Parasitoid wasp venom re-programs host behavior through downmodulation of brain central complex activity and motor output.

The parasitoid wasp Ampulex compressa hunts down its host, the American cockroach (Periplaneta americana), and envenomates its brain to make it a behaviorally compliant food supply for its offspring. The primary target of the wasp sting is a locomotory command center called the central complex (CX). In the present study, we employ, for the first time, chronic recordings of patterned cockroach CX activity in real time as the brain is infused with wasp venom. CX envenomation is followed by sequential changes in the pattern of neuronal firing that can be divided into three distinct temporal phases during the 2 h interval after venom injection: (1) reduction in neuronal activity for roughly 10 min immediately after venom injection; (2) rebound of activity lasting up to 25 min; (3) reduction of ongoing activity for up to 2 h. Long-term reduction of CX activity after venom injection is accompanied by decreased activity of both descending interneurons projecting to thoracic locomotory circuitry (DINs) and motor output. Thus, in this study, we provide a plausible chain of events starting in the CX that leads to decreased host locomotion following brain envenomation. We propose that these events account for the onset and maintenance of the prolonged hypokinetic state observed in stung cockroaches.

Parasitoid wasp venom manipulates host innate behavior via subtype-specific dopamine receptor activation.

The subjugation strategy employed by the jewel wasp is unique in that it manipulates the behavior of its host, the American cockroach, rather than inducing outright paralysis. Upon envenomation directly into the central complex (CX), a command center in the brain for motor behavior, the stung cockroach initially engages in intense grooming behavior, then falls into a lethargic sleep-like state referred to as hypokinesia. Behavioral changes evoked by the sting are due at least in part to the presence of the neurotransmitter dopamine in the venom. In insects, dopamine receptors are classified as two families, the D1-like and the D2-like receptors. However, specific roles played by dopamine receptor subtypes in venom-induced behavioral manipulation by the jewel wasp remain largely unknown. In the present study, we used a pharmacological approach to investigate roles of D1-like and D2-like receptors in behaviors exhibited by stung cockroaches, focusing on grooming. Specifically, we assessed behavioral outcomes of focal CX injections of dopamine receptor agonists and antagonists. Both specific and non-specific compounds were used. Our results strongly implicate D1-like dopamine receptors in venom-induced grooming. Regarding induction of hypokinesia, our findings demonstrate that dopamine signaling is necessary for induction of long-lasting hypokinesia caused by brain envenomation.

Parasitoid Jewel Wasp Mounts Multipronged Neurochemical Attack to Hijack a Host Brain.

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.

Ampulexins: A New Family of Peptides in Venom of the Emerald Jewel Wasp, Ampulex compressa.

The parasitoid wasp Ampulex compressa injects venom directly into the brain and subesophageal ganglion of the cockroach Periplaneta americana, inducing a 7 to 10 day lethargy termed hypokinesia. Hypokinesia presents as a significant reduction in both escape response and spontaneous walking. We examined aminergic and peptidergic components of milked venom with HPLC and MALDI-TOF mass spectrometry. HPLC coupled with electrochemical detection confirmed the presence of dopamine in milked venom, while mass spectrometry revealed that the venom gland and venom sac have distinct peptide profiles, with milked venom predominantly composed of venom sac peptides. We isolated and characterized novel α-helical, amphipathic venom sac peptides that constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin 1, into the subesophageal ganglion of cockroaches resulted in a short-term increase in escape threshold. Neither milked venom nor venom peptides interfered with growth of Escherichia coli or Bacillus thuringiensis on agar plates, and exposure to ampulexins or milked venom did not induce cell death in Chinese hamster ovary cells (CHO-K1) or Hi5 cells ( Trichoplusia ni).

Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling.

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

Behavioral and genomic characterization of molt-sleep in the tobacco hornworm, Manduca sexta.

During the transition from feeding to molting, larval insects undergo profound changes in behavior and patterns of gene expression regulated by the neuroendocrine system. For some species, a distinctive characteristic of molting larvae is presence of a quiescent state sometimes referred to as "molt-sleep". Here, observations of 4th instar Manduca sexta larvae indicate the molting period involves a predominantly quiescent state that shares behavioral properties of adult insect sleep in that it is rapidly reversible and accompanied by a reduced responsiveness to both mildly arousing and noxious stimuli. When subjected to noxious stimuli, molting larvae exhibit locomotory and avoidance behaviors similar to those of inter-molt larvae. Although less consolidated, inter-molt quiescence shares many of the same behavioral traits with molting quiescence. However, when subjected to deprivation of quiescence, inter-molt larvae display a compensatory rebound behavior that is not detected in molting larvae. This suggests that molting quiescence is a specialized form of inactivity that affords survival advantages to molting larvae. RNA-seq analysis of molting larvae shows general reduction in expression of genes encoding GPCRs and down regulation of genes connected with cyclic nucleotide signaling. On the other hand, certain ion channel genes are up-regulated, including transient receptor potential (TRP) channels, chloride channels and a voltage-dependent calcium channel. These findings suggest patterns of gene expression consistent with elevation of quiescent state characteristic of the molt in a model holometabolous insect.

Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

Potent activity of a PK/PBAN analog with an (E)-alkene, trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in insects, including regulation of sex pheromone biosynthesis in moths. A cyclic PK/PBAN analog (cyclo[NTSFTPRL]) retains significant activity on the pheromonotropic HevPBANR receptor from the tobacco budworm Heliothis virescens expressed in CHO-K1 cells. Previous studies indicate that this rigid, cyclic analog adopts a type I beta-turn with a transPro over residues TPRL within the core PK/PBAN region. An analog containing an (E)-alkene, trans-Pro mimetic motif was synthesized, and upon evaluation on the HevPBANR receptor found to have an EC(50) value that is not statistically different from a parent C-terminal PK/PBAN hexapeptide sequence. The results, in aggregate, provide strong evidence for the orientation of Pro and the core conformation of PK/PBAN neuropeptides during interaction with the expressed PBAN receptor. The work further identifies a novel scaffold with which to design mimetic PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated pheromone signaling systems.

Identification, developmental expression, and functions of bursicon in the tobacco hawkmoth, Manduca sexta.

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as crustacean cardioactive peptide in neurons of the ventral nervous system were observed in pharate larval, pupal, and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae expresses ecdysis-triggering hormone (ETH) receptors, suggesting that they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs-like immunoreactivity, whereas burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion.

Strategic expression of ion transport peptide gene products in central and peripheral neurons of insects.

Structurally related ion transport peptides (ITP) and crustacean hyperglycemic hormones (CHH) are increasingly implicated in diverse metabolic and developmental functions in arthropods. We identified a conserved ITP gene encoding two peptides by alternative splicing in Manduca sexta, Bombyx mori, and Aedes aegypti: A C-terminally amidated ITP and a C-terminally unblocked ITP-like peptide (ITPL), which share common N-terminal sequences but have divergent C-termini. In the moth M. sexta, these peptides are expressed in two, regionally distinct neuronal populations in the central and peripheral nervous systems (CNS, PNS). MasITP expression is confined to the brain in five pairs of lateral neurosecretory cells (type Ia(2)) projecting ipsilateral axons into the retrocerebral complex and three to five pairs of adjacent small neurons that arborize extensively within the brain. Expression of MasITPL is comparatively weak in the brain but strong in the ventral ganglia and the PNS, where MasITP is absent. MasITPL occurs in bilaterally paired neurons of all thoracic and abdominal ganglia. In the PNS, MasITPL is coexpressed with crustacean cardioactive peptide in type II link nerve neurons (L1) of abdominal segments 2-7, which project axons into neurohemal transverse nerves. During metamorphosis, additional expression of MasITPL is observed in two pairs of small lateral neurons in the brain and one pair of ventromedial neurons in each of AG2-6. A similar pattern of differential ITP and ITPL expression was observed in the CNS and PNS of B. mori and Schistocerca americana. These distinctive cellular expression patterns suggest that ITP and ITPL have evolved specialized physiological functions in arthropods.

Central peptidergic ensembles associated with organization of an innate behavior.

At the end of each developmental stage, insects perform the ecdysis sequence, an innate behavior necessary for shedding the old cuticle. Ecdysis triggering hormones (ETHs) initiate these behaviors through direct actions on the CNS. Here, we identify the ETH receptor (ETHR) gene in the moth Manduca sexta, which encodes two subtypes of GPCR (ETHR-A and ETHR-B). Expression of ETHRs in the CNS coincides precisely with acquisition of CNS sensitivity to ETHs and behavioral competence. ETHR-A occurs in diverse networks of neurons, producing both excitatory and inhibitory neuropeptides, which appear to be downstream signals for behavior regulation. These peptides include allatostatins, crustacean cardioactive peptide (CCAP), calcitonin-like diuretic hormone, CRF-like diuretic hormones (DHs) 41 and 30, eclosion hormone, kinins, myoinhibitory peptides (MIPs), neuropeptide F, and short neuropeptide F. In particular, cells L(3,4) in abdominal ganglia coexpress kinins, DH41, and DH30, which together elicit the fictive preecdysis rhythm. Neurons IN704 in abdominal ganglia coexpress CCAP and MIPs, whose joint actions initiate the ecdysis motor program. ETHR-A also is expressed in brain ventromedial cells, whose release of EH increases excitability in CCAP/MIP neurons. These findings provide insights into how innate, centrally patterned behaviors can be orchestrated via recruitment of peptide cotransmitter neurons.

Corazonin receptor signaling in ecdysis initiation.

Corazonin is a highly conserved neuropeptide hormone of wide-spread occurrence in insects yet is associated with no universally recognized function. After discovery of the corazonin receptor in Drosophila, we identified its ortholog in the moth, Manduca sexta, as a prelude to physiological studies. The corazonin receptor cDNA in M. sexta encodes a protein of 436 amino acids with seven putative transmembrane domains and shares common ancestry with its Drosophila counterpart. The receptor exhibits high sensitivity and selectivity for corazonin when expressed in Xenopus oocytes (EC(50) approximately 200 pM) or Chinese hamster ovary cells (EC(50) approximately 75 pM). Northern blot analysis locates the receptor in peripheral endocrine Inka cells, the source of preecdysis- and ecdysis-triggering hormones. Injection of corazonin into pharate larvae elicits release of these peptides from Inka cells, which induce precocious preecdysis and ecdysis behaviors. In vitro exposure of isolated Inka cells to corazonin (25-100 pM) induces preecdysis- and ecdysis-triggering hormone secretion. Using corazonin receptor as a biosensor, we show that corazonin concentrations in the hemolymph 20 min before natural preecdysis onset range from 20 to 80 pM and then decline over the next 30-40 min. These findings support the role of corazonin signaling in initiation of the ecdysis behavioral sequence. We propose a model for peptide-mediated interactions between Inka cells and the CNS underlying this process in insect development.

Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster.

Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila gene CG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies.