二种寄生蜂寄生对不同虫龄小菜蛾精巢的影响

为探明寄生蜂引起寄主寄生性去势的机制,本文选取携带不同多分DNA病毒(Polydnavirus,PDV)的2种内寄生蜂与共同寄主小菜蛾Plutella xylostella(L.)为寄生体系,研究不同虫龄小菜蛾被寄生后雄性生精细胞、精子束形态和精巢发育体积变化,系统比较寄生性去势程度和分别拥有2类PDV的寄生蜂在寄主精子发生和形成过程中的作用。结果表明:携带Bracovirus PDV的菜蛾盘绒茧蜂Cotesiave stalis(Haliday)或拥有Ichvovirus PDV的半闭弯尾姬蜂Diadegma semiclausum Hellén寄生对不同虫龄小菜蛾的精子发生和形成过程均产生明显的抑制作用,表现为不能产生精子束或精子束数量减少,但抑制程度以寄生低龄寄主时最明显。2种蜂寄生均能抑制小菜蛾精巢体积的增长,但对低龄寄主的抑制程度明显强于高龄寄主,寄生性去势程度取决于寄生时寄主虫龄。相比而言,寄生不同虫龄小菜蛾时,茧蜂引起小菜蛾寄生性去势的程度均强于姬蜂。     

二种寄生蜂寄生抑制小菜蛾精巢生长和精子束形成的比较研究

我们曾发现菜蛾盘绒茧蜂Cotesia vestalis和半闭弯尾姬蜂Diadegma semiclausum寄生严重阻碍小菜蛾Plutella xylostella幼虫的精子发生。本研究着重比较2种蜂寄生对小菜蛾精巢生长和精子束形成的影响, 以探明寄生因子对昆虫生殖调控的作用途径。 采取过寄生和假寄生方法, 对2种蜂各自寄生后的小菜蛾精巢生长体积, 精子发生和形成过程中生精细胞、精子束的显微形态变化进行了比较。 结果表明: 茧蜂和姬蜂寄生均明显降低小菜蛾精子束的数量, 严重阻碍了寄主幼虫的精子发生和精子形成. 姬蜂寄生造成小菜蛾精巢畸形, 而茧蜂则造成小菜蛾精子束畸形, 且茧蜂对小菜蛾精巢生长的抑制程度明显强于姬蜂。过寄生造成寄主寄生性去势程度加剧, 茧蜂和姬蜂过寄生后的小菜蛾精巢体积分别为0.005 mm³和0.008 mm³, 仅为各自只寄生1次后精巢体积的33.1%和36.3%。假寄生后, 发现只有寄生蜂母代物质存在的前提下, 对小菜蛾精巢生长的抑制程度基本模拟了正常寄生时的状态, 说明多分DNA病毒（polydnavirus, PDV）和毒液发挥了主要作用。 由此推断分属姬蜂属PDV和茧蜂属PDV的2类PDV功能基因对小菜蛾精巢生长发育的调控机制可能存在较大差异。     

Interspecific competition between two endoparasitoids Cotesia vestalis (Hymenoptera: Braconidae) and Oomyzus sokolowskii (Hymenoptera: Eulophidae)

Two endoparasitoids, Cotesia vestalis and Oomyzus sokolowskii, parasitize the same host, larvae of Plutella xylostella. These two species have evolved different parasitization strategies. O. sokolowskii expresses a single factor, venom, and exerts virtually no detrimental effects on the development of its host. C. vestalis, on the other hand, injects polydnavirus (PDV) and venom during oviposition, and teratocytes are released into the host's hemolymph after egg hatching. Parasitization suppresses host immune reactions and redirects its developmental program. Because both these species parasitize the same stage of their hosts, there is the possibility of multiparasitism in nature. Only one species survives multiparasitism and because of its parasitic strategy, we hypothesized that C. vestalis would invariably be the stronger competitor. We designed competition experiments which revealed that C. vestalis is a stronger competitor than O. sokolowskii. We also show that C. vestalis survives intrinsic competition with O. sokolowskii through two mechanisms: physical attack and physiological suppression. We discovered melanized wounds on O. sokolowskii eggs and larvae, which is strong evidence of physical attacks. The physiological suppression is due to PDV and venom injected by C. vestalis. To test this idea more rigorously, we designed a pseudoparasitization experiment which revealed that no O. sokolowskii emerged from multiparasitized hosts when infertile C. vestlais eggs and normal O. sokolowskii larvae are both present inside the same host. These results support our hypothesis that C. vestalis is the stronger competitor and demonstrate two mechanisms that account for the outcome of intrinsic competition between these two endoparasitoids.     

三种内寄生蜂寄生对小菜蛾幼虫精子发生的影响

内寄生蜂寄生可能会引起寄主的寄生性去势。对小菜蛾Plutella xylostella与菜蛾啮小蜂Oomyzus sokolowskii Kurdumov (膜翅目： 姬小蜂科)、半闭弯尾姬蜂Diadegma semiclausum Hellén (膜翅目： 姬蜂科)、菜蛾盘绒茧蜂Cotesia plutellae (Kurdj.) (膜翅目： 茧蜂科) 3个寄生体系,利用形态学方法和蛋白质技术,研究了寄生对小菜蛾幼虫精子发生的影响。结果表明：菜蛾啮小蜂寄生对寄主的精子发生过程没有影响。半闭弯尾姬蜂寄生造成寄主精母细胞的细胞核畸形,精细胞的染色质超浓缩并趋向核膜,但能形成少量的精子;半闭弯尾姬蜂寄生会导致寄主精巢总蛋白的含量显著下降。菜蛾盘绒茧蜂寄生对小菜蛾幼虫精子发生的抑制程度最强,被寄生寄主的精母细胞出现肿胀,核膜皱缩,胞质中的线粒体发生病变;精细胞的染色体也出现超浓缩并趋向核膜,大量的精子溶解,无正常的精子形成;其精巢总蛋白含量的下降程度比姬蜂寄生的更为明显,且导致分子量为63.4 kD的主蛋白缺失。     

Interspecific competition between Diadegma semiclausum Hellen (Hym., Ichneumonidae) and Cotesia plutellae (Kurdjumov) (Hym., Braconidae) in parasitizing Plutella xylostella (L.) (Lep., Plutellidea)

Abstract:  Interspecific competition between Diadegma semiclausum and Cotesia plutellae was investigated at 25°C in the laboratory, by exposing the third instar larvae of the diamondback moth, Plutella xylostella to both species together, either species alone or by exposing the host larvae already parasitized by one species, at different intervals, to the other. When host larvae were exposed simultaneously to two species in one arena, parasitism rates of the host by each species were not reduced by the presence of the other species; joint parasitism rate by two species was not significantly higher than that by either parasitoid alone. Both parasitoids could lay eggs into the host larvae which had previously been parasitized by the other species, leading to the occurrence of multiparasitized hosts. When host larvae were parasitized first by D. semiclausum and then being followed within 1–2 h by exposing to C. plutellae , or vice versa, ensuing parasitoid cocoons from the multiparasitized host larvae were nearly all C. plutellae . When host larvae were parasitized initially by C. plutellae and then being followed by D. semiclausum two or more days later, all parasitoids ensued from the multiparasitized hosts were C. plutellae . In contrast, when host larvae were parasitized initially by D. semiclausum and then being followed by C. plutellae two or more days later, most host larvae could not survive to prepupae and most of the ensuing parasitoid adults from the surviving hosts were D. semiclausum . Dissections of host larvae at various time intervals after parasitization by the two parasitoids showed that development of both parasitoids in multiparasitized hosts were somewhat arrested, and that the first instar larvae of C. plutellae could initiate a physical attack on the larvae of D. semiclausum and remove the latter.     

菜蛾盘绒茧蜂多分DNA病毒的特性及其对小菜蛾幼虫的生理效应

对菜蛾盘绒茧蜂Cotesia plutellae多分DNA病毒的特性及其对寄主小菜蛾Plutella xylostella幼虫的生理效应进行了研究。结果表明：菜蛾盘绒茧蜂雌蜂输卵管萼中含有大量的多分DNA病毒（polydnavirus, PDV）;一个PDV内含多个核衣壳,最多可达16个;核衣壳长40～168 nm,直径39～40 nm;PDV仅在输卵管萼细胞内复制;雌蜂产卵时,随蜂卵将PDV注入寄主血腔,并扩散到寄主的许多组织中;PDV可能先通过脱膜再侵染寄主组织。雌蜂经Co⁶⁰辐射处理后再寄生（即假寄生）小菜蛾2龄、3龄和4龄初期的幼虫,被寄生后的寄主幼虫几乎全部不能化蛹,但末龄（即4龄）幼虫期显著延长,并在寄生后期,幼虫胸部有褐色的短翅芽出现;即将化蛹的4龄末小菜蛾幼虫被假寄生后,即使每头寄主被过寄生9次,依然能正常化蛹,但不能羽化。假寄生与正常寄生后寄主的脂肪体数量和形态结构有明显的不同,推测在正常寄生的情况下蜂卵孵化时释放的畸形细胞及随后的幼蜂可能对脂肪体的结构产生了作用。     

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.     

Isolation and characterization of a novel venom protein from an endoparasitoid,Cotesia rubecula (Hym: Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparasitoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidase. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C. rubecula PDV-protein (Crp32).     

A novel venom peptide from an endoparasitoid wasp is required for expression of polydnavirus genes in host hemocytes

Maternal factors introduced into host insects by endoparasitoid wasps are usually essential for successful parasitism. This includes polydnaviruses (PDVs) that are produced in the reproductive organ of female hymenopteran endoparasitoids and are injected, together with venom proteins, into the host hemocoel at oviposition. Inside the host, PDVs enter various tissue cells and hemocytes where viral genes are expressed, leading to developmental and physiological alterations in the host, including the suppression of the host immune system. Although several studies have shown that some PDVs are only effective when accompanied by venom proteins, there is no report of an active venom ingredient(s) facilitating PDV infection and/or gene expression. In this study, we describe a novel peptide (Vn1.5) isolated from Cotesia rubecula venom that is required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (Pieris rapae), although it is not essential for CrBV entry into host cells. The peptide consists of 14 amino acids with a molecular mass of 1598 Da. In the absence of Vn1.5 or total venom proteins, CrBV genes are not expressed in host cells and did not cause inactivation of host hemocytes.     

Parasitic castration of Pseudaletia separata by Cotesia kariyai and its association with polydnavirus gene expression

Parasitization by the endoparasitoid Cotesia kariyai caused the inhibition of spermatogenesis of Pseudaletia separata. This phenomenon is called parasitic castration. The degree of castration was dependent on the host stage parasitized. Host parasitized on day 1 of the 4th stadium (the time of primary spermatocyte accumulation), had testicular cells with abnormal chromosomes appearing two days after parasitization, and spermiogenesis was completely inhibited. However, when hosts were parasitized on day 0 of the 6th (final) stadium, the degree of castration was less severe, and elongated cells appeared similar to those found in nonparasitized larvae. Results of this study involving injection of C. kariyai polydnavirus (CkPV) and venom suggested that these wasp components caused the appearance of abnormal chromosomes in specific germ cells, which were in mitotic or meiotic prophases. The amount of CkPV gene expression in host testes increased immediately after parasitization and reached a maximum 12h later. The early-expressed CkPV gene(s) may be related to the parasitic castration phenomenon.     

Asobara, braconid parasitoids of Drosophila larvae: unusual strategies to avoid encapsulation without VLPs

Ichneumonoidae parasitoids have been well described for their regulatory effects on host physiology which are usually associated with the activity of polydnaviruses (PDVs) or viruslike-particles (VLPs) injected by the female wasps at oviposition. Among them, parasitoids of the braconid families display specific characteristics like the required activity of secretions from the maternal venom glands or of teratocytes from embryological origin. However, none of these features were observed in two braconid species of the Asobara genus parasitizing Drosophila hosts. In the absence of PDVs and VLPs, the two species A. tabida and A. citri seem to have developed unique strategies to avoid immunity defenses and to succeed in their Drosophila larval hosts. The aim of this study is to report on the complex relationships of braconid parasitoids with their hosts and to present some of the insights from studying Drosophila parasitoids.     

闭弯尾姬蜂与菜蛾盘绒茧蜂寄生菜蛾幼虫时的种间竞争

在室内25℃下,以菜蛾3龄初幼虫作寄主,研究了菜蛾盘绒茧蜂Cotesia plutellae和半闭弯尾姬蜂Diadegma semiclausum的种间竞争。当寄主供2种蜂同时产卵寄生时,2种蜂各自的寄生率与其单独寄生时无显著差异,合计寄生率比一种蜂单独存在时有所提高,但差异不显著。2种蜂均能产卵寄生已被另一种蜂寄生了的寄主幼虫。当寄主被2种蜂寄生的间隔时间很短（少于10 h）时,所育出的蜂绝大部分（80%以上）为绒茧蜂;当寄主先被绒茧蜂寄生,并饲养2天以上再供弯尾姬蜂寄生时,所育出的全为绒茧蜂;当寄主先被弯尾姬蜂寄生,并饲养2天以上再供绒茧蜂寄生时,寄主幼虫绝大部分不能存活,只有少部分能育出寄生蜂,且多为弯尾姬蜂。当2种蜂的幼虫存在于同一寄主体内时,2种蜂的发育均受到另一种蜂的抑制;绒茧蜂1龄幼虫具有物理攻击能力,能将弯尾姬蜂卵或幼虫致死。这些结果表明,菜蛾盘绒茧蜂与半闭弯尾姬蜂在同一寄主中发育时,前者具有明显的竞争优势。     

Parasitism-induced Effects on Host Growth and Metabolic Efficiency in Tobacco Hornworm Larvae Parasitized by Cotesia congregata

Parasitism by the braconid wasp Cotesia congregata affects the growth of Manduca sexta larvae in a parasitoid 'dose-dependent' fashion. Following parasitization of fourth-instar larvae, more heavily parasitized larvae grew larger compared to those containing fewer parasitoids due to an increase in host dry weight. The differences in host mass appeared to arise after oviposition. A 'dose-dependent' enhancement of host dry weight would appear nutritionally beneficial for the parasitoids developing in more 'crowded' hosts. The efficiencies of conversion of ingested and digested food to body mass and the approximate digestibility of the diet ingested by the host caterpillar did not vary significantly with clutch size although parasitoids took slightly longer to develop in the more heavily parasitized hosts. Larval parasitoids developing in the presence of many competitors weighed up to 50% less than those developing in hosts with fewer endoparasitoids, although the weight of adult female parasitoids did not vary significantly with wasp clutch size. The maximum number of emerging wasps was 200 parasitoids, possibly representing the host's 'carrying capacity' for larvae parasitized in the fourth-instar. The ratio of emerging to non-emerging parasitoids decreased as parasitoid clutch size increased, with few or none emerging from very heavily parasitized hosts containing more than 400 parasitoids. Copyright 1997 Elsevier Science Ltd. All right reserved     

WASP‐ASSOCIATED FACTORS ACT IN INTERSPECIES COMPETITION DURING MULTIPARASITISM

Coexistence or displacement of parasitoids in hosts during intrinsic competitive interactions between different parasitoid species (multiparasitism) may depend on their life history traits and behavior. Intense competition for possession of hosts may lead to the elimination of the inferior competitor through physical attack and/or physiological suppression. However, the mechanisms of physiological suppression during multiparasitism remain unclear. Previous work has shown that first instar larvae of the solitary endoparasitoid Meteorus pulchricornis possess well‐developed mandibles that are used to kill competitors. Two gregarious endoparasitoids, Cotesia kariyai and C. rufricus, share host resources especially when the time gap of oviposition is short. Here, we investigated the physiological influence of wasp‐regulatory factors of the three endoparasitoids, M. pulchricornis, C. kariyai, and C. ruficrus, in their common host Mythimna separata. We found that MpVLP alone (or with venom) deleteriously affected the development of the two gregarious species. Similarly, CkPDV plus venom had toxic effect on M. pulchricornis eggs and immature larvae, although they were not harmful to immature stages of C. ruficrus. Cotesia kariyai and C. ruficrus were able to coexist mainly through the expression of regulatory factors and both could successfully emerge from a multiparasitized host. The injection of CkPDV plus venom after oviposition in L5 host larvae facilitated C. ruficrus development and increased the rate of successful parasitism from 9% to 62%. This suggests that the two gregarious parasitoid wasps exhibit strong phylogenetic affinity, favoring their coexistence and success in multiparasitized hosts.     

Parasitism-induced effects on host growth and metabolic efficiency in Plutella xylostella larvae parasitized by Cotesia vestalis or Diadegma semiclausum

The nutritional physiology of the diamondback moth, Plutella xylostella, larvae was examined after parasitization by the solitary endoparasitoids Cotesia vestalis or Diadegma semiclausum. Examinations were performed in two phases, one was examined at the time point of 24 h post‐parasitization, and the other was done at the end of the 4th instar larvae of host. Rates of growth, food consumption, assimilation, excretion, and respiration were calculated as well as approximate digestibility and the rate ratios ECI (percent efficiency of conversion of ingested food to body substance), and ECD (percent efficiency of conversion of digested food to body substance). Parasitization by C. vestalis resulted in significant decrease in the rates of growth, feeding, excretion, assimilation, and respiration, but the final dry rate of respiration at the end of last larval stadium was elevated. The ECI and ECD were also reduced as the result of parasitization, but digestibility was increased. All these parameters in the larvae parasitized by D. semiclausum at 24 h post‐parasitization were also significantly changed compared to the control; however, these differences were quantitatively, but not qualitatively before pupation, similar to those resulted from parasitization by C. vestalis. In spite of the similarities of the parasitism‐induced effects caused by these endoparasitoids, the final metabolic rate, that is, the rate of intake of nutrients required to compensate for metabolism, was much lower in the larvae parasitized by C. vestalis than that of the larvae parasitized by D. semiclausum. All of the results discussed here will contribute toward explaining the different ways these two wasps regulate the parasitoid‐host relationship.     

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.     

Physiological and Biochemical Analysis of Factors in the Female Venom Gland and Larval Salivary Secretions of the Ectoparasitoid Wasp Eupelmus orientalis

The stinging adult female and the biting newly-hatched larva of the solitary ectoparasitoid wasp Eupelmus orientalis can both cause permanent paralysis and stop the development of Callosobruchus maculatus host larvae. These two processes of host envenomation appeared to be independent and complementary in primary parasitism or in hyperparasitism of a distantly related hymenopteran host species. In contrast, the development of larvae as hyperparasites on members of their own species or genus depended completely on the prior injection of female venom. The venoms of the female and the first instar larva had similar effects on the cellular metabolism of the primary hosts. Protein synthesis was blocked in C. maculatus hosts envenomated by a female or a first instar larva of E. orientalis, but the absence of DNA breakdown indicated that these paralysed hosts were alive and quiescent. The venomous secretions injected by adult females and first instar larvae of E. orientalis had distinct electrophoretic profiles. The immunoreactive features of proteins from female venom and larval secretions were also examined. There is evidence for antigenic conservation between some venom proteins of E. orientalis and Apis mellifera. Lastly, the hyaluronidase, phospholipase and lipase activities in the female venom gland and in larval-derived secretions of E. orientalis were assayed. No lipase activity was detected. Phospholipase activity was found in both the female venom and the larval secretions of E. orientalis, whereas hyaluronidase was specific to the female venom.     

Are population differences in plant quality reflected in the preference and performance of two endoparasitoid wasps?

In recent years, increasing attention has been paid in exploring the role of direct plant defence, through the production of allelochemicals, on the performance of parasitoid wasps and their hosts. However, few studies have determined if parasitoids can detect differences in plant quality and thus preferentially attack hosts on which their progeny develop most successfully. In this study we examined the development and preference of two endoparasitoids, Diadegma semiclausum and Cotesia glomerata , developing in larvae of their respective hosts, Plutella xylostella and Pieris brassicae . In turn, these were reared on different wild populations of black mustard Brassica nigra originating in the Netherlands and Sicily (Italy), as well as single cultivated strains of B. nigra and brown mustard, B. juncea . Chemical analyses of foliar glucosinolates and volatile emissions by P. xylostella -damaged plants revealed large differences between B. nigra and B. juncea plants, with smaller differences among the B. nigra populations. The four mustard populations differentially affected development time and body mass of the herbivores and parasitoids. Contrasts among the means revealed significant differences mainly between B. nigra and B. juncea . Both parasitoids, however, preferred to alight on plants in which their progeny developed most successfully. In behavioural bioassays, D. semiclausum did not discriminate among the B. nigra populations and preferred to alight on B. juncea , which was the best plant population for parasitoid development. By contrast, C. glomerata females exhibited the lowest preference for Italian B. nigra populations, on which adult parasitoid size was the smallest. These results reveal that parasitoids can detect even small differences in plant quality presumably through their volatile blends and that plant preference and offspring performance in the two species are 'optimally synchronized'.