cockroach cerebral ganglia, e.g. actin. However, if this were a frequent phenomenon we would expect more housekeeping proteins in the venom fluid such as those found in glycolysis, which we did not observe.

Understanding venom composition is integral to understanding venom action. The venome of A. compressa presents a rich biochemical mixture, whose neuropharmacology exerts a potent long-term, yet reversible suppression of locomotory activity without paralysis. Elucidation of the venome reveals more questions than it answers, and a significant amount of investigation remains to unravel the mechanism of venom action. Each protein or peptide described herein may play some role in venom action and each warrant further investigation. Hypokinesia is a locomotory syndrome, likely caused by concerted action of many venom components, orchestrated temporally to usurp control of cockroach motility for the benefit of A. compressa’s maternal, yet macabre, motives.

Figure.2.8: Schematic representation of venom proteins that could be localized at the synspase. 1. Neuropeptides and novel peptides/proteins can target specific proteins (like receptors, membrane protein and synaptic proteins) in the membrane and carbohydrate targeting enzymes and matrix metalloprotease can interfere with the extracellular matrix. 2. Intercalation of CAMs from the venom into the membrane could affect neurite responses to growth repulsive signals like SLIT or Semaphorin. This could potentially increase sensitivity to repulsive signals and/or cause neurite retraction. 3. Phospholipase A (PLA) could damage membranes by catalysis of lipids and possibly the extracellular matrix. 4. M13 proteases anchored on the extra-cellular surface may deactivate neurotransmitter signaling peptides and iterfere in synaptic signaling.

3. Molecular cross-talk in a unique parasitic manipulation strategy Based on the paper from Kaiser et al. " Molecular cross-talk in a unique parasitoid manipulation strategy", 2018126.

3.1 Abstract Envenomation of cockroach cerebral ganglia by the Jewel wasp induces specific, long lasting behavioral changes. We hypothesized that this prolonged action results from changes in brain neurochemistry. In this chapter, I address this issue by first identifying molecular targets of the venom; that is, proteins to which the venom components bind and interact with to mediate altered behavior. Second, I characterize changes in cerebral ganglia proteins of stung cockroaches at different time points after the sting. Since behavioral changes induced by the sting are long lasting and reversible, I hypothesized that long-term effects of the venom must be mediated by up or down regulation of cerebral ganglia proteins. My results show that the venom binds to synaptic proteins and therefore likely interferes with synaptic processes. I show that numerous proteins are differentially expressed in cerebral ganglia of stung cockroaches, many of which are involved in signal transduction pathways such as the Rho GTPase pathway, which is implicated in synaptic plasticity. Altogether, my data suggest that venom could affect many pathways, causing changes in synaptic efficacy in cockroach cerebral ganglia to induce behavioral changes that benefit the wasp.

3.2 Introduction The Jewel wasp sting does not simply paralyze the cockroach. Instead, it induces a stereotypical behavioral change. After envenomation, the stung cockroach first grooms continuously for 30 minutes and then enters a long-term hypokinetic state, in which spontaneous locomotory activity and normal escape responses to both wind or tactile stimuli cease11. Since the venom is injected directly into the cerebral ganglia, long- lasting behavioral changes observed in stung cockroaches must be the result of a molecular manipulation of neuronal circuits in these ganglia. In order to understand the molecular mechanisms underlying behavioral manipulation, a first step is to identify cerebral ganglion proteins targeted by the venom. As described in the previous chapter, the wasp venom is rich mixture of peptides and proteins, many of them uncharacterized. The venom is unusual, since it is injected directly into the central nervous system and contains many proteins predicted to act inside the cell. Although most venom toxins are known to target membrane proteins such as receptors, ions channels and pumps, some have intracellular targets such as proteins kinases/phosphatases, small GTP binding

46 proteins, nucleic acids, ribosomal proteins and others127. Since the venom contains at least 267 proteins and peptides, identifying each molecular target in the cerebral ganglia is a staggering task. Therefore, in order to identify at least some of the venom targets, I constructed a venom affinity column using a NHS (N-hydroxysuccinimide)-activated Sepharose. The NHS- activated sepharose is typically used for purification of antibodies using a specific antigen. Relevant here is that such a column has also been used to purify antibodies directed against crude snake venom by coupling the NHS- activated Sepharose to the crude venom128, 129.

Many known examples of behavioral manipulation are shedding light on various strategies, which have evolved in parasites, to achieve a desired behavioral manipulation1, 2. Some studies have focused on changes in biogenic amines in the central nervous system of the host1, 130-132. For example, octopamine concentration increases in cerebral ganglia of Manduca sexta parasitized by the wasp Cotesia congregata133. However, no changes in biogenic amines were observed in the cerebral ganglia of stung cockroaches134. A number of studies have looked for changes in protein expression during the behavioral manipulation; for instance, in parasitized M. sexta, distinct changes in biogenic amines are accompanied by an accumulation of neuropeptides in the cerebral neurosecretory system135. The proteomics approach for studying parasite-host interaction has been used in several more recent studies136-138. For example, methods such as two-dimensional gel electrophoresis and mass spectrometry (MS) were used to identify proteins from the grasshopper that are associated with the behavioral manipulation by the nematomorph hairworm Spinochordodes tellinii4. Similar studies found proteins, which are differentially expressed in two gammarid species infected with two altering behavior parasites: the trematode, Microphallus papillorobustus and the acanthocephalan, Polymorphus minutes139. Those studies used 2D gels for comparing proteomes between infected and not-infected host. However, today there are Quantitative MS methods that allow one to directly compare protein levels between groups. This provides a better measurement compared to RNA expression analyses, which do not always correlate with proteins levels140, 141. Although most of the Quantitative MS methods use labeled protein, Label Free methods are less time and resources consuming and still provide relatively reliable and accurate data with high coverage and dynamic range142-146.

3.3 Methods Venom affinity column preparation

In order to identify the venom protein targets in cockroach cerebral ganglia, I used HiTrap NHS-activated HP (GE Healthcare, Product No. 17-0716-01) to create a venom affinity column. Venom was collected in standard coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) using a combination of milking and extraction of venom from the venom sac (A total of approximately 100 milking procedures and 20 venom sac extracts). The venom was then covalently bound to Sepharose beads to construct a HiTrap NHS-activated affinity column by activation with ice cold 1 mM HCl using a syringe as recommended by the product manual. The column was then washed and blocked by alternating washes of pH 4 (sodium acetate) and pH 8.3 (ethanolamine) and stored at 4°C. The binding efficiency was calculated by the measuring absorbance at 280 nm before and after coupling to the NHS column. Two venom columns were prepared and the average binding efficacy was 90%.

Purification of venom targets

For purification of venom targets, I conducted two experiments, each utilizing 12 cockroach cerebral ganglia (SEG or brain) collected in binding buffer (10 mM HEPES, pH 7.3-7.4, containing 0.25% CHAPS). Ganglia were homogenized and centrifuged (60 min, 12000G, 4°C), after which supernatants were loaded onto an AKTA chromatography system. Proteins were eluted with high salt solution (0.5 M NaCl, 10 mM HEPES, pH 7.3-7.4, 0.25% CHAPS). Fractions of 1 ml were collected and separated on a sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis (SDS- PAGE) for protein detection. This procedure was repeated 4 times for brain samples and 3 times for SEG samples. Fractions with visible proteins in the SDS-PAGE were sent for identification by mass spectrometry. Data were analyzed with MaxQuant 1.5.2.8 versus the cerebral ganglia transcriptome database (laboratory of Michael Adams, University of California, Riverside).

Label Free Quantitative Mass Spectrometry analysis

Differentially expressed proteins were identified at 3 different time points, corresponding to different phases of behavioral change: (1) 10 min after the sting, prior to onset of hypokinesia, (2) 24 h after the sting, by which time hypokinesia is fully expressed, and (3) 7 weeks after the sting, when the cockroaches have fully recovered from hypokinesia. For each time point, brain and SEG were surgically removed (after behavioral assay as described below), analyzed separately and compared to age- matched untreated cockroaches. In addition, since most of the venom was found in the region of the central complex (CX), I surgically removed the brain from 3 control and 3 stung cockroaches (24 h after the sting) and isolated the central region of the brain to enrich the contribution of the CX.

All samples were analyzed at the Smoler Proteomic Center. In order to identify changes in protein expression in cockroach cerebral ganglia after the sting, we used quantitative mass spectrometry (Label Free Quantitative MS). Samples were analyzed by LC- MS/MS on a Q Exactive plus mass spectrometer (Thermo). Mass spectrometry data from all biological repeats were analyzed using MaxQuant software 1.5.2.8 (Laboratory of Mathias Mann, Max-Planck Institute 147 vs. a custom cerebral ganglia transcriptome database with 1% FDR. Data were quantified by label free analysis using the same software, based on extracted ion currents (XICs) of peptides enabling quantitation from each LC/MS run for each peptide identified in any of the experiments. Statistical analysis of the identification and quantization results was performed using Perseus 1.5.2.4 software (Mathias Mann's group) 147. To identify differentially expressed proteins, Student's t-test was used for comparing each protein in a group to its age-matched untreated control. A protein group is defined when a set of identified peptides is found to be equal to or completely contained in multiple proteins. Comparison was made between proteins that were identified with at least 3 Razor + unique peptides. Razor peptides are shared among multiple proteins, while being most parsimoniously associated with the group with the highest number of identified peptides, but stay in all groups where they occur. The use of unique and razor peptides is a compromise between unequivocal peptide assignment and most accurate quantification 147, 148. The relative protein quantification across all samples is represented by the LFQ (Label-free quantification), which is a normalized intensity profile 145. 49

Differential expression was defined if the difference between the averaged LFQ of the two groups was of at least 2-fold and if the Student's t-test P-value was less than 0.05 (After applying FDR correction using Benjamini & Hochberg procedure149).

Heat maps for the differentially expressed protein were created using Morpheus, https://software.broadinstitute.org/morpheus.

Protein annotation

Proteins were annotated using BLAST (Basic Local Alignment Search Tool) with the Blast2GO software (Blastp, E-value threshold: 10-3) against the SWISS-PROT database (Manually reviewed, high quality protein sequences and functional annotations produced by UniProt), the Non-redundant (nr) database (All non-redundant GenBank CDS translations, PDB, SWISS-PROT, PIR, PRF excluding environmental samples from WGS projects) and Refseq (NCBI Protein Reference Sequences). In addition, proteins were annotated using the HMMER web server (phmmer) against the SWISS- PROT database with restriction to Drosophila melanogaster proteins only.

Functional enrichment analysis

The annotated list of the D. melanogaster homolog proteins was analyzed by DAVID (The Database for Annotation, Visualization and Integrated Discovery v6.8), against the D. melanogaster well annotated genome. This on-line service is used for ranking functional categories based on co-occurrence with sets of genes in a gene list. This can aid in unraveling new biological processes associated with cellular functions and pathways. The threshold of the EASE Score, a modified Fisher Exact P-Value, for gene- enrichment analysis, was set to include only results with values smaller than 0.05 and the threshold for minimum gene counts belonging to an annotation term was set to at least 2.

Behavioral assays

Behavioral assays were performed as described in Chapters 1 and 2.

Quantitative mass spectrometry analysis: Prior to the wasp sting, behavioral assays were performed on untreated cockroaches (age matched, after the last molt). Next, they were introduced to the wasp for the sting. Wasp sting duration was quantified as a measure of a normal stinging behavior104.

Short term effect of the venom: cockroaches were taken shortly after the sting (10 minutes after) for dissection.

Long term effect of the venom: cockroaches were taken 24 hours after the sting for behavioral assays. In this time point, only cockroaches that showed lack of escape response and decreased spontaneous walking were taken for brain, SEG or CX dissection.

Recovered cockroaches: cockroaches were left to recover after the sting in vials containing sugar water. Once a week, behavioral assays were performed to test if they had recovered. Seven weeks after the sting, all tested cockroaches had fully recovered, as evidenced by return of escape response and spontaneous walking duration back to normal. These cockroaches were taken for brain and SEG dissection.

Effects of a natural sting on recovered cockroaches: tests were performed as mentioned above. However, tested cockroaches recovered faster and 2 weeks after the sting they were introduced for a second sting. Twenty-four hours after the second sting, behavioral assays were performed again. The data from those tests did not pass normality test; therefore, I used Kruskal-Wallis one-way analysis to test for significance difference between groups.

3.3 Results Venom affinity column reveals multiple protein bands of widely ranging of molecular weight from cerebral ganglia

For both SEG and brain samples, peaks eluting in ~3 ml were observed at 280 nm. Separation of these fractions by gel electrophoresis revealed multiple bands ranging from at least 28-250 kDa (figure 3.1). Because of this broad range of protein sizes, all lanes showing visible proteins were sent for identification by mass spectrometry.

Figure 3.1 Venom affinity column revealed multiple bands in a wide range of molecular weights. One chromatogram of one cycle of affinity purification exhibits an absorption peak (fraction 21) following elution with high salt concentration.

Mass spectrometry analysis identifies possible venom protein targets

Mass spectrometry data were screened to limit identification of proteins to those that were identified by at least 3 razor and unique peptides148. This resulted in 967 proteins that were identified in at least one of the brain samples and 438 proteins that were identified in at least one of the SEG samples. 236 proteins were identified in all brain samples and 207 proteins were identified in all SEG samples. In total, 165 proteins were found in both brain and SEG. 71 proteins were unique to the brain samples and 42 proteins were unique to the SEG samples (a total of 278 proteins, table 1). 273 venom target proteins were annotated using Blast against SWISS-PROT database and search against the D. melanogaster SWISS-PROT database identified homolog proteins to 224 of the protein venom targets.

Especially strong identification was found for several proteins in brain and SEG (large number of razor and unique peptides), including: Spectrin, Apolilipophorins and the Microtubule associated Futsch. Proteins with highest intensity in both SEG and brain included: Tubulin, Spectrin and Sodium potassium-transporting ATPase.

Brain SEG Number of repeats (each with 12 ganglia) 4 3 Proteins identifed in at least one sample 967 438 Proteins identifed in all samples 236 207 Unique to ganglion 71 42

Table1. Summary of the mass spectrometry identification of venom targets.

The venom targets database is enriched in proteins associated with synaptic processes

In order to characterize venom protein targets in brain and SEG, I performed functional annotation and gene enrichment analysis using DAVID for each ganglion separately. This analysis revealed that many Gene Ontology (GO) terms are enriched in the database (ease score<0.05). When including only biological processes GO terms (level 5, for high specificity), 240 terms were found enriched in brain and 209 in SEG. 186 of the enriched terms are common between brain and SEG. Using the annotation clustering report on DAVID with manual corrections and adjustments, I constructed clusters, which display similar annotations together, and therefore, genes with similar function or involvement in similar processes and pathways. Two of the most enriched clusters are shown in Figures 3.2-3.5. One of those clusters (figure 3.2, 3.3) contains proteins that are involved in synaptic processes, such as neurotransmitter release and synaptic vesicle endocytosis and exocytosis. This group includes, in both brain and SEG, the following proteins: Clathrin heavy chain, Protein Stoned-B, Phosphatidylinositol- binding clathrin assembly protein LAP, Dynamin, Synapsin, Vesicle-fusing ATPase 1, Synaptobrevin, Protein ROP and Synaptotagmin 1. While Endophilin-A was found only in the brain (figure 3.2), the AP-2 complex alpha was found only in SEG (figure 3.3).

Figure 3.2: The venom targets database is enriched with proteins that are associated with synaptic processes. The Gene ontology terms for brain identified venom targets with their corresponding Ease score (presented as a normalized -log10Pvalue) and their associated proteins (proteins are annotated according to their D. melanogaster homologs description). Star sign ( )marks protein that was found only in brain.

The venom targets database is enriched in proteins associated with cytoskeleton organization and synapse assembly

Another cluster of venom targets includes proteins related to cell or neuron development, cell morphogenesis, cytoskeleton organization, synapse assembly and regulation of those processes (Figures 3.4, 3.5). In both brain and SEG, this group includes: Spectrin, Microtubule-associated protein futsch, G protein alpha o subunit, Failed axon connections, cAMP-dependent protein kinase type-II regulatory subunit,

Figure 3.3: The venom targets database is enriched with proteins that are associated with synaptic processes. The Gene ontology terms for SEG identified venom targets with their corresponding Ease score (presented as the –log10Pvalue) and their associated proteins (proteins are annotated according to their D. melanogaster homologs description). Star sign ( ) marks protein that was found only in SEG

Kinesin heavy chain, Inosine-5'-monophosphate dehydrogenase, Ras-related protein Rab6, Heat shock 70 kDa protein cognate 4, 14-3-3 protein epsilon, Protein unzipped, Moesin/ezrin/radixin homolog 1, Protein hu-li tai shao, Calmodulin, Tropomyosin-1, isoforms 9A/A/B, Proteasome subunit alpha type-6, Proteasome subunit alpha type-6 and Transitional endoplasmic reticulum ATPase TER94. The proteins Heterogeneous nuclear ribonucleoprotein 27C, Cofilin/actin-depolymerizing factor homolog and Dosage compensation regulator from this group were found only in the brain, while Actin-related protein 3 and Protein vav were only found in the SEG.

Figure 3.4: Venom targets in brain show enriched terms that that are associated with cytoskeleton organization and synapse assembly. The Gene ontology terms with their corresponding Ease score (presented as the normalized

–log10Pvalue) and their associated proteins (proteins are annotated according to their D. melanogaster homologs description). Star sign ( ) marks proteins that were found only in brain.

Figure 3.5: Venom targets in SEG show enriched terms that are associated with cytoskeleton organization and synapse assembly. The Gene ontology terms with their corresponding Ease score (presented as the –log10Pvalue) and their associated proteins (proteins are annotated according to their D. melanogaster homologs description). Star sign marks ( ) proteins that were found only in SEG.

Quantitative mass spectrometry identifies changes in protein expression at all time points

In order to find differentially expressed proteins in the cockroach cerebral ganglia, I used quantitative mass spectrometry (Label Free Quantitative MS). I found changes in proteins expression in all time points: For 10 min after the sting, ~0.8% of the proteins are differentially expressed (33 proteins in total); 24h after the sting, ~1% of the proteins are differentially expressed (48 proteins in total); and in recovered cockroaches ~1.2% of the proteins are differentially expressed (50 proteins in total, figure 3.6, Table 2). These results suggest that, although stung cockroaches (10 minutes after the sting) and

recovered cockroaches do not display hypokinesia, their proteome is different from that of control, unstung cockroaches.

1.5

1 % DE proteins 0.5

0 10min 24h Recovered

Figure 3.6: The percentage of differentially expressed (DE) proteins in the cerebral ganglia of stung cockroaches, at the different time points after the wasps sting. P- value<0.05 (after FDR correction), Fold-change>2, Razor + unique peptides>2.

Table 2. Summary of the number of identified differentially expressed proteins at different time points after the wasp’s sting, in the different ganglia. NA- not available, the isolated CX was not tested in those groups.

Changes in protein expression in the CX associated with hypokinesia

In total, 33 proteins were differently expressed in the CX of stung cockroaches 24 hours after the sting (P value<0.05; difference of at least two-fold change). Changes observed were consistent between individual samples (Figure 3.7) and the difference in expression between stung and control for those proteins was remarkable (average fold

58 change of ~1611). Annotation of those proteins indicates that some are involved in the Rho GTPase pathway (Figure 3.7). These proteins include the Guanine nucleotide exchange factor (GEF)150, SLIT-ROBO Rho GTPase-activating protein (GAP)151 , Cappuccino/ Formin152 and Tyrosine kinase Src64B153 (Figure 3.8). Additional differentially expressed proteins are the Protein interacting with C kinase 1 (PICK1), which is implicated in synaptic plasticity and Tyrosine Phosphatase MEG2 which is implicated in regulation of synaptic vesicles (Figure 3.8)154-156. Other proteins that are differentially expressed in the CX are those involved in gene expression regulation such as translation initiation factor eIF-2B (Figure 3.8)157.

Changes in protein expression in the entire brain associated with hypokinesia

In the all brain, a total of 10 proteins were differentially expressed in hypokinesic cockroaches (24 hours after the sting; P value<0.05, at least two-fold change). Similar to the CX, changes observed were consistent between individual samples (Figure 3.9). Expression differences between stung and control for these proteins were lower compared to those found in the CX (an average fold change of ~751), but still constituted a substantial difference. The greatest difference was found for UDP- glucuronosyltransferase, Heterochromatin protein 1 (Or Chromodomain Y 2), Troponin C, Ankyrin repeat KH domain-containing protein mask and Fibroblast growth factor receptor (Figure 3.8). Fibroblast growth factor receptor, Ankyrin repeat KH domain- containing protein mask, which are differentially expressed in brain, are involved in the receptor tyrosine kinase signaling (Figure 3.8) 158-160.

Changes in protein expression in the SEG associated with hypokinesia

Only 5 proteins were differentially expressed in the SEG of hypokinesic cockroaches (P value<0.05, difference of at least two-fold change). Changes observed were consistent between individual samples (Figure 3.10) and expression differences between stung and control for these proteins were lower than those observed in the CX and brain (an average fold change of ~651). The protein identified as Chromodomain Y, which was found to be upregulated in brain, is upregulated in the SEG as well (Figure 3.8).

Changes in protein expression in brain prior to hypokinesia

Cockroaches do not exhibit hypokinesia 10 minutes after the sting, but some behavioral changes are observed, such as grooming22. However, in total, 5 proteins were found to be differentially expressed 10 minutes after sting in the brain (P value<0.05, difference of at least two-fold change). The difference in expression between stung and control for these proteins was ~1445 fold on average. Differentially expressed proteins in the brain 10 minutes after the sting and differentially expressed proteins in the brain or CX, 24h after the sting do not overlap.

Figure 3.7: Heatmap of differentially expressed proteins in CX, 24 hours after the sting. Proteins description are shown next to their normalized LFQ intensities. 61

Figure 3.8: Changes that are associated with the long term effect of the venom. Partial list of proteins with significant differential expression in cerebral ganglia of stung cockroaches, 24 hours after sting sorted by common predicted function. A) Proteins involve in the Rho GTPase pathway, synaptic plasticity, synaptic vesicles and gene expression regulation are differentially expressed in the central complex. B) Proteins involve in the receptor tyrosine kinase signaling, cell cytoskeleton and gene expression regulation are differentially expressed in the brain. C) Protein involve in gene expression regulation is differentially expressed in the SEG. Green marks up regulated proteins and red marks downregulated proteins.

Figure 3.9: Heatmap of differentially expressed proteins in brain, 24 hours after the sting. Proteins description are shown next to their normalized LFQ intensities.

Figure 3.10: Heatmap of differentially expressed proteins in SEG, 24 hours after the sting. Proteins description are shown next to their normalized LFQ intensities.

Figure 3.11: Changes that are associated with the short term effect of the venom. Partial list of proteins with significant differential expression in SEG of stung cockroaches, 10 minutes after sting sorted by common predicted function. Proteins involved in neurotransmitter regulation and Cation channels and regulators are differentially expressed in the SEG. Green marks up regulated proteins and red marks downregulated proteins.

Changes in protein expression in SEG prior to hypokinesia

In the SEG prior to the observed hypokinesia 28 proteins were differentially expressed in the SEG 10 min after the sting: (P value<0.05, difference of at least two-fold). The difference in expression between stung and control for those proteins was ~1159 fold. Among differentially expressed proteins are those related to cation channels and their regulation: Voltage-gated potassium channels Shaw, Sodium channel and clathrin linker 1 and Neurogenic protein big brain. In addition, Acetylcholinesterase, which catalyzes breakdown of acetylcholine, is upregulated in the SEG (Figure 3.11).

Changes in protein expression in the brain associated with recovery from hypokinesia

I hypothesized that, if differentially expressed proteins observed during hypokinesia (24 hours after sting) are different than those observed in recovered cockroaches, they are more likely to be involved in mediating hypokinesia. Surprisingly, recovered cockroaches show a large number of differentially expressed proteins, all of which are different that those of hypokinetic animals. A total of 42 proteins were differentially expressed 7 weeks after sting in the brain (P value<0.05, difference of at least two-fold change). The difference in expression between recovered and control for some of these proteins was remarkably high (average ~3353 fold-change). Among those with the highest difference in the expression were: Hemolymph lipopolysaccharide-binding protein and protease inhibitors such as Serine protease inhibitor and Inter-alpha-trypsin inhibitor.

The Multidrug resistance protein homolog 49, which is involved in defense against toxic compounds, was downregulated in the recovered brain161. Several proteins involved in immune responses were differentially expressed in recovered brain. Among them are gamma-interferon inducible lysosomal thiol reductase 1 (GILT 1,) which was downregulated and the Hemolymph lipopolysaccharide-binding/C-type lectin, which was upregulated162-164.

Changes in protein expression in the SEG associated with recovery from hypokinesia

Only 8 proteins were differentially expressed in SEG of recovered cockroaches (P value<0.05, difference of at least two-fold change). The difference in expression between stung and control for some of these proteins was remarkably high (~3062 fold).

Similar to the recovered cockroach brain, Inter-alpha-trypsin inhibitor is upregulated in the recovered SEG. Like in the recovered brain, the protein gamma-interferon inducible lysosomal thiol reductase 1 (GILT 1) was down regulated in SEG (and in the recovered brain, figure 3.12).

The highest count of enriched Go terms is found in hypokinesic cockroaches

In order to better characterize differentially expressed protein groups that are associated with each time point, I used functional gene enrichment analysis. I found that the highest number of enriched Go terms was found in hypokinesic animals (ease score<0.05, level 5 biological processes for high specificity) (Figure 3.13).

Although some of the Go terms are common between groups, 42 terms are unique to hypokinetic cockroaches (Figure 3.13). Go terms that were found only in hypokinetic cockroaches include Synapse assembly, neuron differentiation and development, and neuron projection morphogenesis (Figure 3.14). Among the proteins involved in those processes are the Rho GTPase-activating protein and Tyrosine-protein kinase Src64B, which are down regulated in the CX 24 hours after the sting, and are involved in the Rho GTPase pathway. In addition, among those proteins are PICK1 and Tyrosine- protein phosphatase MEG2. Ankyrin repeat KH domain-containing protein mask, is also involved in those processes and is differentially expressed in the entire brain 24 hours after the sting. Proteins involved in gene regulation and differentially expressed in the whole brain and SEG 24 hours after the sting, such as the Chromodomain Y 2, were also among the proteins associated with those terms.

Figure 3.12: Changes that are associated with recovery from venom effect. Partial list of proteins with significant differential expression in cerebral ganglia of stung cockroaches, 7 weeks after the sting, sorted by common predicted function. A) Proteins such as protease inhibitors and proteins involved in defense against toxins and immune response are differentially expressed in the brain. B) Proteins such as protease inhibitors and proteins involved in immune response are differentially expressed in the SEG. Green marks up regulated proteins and red marks downregulated proteins.

Figure 3.13: The highest count of enriched Go terms is found 24 hours after the sting. Counts of enriched go terms for differentially expressed protein in different time points after the sting. Ease score<0.05, level 5 biological processes for high specificity, at least 2 proteins for term. Numbers on top of the column (30,42) indicate the number of unique go terms for group.

Go terms common between hypokinetic and recovered cockroaches include cytoskeleton organization. All enriched go terms in recovered cockroaches are also found in hypokinetic cockroaches and/or recently stung cockroaches (10 minutes after sting group).

Among the go terms associated with the short term effect of the venom, 30 terms are unique to this time. Among them, cell surface receptor signaling pathway and STAT cascade (JAK-STAT signaling pathway).

Figure 3.14: Functional enrichment analysis of the differentially expressed proteins reveals common and unique processes for the different time point. Enriched Go terms for the differentially expressed proteins in the cerebral ganglia of stung cockroaches (Ease score<0.05, level 5 biological processes for high specificity, at least 2 proteins), at the different time points after the wasps sting. The different colors mark terms that are in common between groups (as detailed at the legend), terms which are not marked are unique for each time point.

Recovered cockroaches are immune to the wasp sting

Since my results demonstrate many differentially expressed proteins in cerebral ganglia of recovered cockroaches, I hypothesized that these might be involved in some sort of adaptation or immune response to wasp venom. In order to test this, I exposed recovered cockroaches to a second sting by the wasp and found a total failure of the venom to induce hypokinesia, as indicated by no change in escape distance following tactile stimuli (Figure 3.15 A). In addition, swimming tests show no significant difference in all tested groups (Figure 3.15 B).

Figure 3.15: Recovered cockroaches are immune to the wasp sting. A) After sting (24h), the escape distance is significantly decreased in stung cockroach (*=p<0.05) and two weeks after the sting, this effect wears off. However, 24h after the second sting, there is no effect on the escape distance. B) The first sting or the second sting had no significant effect on swimming duration. (n=5, n=9, n=8, n=9 for 'control', 'after sting', 'recovered' and '2nd sting', respectively).

3.4 Discussion Using NHS- activated sepharose I was able to construct a venom affinity column, for purification of venom protein targets from cockroach cerebral ganglia. Although I cannot ascertain which venom proteins were bound to the column, the calculated binding efficiency was high and it is therefore reasonable to consider that proteins isolated from the cerebral ganglia using this column are candidate venom targets.

As expected from the large number and diversity of venom proteins, the same trend is observed for venom targets. Considering that there are at least 264 proteins in the venom, among them, proteases or proteins that are subject to post-translational modifications, that could bind non-specifically to different protein targets, it is not surprising to have found so many protein targets in cockroach cerebral ganglia. Due to the large number of identified target proteins, I focused on those that were found repeatedly in all brain or SEG samples, which are more likely to be venom targets.

Most proteins that were found in all brain samples were found also in all SEG samples, while a small set of proteins were found only in the brain or only in SEG. Although the number of identified proteins is quite large, many are isoforms or subunits of the same protein. Proteins with the strongest intensity and strongest identification by mass spectrometry include spectrins and microtubule associated Futsch (homolog to the mammalian family of microtubule-associated proteins (MAPs)165, 166). Those proteins are synaptic cytoskeletal elements that can affect synaptic function167. Spectrins (alpha and beta) are membrane-associated proteins implicated in several aspects of synaptic function, such as presynaptic neurotransmitter release. Absence of spectrins disrupts subcellular localization of numerous synaptic proteins, suggesting that defects in presynaptic neurotransmitter release result from inappropriate assembly, transport, or localization of proteins required for synaptic function168. Studies in D. melanogaster show that the microtubule associated Futsch has a role in microtubule stability, regulation of active zone and neurotransmitter release165, 169, 170.

Because of the large number of affinity-isolated proteins, I propose that it might be useful to characterize candidate venom targets as a group. For this purpose, I used functional enrichment analysis against the D. melanogaster well annotated genome. If the isolated proteins were simply randomly bound to the column by non-specific binding, I would expect that no specific term would be enriched in the database.

However, my analysis revealed that many Go terms are enrichment in the venom targets database. This indicates that the isolated proteins were not just randomly bound to the column, but rather that a specific set of proteins known as Go terms were bound to the venom column. These include synaptic signaling, vesicle mediated transport at the synapse (or synaptic vesicle transport), establishment of synaptic vesicle localization (or synaptic vesicle localization), neurotransmitter secretion and transport, signal release, exocytosis, endocytosis, and synaptic vesicle and membrane budding. Those enriched terms show that the venom targets specifically synaptic processes. The proteins involved in those terms are mainly presynaptic proteins such as Clathrin, Stoned proteins, Dynamin, Synapsin, Vesicle-fusing ATPase 1, Synaptobrevin and Synaptotagmin 1. All those proteins are implicated in the process of endocytosis or exocytosis of synaptic vesicles (Fig 3.16) 171-177. Even if the venom binds only one of those proteins, it could dramatically affect synaptic transmission, by inhibiting protein function or by interfering with protein-protein interactions. If the wasp venom targets those synaptic proteins, it could interfere with exocytosis or/and endocytosis in all synapses of the CX or SEG, rather than affecting a specific type of synapse. It could also serve as a mechanism for internalization of venom proteins. If a venom protein targets the endocytosis machinery, it could "hijack" this naturally occurring process to the advantage of the wasp. This could explain why most of the venom targets (and venom proteins) were identified as intracellular proteins (I will elaborate on this subject at the general discussion section). A similar "hijacking" of endocytosis machinery is used by botulinum toxins, metalloproteases that bind Synaptotagmin to penetrate neurons127, 178. The functional enrichment analysis also revealed another group of biological processes, such as: Microtubule cytoskeleton organization, axon development, cell or neuron differentiation and morphogenesis, neurogenesis and synaptic assembly. Many of the proteins associated with those terms (as I mentioned above: Spectrins and microtubule associated Futsch) appear to provide a link between synaptic function and morphological changes (caused by changes in cytoskeleton organization) at the synapse.

Although I can only predict the function of isolated proteins based in sequence similarity, my results suggest that the wasp venom targets synaptic proteins and synaptic cytoskeleton-related proteins in cockroach cerebral ganglia, thereby leading to decreased synaptic efficacy (Fig 3.16).

The second step for identifying proteins mediating venom actions was to characterize changes in protein expression in cerebral ganglia. To find differentially expressed proteins, I used quantitative mass spectrometry (Label free quantitative MS). Although I refer throughout this chapter to the identified changes in the relative measured proteins level as differential protein expression, those changes can occur not only by changes of gene expression, but also by changes in the overall turnover rate of the proteins.

Figure 3.16: Representation of the venom’s molecular targets at the synapse. A representation of some of venom's molecular targets at the synapse. Many of the venom targets are located at the synapse and could interfere with processes such as endocytosis and exocytosis.

This analysis revealed changes in protein expression in all tested time points (10 minutes, 24 hours and 7 weeks after the sting). These results suggest that, although cockroaches immediately after the sting (10 minutes after the sting) and recovered cockroaches (7 weeks after the sting) do not present symptoms of hypokinesia, they display a protein expression profile different from that of control cockroaches. This means that the wasp venom causes directly or indirectly changes in protein expression in the cerebral ganglia and that some changes occur quickly (proteins associated with the short term effect of the venom), while other changes are associated with the long term hypokinetic state (proteins associated with the long term effect of the venom). The

73 observed recovery in cockroaches is not necessarily due to abolition of those changes, but may involve more complex mechanisms related to up or down regulation of additional proteins.

In most cases, the brain and SEG have different protein expression profiles; most proteins that are differentially expressed in the brain are not the same proteins that are differentially expressed in the SEG (and vice versa). This is in contrast to venom targets, which are mostly common to the brain and SEG, implying that although the targets are the same, the downstream effect of the venom is different in each ganglion.

Few changes in protein expression were identified shortly following the sting (10 minutes after). Main changes are in SEG and are related to neurotransmitter release, cation channels, and their regulators (Figure 3.11). This could indicate that shortly after the sting, proteins that could have direct effect on the neuronal activity in SEG are differentially expressed and that the response to the venom is divided to different phases, quick and short lasting changes followed by slow and long lasting changes.

Since the hypokinetic state is a long lasting and drastic change in cockroach behavior, I expected to find changes in protein expression in the cerebral ganglia underlying such a behavioral change. Differentially expressed proteins associated with long-term effects of the venom were different between the entire brain and the isolated CX (24 hours after the sting). Most of the venom is injected into the CX, which is a relatively small central region of the brain8. Isolating this region for a more focused analysis revealed changes that were not apparent through analysis of the entire brain, since such changes in the CX were possibly masked by other regions of the brain.

Several differentially expressed proteins in the CX are involved in the Rho GTPase signaling pathway. Rho GTPases are known to be directly involved in synaptic plasticity through direct interaction with actin and microtubule organizing proteins for regulation of axonal growth and dendritic spine formation179-181.

These small GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). GAPs are regulatory proteins that bind to activated G proteins and stimulate their GTPase activity to turn them inactive, while GEFs are proteins that activate small GTPases by stimulating release of GDP to allow binding of GTP, pushing the GTPase to an active state180. More specifically, differentially

74 expressed proteins in the CX seem to be related to the Robo-Slit signaling pathway. Roundabout receptors (Robo) and their Slit ligands form one of the most important ligand-receptor pairings among axon guidance molecules182, 183. Robo signaling depends upon a range of secondary molecules, which affect neurite growth through control of cytoskeletal rearrangements. I found that a SLIT-ROBO Rho GTPase- activating protein (srGAP), which is known as a downstream effector of the Slit- Robo signaling pathway, is upregulated in the CX151, 184. This protein has a higher similarity to the vertebrates srGAPs (Identity of 45-46%) than to Drosophila GAPs (33%). While the Slit-Robo pathway was first discovered in Drosophila, most of what is known about srGAPs comes from studies on vertebrates151. The closest studied relatives in Drosophila are RhoGAP93B and RhoGAP92B185, 186. Those proteins, similarly to vertebrate srGAPs, seem to target Cdc42 Rho GTPase184-186. Therefore, it is likely that increased expression of this specific GAP in the CX increases the intrinsic GTPase activity of Cdc42, which converts the GTP-bound form of Cdc42 into its GDP-bound form, thereby inactivating Cdc42 (Fig. 3.17). Inactivation of Cdc42 in turn leads to reduced activation of the Neuronal Wiskott-Aldrich Syndrome protein (N-WASP), thus decreasing the level of active Arp2/3 complex. Because active Arp2/3 promotes actin polymerization, reduction of active Cdc42 eventually decreases actin polymerization184.

Also significantly down-regulated in the CX of stung cockroaches is a GEF protein, which contains a conserved RasGEF domain (found in the CDC25 family of guanine nucleotide exchange factors for Ras-like small GTPases) and a REM domain (GEF for Ras-like GTPases; N-terminal domain). This protein shares 61% of the amino acid sequence with the C3G guanyl-nucleotide exchange factor of Drosophila. While some GEFs are specific to a single GTPase, others have multiple GTPase substrates187. The functional domains of these GEF families are not structurally related and do not share sequence homology. These GEF domains appear to be evolutionarily unrelated, despite similar function and substrates. Thus, it is harder to predict GEF substrates based on their sequence187. However, it is possible that this GEF, which is downregulated in the CX, targets GTPases such as CDC42, similar to the way a familiar member of the Ras- GEF, Son of Sevenless (SOS) regulates slit-robo signaling150. Downregulation of this GEF could lead to inactivation of Cdc42 (Fig 3.17).

More down-regulated proteins in the CX are involved in the Rho GTPase pathway, such as Tyrosine kinase Src64B and Cappuccino/Formin. Tyrosine kinase Src64B has a strong homology to Drosophila Tyrosine-protein kinase Src64B (73% identity) and similarity to Tyrosine protein kinase Fyn in vertebrates (58-59% identity). It contains a catalytic domain of Src kinase-like Protein Tyrosine Kinases and a Src homology 2 (SH2) domain found in the Src family of non-receptor tyrosine kinases. This Src kinase was shown to negatively regulate p109 RhoGAP in Drosophila and therefore affects the Rho GTPase pathway153. The protein formin Cappuccino is similar to Drosophila Cappuccino (46% identity) and vertebrate Formin (32-34%). The formin Cappuccino protein is known to be an effector of the Rho GTPase pathway and acts as an actin nucleation factor to promote assembly of actin filaments152.

In addition, PICK1, which is involved in regulation of Rho GTPase and synaptic plasticity, is differentially expressed in the CX156, 188. The identified PICK1 protein has high homology to Drosophila and vertebrate PICK1 (68-69% identity). It contains Bin/Amphiphysin/Rvs (BAR) and PDZ conserved domains. Although PICK1 has not been thoroughly studied in Drosophila, a study suggests that PICK1 could regulate levels of glutamate receptors at the neuromuscular junction (NMJ). PICK1 reduction leads to smaller synapse size and reduced number of boutons per unit muscle area at the NMJ189. In mammals, PICK1 interacts directly with AMPA receptors and is involved in their regulated removal from the synaptic membrane. PICK1 has the ability to functionally interact with a number of cellular processes, including calcium signaling, actin polymerization and phospholipid membrane architecture156, 188, 190, 191.

Figure 3.17: Representation of differentially expressed proteins in the CX of stung cockroaches. A representation of some of differentially expressed proteins in the CX of stung cockroaches (24h after the sting). Many of the of differentially expressed proteins in CX are involve in the Rho GTPase pathway and are implicated in synaptic plasticity.

Functional enrichment analysis of groups of differentially expressed proteins in each of the tested time points shows that the highest count of enriched Go terms is found 24 hours after the sting. This implies that, although protein levels are different compared to controls in all tested groups, the number of involved processes seem to be highest 24 hours after the sting, when hypokinetic behavior is easily observed. Among the processes enriched only during the long-term effect are: Synapse assembly, neuron differentiation and development, and neuron projection morphogenesis. Those results further support the hypothesis that the long-term effects of the venom could be associated with changes in synaptic structure or assembly (as a result of modifying the rho GTPase pathway).

Surprisingly, recovered cockroach shows a large number of differentially expressed proteins (similar to the number found in hypokinetic cockroaches). Notable among the differentially expressed proteins associated with behavioral recovery are protease inhibitors (such as Serine protease inhibitor and Inter-alpha-trypsin inhibitor) and proteins which are involved in the immune response (such as The Multidrug resistance

77 protein homolog161, 192 and GILT163). The presence of a large difference between the proteome of recovered cockroaches and control cockroaches could be explained if those differentially expressed proteins are a part of some immune “memory” or a compensatory mechanism to resist effects of the venom.

In order to test if those proteomics changes can create a "resistance" to the wasp venom, I introduced recovered cockroaches for a second sting by the wasp and the behavioral effect of the sting was tested. The results show that in contrast to the first sting, which dramatically decreased the escape response of the cockroaches, a second sting does not affect the escape response. This suggests that that the differentially expressed proteins in the recovered cockroach could somehow confer resistance to the venom at least in terms of the escape response. At this point I can only speculate about the reason that recovered cockroaches are resistant to the second sting. One possibility is that differentially expressed proteins in recovered cockroaches are involved in an immune response to the wasp venom. Although invertebrates do not possess an antibody-based immune response similar to vertebrates, they show adaptive and specific responses to pathogens193-195. In 1980, Richard D. Karp and colleagues reported that an adaptive humoral response was induced in the American cockroach to soluble protein complexes from Honeybee toxin and Cottonmouth Moccasin venom196, 197. Though most of those studies focused on the humoral immune system, others described a cerebral innate immune response198. In addition, since the venom contains many proteases, it is possible that upregulation of protease inhibitors in recovered cockroach cerebral ganglia inhibit those proteases and hence, reduce the effect of the venom. Another possibility is that compensatory mechanisms resulting from the first sting blunt the effects of the second sting. This is possible, since many differentially expressed proteins in the recovered cerebral ganglia are involved in some processes observed in stung cockroaches (such as actin cytoskeleton organization, Fig 3.14), which could perhaps mask the effect of the venom.

To conclude, in this chapter I show that wasp venom binds synaptic proteins and thus could interfere with synaptic processes. In addition, proteins such as spectrins and microtubule associated Futsch could also interfere with synaptic processes, synaptic protein localization, and/or induce changes in the cell cytoskeleton (Fig 3.16). Proteins associated with long term effects, mainly in the CX of stung cockroaches, are involved in signal transduction pathways, such as the Rho GTPase pathway, which is implicated

78 in synaptic plasticity (Fig 3.17). Taken together, I suggest that venom could interfere with synaptic efficacy in cockroach cerebral ganglia to induce a behavioral change that benefits the wasp.

4. General Discussion and Future Research Directions Although the wasp-cockroach association is certainly a well-studied behavioral manipulation prior to this study, not much was known about molecular mechanisms underlying this parasitoid-host interaction. This study has revealed the role of the cerebral ganglia in the behavioral manipulation, the components of the venom, their targets and the associated changes in proteins expression.

An integrated model for the molecular mechanisms underlying the behavioral manipulation

This extensive database has allowed me to build a model that combines all this information and could account for the stung cockroach behavioral change. In this model (Fig 4), I focused on some processes or effects that rise from the collected data.

The venom is a rich mixture of different proteins, many of which could affect synaptic efficacy. For example, neuropeptides and novel peptides/proteins can target specific proteins in the membrane (such as the Na/K ATPase) and Carbohydrate targeting enzymes and matrix metalloprotease can interfere with the extracellular matrix. Some venom proteins are expected to be immunoglobulin-like (Ig) cell adhesion molecules (CAMs). Among CAMs in the venom, I found homologs to OBCAM, Plexin (Semaphorin receptor) and Roundabout (SLIT receptor). These proteins can potentially intercalate into the membrane (Fig 2.8 and 4) and affect neurite responses to growth repulsive signals like SLIT or Semaphorin. Their incorporation into the membrane at synapses of the CX or SEG could increase their sensitivity to repulsive signals and/or cause neurite retraction. Even a slight neurite retraction could affect synaptic efficacy and therefore reduce neuronal activity in the CX and SEG, which would cause an aforementioned decrease in spontaneous walking. Interestingly, a CX slit-ROBO GAP (srGAP, Fig 4), a downstream effector in the SLIT signaling pathway, is upregulated 24 h after the sting. Upregulation of srGAP could lead to less active Rho GTPase (probably CDC42), and therefore to decreased actin polymerization, changes in synaptic plasticity, and possible neurite retraction. Spectrins, here identified as venom targets, might be involved in regulation of Slit-Robo pathway components and therefore are possible mediators of this signaling cascade199. Spectrin were shown to mediate the effect of CAMs and might be a targets of the CAMs from the venom. CAMs signal transduction pathways could have long lasting effect at least in the CX where the Rho

GTPase pathway seem to be effected. I suggest this pathway could lead to changes in synaptic plasticity, synaptic protein assembly, neurite retraction and decreased actin polymerization (Fig 4). This hypothesis is supported by the downregulation of other proteins in the CX, namely PICK1 and the formin Cappuccino, which could lead ultimately to decreased synaptic efficacy. Reduced synaptic efficacy in the CX fits perfectly with the stung cockroach phenotype, as I showed in Chapter 1; that is, decreased neuronal activity in the CX leads to decreased spontaneous walking.

Figure 4: An integrated model for the molecular mechanisms underlying the venom- induced behavioral manipulation. Possible mechanisms based on the proteomics data from venom, venom targets and changes in protein expression at the cerebral ganglia. 1. Neuropeptides and novel peptides/proteins can target specific proteins (like receptors, membrane protein and synaptic proteins) in the membrane and carbohydrate targeting enzymes and matrix metalloprotease can interfere with the extracellular matrix. 2. Intercalation of CAMs from the venom into the membrane could affect neurite responses to growth repulsive signals like SLIT or Semaphorin. This could potentially increase sensitivity to repulsive signals and/or cause neurite retraction. 3. Spectrins might be involved in regulation of Slit-Robo pathway components and therefore are possible mediators of this signaling cascade and candidate targets of the CAMs from the venom. Novel peptides/proteins from the venom could targets synaptic proteins and interference with exocytosis and endocytosis of synaptic vesicles. 4. In the CX, less active Rho GTPase (CDC42) and downregulation of PICK1 and formins could lead to

81 decreased actin polymerization, changes in synaptic plasticity, and possible neurite retraction. Taken together, molecular cross-talk between venom components and CNS targets depicted here could lead ultimately to decreased neuronal activity and synaptic efficacy in the CX, the outcome of which is hypokinesia.

Entering the cell by 'hijacking' endocytosis machinery

Notable among venom targets are synaptic proteins, many of which are involved in endocytosis and exocytosis of synaptic vesicles. I suggest that wasp venom proteins target these synaptic proteins with two possible outcomes: 1. Interference with exocytosis and/or endocytosis; 2. 'hijacking' the endocytosis machinery. In both cases, targeting synaptic vesicle proteins could have a devastating effect on neuronal activity.

This leads to one important question in this behavioral manipulation strategy: do venom components enter and act inside target cells? The data collected in this study suggests that they do. Many venom proteins are predicted to act inside the cell and most of the identified venom targets are cytoplasmic proteins. It seems possible, if not likely that wasp venom components could 'hijack' endocytosis mechanisms to enter the cell as does one of the most potent toxins known: Botulinum toxin178, 200, 201. For example, it is possible that, similar to Botulinum toxin, proteins from the wasp venom bind Synaptotagmin when it is exposed to the extracellular space during exocytosis. However, it remains uncertain which venom proteins target these endocytosis/ exocytosis proteins. Novel uncharacterized venom peptides/proteins are promising candidates for this function, but so are several other candidates. The venom vesicular glutamate transporter interacts with Endophilin and Clathrin adaptor protein 2 (AP-2 complex)202, 203. The FXNPXY motif in the Low-density lipoprotein receptor-related protein 2 (LRP2) from the venom binds directly to Clathrin204, which is known to mediate internalization of a variety of ligands/cargos205. Another protein, which contains a seven transmembrane receptor domain from the venom, bears similarity to vertebrate GPR107, a G-protein coupled receptor that is involved in Golgi-to-ER retrograde transport. It functions as a host factor required for infection by Pseudomonas aeruginosa exotoxin A and Campylobacter jejuni CDT toxins206. Similar to LRP2, this protein was shown to interact with Clathrin207. In effect, Clathrin-mediated endocytosis is exploited by many pathogens, such as toxins, bacteria and viruses208. For example,

82 tetanus, shiga, diphtheria and anthrax toxins utilize clathrin-mediated endocytosis to enter cells209. It is therefore possible that receptors from the venom could 'hijack' Clathrin mediated endocytosis and to mediate the entrance of venom proteins into the cell. In this parasitic-host evolutionary arms race, the wasp evolved a powerful arsenal in order to defeat the cockroach. Indeed, if the wasp targets the endocytosis/exocytosis pathway, similarly to the most lethal toxins, it should be powerful tool to control the behavior of the cockroach for the benefit of its offspring.

'Immunity' in recovered cockroaches

Sting-induced changes in the host proteome and behavioral assays reveal that recovered cockroaches gain immunity against the wasp venom. We cannot, however, exclude the possibility that the wasp venom damages, injures or kills neurons in the cockroach cerebral ganglia. Although previous studies showed that milked venom does not induce cell death and probably does not damage the membrane of Chinese hamster ovary cells (CHO-K1) or Hi5 cells (Trichoplusia ni), the venom was not tested on neurons or glia53. It is possible that the wasp launches a massive multi-pronged attack on cockroach cerebral ganglia with a focal injection to specific regions that control spontaneous walking and escape responses, thereby modifying these specific behavioral aspects for a relatively long period. The 'immunity' that recovered cockroaches show to a second sting could result from many proteins working together for repair of membrane/cell/neurite damage for restoration of synaptic connections. However, while peripheral neurite regeneration was shown in many studies, whether neurite regeneration appears in the central nervous system of insects (mainly studied in Drosophila), is still controversial210-213. On the other hand, understanding the basis of 'immunity' in recovered cockroaches to subsequent envenomation could increase our understanding of immune mechanisms of the cockroach central nervous system and also provide further insight into molecular mechanisms underlying the long term behavioral change.

Future directions and experiments

Completing this molecular database for the wasp-cockroach interaction opens new avenues for deciphering molecular mechanisms underlying the behavioral manipulation. The challenge now will be determining a causal relationship between individual venom components, their protein targets, and their impacts on host behavior. Advances in the field of mass spectrometry and bioinformatics have allowed me to conduct this study and rapid developments in this field hold promise for future studies. While writing this thesis, the genome of the American cockroach was finally completed214. Publication of this genome provides great opportunities for future research. Understanding such mechanisms is at the heart of applied aspects of parasitology such as epidemiology and medicine and is important for evolutionary and ecological reasons. In addition, this study contributes to the field of Neuroethology by uncovering basic neural mechanisms underlying decision making. Specifically, since the wasp seems to alter decision making in the cockroach or the motivation of the cockroach to escape, uncovering the mechanisms responsible will shed light on how cockroaches make decisions, from the neuronal site of this process to the molecules and pathways that are involved. There are many directions in which to continue this study. Use the molecular database provided here can lead to new insights in this story. I will describe briefly some of those directions.

1. Testing the hypothesis of the integrated model for long term behavioral manipulation:

My integrated model is based on correlation between identified proteins in the venom, venom targets and differentially expressed proteins. I used thorough literature screening and bioinformatics tools to predict functions for these proteins. Based on my work, many experiments can be conducted in order to support or disprove my hypothesis. Here are several examples:

a. Although the quantitative mass spectrometry results are reliable, additional methods need to be used to validate the results. For this reason, immunohistochemistry and quantitative western blotting analysis for some of the identified differentially expressed protein are useful. One possible drawback inherent in this approach is that commercial antibodies for some of the interesting proteins may not cross-react with the cockroach homolog proteins.

Another possible difficulty lies in the comparison between brain sections of control and stung cockroaches, which is almost impossible due to the small size of the cockroach brain. Namely, it is rather challenging to standardize the plane of sections between samples. Therefore, in order to compare stung and control brains, whole-brain staining is proposed. A recently developed method called “CLARITY" enhances tissue clearing, thereby increasing image resolution of proteins in the brain of mammals without the additional disadvantage of tissue shrinkage215-217. I have adapted this technique to the invertebrate brain of cockroaches and this technique could be used in future experiments in order to compare immunolabeling between whole-brain samples. Preliminary results of immunohistochemistry and quantitative Western blotting for PICK1 showed it is downregulated in the CX of stung cockroaches, in agreement with the quantitative mass spectrometry results (Appendix 4). b. Although it was shown that the venom decreases neuronal activity in SEG and that procaine injection into CX, which decreases neuronal activity, mimics the hypokinetic phenotype, there is a need to show that the venom decreases neuronal activity in CX. An important step in testing this hypothesis will be to test whether the neuronal activity in the CX decreases after venom injection/natural sting. Extracellular electro-physiological recording from CX, after natural sting or after artificial venom injection could be used for this purpose. c. Immuno-labeling of synaptic markers could be used to determine if there is altered localization of those proteins in stung cockroaches (which will be the results of interference with Spectrin function). Determining neurite retraction could be difficult using immunohistochemistry and transmission electron microscopy might be necessary to test this. d. To test whether the Slit-Robo signaling pathway is necessary for behavioral changes, inhibitors of this signaling pathway could be injected into stung cockroach CX and test whether it rescues the stung cockroach behavior. Blocking of this pathway can be done by commercially available inhibitors (RoboN and R5)218, 219, or if not applicable, inhibition of this pathway can be achieved using antibodies-specific inhibition. Similar strategy could be used to test the involvement of OBCAM and Plexin.

e. To test whether there is less actin polymerization in the stung cockroach CX, Western blot quantitation can be used to determine the amount of F actin content versus free G actin content220-222.`

2. Entering the cell by 'hijacking' endocytosis machinery

The results described here suggest that venom components could target intracellular proteins in a manner similar to some bacterial toxins, but in contrast to other venoms which target mainly membrane proteins. In order to test this, two methods can be used. The first is to use radiolabeled amino acid injection into wasps as described previously8. Second, crude venom could be fluorescently labeled, based on covalent interactions with NHS, followed by injections into the cockroach brain, sections or neuron cultures, and detection of fluorescent signals inside cells using confocal microsocopy223.

Another interesting possibility regarding ‘hijacking’ of the endocytosis machinery will be to isolate venom proteins that may interact with Synaptotagmin or other synaptic vesicles proteins. This can be done by constructing a Synaptotagmin affinity column using an NHS- activated column.

3. 'Immunity' in recovered cockroaches

An important factor in need of testing is whether the venom could cause membrane damage to neurons or glia. This can be done by using Trypan blue staining on neuron cultures or directly on cockroach brain slices.

I suggest that the upregulation of some protease inhibitors could contribute to the observed ‘immunity’ of recovered cockroaches; injection of protease inhibitors prior to stinging could be used to test this.

In addition, I suggest that further investigation of differentially expressed protein during different time points after the sting (such as 1 hour, 6 hours and 2 weeks) could shed light on the difference between the fast short lasting effect of the venom and the slow long lasting effect.

The data described here offers new possibilities, that combined with advanced molecular tools (such as RNA interference and CRISPR), can lead to new research avenues, with the hope of understanding molecular mechanisms underlying this unique behavioral manipulation.

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Appendix 1 Procaine injections into CX/MB protocol

Step 1 pre- injection 1. Prepare a glass capillary for the Nano-injector filled with janus green (J.G) in saline (0.5%) or procaine (500mg/ml) in J.G saline. 2. Clean the arena with ethanol and water and let a cockroach acclimate in the arena for 5 minutes. 3. Observe and quantify baseline of the behavior of interest.

Step 2 injection 1. Anesthetize with CO2 and open a flap in the cockroach head (between ocelli). 2. Inject (4.9 nl*2) into central complex or mushroom bodies*, wait 30 seconds before you pull out the electrode and then seal the cuticular flap with wax.

Step 3 after- injection (use the work sheet for this part). 1. Put the cockroach in the arena and start timing. Let the cockroach recover for 10min. 2. Observe and quantify behavior of interest.

Step 4 fixation 1. Prepare a fixation plate (formalin, 10%) in the hood (or use a modified syringe). 2. Cut the cockroach head and place the head in the fixative plate (open the flap for better fixative circulation). Fix the head to the plate under a binocular (put the plate in a larger plate to take it out of the hood). Mark the head sample as you will. 3. Seal the plate and the larger plate well inside the hood. 4. Put in the fridge for overnight fixation or seal well and put at room temperature for 4 hours.

Step 5 slicing 1. Prepare 6% agar in PBS or Saline. 2. Rinse the fixation plate in the hood with buffer (PBS or Saline) - spill the fixative into the appropriate garbage can, rinse with buffer 3-5 times. 3. Wash the plate with the buffer 3 times, each for 10-15min. 4. Place under the binocular and dissect the brain out. 5. Put in the agar (use the lid of Eppendorf tube as a mold), wait until the agar becomes solid and slice in the Vibratome (50-70µm thickness, speed 5, frequency 6, use a few drops of water for collecting sections).

*The tricky part in this protocol is locating the injections site. For CX injections, it depends on the angle that the cockroach is placed, but with a bit of adjusting, you should see small tracheae that make a X sign on the brain. This marks the place of injection.

Appendix 2 Venom injections protocol (modified procaine injection protocol)

Step 1 pre- injection 1. Prepare a glass capillary for the Nano-injector, filled with Janus green (J.G) in saline (0.1%). 2. Clean the arena with ethanol and water and let a cockroach acclimate in the arena for 5 minutes. 3. Observe and quantify baseline of the behavior of interest.

Step 2 milking* 1. Anesthetize the wasps in ice for ~3 minutes. 2. Prepare the wasps for milking as described in the milking protocol 3. Inject a few drops of 10nl Saline +J.G on the Parafilm (for use at next step and to serve as an estimate of the volume of the venom drops). Empty all remaining saline from the electrode. 4. Fill the electrode with 10nl Saline (using one of the drops). 5. Start milking. Collect all drops until the venom's volume reaches 10nl (1:1 – venom: saline, using the 10nl drops as reference).

* In many cases, it is better to use crude not-diluted venom. In this case collect venom directly with the Nano-injector and keep the glass capillary in ice until use.

Step 3 injection (this part needs to be done as quick as possible after part 2) 1. Anesthetize the cockroach with CO2 and open a flap in the cockroach head. 2. Inject 20nl to the Central Complex (press empty), wait 30 secs before you pull out the electrode. 3. Seal the flap with wax.

Continue as describes for procaine injection protocol

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Appendix 3 CLARITY modified protocol (This protocol shows Bruchpilot staining and should be adjusted for use with other staining)

Step Temp How long Notes

1 Dissection and fixation: Remove the 4 °C 1 day brain (or other tissues) and place it in 4% HM solution. Incubate the sample at 4 °C for a minimum of 1 d.

2 Polymerization: Transfer to air-tight 37 °C 3-4h in Bath closed tube, cover with Mineral oil and close lid. Keep in 37 °C for 3–4 h.

3 Wash: Carefully extract the brain from room 1 day solidified hydrogel by softly rubbing temperature all the hydrogel from the surface. Kimwipes can be used to remove the residual gel from the tissue surface. Wash the tissue sample with SBC buffer for 24 h at room temperature to dialyze the remaining PFA, initiator and monomer.

4 Passive clearing: move the samples to 37 °C Until tissue is Change incubator and keep the sample at 37 transparent. solution °C. Maximum one every 1 or week (Fig. 1). 2 days.

5 Wash: Change to boric acid buffer (0.2 37 °C 2 d Change M/pH 8.5 with 0.1% (vol/vol) Triton solution X-100). keep for 2 days at 37 °C. after 1 d. This step is important for the removal of the SDS.

6 Immunostaining preparation: Incubate 37 °C 30 min Tissue will with PBST (0.1%) for 30 min become less transparent after

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incubation with PBST

7 Immunostaining primary antibody: 37 °C 4 days Incubate with primary antibody in PBST (1:500, NC82)

8 Wash: PBST 37 °C 1 day

9 Immunostaining secondary antibody: 37 °C 2 days Incubate with secondary antibody (3:500, anti-mouse 488) and Hoechst (1:500) in PBST

10 Wash: PBST 37 °C at least 1/2 day, 3 washes

11 Sample mounting and refractive index Tissue homogenization: Incubate in glycerol should 75% and mount sample in custom become aluminium slides. clearer in Glycerol

Fig 1. CLARITY clearing. Effective clearing of the tissue can be achieved via the removal of lipids with SDS. The brain original brain (A) gets more transparent after three (B) and seven (C) days of clearing

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Appendix 4 Preliminary results of immunohistochemistry and western blotting for PICK1

No major differences in the observed intensity between stung and control brains were found when staining with Pick1 antibodies using the CLARITY method, although specific cell bodies were marked in control but not in stung brains (Fig. 1). Pick1 expression in stung cockroaches was lower than in control cockroaches (in 3 trials out of 4), but this result was not significant due to small sample size (Fig. 6).

Figure 1: PICK1 staining. Representing confocal microscopy images for PICK1 using the 4% acrylamide CLARITY protocol for brains of control (Top images) and stung (bottom images) cockroaches. Images show Pick1 staining in two different depth points. Observed intensity is comparable but in control brains specific cell bodies may have been marked (red arrows).

Figure 2: Quantitative western blotting. Apparent downregulation of both isoforms of PICK1. Error bars represent standard error.

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תקציר העבודה

הצרעה הטפילית, צרעת התכשיט, Ampulex compressa צדה תיקנים כמקור מזון חי לצאצאיה. הצרעה עוקצת תיקן אל תוך ראשו ומזריקה קוקטייל של ארס ישירות אל תוך גנגליוני הראש של התיקן ,לתוך הגנגליון התת ושטי )SEG( לתוך הגנגליון העל ושטי )מוח התיקן(. רוב הארס נמצא בעיקר בתוך ובאזור הקומפלקס המרכזי במוח )CX(, אזור במוח התיקן החשוב ליוזמת ושמירת ההליכה. על מנת לספק לצאצאיה מזון טרי, עקיצת הצרעה משרה שינוי התנהגותי בתיקן, שהופך לאחר העקיצה להיפוקינטי ואינו מסוגל ליזום תנועה רצונית במשך מספר ימים. אולם, על אף שאינו נע בעצמו, התיקן העקוץ אינו משותק. במובן זה, עקיצת צרעת התכשיט הינה ייחודית לעומת צרעות טפיליות אחרות, אשר בדרך כלל משתקות את טרפן. אך במובן רחב יותר, יש דוגמאות נוספות לטפילים המשנים את התנהגות מאחסנם. תחום זה הנקרא נוירו-פריזטולוגיה, הינו תחום חדש יחסית, העוסק במנגנונים המאפשרים לטפילים לשלוט בהתנהגות מאחסנם, לטובת הטפיל )או צאצאיו(. היכולת של טפילים לשנות התנהגות אצל המאחסנים שלהם, מעוררת עניין רב בציבור המדעי וכמו כן, בציבור הלא מדעי. אך עדיין, מעט מאוד ידוע על המנגנונים בהם טפיל יכול לשלוט במערכת העצבים של המאחסן שלו.

בעבודה המתוארת במחקר זה אני מראה שהזרקת ארס בSEG או ב ,CX מספיקה כדי להוריד את התנהגות ההליכה סהפונטנית בתיקן, לפחות לטווח הקצר. עוד על כך, אני מראה שחלבונים בארס הכרחיים לשינוי ההתנהגותי בתיקן וכמו כן, אני מראה שהארס מורכב ממארג רב של חלבונים, אניזמים ופפטידים. ביניהם בולטים במיוחד: פרוטאזות, פוספוליפאזות, אמפולקסינים וטאקיקינינים, בנוסף למספר רב של פפטידים וחלבונים החדשים למדע. תוצאות אלו מצביעות על כך שהצרעה משתמשת בארס כתקיפה מרובת מטרות על גנגליוני הראש של התיקן.

מכיוון שהשפעת הארס על התיקן היא ארוכת טווח והפיכה, שערתי שההשפעה ארוכת הטווח מתווכת ע"י שינויים בביטוי חלבונים בגנגליוני הראש. התוצאות המתוארות כאן, מראות כי הארס קושר חלבונים סינפטיים ולכן יתכן שמשפיע על תהליכים סינפטיים. אני מראה בעבודה זו, שיש שינויים במספר רב של חלבונים בגנגליוני הראש לאחר עקיצה, רבים מעורבים בתהליכי מעבר אותות בתא, כמו מסלול ה- Rho GTPase, אשר מעורב בתהליכי גמישות סינפטית.

תוצאות מחקרי מצביעות על כך שהארס יכול להשפיע על מספר רב של תהליכים ובכך לגרום שינויים ביעלות הסינפטית בגנגליוני הראש ולכן לירידה בהליכה הספונטנית בתיקן. ממצאים אלו מהווים את הבסיס לבניית השערות ספציפיות וחקר המנגנונים שבבסיס המניפולציה ההתנהגותית.

מילות מפתח: צרעה; תיקן; נוירואתולוגיה; פרזיטואיד; נוירו-פרזיטולוגיה; ארס; הקומפלקס המרכזי; יעילות סינפטית.

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סיוון תשע"ח יוני 2018

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