Advertisement

Parasitoid Jewel Wasp Mounts Multipronged Neurochemical Attack to Hijack a Host Brain*[S]

  • Ryan Arvidson
    Affiliations
    From the ‡Graduate Program in Biochemistry and Molecular Biology, University of California, Riverside, California 92521;

    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Maayan Kaiser
    Affiliations
    §Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel;
    Search for articles by this author
  • Sang Soo Lee
    Affiliations
    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;

    ‖Graduate Program in Neuroscience, University of California, Riverside, California 92521;
    Search for articles by this author
  • Jean-Paul Urenda
    Affiliations
    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Christopher Dail
    Affiliations
    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Haroun Mohammed
    Affiliations
    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Cebrina Nolan
    Affiliations
    **Department of Entomology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Songqin Pan
    Affiliations
    ‡‡Institute for Integrated Genome Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Jason E. Stajich
    Affiliations
    §§Department of Microbiology & Plant Pathology, University of California, Riverside, California 92521
    Search for articles by this author
  • Frederic Libersat
    Affiliations
    §Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel;
    Search for articles by this author
  • Michael E. Adams
    Correspondence
    To whom correspondence should be addressed: Department of Entomology, University of California, Riverside, CA 92521; Tel.: +1-951-827-4746;
    Affiliations
    From the ‡Graduate Program in Biochemistry and Molecular Biology, University of California, Riverside, California 92521;

    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;

    ‖Graduate Program in Neuroscience, University of California, Riverside, California 92521;

    **Department of Entomology, University of California, Riverside, California 92521;

    ‡‡Institute for Integrated Genome Biology, University of California, Riverside, California 92521;

    ¶Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521;
    Search for articles by this author
  • Author Footnotes
    * This work was supported by BSF grant number (2015161).
    >[S] This article contains supplemental Figures, Tables, and Movie.
    >1 The abbreviations used are:SEGsubesophageal ganglionMudPITmultiple dimension Protein Identification TechnologyemPAIexponentially modified Protein Abundance IndexPSMProtein spectral countVSvenom sacDVductus venatus.
Open AccessPublished:October 06, 2018DOI:https://doi.org/10.1074/mcp.RA118.000908
      The parasitoid emerald jewel wasp Ampulex compressa induces a compliant state of hypokinesia in its host, the American cockroach Periplaneta americana through direct envenomation of the central nervous system (CNS). To elucidate the biochemical strategy underlying venom-induced hypokinesia, we subjected the venom apparatus and milked venom to RNAseq and proteomics analyses to construct a comprehensive “venome,” consisting of 264 proteins. Abundant in the venome are enzymes endogenous to the host brain, including M13 family metalloproteases, phospholipases, adenosine deaminase, hyaluronidase, and neuropeptide precursors. The amphipathic, alpha-helical ampulexins are among the most abundant venom components. Also prominent are members of the Toll/NF-κB signaling pathway, including proteases Persephone, Snake, Easter, and the Toll receptor ligand Spätzle. We find evidence that venom components are processed following envenomation. The acidic (pH∼4) venom contains unprocessed neuropeptide tachykinin and corazonin precursors and is conspicuously devoid of the corresponding processed, biologically active peptides. Neutralization of venom leads to appearance of mature tachykinin and corazonin, suggesting that the wasp employs precursors as a prolonged time-release strategy within the host brain post-envenomation. Injection of fully processed tachykinin into host cephalic ganglia elicits short-term hypokinesia. Ion channel modifiers and cytolytic toxins are absent in A. compressa venom, which appears to hijack control of the host brain by introducing a “storm” of its own neurochemicals. Our findings deepen understanding of the chemical warfare underlying host-parasitoid interactions and in particular neuromodulatory mechanisms that enable manipulation of host behavior to suit the nutritional needs of opportunistic parasitoid progeny.

      Graphical Abstract

      The parasitoid jewel wasp Ampulex compressa (Aculeata: Ampulicidae) injects venom directly into the central nervous system of its host, the American cockroach (Periplaneta americana) to induce a week-long lethargic state known as hypokinesia (
      • Haspel G.
      • Rosenberg L.A.
      • Libersat F.
      Direct injection of venom by a predatory wasp into cockroach brain.
      ). Following envenomation, the stung host becomes compliant, allowing the wasp to physically manipulate and maneuver it into its burrow. Upon first encountering its victim, the wasp promptly and aggressively attacks, first stinging into the prothoracic ganglion to cause a 2- to 3-min flaccid paralysis of the prothoracic legs (
      • Haspel G.
      • Libersat F.
      Wasp venom blocks central cholinergic synapses to induce transient paralysis in cockroach prey.
      ). This facilitates subsequent, precise stings into two cephalic ganglia: the brain–specifically the central complex (
      • Haspel G.
      • Rosenberg L.A.
      • Libersat F.
      Direct injection of venom by a predatory wasp into cockroach brain.
      )–and the subesophageal ganglion (SEG)

      The abbreviations used are:

      SEG
      subesophageal ganglion
      MudPIT
      multiple dimension Protein Identification Technology
      emPAI
      exponentially modified Protein Abundance Index
      PSM
      Protein spectral count
      VS
      venom sac
      DV
      ductus venatus.
      . Upon completion of stings into cephalic ganglia, the cockroach engages in vigorous grooming behavior for ∼20 min while the wasp readies its burrow (
      • Weisel-Eichler A.
      • Haspel G.
      • Libersat F.
      Venom of a parasitoid wasp induces prolonged grooming in the cockroach.
      ). Once the stung cockroach is led into the burrow, the wasp deposits a single egg on the femur of the host leg. The wasp larva hatches within 3 days and begins feeding on host hemolymph by inserting its mandibles through soft cuticle at the base of the leg. At the end of the second instar, the larva enters the host body cavity and consumes internal tissues selectively during the third instar; pupation occurs around day eight. After 4 weeks, the adult wasp emerges and the life cycle is completed (
      • Arvidson R.
      • Landa V.
      • Frankenberg S.
      • Adams M.E.
      Life history of the emerald jewel wasp, Ampulex compressa.
      ,
      • Haspel G.
      • Gefen E.
      • Ar A.
      • Glusman J.G.
      • Libersat F.
      Parasitoid wasp affects metabolism of cockroach host to favor food preservation for its offspring.
      ).
      Hypokinesia is a specific, venom-induced behavioral state characterized by increased threshold for the escape response and reduced spontaneous walking; other motor functions remain intact, such as the righting response, swimming, eating and drinking. Thus, stung, hypokinesic cockroaches walk if pulled, swim if submerged in water and fly in a wind tunnel. The venom appears to modulate descending signals from cephalic ganglia, resulting in suppression of host escape behavior, without affecting other behaviors (
      • Libersat F.
      Wasp uses venom cocktail to manipulate the behavior of its cockroach prey.
      ).
      Whereas many parasitoid venoms are simply paralytic, A. compressa venom targets cephalic ganglia specifically and modifies a specific subset of behaviors related to escape; this is particularly interesting and unique among host-parasitoid interactions (
      • Asgari S.
      • Rivers D.B.
      Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.
      ,
      • Piek T.
      Neurotoxins from venoms of the Hymenoptera - twenty-five years of research in Amsterdam.
      ). Hypokinesia is reversible: if egg deposition is prevented following the sting, the escape response of a stung cockroach returns to normal within 5–7 days. Interestingly, this corresponds closely to the duration of larval development. Furthermore, the venom lacks necrotic or lethal effects, so that the hypokinesic host remains in good condition as a food source for the wasp larva (
      • Gal R.
      • Rosenberg L.A.
      • Libersat F.
      Parasitoid wasp uses a venom cocktail injected into the brain to manipulate the behavior and metabolism of its cockroach prey.
      ).
      Regarding the host-parasitoid interaction described here, the biochemical bases for some behavioral sequelae of post-envenomation have been described previously. For example, short-term paralysis of prothoracic legs induced by the initial sting into the prothoracic ganglion, is caused by venom components GABA and GABAA receptor agonists β-alanine and taurine (
      • Moore E.L.
      • Haspel G.
      • Libersat F.
      • Adams M.E.
      Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host.
      ). The vigorous grooming response induced by stings into cephalic ganglia likely result from venom component dopamine (
      • Weisel-Eichler A.
      • Haspel G.
      • Libersat F.
      Venom of a parasitoid wasp induces prolonged grooming in the cockroach.
      ,
      • Moore E
      • et al.
      Ampulexins: A new family of peptides in venom of the emerald jewel wasp, Ampulex compressa.
      ).
      Venom-induced hypokinesia raises an interesting biological question: How can such a potent biochemical mixture cause such 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. Recent advances in nucleotide sequencing and mass spectrometry technologies have greatly facilitated protein discovery in nonmodel systems, thus advancing the field of venomics (
      • Escoubas P.
      • Quinton L.
      • Nicholson G.M.
      Venomics: unravelling the complexity of animal venoms with mass spectrometry.
      ). In this study, transcriptomes and differential expression analysis of the venom apparatus were generated de novo, using Illumina short read sequencing and the Trinity pipeline (
      • Grabherr MG
      • et al.
      Full-length transcriptome assembly from RNA-Seq data without a reference genome.
      ,
      • Haas BJ
      • et al.
      De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.
      ). 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 Multiple dimension Protein Identification Technology (MudPIT), has been used to profile complex proteomes, including venoms (
      • Batista CV
      • et al.
      Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins.
      ,
      • dos Santos L.D.
      • Dias N.B.
      • Roberto J.
      • Pinto A.S.
      • Palma M.S.
      Brown recluse spider venom: proteomic analysis and proposal of a putative mechanism of action.
      ,
      • Haney R.A.
      • Ayoub N.A.
      • Clarke T.H.
      • Hayashi C.Y.
      • Garb J.E.
      Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.
      ) (supplemental Fig. S1).
      Although 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.

      DISCUSSION

      Hypokinesia induced by A. compressa in its envenomated cockroach host is remarkable for its specificity, duration, and reversibility. Our objective in this study was to begin unraveling how components of the venom mixture induce a sleep-like lethargic state lasting for about a week. In the absence of genomic data, we constructed a comprehensive venome, consisting of venom apparatus transcriptomics and proteomic analysis of milked venom.
      Combined transcriptomics and proteomics greatly facilitate protein discovery and annotation. Transcript open reading frames (ORFs) were validated as venom-specific via mass spectroscopy-derived peptides isolated from milked venom. The putative function or novelty of each identified ORF can then be determined by searching global databases. Whereas trypsinized venom samples allowed for analysis of larger venom proteins by aligning digestive peptides unto its ORF, the analysis of nontrypsinized samples revealed many small, novel peptides as well as the notable absence of mature neurotransmitter peptides. Consisting of 69,009 transcripts and ∼264 proteins, the A. compressa venome represents one of the most comprehensive descriptions of a parasitoid venom to date (
      • Moreau S.J.
      • Asgari S.
      Venom proteins from parasitoid wasps and their biological functions.
      ,
      • Sim A.D.
      • Wheeler D.
      The venom gland transcriptome of the parasitoid wasp Nasonia vitripennis highlights the importance of novel genes in venom function.
      ). It reveals a plethora of potential biochemical actions on the host brain that is stimulating hypothesis testing of this interspecific neuromodulation. A salient feature of the venome is absence of conventional ion channel-directed toxins or necrotic enzymes. Instead, the chemical biology of the venom appears to create a “neurochemical storm” in the envenomated host brain, re-ordering its function with its own constituents for the benefit of the parasitoid larva.
      Transcriptomics of the VG and VS confirm that they both serve as glandular sources of the overall protein repertoire of the venom, precluding the notion that the venom sac serves strictly as a passive venom reservoir. Although VG and VS differ greatly in expression levels of certain venom transcripts, each has at least some level of expression for the great majority of venom proteins. For example, the ampulexins, among the most abundant venom components, are products primarily of the VS, confirming its functional role as a venom gland-independent contributor to the venom (
      • Moore E
      • et al.
      Ampulexins: A new family of peptides in venom of the emerald jewel wasp, Ampulex compressa.
      ). We have demonstrated the acidic nature of A. compressa venom and that VS contents are more acidic than those of the venom gland, i.e. in the pH range 4–5 (supplemental Fig. S8). It is reasonable to infer that venom gland products are translocated into the VS, where it is supplemented with additional proteins and peptides and maintained under acidic conditions. The muscle-bound VS is innervated and contracts in response to calcium entry; it is thus ready to expel venom in response to neural inputs, presumably induced by mechanoreceptors on the stinger shaft that help target the sting by testing the density of the neural tissue (
      • Gal R.
      • Kaiser M.
      • Haspel G.
      • Libersat F.
      Sensory arsenal on the stinger of the parasitoid jewel wasp and its possible role in identifying cockroach brains.
      ).
      We propose that the acidic nature of the venom serves several purposes, including preservation of protein integrity in the mixture until injected into the host brain and delayed biosynthesis and processing of neuropeptide precursors. Once envenomation occurs, the diverse range of proteases in the venom may contribute to: (1) destruction of the extracellular matrix, facilitating penetration of the venom in the host brain, (2) loss of synapse integrity, perhaps contributing to hypokinesia, (3) processing of venom protein precursors into active form, including zymogens and propeptide precursors, leading to disruption of host synaptic signaling, and (4) activation of the Toll signaling pathway.
      The large representation of M13 proteases, especially members of the neprilysin and endothelin-converting enzyme families is particularly noteworthy. Indeed, Hmmscan of the venom against Swiss-Prot and PfamA databases shows that 10% of all proteins contain M13 protease domains. These proteases are reported to be anchored on the extracellular surface of expressing cells (
      • Turner A.J.
      • Isaac R.E.
      • Coates D.
      The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function.
      ), 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.
      Besides proteases that activate zymogens and propeptide convertases, the venom contains enzymes involved in post-translational modification of neuropeptides, including amidation and pyroglutamyl capping. Our evidence suggests that these are bona fide venom enzymes rather than originating in the venom gland ER and “hitch-hiking” into the venom in trace amounts (i.e. ER retention signals KDEL or HDEL are absent). Venom neuropeptide precursors tachykinin and corazonin, prominent in the venom mixture, 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. In honeybees, phospholipases have cytolytic activity, especially in the presence of melittin, although we reported previously that A. compressa venom is not lytic (
      • Moore E
      • et al.
      Ampulexins: A new family of peptides in venom of the emerald jewel wasp, Ampulex compressa.
      ). Phospholipase A2 activity may also interfere with endogenous lipid signaling systems by releasing lipid secondary messengers (e.g. arachidonic or lysophosphatidic acids) from membranes. The toxicity of phospholipase A2 in some snake venoms is attributed to agonism of secretory phospholipase A2 receptors, rather than their hydrolysis of membrane lipids (
      • Lambeau G.
      • Lazdunski M.
      Receptors for a growing family of secreted phospholipases A2.
      ,
      • Rouault M
      • et al.
      Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.
      ).
      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 matrix (
      • King T.P.
      • Wittkowski K.M.
      Hyaluronidase and hyaluronan in insect venom allergy.
      ,
      • Girish K.S.
      • Jagadeesha D.K.
      • Rajeev K.B.
      • Kemparaju K.
      Snake venom hyaluronidase: an evidence for isoforms and extracellular matrix degradation.
      ). Hyaluronan, a major component of the extracellular matrix, is important in maintaining synapse connectivity (
      • Bikbaev A.
      • Frischknecht R.
      • Heine M.
      Brain extracellular matrix retains connectivity in neuronal networks.
      ,
      • Pyka M
      • et al.
      Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.
      ). Phospholipase A2 and hyaluronidase have been characterized as venom spreading factors through “loosening” of the extracellular space to allow penetration deeper into the tissue (
      • Girish K.S.
      • Shashidharamurthy R.
      • Nagaraju S.
      • Gowda T.V.
      • Kemparaju K.
      Isolation and characterization of hyaluronidase a “spreading factor” from Indian cobra (Naja naja) venom.
      ,
      • Kemparaju K.
      • Girish K.S.
      Snake venom hyaluronidase: a therapeutic target.
      ,
      • Tu A.T.
      • Hendon R.R.
      Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor.
      ,
      • Bordon K.C.
      • Wiezel G.A.
      • Amorim F.G.
      • Arantes E.C.
      Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology.
      ). It is interesting to consider what the effect of “loosening” cellular connectivity of a brain, without killing the cells, would have on synaptic transmission. 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.
      One of our more striking findings is presence in the venom of the Toll receptor activator Spätzle, along with upstream serine proteases that process it into active form, including Easter, Persephone, and Snake in the venom proteome, and gastrulation defective (Gd) expressed in the venom apparatus (
      • Jang IH
      • et al.
      ASpatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity.
      ). Activation of the Toll pathway triggers expression of the transcription factor NF-κB, which is well-known to have functional roles in neuroprotection and synaptic plasticity (
      • Engelmann C.
      • Haenold R.
      Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-kappaB.
      ,
      • Shih R.H.
      • Wang C.Y.
      • Yang C.M.
      NF-kappaB Signaling Pathways in Neurological Inflammation: A Mini Review.
      ). Although our phylogenetic analysis indicates that these proteases are bona fide Spätzle processing enzymes, they could have other functions as well; for example, serine protease Bi-VSP in bee venom activates the phenoloxidase cascade, but also targets fibrinogen, affecting blood clotting in mammals (
      • Choo YM
      • et al.
      Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.
      ).
      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. This is perhaps surprising considering its unique target location, the cockroach central nervous system, and the specific behavioral modification caused by the venom. For example, venom of another parasitoid wasp, N. vitripennis, contains many protein classes in common with A. compressa, including metalloprotease, serine protease and serine protease inhibitors, chitinase and trehalase, phosphatases, and lipases. We also found high representation of A. compressa venom homologs in genomes of the king cobra (91), black widow (91) and brown recluse (93) spiders, bark scorpion (103), and centipede (105), demonstrating conservation of certain venom proteins, the protease and lipase families, 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 animals, with a more limited representation in the nonvenomous 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 nonvenomous insects.
      The large molecular weight fraction of A. compressa venom contains proteins 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 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 peptides (
      • Pringos E.
      • Vignes M.
      • Martinez J.
      • Rolland V.
      Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on omega-agatoxins.
      ,
      • Dutertre S.
      • Lewis R.J.
      Use of venom peptides to probe ion channel structure and function.
      ,
      • Adams M.E.
      Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta.
      ,
      • Yoshikami D.
      • Bagabaldo Z.
      • Olivera B.M.
      The inhibitory effects of omega-conotoxins on Ca channels and synapses.
      ). 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 system (
      • Moore E.L.
      • Haspel G.
      • Libersat F.
      • Adams M.E.
      Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host.
      ). A. compressa venom contains several novel small peptide toxins whose role in hypokinesia is yet to be determined. Coding sequences and read counts are provided in the supplementary metadata.
      The presumed target of venom tachykinin is the cockroach tachykinin receptor. The role of tachykinin in induction of hypokinesia is supported by in vivo injection into the SEG. Mature A. compressa tachykinins can activate the cockroach tachykinin receptor with comparable affinities to endogenous tachykinins in vitro. These data further support the role of tachykinin in modulating locomotion and establish that tachykinin may module escape threshold in the subesophageal ganglion. The wasp also targets the central complex of the cockroach brain, a region known to regulate locomotion. This area of the brain contains tachykinin positive cells and is reported to express tachykinin receptors (
      • Vitzthum H.
      • Homberg U.
      Immunocytochemical demonstration of locustatachykinin-related peptides in the central complex of the locust brain.
      ,
      • Johard H.A.
      • Muren J.E.
      • Nichols R.
      • Larhammar D.S.
      • Nassel D.R.
      A putative tachykinin receptor in the cockroach brain: molecular cloning and analysis of expression by means of antisera to portions of the receptor protein.
      ). Venom induced hypokinesia is most likely caused by the concerted action of many elements in the venom, in which tachykinin and its processing may play an interesting and critical part.
      Tachykinin deficiency has been associated with hyperactivity in Drosophila, suggesting that elevated levels of the peptide may suppress locomotory activity (
      • Winther A.M.
      • Acebes A.
      • Ferrus A.
      Tachykinin-related peptides modulate odor perception and locomotor activity in.
      ,
      • Nassel D.R.
      • Winther A.M.
      Drosophila neuropeptides in regulation of physiology and behavior.
      ). The sub-esophageal ganglion, a target of the wasp venom, regulates locomotion in cockroaches (
      • Kaiser M.
      • Libersat F.
      The role of the cerebral ganglia in the venom-induced behavioral manipulation of cockroaches stung by the parasitoid jewel wasp.
      ,
      • Gal R.
      • Libersat F.
      A wasp manipulates neuronal activity in the sub-esophageal ganglion to decrease the drive for walking in its cockroach prey.
      ), and we demonstrate in this work that injection of tachykinin into the subesophageal ganglion of cockroaches causes a reversible effect on its escape response. Tachykinin has been implicated in affecting presynaptic inhibition in crayfish amacrine neurons and inhibits responses in cockroach olfactory receptor neurons (
      • Glantz R.M.
      • Miller C.S.
      • Nassel D.R.
      Tachykinin-related peptide and GABA-mediated presynaptic inhibition of crayfish photoreceptors.
      ,
      • Jung JW
      • et al.
      Neuromodulation of olfactory sensitivity in the peripheral olfactory organs of the American cockroach, Periplaneta americana.
      ). Functional analysis of venom tachykinin implicates tachykinin as a regulator of locomotion in the central nervous system and serves as a good example of how venomics of A. compressa venom generates testable hypotheses that lead to greater insight into the behavioral manipulation of its host.
      Corazonin activity in the venom was assessed using R. prolixus corazonin receptor expressing cells. Venom was milked in 0.5% aq. trifluoroacetic acid (TFA) to establish the corazoin activity of just-injected venom, assuming the solution would preclude any further activation of corazonin peptide from precursor. Simulating the acidic environment of the venom-sac, milked venom was also incubated in pH 4 and had an activity like venom milked in TFA. However, if incubated at pH 7, the venom had three times the corazonin activity as pH 4 and TFA. This supports the hypothesis that there is enough enzyme activity in the venom to process corazonin from precursor once injected. Further, this processing appears pH-sensitive, increasing in activity if at neutral pH, as would be found in the cockroach brain. This could serve as a time-release mechanism, where these neuropeptides are continuously generated at the injection site, as long as precursor or enzyme activity remain.
      This analysis reveals a multi-pronged attack on the envenomated cockroach CNS targeting endogenous signaling systems, and likely structural alterations of the synapse. Besides revealing mechanisms of hypokinesia induction, analysis of this venom can also inform about previously unrecognized signaling systems present in an adult insect brain. For example, presence of eclosion hormone and corazonin in the venom suggests that these signaling systems are present in the adult cockroach brain.
      Understanding venom composition is integral to deciphering the elements of venom action on the host brain. 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 to serve A. compressa's maternal, yet macabre, motives.

      DATA AVAILABILITY

      Raw RNA sequencing data was submitted to NCBI under BioProject PRJNA356979; https://www.ncbi.nlm.nih.gov/. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006340 (22); http://www.proteomexchange.org/.

      Acknowledgments

      We thank Robert Hice for his RNA sequencing library expertise and assistance with methods development, and to Peter Arensburger for preliminary bioinformatics support and expertise. We thank undergraduates Victor Landa for the sting photo in Figure 1, and Alex Nguyen for molecular biology support, and Sarah Frankenberg for assistance with the Toll pathway phylogeny and design of the graphical abstract. We are grateful to Ian Orchard and colleagues for providing the R. prolixus corazonin receptor mammalian expression plasmids. We acknowledge the Smoler Proteomics Center at the Technion-Israel Institute of Technology and the Mass Spectrometry Core facility of the Institute for Integrative Genome Biology. The Orbitrap Fusion mass spectrometer was purchased from an NIH shared instrumentation grant (S10 OD010669).

      REFERENCES

        • Haspel G.
        • Rosenberg L.A.
        • Libersat F.
        Direct injection of venom by a predatory wasp into cockroach brain.
        J. Neurobiol. 2003; 56: 287-292
        • Haspel G.
        • Libersat F.
        Wasp venom blocks central cholinergic synapses to induce transient paralysis in cockroach prey.
        J. Neurobiol. 2003; 54: 628-637
        • Weisel-Eichler A.
        • Haspel G.
        • Libersat F.
        Venom of a parasitoid wasp induces prolonged grooming in the cockroach.
        J. Exp. Biol. 1999; 202: 957-964
        • Arvidson R.
        • Landa V.
        • Frankenberg S.
        • Adams M.E.
        Life history of the emerald jewel wasp, Ampulex compressa.
        J. Hymenoptera Res. 2018; 63: 1-13
        • Haspel G.
        • Gefen E.
        • Ar A.
        • Glusman J.G.
        • Libersat F.
        Parasitoid wasp affects metabolism of cockroach host to favor food preservation for its offspring.
        J. Comparative Physiol. A. 2005; 191: 529-534
        • Libersat F.
        Wasp uses venom cocktail to manipulate the behavior of its cockroach prey.
        J. Comparative Physiol. A. 2003; 189: 497-508
        • Asgari S.
        • Rivers D.B.
        Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.
        Ann. Rev. Entomol. 2011; 56: 313-335
        • Piek T.
        Neurotoxins from venoms of the Hymenoptera - twenty-five years of research in Amsterdam.
        Comp. Biochem. Physiol. C. 1990; 96: 223-233
        • Gal R.
        • Rosenberg L.A.
        • Libersat F.
        Parasitoid wasp uses a venom cocktail injected into the brain to manipulate the behavior and metabolism of its cockroach prey.
        Arch. Insect Biochem. Physiol. 2005; 60: 198-200
        • Moore E.L.
        • Haspel G.
        • Libersat F.
        • Adams M.E.
        Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host.
        J. Neurobiol. 2006; 66: 811-820
        • Moore E
        • et al.
        Ampulexins: A new family of peptides in venom of the emerald jewel wasp, Ampulex compressa.
        Biochemistry. 2018; 57
        • Escoubas P.
        • Quinton L.
        • Nicholson G.M.
        Venomics: unravelling the complexity of animal venoms with mass spectrometry.
        J. Mass Spectrometry. 2008; 43: 279-295
        • Grabherr MG
        • et al.
        Full-length transcriptome assembly from RNA-Seq data without a reference genome.
        Nat. Biotechnol. 2011; 29: 644-652
        • Haas BJ
        • et al.
        De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.
        Nat. Protocols. 2013; 8: 1494-1512
        • Batista CV
        • et al.
        Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins.
        J. Chromatography B. 2004; 803: 55-66
        • dos Santos L.D.
        • Dias N.B.
        • Roberto J.
        • Pinto A.S.
        • Palma M.S.
        Brown recluse spider venom: proteomic analysis and proposal of a putative mechanism of action.
        Protein & Peptide Lett. 2009; 16: 933-943
        • Haney R.A.
        • Ayoub N.A.
        • Clarke T.H.
        • Hayashi C.Y.
        • Garb J.E.
        Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.
        BMC Genomics. 2014; 15: 366
        • Li B.
        • Dewey C.N.
        RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.
        BMC Bioinformatics. 2011; 12: 323
        • Love M.I.
        • Huber W.
        • Anders S.
        Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
        Genome Biol. 2014; 15: 550
        • Drakakaki G
        • et al.
        Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis.
        Cell Res. 2012; 22: 413-424
        • Hebert AS
        • et al.
        The one hour yeast proteome.
        Mol. Cell Proteomics. 2014; 13: 339-347
        • Vizcaino JA
        • et al.
        The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.
        Nucleic Acids Res. 2013; 41: D1063-D1069
        • Ishihama Y
        • et al.
        Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.
        Mol. Cell. Proteomics. 2005; 4: 1265-1272
        • Stamatakis A.
        RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies.
        Bioinformatics. 2014; 30: 1312-1313
        • Werren JH
        • et al.
        Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.
        Science. 2010; 327: 343-348
        • Poelchau M
        • et al.
        The i5k [email protected] - enabling genomic data access, visualization and curation of arthropod genomes.
        Nucleic Acids Res. 2015; 43: D714-D719
        • Wurm Y
        • et al.
        The genome of the fire ant Solenopsis invicta.
        Proc. Natl. Acad. Sci. USA. 2011; 108: 5679-5684
        • Smith CR
        • et al.
        Draft genome of the red harvester ant Pogonomyrmex barbatus.
        Proc. Natl. Acad. Sci. USA. 2011; 108: 5667-5672
        • Smith CD
        • et al.
        Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).
        Proc. Natl. Acad. Sci. USA. 2011; 108: 5673-5678
        • Bonasio R
        • et al.
        Genomic comparison of the ants Camponotus floridanusHarpegnathos saltator.
        Science. 2010; 329: 1068-1071
        • Suen G
        • et al.
        The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle.
        PLo Genet. S. 2011; 7: e1002007
        • Elsik CG
        • et al.
        Hymenoptera Genome Database: integrating genome annotations in HymenopteraMine.
        Nucleic Acids Res. 2016; 44: D793-D800
        • Nygaard S
        • et al.
        The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming.
        Genome Res. 2011; 21: 1339-1348
        • Sadd BM
        • et al.
        The genomes of two key bumblebee species with primitive eusocial organization.
        Genome Biol. 2015; 16: 76
        • Elsik CG
        • et al.
        Finding the missing honey bee genes: lessons learned from a genome upgrade.
        BMC Genomics. 2014; 15: 8
        • Honeybee Genome Sequencing Consortium
        Insights into social insects from the genome of the honeybee Apis mellifera.
        Nature. 2006; 443: 931-949
        • Attrill H
        • et al.
        FlyBase: establishing a Gene Group resource for Drosophila melanogaster.
        Nucleic Acids Res. 2016; 44: D786-D792
        • Tribolium Genome Sequencing Consortium
        The genome of the model beetle and pest Tribolium castaneum.
        Nature. 2008; 452: 949-955
        • Mouse Genome Sequencing Consortium
        Initial sequencing and comparative analysis of the mouse genome.
        Nature. 2002; 420: 520-562
        • Church DM
        • et al.
        Lineage-specific biology revealed by a finished genome assembly of the mouse.
        PLos Biol. 2009; 7: e1000112
        • Vonk FJ
        • et al.
        The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.
        Proc. Natl. Acad. Sci. USA. 2013; 110: 20651-20656
        • Vleugels R.
        • Lenaerts C.
        • Baumann A.
        • Vanden Broeck J.
        • Verlinden H.
        Pharmacological characterization of a 5-HT1-type serotonin receptor in the red flour beetle, Tribolium castaneum.
        PLoS ONE. 2013; 8: e65052
        • Park Y.
        • Kim Y.J.
        • Dupriez V.
        • Adams M.E.
        Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster.
        J. Biol. Chem. 2003; 278: 17710-17715
        • Torfs H
        • et al.
        Analysis of C-terminally substituted tachykinin-like peptide agonists by means of aequorin-based luminescent assays for human and insect neurokinin receptors.
        Biochem. Pharmacol. 2002; 63: 1675-1682
        • Hamoudi Z.
        • Lange A.B.
        • Orchard I.
        Identification and Characterization of the Corazonin Receptor and Possible Physiological Roles of the Corazonin-Signaling Pathway in Rhodnius prolixus.
        Front. Neurosci. 2016; 10: 35
        • Gavra T.
        • Libersat F.
        Involvement of the opioid system in the hypokinetic state induced in cockroaches by a parasitoid wasp.
        J. Comparative Physiol. A. 2011; 197: 279-291
        • Gal R.
        • Libersat F.
        A parasitoid wasp manipulates the drive for walking of its cockroach prey.
        Curr. Biol. 2008; 18: 877-882
        • Gnatzy W.
        • Michels J.
        • Volknandt W.
        • Goller S.
        • Schulz S.
        Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta, Hymenoptera, Sphecidae): structural and biochemical aspects.
        Arthropod Structure Development. 2015; 44: 491-507
        • Piek T
        • et al.
        The venom of Ampulex compressa - effects on behaviour and synaptic transmission of cockroaches.
        Comp. Biochem. Physiol. 1989; 92: 175-183
        • Parra G.
        • Bradnam K.
        • Korf I.
        CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes.
        Bioinformatics. 2007; 23: 1061-1067
        • King T.P.
        • Spangfort M.D.
        Structure and biology of stinging insect venom allergens.
        Int. Arch. Allergy Immunol. 2000; 123: 99-106
        • Matsui T.
        • Fujimura Y.
        • Titani K.
        Snake venom proteases affecting hemostasis and thrombosis.
        Biochim. Biophys. Acta. 2000; 1477: 146-156
        • Frischknecht R.
        • Seidenbecher C.I.
        The crosstalk of hyaluronan-based extracellular matrix and synapses.
        Neuron Glia Biol. 2008; 4: 249-257
        • Zimmermann D.R.
        • Dours-Zimmermann M.T.
        Extracellular matrix of the central nervous system: from neglect to challenge.
        Histochem. Cell Biol. 2008; 130: 635-653
        • Peters RS
        • et al.
        Evolutionary history of the Hymenoptera.
        Curr. Biol. 2017; 27: 1013-1018
        • Moreau S.J.
        • Asgari S.
        Venom proteins from parasitoid wasps and their biological functions.
        Toxins. 2015; 7: 2385-2412
        • Sim A.D.
        • Wheeler D.
        The venom gland transcriptome of the parasitoid wasp Nasonia vitripennis highlights the importance of novel genes in venom function.
        BMC Genomics. 2016; 17: 571
        • Gal R.
        • Kaiser M.
        • Haspel G.
        • Libersat F.
        Sensory arsenal on the stinger of the parasitoid jewel wasp and its possible role in identifying cockroach brains.
        PLoS ONE. 2014; 9: e89683
        • Turner A.J.
        • Isaac R.E.
        • Coates D.
        The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function.
        Bioessays. 2001; 23: 261-269
        • Lambeau G.
        • Lazdunski M.
        Receptors for a growing family of secreted phospholipases A2.
        Trends Pharmacol. Sci. 1999; 20: 162-170
        • Rouault M
        • et al.
        Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.
        Biochemistry. 2006; 45: 5800-5816
        • King T.P.
        • Wittkowski K.M.
        Hyaluronidase and hyaluronan in insect venom allergy.
        Int. Arch. Allergy Immunol. 2011; 156: 205-211
        • Girish K.S.
        • Jagadeesha D.K.
        • Rajeev K.B.
        • Kemparaju K.
        Snake venom hyaluronidase: an evidence for isoforms and extracellular matrix degradation.
        Mol. Cell. Biochem. 2002; 240: 105-110
        • Bikbaev A.
        • Frischknecht R.
        • Heine M.
        Brain extracellular matrix retains connectivity in neuronal networks.
        Sci. Reports. 2015; 5: 14527
        • Pyka M
        • et al.
        Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.
        Eur. J.Neurosci. 2011; 33: 2187-2202
        • Girish K.S.
        • Shashidharamurthy R.
        • Nagaraju S.
        • Gowda T.V.
        • Kemparaju K.
        Isolation and characterization of hyaluronidase a “spreading factor” from Indian cobra (Naja naja) venom.
        Biochimie. 2004; 86: 193-202
        • Kemparaju K.
        • Girish K.S.
        Snake venom hyaluronidase: a therapeutic target.
        Cell Biochem. Function. 2006; 24: 7-12
        • Tu A.T.
        • Hendon R.R.
        Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor.
        Comp. Biochem. Physiol. 1983; 76: 377-383
        • Bordon K.C.
        • Wiezel G.A.
        • Amorim F.G.
        • Arantes E.C.
        Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology.
        J. Venomous Animals Toxins Including Tropical Dis. 2015; 21: 43
        • Jang IH
        • et al.
        ASpatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity.
        Developmental Cell. 2006; 10: 45-55
        • Engelmann C.
        • Haenold R.
        Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-kappaB.
        Neural Plasticity. 2016; 2016: 7027949
        • Shih R.H.
        • Wang C.Y.
        • Yang C.M.
        NF-kappaB Signaling Pathways in Neurological Inflammation: A Mini Review.
        Front. Mol. Neurosci. 2015; 8: 77
        • Choo YM
        • et al.
        Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.
        PLoS ONE. 2010; 5: e10393
        • Pringos E.
        • Vignes M.
        • Martinez J.
        • Rolland V.
        Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on omega-agatoxins.
        Toxins. 2011; 3: 17-42
        • Dutertre S.
        • Lewis R.J.
        Use of venom peptides to probe ion channel structure and function.
        J. Biol. Chem. 2010; 285: 13315-13320
        • Adams M.E.
        Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta.
        Toxicon. 2004; 43: 509-525
        • Yoshikami D.
        • Bagabaldo Z.
        • Olivera B.M.
        The inhibitory effects of omega-conotoxins on Ca channels and synapses.
        Ann. N.Y. Acad. Sci. 1989; 560: 230-248
        • Vitzthum H.
        • Homberg U.
        Immunocytochemical demonstration of locustatachykinin-related peptides in the central complex of the locust brain.
        J. Comparative Neurol. 1998; 390: 455-469
        • Johard H.A.
        • Muren J.E.
        • Nichols R.
        • Larhammar D.S.
        • Nassel D.R.
        A putative tachykinin receptor in the cockroach brain: molecular cloning and analysis of expression by means of antisera to portions of the receptor protein.
        Brain Res. 2001; 919: 94-105
        • Winther A.M.
        • Acebes A.
        • Ferrus A.
        Tachykinin-related peptides modulate odor perception and locomotor activity in.
        Drosophila. Mol. Cell. Neurosci. 2006; 31: 399-406
        • Nassel D.R.
        • Winther A.M.
        Drosophila neuropeptides in regulation of physiology and behavior.
        Prog. Neurobiol. 2010; 92: 42-104
        • Kaiser M.
        • Libersat F.
        The role of the cerebral ganglia in the venom-induced behavioral manipulation of cockroaches stung by the parasitoid jewel wasp.
        J. Exp. Biol. 2015; 218: 1022-1027
        • Gal R.
        • Libersat F.
        A wasp manipulates neuronal activity in the sub-esophageal ganglion to decrease the drive for walking in its cockroach prey.
        PLoS ONE. 2010; 5: e10019
        • Glantz R.M.
        • Miller C.S.
        • Nassel D.R.
        Tachykinin-related peptide and GABA-mediated presynaptic inhibition of crayfish photoreceptors.
        J. Neurosci. 2000; 20: 1780-1790
        • Jung JW
        • et al.
        Neuromodulation of olfactory sensitivity in the peripheral olfactory organs of the American cockroach, Periplaneta americana.
        PLoS ONE. 2013; 8: e81361