Analysis of endoparasitoid-released proteins and their effects on host development in the system Chelonus inanitus (Braconidae)-Spodoptera littoralis (Noctuidae)

Having shown earlier that the larva of C. inanitus is essential in inducing the precocious onset of metamorphosis in polydnavirus/venom containing S. littoralis, we here analysed release of proteins by parasitoid larvae and their effects on host development. Parasitoid larvae released proteins in vivo and in vitro in a stage dependent manner. An approximately 212 kD protein was released from the mid 1st instar onwards and additional smaller proteins could be associated mainly with the 2nd and 3rd instar. When parasitoids were implanted into S. littoralis larvae, parasitoid-released proteins were seen 6 hr later. When parasitoids were removed from hosts, parasitoid-released proteins persisted in the host haemolymph for some time. Injection of antiserum against parasitoid-released proteins after removal of the parasitoid larva accelerated the disappearance of the 212 kD protein and reduced the number of larvae entering metamorphosis precociously. Repeated injections of concentrated parasitoid medium into polydnavirus/venom containing larvae caused a reduction of the head capsule width and formation of miniature 6th instar larvae; this effect was not seen in the absence of polydnavirus/venom. These observations suggest that proteins released by the parasitoid might play a role in modifying host metamorphosis in the presence of polydnavirus/venom, and the temporal appearance of the 212 kD protein makes it the most interesting candidate for being involved in such an effect.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

Bioinformatic analysis suggests potential mechanisms underlying parasitoid venom evolution and function

Hymenopteran parasitoid wasps are a diverse collection of species that infect arthropod hosts and use factors found in their venoms to manipulate host immune responses, physiology, and behaviour. Whole parasitoid venoms have been profiled using proteomic approaches, and here we present a bioinformatic characterization of the venom protein content from Ganaspis sp. 1, a parasitoid that infects flies of the genus Drosophila. We find evidence that diverse evolutionary processes including multifunctionalization, co-option, gene duplication, and horizontal gene transfer may be acting in concert to drive venom gene evolution in Ganaspis sp.1. One major role of parasitoid wasp venom is host immune evasion. We previously demonstrated that Ganaspis sp. 1 venom inhibits immune cell activation in infected Drosophila melanogaster hosts, and our current analysis has uncovered additional predicted virulence functions. Overall, this analysis represents an important step towards understanding the composition and activity of parasitoid wasp venoms.     

Host castration by Aphidius ervi venom proteins

The braconid Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae). Parasitized host aphids show different degrees of castration, a response that is total when parasitoid oviposition takes place in first instar hosts. Deleterious effects on the host reproductive system are already evident by 24h following parasitization, before egg hatching. The effect of parasitoid venom on A. pisum ovaries has been studied by performing microinjections in non-parasitized host aphids and observing the cellular alterations of the apical germaria of ovarioles. Venom injection reproduced the same alterations observed in parasitized aphids, while injections of saline solution did not induce any detectable change. By 24h, the germarial cells of both venom-treated aphids and parasitized aphids showed the absence of the nucleolus and of electron-dense material around the nucleus, frequently referred to as "nuage material". By 48h more evident signs of degeneration were observed, suggesting the possible occurrence of apoptosis. The bioactive component of the venom was both heat- and protease-sensitive. The activity was found in purified fractions that were highly enriched in two proteins with an approximate molecular mass of 21kD and 36kD, respectively. These macromolecules are the most abundant components of A. ervi venom and, unlike many venom proteins of studied parasitic Hymenoptera, are not glycosylated and appear to be subunits of an oligomeric protein. The adaptive significance of host castration is discussed.     

Role of venom and ovarian proteins in immune suppression of Ostrinia furnacalis （Lepidoptera： Pyralidae） larvae parasitized by Macrocentrus cingulum （Hymenoptera： Braconidae）, a polyembryonic parasitoid

Venom and ovarian proteins in braconid and ichneumonid wasps play an important role in the successful parasitism of their host, especially for immune suppression immediately after oviposition. In this study, we compared the effect of venom and ovarian proteins collected from the female wasps of Macrocentrus cingulum, a polyembryonic parasitoid of the larvae of Ostriniafunacalis, on the encapsulation capacity of Sephadex A- 25 beads at 4 h and 24 h post-injection both in vivo and in vitro. The results showed that the ovarian proteins significantly interfered with the encapsulation capacity of hemocytes in a dose-dependent manner. Spreading and viability of hemocytes in O. furnacalis was not disrupted by venom and ovarian proteins at various concentrations injected. It seems likely that the ovarian proteins from M. cingulum play a role in suppressing the encapsulation capacity of host hemocytes.     

Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins

The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became redundant following the acquisition of polydnaviruses by the lineage.     

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.     

Chelonus sp. near curvimaculatus venom proteins: Analysis of their potential role and processing during development of host Trichoplusia ni

The venom that Chelonus sp. near curvimaculatus injects into each parasitized Trichoplusia ni egg is entirely injected within the first 8 s of the 19-s oviposition period, before deposition of the parasitoid egg that is injected during the final 1-2 s of the oviposition. The parasitization factor, causing precocious metamorphosis of the host, is injected after the venom, but before the parasite egg. The venom by itself does not cause developmental redirection of the host. Chelonus venom proteins are very stable in the host egg during the first 2 days of egg development. Then, on the last day before hatching, they are rapidly degraded by the proteolytic enzymes appearing in 3-day-old T. ni eggs. Among those that degrade the venom proteins are serine-type proteinases, and at least one seems to be a trypsin-like enzyme.     

Host regulation effects of ovary fluid and venom of Aphidius ervi (Hymenoptera: Braconidae)

The host regulation effects of venom and ovary fluid of the endophagous braconid Aphidius ervi Haliday on the pea aphid, Acyrthosiphon pisum (Harris), have been studied. Extracts of ovaries and of venom glands were injected into nonparasitized 4th instar pea aphids, both separately and mixed. Aphids treated with parasitoid material died as 4th instars, often showing developmental arrest. In contrast, control aphids that received an injection of Pringle's saline solution regularly moulted to the adult stage and reproduced. Venom alone was as effective as the combined extracts in determining developmental arrest and death. Separate heat and protease treatments of these parasitoid's reproductive secretions significantly reduced their biological activity, suggesting that the active component(s) involved is a protein(s). SDS-PAGE analysis of haemolymph samples obtained from pea aphids which had received an injection of combined venom and ovary extract revealed an increase of the titre of various proteins, particularly in the 43-47kDa interval, as registered for truly parasitized hosts. This altered protein profile was first detected 48h following injection. Based on this information a tentative physiological model is proposed. The apical tract of host ovarioles, where the germarium and growing oocytes are located, is suggested to be one of the major targets of female parasitoid secretions injected with the egg.     

Effects of Asobara japonica venom on larval survival of host and nonhost Drosophila species

Abstract Ovipositing Asobara japonica females inject venom (containing paralysis‐inducing factors) immediately after the insertion of their ovipositors into Drosophila larvae, and lay eggs a little later. Interruption of their oviposition behaviour before egg laying causes high larval mortality in host Drosophila species, whereas normal oviposition does not. This suggests that venom of this parasitoid is toxic to larvae of these host species but its toxicity is suppressed by factor(s) provided by parasitoid females at the time of laying egg or by parasitoid embryos developing in the hosts. On the other hand, venom does not show toxicity to larvae of nonhost Drosophia species. Possible functions of venom are discussed.     

Influence of nutrient deficiency caused by host developmental arrest on the growth and development of a koinobiont parasitoid

Koinobiont parasitoids utilize nutrients obtained from hosts that contine to feed and grow after parasitization. However, if the ecdysis of early host instars is prevented, parasitized larvae will fail to grow large enough to support the development of the parasitoid brood and both organisms will perish. When L5 instar larvae (the penultimate stage) of Pseudaletia separata were parasitized by Cotesia kariyai and injected with Euplectrus separatae venom (5PV), the development of these hosts was arrested before molting to the next stage and the caterpillars thus failed to gain weight. These hosts remained at approximately 300 mg until parasitoid emergence. In contrast, hosts parasitized as L5 but without the injection of venom (5P) exhibited an increase in weight after molting to the next stage and ultimately grew to approximately 700 mg. The inhibition of ecdysis reduced the amount of food resource (e.g. fat body) for the parasitoid larvae. On the other hand, when final (= L6) host instars were parasitized and injected with E. separatae venom (6PV), the maximum weight attained by these larvae was about 710 mg, although weight gain was depressed compared to hosts parasitized without the injection of E. separatae venom (6P). The adult weight of C. kariyai that emerged from 5PV hosts was less than conspecifics that emerged from 5P, 6P, and 6PV respectively, although the egg-pupal period of the parasitoid from 5PV hosts was extended. The offspring sex ratio (percentage males) of adult wasps did not vary significantly with treatment. Female parasitoids that eclosed from 5PV hosts laid almost the same number of eggs in day 0-6th host instars as those emerging from 5P, 6P, 6PV hosts. Their egg-pupal period was extended and the cocoon cluster mass and the parasitoid body mass on subsequent generations was lighter than those reared from 5P, 6P, 6PV hosts. The sex ratio of F2 C. kariyai wasps that eclosed from 5PV increased more than in wasps that eclosed from the other host treatments (5P, 6P, 6PV). Our results reveal that a reduction in host quality and offspring fitness in the first generation negatively impacted female fitness in the second generation. An early arrestment of host growth, mediated by the addition of E. separatae venom, has severe implications on parasitoid fitness by reducing host quality, especially in smaller hosts.     

Venom of the egg-larval parasitoid Chelonus inanitus is a complex mixture and has multiple biological effects

The egg-larval parasitoid Chelonus inanitus injects bracoviruses (BVs) and venom along with the egg into the host egg; both components are essential for successful parasitoid development. All stages of eggs of its natural host, Spodoptera littoralis, can be successfully parasitized, i.e. from mainly a yolk sphere to a fully developed embryo. Here, we show that the venom contains at least 25 proteins with masses from 14 kDa to over 300 kDa ranging from acidic to basic. The majority is glycosylated and their persistence in the host is short when old eggs are parasitized and much longer when young eggs are parasitized. Physiological experiments indicated three different functions. (1) Venom synergized the effect of BVs in disrupting host development when injected into third instar larvae. (2) Venom had a transient paralytic effect when injected into sixth instar larvae. (3) In vitro experiments with haemocytes of fourth instar larvae suggested that venom alters cell membrane permeability. We propose that venom promotes entry of BVs into host cells and facilitates placement of the egg in the embryo's haemocoel when old eggs are parasitized. The multifunctionality of the venom might thus be essential in enabling parasitization of all stages of host eggs.     

Overview of parasitism associated effects on host haemocytes in larval parasitoids and comparison with effects of the egg-larval parasitoid Chelonus inanitus on its host Spodoptera littoralis

In the first part we review the effects of larval endoparasitoids and their polydnavirus and venom on the immune system of their hosts. In all systems investigated, haemocyte spreading and encapsulation activity was reduced; in some cases effects on total (THC) or differential (DHC) haemocyte count as well as modification of haemocyte morphology and ultrastructure were also documented. In many cases polydnavirus (and venom) were shown to play a major role in abrogation of the host's immune reaction. In the second part we present the first investigation of effects of parasitism and polydnavirus/venom on the immune system of the host for an egg-larval parasitoid, Chelonus inanitus. We observed that in 4th and 5th instar larvae, i.e. 7 to 10 days after parasitization, neither haemocyte spreading and encapsulation activity, nor DHC, nor haemocyte ultrastructure were altered. After parasitization with X-ray irradiated wasps, which inject polydnavirus and venom and infertile eggs, there was no alteration of the above mentioned parameters. Nevertheless, parasitoid larvae implanted into 4th instar larvae which developed from eggs parasitized with X-ray irradiated wasps were not encapsulated, whereas co-injected latex beads were. These results show that parasitism by this egg-larval parasitoid does not generally suppress the host's immune system but that polydnavirus/venom injected at oviposition prevent, by, as yet unknown mechanisms, encapsulation of the parasitoid larva.     

Natural and artificial envenomation of Ceratitis capitata by Eupelmus urozonus and the search for new bioinsecticides

This study was designed to indentify novel bioactive molecules in the venom of the parasitoid Eupelmus urozonus Dalman (Hymenoptera: Eupelmidae). Parasitism by E. urozonus induces apparent paralysis in the larvae of the host Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) and it arrests the development of host pupae. Parasitoid eggs were transferred from stung to unparasitized host pupae to determine whether adult female stings or bites from the first instars were responsible for the above effects. The two treatments gave the same results, indicating that both parasitoid adults and larvae produced venoms capable to compromise host development. A protocol was developed to artificially microinject E. urozonus venom into healthy host pupae and adults at known concentrations to study the effects. The microinjection of venom was found to produce the same macroscopic result as natural parasitization, indicating that host developmental arrest was caused by molecules produced by the parasitoid venom glands. One‐tenth, one‐twentieth, and one‐hundredth of the contents of a female venom reservoir was sufficient to compromise the development of 100, 90, and 50% of the microinjected host pupae, respectively. The microinjection of 0.1 female venom equivalents into host adults always caused death within 24 h. Extraction and freezing did not affect the activity of the E. urozonus venom, which facilitates its storage, whereas denaturation treatments demonstrated that the bioactive molecules were proteins. The venom was also found to prevent the hosts from decaying for over 2 weeks and it promoted the accumulation of unknown subspherical granules in the host haemocoel. These results suggest the potential identification of novel molecules with interesting biological activity with various possible applications.     

Effect of the venom of the endoparasitoid,Apanteles kariyai Watanabe,on the cellular defence reaction of the host,Pseudaletia separata Walker

Although a venom apparatus is present in all female braconid wasps, the function of the venom injected into the host at the time of oviposition by the endophagous members of this group is unknown. Eggs laid by females of Apanteles kariyai, from which the venom apparatus had been removed, were encapsulated, which suggests that the fluid is necessary to enable the parasitoid eggs to escape the cellular defence reaction of the host. Studies with anti-venom serum demonstrated that the venom is attached to the surface of the egg. However, injection of DEAE-Sephadex A-50 particles (Sephadex particles) revealed that the venom alone is insufficient to inhibit the encapsulation reaction. Calyx and venom fluids together seem to be essential for evasion of the host defence reaction by the parasitoid eggs. Eighty-five % Sephadex particles, injected together with calyx and venom fluids, fail to become fully encapsulated, whereas 46% of particles injected with only the calyx fluid avoided encapsulation. Furthermore, when eggs from the lateral oviduct were injected into unparasitized larvae, together with the calyx and venom fluids, a few eggs developed successfully although they had undergone no mechanical distortion.     

Avoidance of the host immune response by a generalist parasitoid, Compsilura concinnata Meigen

Abstract.  1. Ecological interactions between parasitoids and their hosts are extremely strong as parasitoid offspring rely entirely on an individual host to complete development. The ability of a parasitoid to use a host is influenced directly by the degree to which the parasitoid can overcome host defences and grow within the host.
2. Hymenopteran parasitoids have evolved different host-specific strategies to defeat the host immune system, such as the use of venom, endosymbiont virus, or mimicking the host tissue. Dipteran parasitoids from the Tachinidae family do not use these subterfuges and rely mainly on avoiding the host immune system by hiding in specific tissues.
3. Little is known of the effect of this strategy on the host immune system, the absorption of nutrients by the parasitoid larvae, or the implications for parasitoid host range.
4. In this study, the impact of a polyphagous tachinid parasitoid Compsilura concinnata Meigen on a pest lepidopteran Trichoplusia ni Hübner are assessed. Phenoloxidase levels and haemolymph proteins were measured in parasitised T. ni as a function of host immune response.
5. Haemolymph phenoloxidase in the host did not vary with parasitisation but was triggered when a piece of monofilament was implanted in the haemocoel. Haemolymph proteins were depleted in heavily parasitised T. ni .
6. These results indicate that C. concinnata has a strategy that avoids the host immune system, and accesses the necessary nutrients for larval growth. This strategy could explain the success of this tachinid and its wide host range.     

Venom gland and reservoir morphology in cynipoid wasps

The venom apparatus morphology was examined in 25 species of Cynipoidea, representing 11 parasitoid taxa; 12 gall inducers and two inquilines. Typically the venom apparatus consists of an oval or bilobed reservoir connected to the ovipositor apparatus by a very short venom duct at the anterior end and to a single elongate unbranched venom gland at or near its posterior end. The Dufour's gland was not found in any of the examined species. The elongate unbranched venom gland and the absence of the Dufour's gland are putative cynipoid synapomorphies. The shape and size of especially the venom reservoir were found to vary considerably within the Cynipoidea. It is typically less prominent in the parasitoid taxa than in the gall inducers. Exceptions include the poppy gallers Barbotinia and Aylax, in which the venom reservoirs were remarkably small and in the rose galler Diplolepis, where only a rudimentary venom apparatus was found. Possible functional and phylogenetic implications of cynipoid venom apparatus features are discussed.