Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera: Braconidae) suggest a high level of specificity in their associated bracoviruses

The immunological and developmental effects of bracoviruses (BVs) from three parasitoids in the genus Microplitis (Braconidae: Microgastrinae) were compared in the hosts Pseudoplusia includens and Heliothis virescens (Lepidoptera: Noctuidae). Southern blotting experiments indicated that viral DNAs from Microplitis demolitor bracovirus (MdBV) cross-hybridized with viral DNAs from Microplitis croceipes bracovirus (McBV) and Microplitis mediator bracovirus (MmBV) under conditions of high stringency. Injection of calyx fluid plus venom from each parasitoid species dose-dependently delayed development of P. includens and H. virescens. Each virus also inhibited pupation of P. includens but not H. virescens. In situ hybridization experiments indicated that MdBV and McBV persistently infect hemocytes in both hosts while MmBV persistently infects hemocytes in P. includens but not H. virescens. While MdBV infection induced a loss of adhesion by most plasmatocytes, McBV and MmBV infection induced a loss of adhesion in less than 50% of cells. Cross-protection experiments indicated that calyx fluid plus venom from one species usually protected progeny of another species from encapsulation but did not always promote successful development.     

Interaction of calyx fluid and venom from Microplitis croceipes (Braconidae) on developmental disruption of the natural host,Heliocoverpa zea,and two atypical hosts,Galleria mellonella and Spodoptera exigua

Polydnaviruses of many braconid and ichneumonid endoparasitoids play an important role in the successful parasitism of their hosts. The host's development is altered and its immune response is also suppressed. In this study, we compared the effects of calyx fluid and venom on the development of the natural host, Helicoverpa zea, and two atypical hosts that the parasitoid does not normally attack in nature, Galleria mellonella and Spodoptera exigua. The levels of calyx fluid and\or venom injected was 0.05, 0.1 and 0.2 female equivalents (FE)/larva. In H. zea, calyx fluid significantly reduced larval growth on day 5 post injection. Venom alone did not affect larval growth but it synergized the action of calyx fluid by reducing growth earlier and for a longer period after injection. Other effects of calyx fluid on the host, either alone or in combination with venom, were an increase in developmental period, and a reduction in percent emergence and weight of adult moths. The percentage of H. zea larvae that pupated was not affected by calyx fluid or venom. In Galleria mellonella, venom alone reduced larval growth comparable to calyx fluid and both tissues induced the effects on day 1 post injection. Other effects caused by calyx fluid or venom alone or the combination were a reduction in percent pupation and emergence, and the average adult weight. In S. exigua, high mortality occurred when 4th instar larvae were injected. Although the injection of larger fifth instars reduced overall mortality, the sham-injected larvae only gained weight during the first 24 hours after injection (from day 0 to day 1). However, adults were produced at all doses of calyx fluid or venom. The effects of the virus on development in this species were a prolongation of the larval stage and reduction of adult weight by calyx fluid in combination with venom. In conclusion, injections of calyx fluid and venom of Microplitis croceipes can differentially affect the growth and development of its natural host H. zea, and atypical host, G. mellonella, but only a minimal effect was observed in S. exigua.     

Retardation of testis development in the armyworm,Pseudaletia separata,parasitized by the braconid wasp,Cotesia kariyai

Summary

In order to complete growth and development, the endoparasitoid wasp, Cotesia (=Apanteles) kariyai, inhibits pupation of its armyworm host, Pseudaletia (=Leucania) separata. In host larvae retardation of testis and spermatocyst development caused by the parasitoid was also observed. The agents causing the retardation were found in the ovaries and venom of the female adult parasitoid. When an unparasitized male host larva was artificially injected with calyx fluid obtained from ovaries together with venom, it showed the same degree of developmental retardation of testes and spermatocysts as in natural parasitization. Testes implanted in isolated abdomens of healthy larvae did not increase in size by ecdysteroid stimulation after exposure to calyx fluid plus venom. It is suggested that both symbiotic polydnavirus existing in calyx fluid and venom in the parasitoid, C. kariyai, are responsible for the parasitic retardation of the male reproductive organs in the host, P. separata.     

The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus

Campoletis sonorensis is a habitual parasitoid of 3rd-instar larvae of Heliothis virescens. C. sonorensis eggs and small glass rods were encapsulated in 5th-instar host larvae implanted in the absence of wasp calyx fluid; prior injection of calyx fluid into larvae suppressed the encapsulation response. Within 8 h of calyx fluid injection there was a removal of approx. 75% of the circulating capsule-forming haemocytes (plasmatocytes). The remaining subpopulation of plasmatocytes, in addition to being incapable of encapsulating targets in vivo, spread at a significantly reduced rate in vitro. Identical changes in plasmatocyte count and behaviour were observed after injection of virus purified from calyx fluid. Additionally, the activity of calyx fluid was abolished after ultraviolet irradiation. The onset of haemocytic abnormalities occurred more rapidly after natural parasitism of 3rd-instar host larvae. The cell-free haemolymph of calyx fluid-injected 5th-instar larvae also retarded the spreading of plasmatocytes from non-injected control larvae in vitro. We conclude that the abnormalities induced in H. virescens plasmatocytes by C. sonorensis virus contribute to the suppression of encapsulation.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

腰带长体茧蜂寄生对亚洲玉米螟幼虫体内酚氧化酶活性的影响

通过对被腰带长体茧蜂Macrocentrus cingulum Brischke寄生的5龄亚洲玉米螟Ostrinia furnacalis Guenée幼虫体内不同组织中酚氧化酶活性的测定,采用体外注射腰带长体茧蜂雌性成蜂的萼液成分、毒液成分、萼液与毒液混合物的方法,研究了寄生蜂各种主要生理因子对寄主血清中酚氧化酶活性的影响。结果表明： 寄生蜂寄生可明显抑制寄主体内的酚氧化酶活性,减少黑色素产生;被寄生组FITC标记的血细胞阳性百分率低于未被寄生组,差异极显著( P<0.01）;萼液成分可明显地抑制亚洲玉米螟幼虫血清中酚氧化酶的活性 （P<0.01）;萼液与毒液混合物对酚氧化酶活性也有明显抑制作用（P<0.01）。研究认为寄生蜂产卵时注入的萼液、毒液可对寄主昆虫酚氧化酶活性产生明显的抑制作用,其中萼液是抑制寄主免疫能力的主要因素。     

Effects of venom/calyx fluid from the endoparasitic wasp Cotesia plutellae on the hemocytes of its host Plutella xylostella in vitro

Crude venom and calyx fluid from Cotesia plutellae (Hymenoptera Braconidae) were assayed for biological activity toward hemocytes of Plutella xylostella (Lepidoptera Plutellidae). Venom from C. plutellae displayed high activity toward the spreading of plasmatocytes of P. xylostella early in the incubation period, and the inhibition was more severe as the concentration of venom increased. However, most inhibited hemocytes spread normally after being incubated for 4h. No effects were found toward granular cells from the host. Additionally, the venom from C. plutellae had some lethal effects on hemocytes of P. xylostella at high concentrations. In contrast, when incubated with different concentrations of calyx fluid, the spreading of some hemocytes was inhibited, some began to disintegrate, and some were badly damaged with only the nucleus left. After 4h, the majority of hemocytes died. The same results were observed when hemocytes were incubated in calyx fluid together with venom. These results show that calyx fluid from C. plutellae may play a major role in the suppression of the host immune system, whereas venom from C. plutellae has a limited effect on hemocytes and probably synergizes the effect of calyx fluid or polydnavirus.     

Biochemical interaction between chelonine wasps and their lepidopteran hosts: after a decade of research - the parasite is in control

A decade of research on the biochemical interaction between chelonine wasps and their lepidopteran hosts has yielded considerable data on the underlying basis for the developmental, immunological and reproductive effects that these parasites inflict upon their hosts. These egg-larval parasites induce their immunologically compromised host larvae to precociously initiate metamorphosis, followed by suppression of development of the precocious prepupa, in addition to castration of the host. The results from numerous laboratories have shown that the parasite egg that is normally injected by the adult female into the host along with venom, polydnavirus and calyx fluid proteins need not hatch or even be present for the host to exhibit each of these alterations. In addition to these aspects the parasite larva, when present, itself releases hormones and proteins into the hemolymph of the host. A review of the data amassed to date leads inexorably to the conclusion that it is the chelonine wasp that is the biochemically dominant partner. Thus, after 10 years of research, it still appears that in chelonine-lepidopteran parasite-host systems, the parasite is in control of specific points of the biochemistry and development of its host.     

Venom of ectoparasitoid,Euplectrus sp near plathypenae (Hymenoptera: Eulophidae) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource

Euplectrus sp. near plathypenae is an ectoparasitoid that can parasitize from 3rd to day 0-6th instar Pseudaletia separata. The developmental period of the parasitoid from the egg to the pupal stage is about 13 days. Parasitized hosts are developmentally arrested and never molt to the next stadium. The injection of venom fluid results in similar effects on P. separata larvae as does parasitization. The inhibitory effect of the venom on molting was dose dependent. Injection of 0.3 female equivalents of venom into day 0-5th host instar resulted in a similar developmental arrest as seen in parasitized hosts. The amount of total lipid in the hemolymph of the host increased as a function of the amount of venom injected, while the lipid content of the fat body was similar to lipid levels in the fat body of parasitized larvae. The amount of total protein in the hemolymph also increased when venom was injected, whereas the protein level of the fat body did not increase. The lipid concentration within the parasitoid larva was maintained at the same level throughout larval development, but increased before pupation. We conclude that the injected venom increased the hemolymph content of lipid and protein to support the growth and development of the ectoparasitoid larva.     

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Development of the solitary endoparasitoid Microplitis demolitor: host quality does not increase with host age and size

Abstract.  1. Many studies examining the relationship between host size, an index of host quality, and parasitoid fitness use development time and/or adult parasitoid size as currencies of fitness, while ignoring pre-adult mortality. Because the physiological suitability of the host may vary in different stages, sizes, or ages of hosts, a misleading picture of host quality may therefore be obtained in cases where fitness is based on only one or two developmental traits.
2. The development of the solitary koinobiont endoparasitoid Microplitis demolitor is examined in different larval age-classes of its host the soybean looper Pseudoplusia includens . Hosts were parasitised on days 1–8 after hatching from the egg, and development time, adult body size, and mortality of the parasitoid were compared.
3. A comparison of larval growth trajectories (using dry body mass) of M. demolitor revealed that parasitoid larvae attained over twice as much body mass in old hosts than in younger hosts. Similarly, adult parasitoid size at eclosion generally increased with host size, although parasitoids developing in smaller hosts lost a much lower proportion of mass between pupation and eclosion.
4. Overall egg-to-adult development was most rapid in intermediate-aged hosts, and longer in hosts at opposite ends of the age continuum. Moreover, parasitoid mortality varied non-linearly with host stage, and was generally higher in very young and older hosts.
5. Based on these results and other empirical data for koinobionts, it is argued that fitness functions in this group of parasitoids are not simply a positive function of host size or age, but instead may be distinctly dome-shaped, both patterns reflecting the degree of physiological and nutritional compatibility between the two organisms.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Penetration into caterpillar cells of virus-like particles injected during oviposition by parasitoid ichneumonid wasps.

Particles originating from the ovarial calyx epithelium of two different species of ichneumonid wasp are injected into host caterpillars during oviposition. At 1 3/4 h post oviposition, many calyx fluid particles are either associated with or have oenetrated through the basement membranes surrounding various tissues. Shortly thereafter, apparently intact particle nucleocapsids are observed in both the cytoplasm and nucleus of host cells. An unusual tubular protrusion of the viral envelope appears to be involved in either or both of penetration of basement membranes and entry of nucleocapsids into host cells.     

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.     

中红侧沟茧蜂雌蜂输卵管萼中病毒样纤丝的特征和功能

中红侧沟茧蜂Microplitis mediator雌蜂输卵管萼中有一种病毒样纤丝（virus-like filaments, VLFs）。在蜂卵从卵巢管通过输卵管萼产出的过程中,VLF包裹在蜂卵的表面,随蜂卵进入寄主体内。透射电镜显示,VLF中心是电子致密物质,外有单层膜包被,直径约35 nm。负染技术表明,VLF是具有左螺旋结构的纤丝,负染时的直径约25 nm。不含VLF的蜂卵进入3龄初寄主后,全部被寄主血细胞包囊;含有VLF的蜂卵进入同样的寄主后,88.2%受到保护。VLF对蜂卵的保护作用在不同发育期的寄主中不同,在3龄初的粘虫体内,平均有64.7%的初产卵不被包囊,而在4龄初的粘虫体内,只有9.5%的初产卵受到保护。这一结果说明,VLF只能为蜂卵提供部分的保护作用,需配合其它寄生蜂因子（萼液、蜂毒等）共同作用于寄主的免疫系统。