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.

Neuroparasitology of Parasite-Insect Associations.

Insect behavior can be manipulated by parasites, and in many cases, such manipulation involves the central and peripheral nervous system. Neuroparasitology is an emerging branch of biology that deals with parasites that can control the nervous system of their host. The diversity of parasites that can manipulate insect behavior ranges from viruses to macroscopic worms and also includes other insects that have evolved to become parasites (notably, parasitic wasps). It is remarkable that the precise manipulation observed does not require direct entry into the insect brain and can even occur when the parasite is outside the body. We suggest that a spatial view of manipulation provides a holistic approach to examining such interactions. Integration across approaches from natural history to advanced imaging techniques, omics, and experiments will provide new vistas in neuroparasitology. We also suggest that for researchers interested in the proximate mechanisms of insect behaviors, studies of parasites that have evolved to control such behavior is of significant value.

The role of the cerebral ganglia in the venom-induced behavioral manipulation of cockroaches stung by the parasitoid jewel wasp.

The jewel wasp stings cockroaches and injects venom into their cerebral ganglia, namely the subesophageal ganglion (SOG) and supraesophageal ganglion (SupOG). 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 SOG reduces neuronal activity, thereby suggesting a similar effect of venom injection in the SupOG. Paradoxically, SupOG-ablated cockroaches show increased spontaneous walking in comparison with control. Yet most of the venom in the SupOG 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. Our first aim was to resolve this discrepancy by using a combination of behavioral and neuropharmacological tools. Our 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 SOG decreases walking. Hence our second aim was to test the interactions between the SupOG and SOG in the venom-induced behavioral manipulation. We show that, in the absence of the inhibitory control of the SupOG on walking initiation, injection of venom in the SOG alone by the wasp is sufficient to induce the hypokinetic state. To summarize, we show that venom injection to either the SOG or the CX of the SupOG is, by itself, sufficient to decrease walking.

What can parasitoid wasps teach us about decision-making in insects?

Millions of years of co-evolution have driven parasites to display very complex and exquisite strategies to manipulate the behaviour of their hosts. However, although parasite-induced behavioural manipulation is a widespread phenomenon, the underlying neuronal mechanisms are only now beginning to be deciphered. Here, we review recent advancements in the study of the mechanisms by which parasitoid wasps use chemical warfare to manipulate the behaviour of their insect hosts. We focus on a particular case study in which a parasitoid wasp (the jewel wasp Ampulex compressa) performs a delicate brain surgery on its prey (the American cockroach Periplaneta americana) to take away its motivation to initiate locomotion. Following a brief background account of parasitoid wasps that manipulate host behaviour, we survey specific aspects of the unique effects of the A. compressa venom on the regulation of spontaneous and evoked behaviour in the cockroach host.

Suppression of host nocifensive behavior by parasitoid wasp venom.

The parasitoid wasp Ampulex compressa envenomates the brain of its host the American cockroach (Periplaneta americana), thereby making it a behaviorally compliant food supply for its offspring. The target of venom injection is a locomotory command center in the brain called the central complex. In this study, we investigate why stung cockroaches do not respond to injuries incurred during the manipulation process by the wasp. In particular, we examine how envenomation compromises nociceptive signaling pathways in the host. Noxious stimuli applied to the cuticle of stung cockroaches fail to evoke escape responses, even though nociceptive interneurons projecting to the brain respond normally. Hence, while nociceptive signals are carried forward to the brain, they fail to trigger robust nocifensive behavior. Electrophysiological recordings from the central complex of stung animals demonstrate decreases in peak firing rate, total firing, and duration of noxious-evoked activity. The single parameter best correlated with altered noxious-evoked behavioral responses of stung cockroaches is reduced duration of the evoked response in the central complex. Our findings demonstrate how the reproductive strategy of a parasitoid wasp is served by venom-mediated elimination of aversive, nocifensive behavior in its host.

A parasitoid wasp manipulates the drive for walking of its cockroach prey.

The parasitoid wasp A. compressa hunts cockroaches as a live food supply for its offspring. The wasp selectively injects venom into the cerebral ganglia of the prey to induce long-term hypokinesia [1-5], during which the stung cockroach, although not paralyzed, does not initiate spontaneous walking and fails to escape aversive stimuli. This allows the wasp to grab the cockroach by the antenna and walk it to a nest much like a dog on a leash. There, the wasp lays an egg on the prey, seals the nest, and leaves. The stung cockroach, however, does not fight to escape its tomb but rather awaits its fate, being consumed alive by the hatching larva over several days. We investigated whether the venom-induced hypokinesia is a result of an overall decrease in arousal or, alternatively, a specific decrease in the drive to initiate or maintain walking. We found that the venom specifically affects both the threshold for the initiation and the maintenance of walking-related behaviors. Nevertheless, the walking pattern generator itself appears to be intact. We thus report that the venom, rather than decreasing overall arousal, manipulates neuronal centers within the cerebral ganglia that are specifically involved in the initiation and maintenance of walking.

Involvement of the opioid system in the hypokinetic state induced in cockroaches by a parasitoid wasp.

The parasitoid wasp Ampulex compressa stings and injects venom into the cockroach brain to induce a long-lasting hypokinetic state. This state is characterized by decreased responsiveness to aversive stimuli, suggesting the manipulation of a neuromodulatory system in the cockroach's central nervous system. A likely candidate is the opioid system, which is known to affect responsiveness to stimuli in insects. To explore this possibility, we injected cockroaches with different opioid receptor agonists or antagonists before they were stung by a wasp and tested the escape behavior of these cockroaches to electric foot shocks. Antagonists significantly decreased the startle threshold in stung individuals, whereas agonists led to an increased startle threshold in controls. Yet, neither agonists nor antagonists had any effect on grooming. To further characterize the interaction between the venom and opioid receptors, we used an antenna-heart preparation. In un-stung individuals external application of crude venom completely inhibits antenna-heart contractions. In stung individuals the antenna-heart showed no contractions. Although acetylcholine restored contractions, the opioid receptor antagonist naloxone was unable to antagonize the venom inhibition. These results suggest that the venom of A. compressa might contribute to the manipulation of cockroach behavior by affecting the opioid system.

Why behavioral neuroscience still needs diversity?: A curious case of a persistent need.

In the past few decades, a substantial portion of neuroscience research has moved from studies conducted across a spectrum of animals to reliance on a few species. While this undoubtedly promotes consistency, in-depth analysis, and a better claim to unraveling molecular mechanisms, investing heavily in a subset of species also restricts the type of questions that can be asked, and impacts the generalizability of findings. A conspicuous body of literature has long advocated the need to expand the diversity of animal systems used in neuroscience research. Part of this need is utilitarian with respect to translation, but the remaining is the knowledge that historically, a diverse set of species were instrumental in obtaining transformative understanding. We argue that diversifying matters also because the current approach limits the scope of what can be discovered. Technological advancements are already bridging several practical gaps separating these two worlds. What remains is a wholehearted embrace by the community that has benefitted from past history. We suggest the time for it is now.

On the Role of the Head Ganglia in Posture and Walking in Insects.

In insects, locomotion is the result of rhythm generating thoracic circuits and their modulation by sensory reflexes and by inputs from the two head ganglia, the cerebral and the gnathal ganglia (GNG), which act as higher order neuronal centers playing different functions in the initiation, goal-direction, and maintenance of movement. Current knowledge on the various roles of major neuropiles of the cerebral ganglia (CRG), such as mushroom bodies (MB) and the central complex (CX), in particular, are discussed as well as the role of the GNG. Thoracic and head ganglia circuitries are connected by ascending and descending neurons. While less is known about the ascending neurons, recent studies in large insects and Drosophila have begun to unravel the identity of descending neurons and their appropriate roles in posture and locomotion. Descending inputs from the head ganglia are most important in initiating and modulating thoracic central pattern generating circuitries to achieve goal directed locomotion. In addition, the review will also deal with some known monoaminergic descending neurons which affect the motor circuits involved in posture and locomotion. In conclusion, we will present a few issues that have, until today, been little explored. For example, how and which descending neurons are selected to engage a specific motor behavior and how feedback from thoracic circuitry modulate the head ganglia circuitries. The review will discuss results from large insects, mainly locusts, crickets, and stick insects but will mostly focus on cockroaches and the fruit fly, Drosophila.

Nociceptive Pathway in the Cockroach Periplaneta americana.

Detecting and avoiding environmental threats such as those with a potential for injury is of crucial importance for an animal's survival. In this work, we examine the nociceptive pathway in an insect, the cockroach Periplaneta americana, from detection of noxious stimuli to nocifensive behavior. We show that noxious stimuli applied to the cuticle of cockroaches evoke responses in sensory axons that are distinct from tactile sensory axons in the sensory afferent nerve. We also reveal differences in the evoked response of post-synaptic projection interneurons in the nerve cord to tactile versus noxious stimuli. Noxious stimuli are encoded in the cockroach nerve cord by fibers of diameter different from that of tactile and wind sensitive fibers with a slower conduction velocity of 2-3 m/s. Furthermore, recording from the neck-connectives show that the nociceptive information reaches the head ganglia. Removing the head ganglia results in a drastic decrease in the nocifensive response indicating that the head ganglia and the nerve cord are both involved in processing noxious stimuli.

Parasite manipulation of host behavior.

Some parasites have evolved the ability to precisely control the behavior of animals in ways that enhance the transmission of parasite genes into the next generation. This is the concept of the 'extended phenotype' first conceived by Richard Dawkins in 1982. It states that the behavior we observe in animals is due not only to the expression of their genes, but also to the genes of parasites infecting them. In such cases, the behavior is an extended phenotype of the parasite.

Molecular cross-talk in a unique parasitoid manipulation strategy.

Envenomation of cockroach cerebral ganglia by the parasitoid Jewel wasp, Ampulex compressa, induces specific, long-lasting behavioural changes. We hypothesized that this prolonged action results from venom-induced changes in brain neurochemistry. Here, we address this issue by first identifying molecular targets of the venom, i.e., proteins to which venom components bind and interact with to mediate altered behaviour. Our results show that venom components bind to synaptic proteins and likely interfere with both pre- and postsynaptic processes. Since behavioural changes induced by the sting are long-lasting and reversible, we hypothesized further that long-term effects of the venom must be mediated by up or down regulation of cerebral ganglia proteins. We therefore characterize changes in cerebral ganglia protein abundance of stung cockroaches at different time points after the sting by quantitative mass spectrometry. Our findings indicate that numerous proteins are differentially expressed in cerebral ganglia of stung cockroaches, many of which are involved in signal transduction, such as the Rho GTPase pathway, which is implicated in synaptic plasticity. Altogether, our data suggest that the Jewel wasp commandeers cockroach behaviour through molecular cross-talk between venom components and molecular targets in the cockroach central nervous system, leading to broad-based alteration of synaptic efficacy and behavioural changes that promote successful development of wasp progeny.

Mind Control: How Parasites Manipulate Cognitive Functions in Their Insect Hosts.

Neuro-parasitology is an emerging branch of science that deals with parasites that can control the nervous system of the host. It offers the possibility of discovering how one species (the parasite) modifies a particular neural network, and thus particular behaviors, of another species (the host). Such parasite-host interactions, developed over millions of years of evolution, provide unique tools by which one can determine how neuromodulation up-or-down regulates specific behaviors. In some of the most fascinating manipulations, the parasite taps into the host brain neuronal circuities to manipulate hosts cognitive functions. To name just a few examples, some worms induce crickets and other terrestrial insects to commit suicide in water, enabling the exit of the parasite into an aquatic environment favorable to its reproduction. In another example of behavioral manipulation, ants that consumed the secretions of a caterpillar containing dopamine are less likely to move away from the caterpillar and more likely to be aggressive. This benefits the caterpillar for without its ant bodyguards, it is more likely to be predated upon or attacked by parasitic insects that would lay eggs inside its body. Another example is the parasitic wasp, which induces a guarding behavior in its ladybug host in collaboration with a viral mutualist. To exert long-term behavioral manipulation of the host, parasite must secrete compounds that act through secondary messengers and/or directly on genes often modifying gene expression to produce long-lasting effects.

Do Quiescence and Wasp Venom-Induced Lethargy Share Common Neuronal Mechanisms in Cockroaches?

The escape behavior of a cockroach may not occur when it is either in a quiescent state or after being stung by the jewel wasp (Ampulex compressa). In the present paper, we show that quiescence is an innate lethargic state during which the cockroach is less responsive to external stimuli. The neuronal mechanism of such a state is poorly understood. In contrast to quiescence, the venom-induced lethargic state is not an innate state in cockroaches. The Jewel Wasp disables the escape behavior of cockroaches by injecting its venom directly in the head ganglia, inside a neuropile called the central complex a 'higher center' known to regulate motor behaviors. In this paper we show that the coxal slow motoneuron ongoing activity, known to be involved in posture, is reduced in quiescent animals, as compared to awake animals, and it is further reduced in stung animals. Moreover, the regular tonic firing of the slow motoneuron present in both awake and quiescent cockroaches is lost in stung cockroaches. Injection of procaine to prevent neuronal activity into the central complex to mimic the wasp venom injection produces a similar effect on the activity of the slow motoneuron. In conclusion, we speculate that the neuronal modulation during the quiescence and venom-induced lethargic states may occur in the central complex and that both states could share a common neuronal mechanism.

Mechanisms of dendritic maturation.

The highly complex geometry of dendritic trees is crucial for neural signal integration and the proper wiring of neuronal circuits. The morphogenesis of dendritic trees is regulated by innate genetic factors, neuronal activity, and external molecular cues. How each of these factors contributes to dendritic maturation has been addressed in the developing nervous systems of animals ranging from insects to mammals. The results of such investigations have shown that the contribution of intrinsic and extrinsic factors and activity, however, appear to be weighted differentially in different types of neurons, in different brain areas, and especially in different species. Moreover, it appears that dozens of molecules have been found to regulate dendritic maturation, but it is almost certain that each molecule plays only a specific role in this formidable cooperative venture. This article reviews our current knowledge and understanding of the role of various factors in the establishment of the architecture of mature dendritic trees.

Morphometric analysis of dendritic remodeling in an identified motoneuron during postembryonic development.

A detailed quantitative description of modifications in neuronal architecture is an important prerequisite to investigate the signals underlying behaviorally relevant changes in neuronal shape. Extensive morphological remodeling of neurons occurs during the metamorphosis of holometabolous insects, such as Manduca sexta, in which new adult behaviors develop postembryonically. In this study, a morphometric analysis of the structural changes of an identified Manduca motoneuron, MN5, was conducted by sampling its metric parameters at different developmental stages. The remodeling of MN5 is divided into three main phases. The regression of most larval dendrites (1) is followed by the formation of dendritic growth-cones (2), and subsequently, adult dendrite formation (3). In contrast, the cell body and link segment surface increase during dendritic regression and regrowth, indicating that different cell compartments receive different signals, or respond differently to the same signal. During dendritic growth-cone formation, the growth of the cell body and the link segment are arrested. Sholl and branch frequency analysis suggest two different modes of dendritic growth. During a first growth-cone-dependent phase, new branch formation occurs at all dendrites. The maximum path length of the major dendritic tree changes little, whereas branch order increases from 20 to 45. Changes in total dendritic length are correlated with strong changes in the number of nodes but with minor changes in the average dendritic segment length, indicating a mode of growth similar to that induced by steroid hormone application to cultured motoneurons. The second phase is growth-cone-independent, and branching is limited to high order dendrites.

Afferent input regulates the formation of distal dendritic branches.

During postembryonic development, the dendritic arbors of neurons grow to accommodate new incoming synaptic inputs. Our goal was to examine which features of dendritic architecture of postsynaptic interneurons are regulated by these synaptic inputs. To address this question, we took advantage of the cockroach cercal system where the morphology of the sensory giant interneurons (GIs) is uniquely identified and, therefore, amenable to quantitative analysis. We analyzed the three-dimensional architecture of chronically deafferented vs. normally developed dendritic trees of a specific identified GI, namely GI2. GI2 shows five prominent dendrites, four of which were significantly altered after deafferentation. De-afferentation induced an average of 55% decrease in metric measures (number of branch points, total length, and total surface area) on the entire dendritic tree. Sholl and branch order analysis showed a decrease in the most distal and higher order branches. We suggest that afferent input plays a specific role in shaping the morphology of dendritic trees by regulating the formation or maintenance of high-order distal branches.

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.