Parasitoid wasp virulence

In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the antiwasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp.1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp.1 virulence. This venom SERCA antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes.     

Lipidomics reveals how the endoparasitoid wasp Pteromalus puparum manipulates host energy stores for its young

Endoparasitoid wasps inject venom along with their eggs to adjust the physiological and nutritional environment inside their hosts to benefit the development of their offspring. In particular, wasp venoms are known to modify host lipid metabolism, lipid storage in the fat body, and release of lipids into the hemolymph, but how venoms accomplish these functions remains unclear. Here, we use an UPLC-MS-based lipidomics approach to analyze the identities and concentrations of lipids in both fat body and hemolymph of host cabbage butterfly (Pieris rapae) infected by the pupal endoparasitoid Pteromalus puparum. During infection, host fat body levels of highly unsaturated, soluble triacylglycerides (TAGs) increased while less unsaturated, less soluble forms decreased. Furthermore, in infected host hemolymph, overall levels of TAG and phospholipids (the major component of cell membranes) increased, suggesting that fat body cells are destroyed and their contents are dispersed. Altogether, these data suggest that wasp venom induces host fat body TAGs to be transformed into lower melting point (more liquid) forms and released into the host hemolymph following infection, allowing simple absorption and nutritional acquisition by wasp larvae. Finally, cholesteryl esters (CEs, a dietary lipid derived from cholesterol) increased in host hemolymph following infection with no concomitant decrease in host cholesterol, implying that the wasp may provide this necessary food resource to its offspring via its venom. This study provides novel insight into how parasitoid infection alters lipid metabolism in insect hosts, and begins to uncover the wasp venom proteins responsible for host physiological changes and offspring development.     

Deadly venom of Asobara japonica parasitoid needs ovarian antidote to regulate host physiology

Asobara japonica (Braconidae) is an endophagous parasitoid developing in Drosophila larvae. The present study shows that A. japonica was never encapsulated in Drosophila melanogaster, and that it caused an overall inhibition of the host encapsulation reaction since injected foreign bodies were never encapsulated in parasitized hosts. Both the number of circulating hemocytes and the phenoloxidase activity decreased in parasitized larvae, and the hematopoietic organ appeared highly disrupted. We also found that A. japonica venom secretions had atypical effects on hosts compared to other braconid wasps. A. japonica venom secretions induced permanent paralysis followed by death of D. melanogaster larvae, whether injected by the female wasp during an interrupted oviposition, or manually injected into unparasitized larvae. More remarkably, these effects could be reversed by injection of ovarian extracts from female wasps. This is the first report that the venom of an endophagous braconid parasitoid can have a deadly effect on hosts, and moreover, that ovarian extracts can act as an antidote to reverse the effects of the wasp's venom. These results also demonstrate that A. japonica secretions from both venom gland and ovary are required to regulate synergistically the host physiology for the success of the parasitoid.     

Nasonia vitripennis venom causes targeted gene expression changes in its fly host

Parasitoid wasps are diverse and ecologically important insects that use venom to modify their host's metabolism for the benefit of the parasitoid's offspring. Thus, the effects of venom can be considered an ‘extended phenotype’ of the wasp. The model parasitoid wasp Nasonia vitripennis has approximately 100 venom proteins, 23 of which do not have sequence similarity to known proteins. Envenomation by N. vitripennis has previously been shown to induce developmental arrest, selective apoptosis and alterations in lipid metabolism in flesh fly hosts. However, the full effects of Nasonia venom are still largely unknown. In this study, we used high throughput RNA sequencing (RNA‐Seq) to characterize global changes in Sarcophaga bullata (Diptera) gene expression in response to envenomation by N. vitripennis. Surprisingly, we show that Nasonia venom targets a small subset of S. bullata loci, with ~2% genes being differentially expressed in response to envenomation. Strong upregulation of enhancer of split complex genes provides a potential molecular mechanism that could explain the observed neural cell death and developmental arrest in envenomated hosts. Significant increases in antimicrobial peptides and their corresponding regulatory genes provide evidence that venom could be selectively activating certain immune responses of the hosts. Further, we found differential expression of genes in several metabolic pathways, including glycolysis and gluconeogenesis that may be responsible for the decrease in pyruvate levels found in envenomated hosts. The targeting of Nasonia venom effects to a specific and limited set of genes provides insight into the interaction between the ectoparasitoid wasp and its host.     

Venom is beneficial but not essential for development and survival of Nasonia

1. Parasitoid wasps sting and inject venom into arthropod hosts, which alters host metabolism and development while keeping the host alive for several days, presumably to induce benefits for the parasitoid young. 2. This study investigates the consequences of host envenomation on development and fitness of wasp larvae in the ectoparasitoid Nasonia vitripennis, by comparing wasps reared on live unstung, previously stung, and cold‐killed hosts. Developmental arrest and suppression of host response to larvae are major venom effects that occur in both stung and cold‐killed hosts, but not in unstung hosts, whereas cold‐killed hosts lack venom effects that require a living host. Thus, cold‐killed hosts mimic some of the effects of venom, but not others. 3. Eggs placed on live unstung hosts have significantly higher mortality during development; however, successfully developing wasps from these hosts have similar lifetime fecundity to that of wasps from cold‐killed or stung hosts. Therefore, although venom is beneficial, it is not required for wasp survival. 4. While wasps developing on both cold‐killed and stung hosts have similar fitness levels, rearing multiple generations on cold‐killed hosts results in significant fitness reductions of wasps. 5. It is concluded that the largest benefits of venom are induction of host developmental arrest and suppression of host response to larva (e.g. immune responses), although more subtle benefits may accrue across generations or under stressful conditions.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

With more than 150,000 species, parasitoids are a large group of hymenopteran insects that inject venom into and then lay their eggs in or on other insects, eventually killing the hosts. Their venoms have evolved into different mechanisms for manipulating host immunity, physiology and behavior in such a way that enhance development of the parasitoid young. The venom from the ectoparasitoid Nasonia vitripennis inhibits the immune system in its host organism in order to protect their offspring from elimination. Since the major innate immune pathways in insects, the Toll and Imd pathways, are homologous to the NF-κB pathway in mammals, we were interested in whether a similar immune suppression seen in insects could be elicited in a mammalian cell system. A well characterized NF-κB reporter gene assay in fibrosarcoma cells showed a dose-dependent inhibition of NF-κB signaling caused by the venom. In line with this NF-κB inhibitory action, N. vitripennis venom dampened the expression of IL-6, a prototypical proinflammatory cytokine, from LPS-treated macrophages. The venom also inhibited the expression of two NF-κB target genes, IκBα and A20, that act in a negative feedback loop to prevent excessive NF-κB activity. Surprisingly, we did not detect any effect of the venom on the early events in the canonical NF-κB activation pathway, leading to NF-κB nuclear translocation, which was unaltered in venom-treated cells. The MAP kinases ERK, p38 and JNK are other crucial regulators of immune responses. We observed that venom treatment did not affect p38 and ERK activation, but induced a prolonged JNK activation. In summary, our data indicate that venom from N. vitripennis inhibits NF-κB signaling in mammalian cells. We identify venom-induced up regulation of the glucocorticoid receptor-regulated GILZ as a most likely molecular mediator for this inhibition.     

Functional analysis of the venom of mealybug parasitoid Aenasius bambawalei (Hymenoptera: Encyrtidae)

Aenasius bambawalei (Hymenoptera: Encyrtidae) is a koinobiont nymphal endoparasitoid of cotton mealybug, Phenacoccus solenopsis (Hemiptera: Pseudocccidae). Functional analysis of the venom of the wasp was performed by artificial microinjections of both crude and treated venom (heat and proteinase) of the wasp containing 0.3 and 0.5 μl in non-parasitized and synchronized adult hosts (mealybugs) and the mortality data were recorded 24, 48, 72 and 96 hours post injecton while mealybugs receiving saline injections were acted as control. The main effects for artificially envenomated mealybugs were observed on their mortality and survival. The biological activity of crude venom was also evaluated by heat and protease treatment. Here, we demonstrate that maximum mortality (82 ± 2.0%) was achieved by microinjections containing higher volume (0.5 μl) of crude venom while lower mortality (68 ± 4.0%) was achieved with lower volume of venom (0.3 μl). On the other hand, heat and proteinase K treated venom did not show any significant effect on mortality of the host insect. Our findings suggest that bioactive components of the crude venom are proteins which lost their activity upon heat and protease treatment. This basic information regarding the functional role of the venom of A. bambawalei serves as a starting point for comprehensive analysis of the role of the venom of the parasitoid on the regulation processes in its host.     

Does host age affect progeny production and progeny fitness in Trichopria sp.?

The gregarious endoparasitoid Trichopria sp. (Hymenoptera: Diapriidae) is currently under evaluation for its potential to suppress the levels of parasitism in larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) caused by the tachinid fly Exorista bombycis (Louis) (Diptera: Tachinidae) in the rearing houses of sericulture farmers in southern India. Parasitism by E. bombycis currently results in cocoon yield reductions of 10–20 %. Prior to the mass production and release of Trichopria sp., there is a need to understand the factors that affect progeny production and fitness. Within this framework, we have attempted to determine whether host age has an impact on the numbers of Trichopria sp. progeny produced and on the subsequent fitness of the adults of the wasp. Two day-old adult females of the parasitoid were allowed to parasitize day (D) 1, 3, 5, 7, and 9 puparia of E. bombycis at a ratio of 1:4 (wasp:host). After eclosion of the parasitoid adults, data on the rate of parasitism, developmental period, brood allocation, progeny production, sex ratio, female progeny longevity and adult size were recorded. The reproductive performance of the adult progeny was also recorded by allowing them to parasitize three day-old E. bombycis puparia. Host age was seen to have an effect on several of the parameters recorded, such that while the most progeny were produced by the youngest (D1) hosts, larger wasps emerged from older E. bombycis puparia. It was, however, apparent that all but the oldest hosts (D9) examined were able to support adequate parasitoid development. Further, only relatively minor impacts on progeny fitness were observed between wasps deriving from hosts of different ages. However, the results strongly indicate that optimal production and parasitoid performance will be obtained by using young hosts of less than three days old.     

Quantitative trait locus analysis of parasitoid counteradaptation to symbiont-conferred resistance

Insect hosts and parasitoids are engaged in an intense struggle of antagonistic coevolution. Infection with heritable bacterial endosymbionts can substantially increase the resistance of aphids to parasitoid wasps, which exerts selection on parasitoids to overcome this symbiont-conferred protection (counteradaptation). Experimental evolution in the laboratory has produced counteradapted populations of the parasitoid wasp Lysiphlebus fabarum. These populations can parasitize black bean aphids (Aphis fabae) protected by the bacterial endosymbiont Hamiltonella defensa, which confers high resistance against L. fabarum. We used two experimentally evolved parasitoid populations to study the genetic architecture of the counteradaptation to symbiont-conferred resistance by QTL analysis. With simple crossing experiments, we showed that the counteradaptation is a recessive trait depending on the maternal genotype. Based on these results, we designed a customized crossing scheme to genotype a mapping population phenotyped for the ability to parasitize Hamiltonella-protected aphids. Using 1835 SNP markers obtained by ddRAD sequencing, we constructed a high-density linkage map consisting of six linkage groups (LGs) with an overall length of 828.3 cM and an average marker spacing of 0.45 cM. We identified a single QTL associated with the counteradaptation to Hamiltonella in L. fabarum on linkage group 2. Out of 120 genes located in this QTL, several genes encoding putative venoms may represent candidates for counteradaptation, as parasitoid wasps inject venoms into their hosts during oviposition.Subject terms: Experimental evolution, Evolutionary genetics, Evolutionary ecology, Genetic linkage study     

Host Handling Time in a Polyembryonic Wasp is Affected both by Previous Experience and by Host State (Parasitized or Not)

Foraging behavior for hosts in parasitoids resembles that of predators with respect to finding, evaluating and manipulating of the prey. Host handling time may depend on the life history of the parasitoid and can be affected by oviposition experience. Additionally, handling time can be affected by host aggregation, species, size and state (parasitized or not). We studied handling times in the egg-larval parasitoid wasp Copidosoma koehleri. We allowed naïve female wasps to oviposit into three consecutive unparasitized hosts, and measured time until oviposition, and the duration of ovipositor insertion. We recorded the same data for naïve females ovipositing into already parasitized hosts. We found that both previous experience by females and previous parasitism of hosts reduced handling time. The results suggest that host handling durations reflect the interplay between host state and parasitoid internal state.     

Effects of Intraspecific Competition and Host-Parasitoid Developmental Timing on Foraging Behaviour of a Parasitoid Wasp

In a context where hosts are distributed in patches and susceptible to parasitism for a limited time, female parasitoids foraging for hosts might experience intraspecific competition. We investigated the effects of host and parasitoid developmental stage and intraspecific competition among foraging females on host-searching behaviour in the parasitoid wasp Hyposoter horticola. We found that H. horticola females have a pre-reproductive adult stage during which their eggs are not mature yet and they forage very little for hosts. The wasps foraged for hosts more once they were mature. Behavioural experiments showed that wasps’ foraging activity also increased as host eggs aged and became susceptible to parasitism, and as competition among foraging wasps increased.     

Preferential infectivity of entomopathogenic nematodes in an envenomed host

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO₂ emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO₂ than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.     

肿腿蜂类寄生蜂室内控害效能评价——以松脊吉丁肿腿蜂为例

为了开发利用天敌资源防治害虫,科学而客观地评价肿腿蜂对蛀干害虫的控害作用,研究了松脊吉丁肿腿蜂(Sclerodermus sp.)对松褐天牛3龄幼虫的功能反应,建立了肿腿蜂类寄生蜂室内控害效能评价体系,并证明了其可行性。该评价体系包括供试虫源标准、寄生蜂生物学与寄生行为学观察、寄生蜂对寄主的功能反应和寻找寄主效应以及寄生蜂的忠岐指数(Y)4个部分。研究结果表明:松脊吉丁肿腿蜂具有较强的控害潜力,单头雌成虫能防治3—4头松褐天牛3龄幼虫,21d内最多可致死9.07头寄主幼虫;寄主密度对单头寄生蜂寄生作用功能反应的测定结果与对寄生作用寻找效应的测定结果以及对产卵量影响的测定结果一致;综合评价该蜂对松褐天牛3龄幼虫控制效能,释放比为1∶1(即1头肿腿蜂雌蜂:1头3龄松褐天牛幼虫)时最高,其忠岐指数Y为39.63,这可能是肿腿蜂类天敌特有的抚幼习性所致,结论也与寄生蜂生物学特性及寄生行为学观察结果相同。研究结论证明该评价体系是稳定可靠的。评价体系在利用天敌昆虫生物防治害虫时为筛选最佳寄生性天敌种类提供了可行的方法和依据。     

Drosophila parasitoids (Hymenoptera) of Japan

The Drosophila parasitoid system is an excellent model for the study of biological and ecological interactions. In Japan, extensive studies have been carried out on Drosophila parasitoids since the early 2000s. Here we provide up-to-date information on their diversity, distributions, host use and reproductive mode. Until now, 45 wasp species have been recorded. In addition, four genetically distinct groups (tentatively assigned as “races”) have been found in Ganaspis brasiliensis (Figitidae). Most of them are larvo-pupal parasitoids belonging to the families Figitidae and Braconidae, and some are pupal parasitoids of the families Pteromalidae and Diapriidae. A species of Encyrtidae was also recorded. Their major hosts are indicated with information on the host's breeding sites. Except Ganaspis xanthopoda and two races of G. brasiliensis that are distributed both in tropical and temperate regions, they are divided into tropical and temperate species. The tropical species are mainly distributed in Southeast Asia, and their distributions in Japan are restricted to the Ryukyu islands, particularly Iriomote-jima. The temperate species have been recorded from the main islands of Japan and surrounding islands but not from the Ryukyu islands, except two braconid species, Asobara japonica and Phaenocarpa sp. TK1. Some of the temperate species have also been recorded from Korea, China, Europe and/or North America. Among the 45 Japanese parasitoid species, at least seven are assumed to be thelytokous.     

VENOM COMPONENTS OF Asobara japonica IMPAIR CELLULAR IMMUNE RESPONSES OF HOST Drosophila melanogaster

The endoparasitoid wasp Asobara japonica has highly poisonous venom: the host Drosophila larvae are killed by envenomation at a dose that is naturally injected by the female wasp at parasitism. This insecticidal venom is neutralized, however, because A. japonica introduces lateral oviduct components soon after venom injection at oviposition. Although the venom and lateral oviduct components of this parasitoid have been partially characterized, how the venom components favor successful development of wasp eggs and larvae in the host remains ambiguous. Here, we demonstrated that A. japonica venom did not affect host humoral immune responses, determined as expression of antimicrobial peptide (AMP) genes, but significantly diminished two cellular responses, spreading and phagocytosis, by host hemocytes. Moreover, venom components drastically elevated a serine protease‐like activity 4 h after its injection. The lateral oviduct components did not negate the detrimental effects of the venom on host cellular immunities, but significantly reduced the venom‐induced elevation of protease activity. Both active factors in venom and lateral oviduct components were roughly characterized as heat‐labile substances with a molecular mass of at least 10 kDa. Finally, venom of A. japonica, with a wide host range, was found to be much more toxic than that of Asobara rossica, which has a limited host range. These results reveal that A. japonica venom toxicity allows exploitation of a broader range of host insects because it is essential to overcome cellular immune responses of the host for successful parasitism.     

Effects of parasitism by Asobara tabida (Hymenoptera: Braconidae) on the development,survival and activity of Drosophila melanogaster larvae

The impact of parasitism by Asobara tabida on Drosophila melanogaster larval development, survival features and larval activity has been investigated using two strains of the parasitoid. The successful parasitism rate of the A1 strain was four times greater than that of the WOPV strain. Both strains induced equivalent mortality rates but hosts parasitized by A1 predominantly died as pupae. The time necessary for the host pupariation and emergence, and the larval weight at 72, 96 and 120 h post-parasitization were measured. Parasitized larvae exhibited longer periods of development and lower weights than controls, especially when parasitized by A1. These results suggest that hosts underwent physiological costs varying with respect to the outcome of the parasitic relationship. Of the parasitoid factors possibly responsible for these costs, we examined venoms for their impact on host mortality. Artificial injections of WOPV venoms induced higher mortality rates than did A1 venoms. Venoms were also found responsible for the induction of a transient paralysis, naturally occuring after parasitization. Again, the strongest effect was observed after parasitization by WOPV or injections of its venoms. This study gives new insights into the intriguing features of A. tabida and constitutes the first report of the paralysing properties of the venoms.