The reproductive strategy of the gregarious parasitoid,Pteromalus puparum (Hymenoptera: Pteromalidae)

Summary Pteromalus puparum is a gregarious parasitoid of lepidopterous pupae. To determine in which phase of a host outbreak superparasitism occurs, field investigations were carried out on seasonal prevalence of the host, Papilio xuhtus, and parasitism by P. puparum in a citrus grove in Fukuoka, Japan in 1974. Host pupation occurred from May to November and the parasitoid attacked the host throughout this period. Pupal density increased rapidly after mid-August and the percentage parasitism decreased during this period. A high level of parasitism was attained after one or more parasitoid generations in mid-September. The superparasitism was observed after mid-September when the parasitoid attained extremely high density, and resulted in an increase in the proportion of males, high mortality, and a decrease in the size of the progeny.     

The reproductive strategy of the gregarious parasitoid,Pteromalus puparum (Hymenoptera: Pteromalidae)

Summary Host size of Pteromalus puparum, a gregarious pupal parasitoid, shows a wide inter- and intraspecific variation. Experiments were made to study the regulation of the number and sex ratio of progeny per host by the parasitoid. The parasitoid could discriminate inter- and intraspecific size differences of the host and regulate the number of eggs according to the host size when a single female attacked the host. The sex ratio of progeny (proportion males) was about 0.1. The number of progeny laid by the female agreed with the energetically most efficient number og eggs in order to maximize total weight of progeny per host but not with the reproductively most efficient number of eggs to maximize the total fecundity of the progeny. The parasitoid laid smaller number of eggs in a half buried host, but the number was much larger than a half of those in a fully exposed host. When more than one female attacked a single host, the number and sex ratio of progeny per host increased with the number of females attacking the host, but the number of progeny per female decreased. The change of the sex ratio agreed with the prediction of the local mate competition model.     

聚寄生性蝶蛹金小蜂雌蜂体型大小对产卵策略的影响

在寄生蜂行为生态学研究中, 通常将寄主体型大小作为寄主品质的主要性状来探究寄生蜂的搜寻行为机理, 而忽略寄生蜂体型大小的意义。为揭示聚寄生蜂雌蜂体型大小对其产卵决策的影响, 在严格控制寄主菜粉蝶Pieris rapae蛹体型大小（体重）的情况下, 于室内观察了不同体型大小的蝶蛹金小蜂Pteromalus puparum雌蜂的产卵行为, 并调查了子代蜂数量（窝卵数）、 性比和体型大小的变化。结果表明： 雌蜂在寄主上的驻留时间随其自身体型增大而缩短, 但随寄主体重增大而延长。窝卵数和余卵量受到雌蜂体型大小的显著影响, 均随雌蜂体型增大而显著增加（P<0.05）; 但子代蜂性比不受雌蜂体型大小的显著影响 (P>0.05)。子代雌、 雄性体型大小均与雌蜂体型大小无关, 但子代雌蜂体型随寄主体重增大而增大。结果证实, 雌性蝶蛹金小蜂体型大小影响其部分产卵决策。因此, 在建立聚寄生蜂产卵决策模型中应考虑雌蜂体型大小这一重要变量因素。     

菜粉蝶蛹体型大小对蝶蛹金小蜂后代数量、性比及体型大小的影响

为检验“寄主体型大小-质量假说”,采取饥饿方法处理菜粉蝶(Pieris rapae)高龄幼虫,以获得体型大小(用体质量表示)差异大于同一自然地理种群内个体间差异的寄主蛹,然后供聚寄生蜂-蝶蛹金小蜂(Pteromalus puparum)寄生,观察寄生蜂在不同大小寄主蛹内的后代数量、性比以及体型大小。结果表明,蝶蛹金小蜂1次攻击产出的后代蜂数随寄主蛹体质量增加而显著增多,蛹中平均出蜂314.97头·g^-1。蝶蛹金小蜂后代的雌性比例随寄主蛹质量的增大而显著提高,后代雌蜂和雄蜂的体型大小(后足胫节长度)均随寄主蛹质量的增大而显著增大。可见,蝶蛹金小蜂母蜂能够根据寄主蛹质量来调整后代数量和性比,以使后代适应度最大化。最后,就蝶蛹金小蜂产卵策略及其影响因素进行了讨论。     

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT₇ genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT_1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.     

蝶蛹金小蜂热激蛋白家族基因表达与热保护功能

作为分子伴侣, 热激蛋白可起修复变性蛋白与阻止其他蛋白质聚集的作用。为进一步理解蝶蛹金小蜂Pteromalus puparum热激蛋白家族的分子伴侣功能, 本研究对来自该寄生蜂的热激蛋白Pphsp90, Pphsp70, Pphsc70, Pphsp60, Pphsp40和Pphsp20的基因在大肠杆菌Escherichia coli BL21菌株中进行了过表达。结果表明： 除Pphsp40外, 其余5个基因均得到高效表达, 且表达的重组热激蛋白在高温下（80℃）具可溶性与热稳定性。其中, Pphsp20与Pphsp90在大肠杆菌中的表达显著提高了高温下（50℃, 1 h）细胞的存活率。离体活性实验证实, 利用纯化的融合蛋白Pphsp20可减少高温下荧光素酶的聚集现象。据此认为, Pphsp20与Pphsp90均具有大肠杆菌细胞的热保护功能, 但Pphsp20可以单独发挥作用, 而Pphsp90可能需其他因子协同作用才有保护活性。     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.     

蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立

为深入研究寄生蜂卵黄发生及其内分泌调控,特采用杂交瘤细胞技术,制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin, Vt）的单克隆抗体（mAb）,即PpVt mAb1,PpVt mAb2,PpVt mAb3和PpVt mAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型,不仅特异性识别Vt,而且识别雌蜂血淋巴中卵黄原蛋白（vitellogenin,Vg）,但与雄蜂体液无反应。通过比较4种不同的ELISA方法,确定了微量检测蝶蛹金小蜂体内Vg/Vt的最适ELISA法,即双夹心ELISA法。该方法可用于单头雌蜂体内Vg/Vt的检测,其检测灵敏度为20 ng/mL。用Western 免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫,并在羽化后12～36 h内含量达到高峰。     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

蝶蛹金小蜂寄生对菜粉蝶血细胞骨架相关蛋白基因的转录调控

为揭示蝶蛹金小蜂Pteromalus puparum抑制寄主菜粉蝶Pieris rapae血细胞免疫反应的分子机制, 克隆了菜粉蝶肌动蛋白、肌动蛋白解聚因子及微管蛋白cDNA, 并研究了蝶蛹金小蜂寄生对其转录水平的影响。结果显示： 菜粉蝶肌动蛋白cDNA ORF为1 131 bp, 编码377 aa, 预测分子量为41.78 kDa, pI为5.29, 与其他昆虫肌动蛋白的相似性非常高, 在90%以上。氨基酸组成特点和系统进化分析表明, 克隆到的菜粉蝶肌动蛋白基因属细胞质型肌动蛋白。菜粉蝶肌动蛋白解聚因子cDNA全长为1 243 bp, ORF为 447 bp, 编码149 aa, 预测分子量为16.97 kDa, pI为7.11,与家蚕Bombyx mori、赤拟谷盗Tribolium castaneum、意大利蜜蜂Apis mellifera和桑粉介壳虫Maconellicoccus hirsutus肌动蛋白解聚因子的相似性分别为97%, 87%, 89%和72%。菜粉蝶微管蛋白cDNA全长为1 757 bp, ORF为1 344 bp, 编码448 aa, 预测分子量为50.38 kDa, pI为4.86, 与家蚕β型微管蛋白1, 2, 3和4的相似性分别为97%, 97%, 87%和93%。系统进化分析表明, 克隆到的菜粉蝶微管蛋白基因属β型微管蛋白。RT-PCR分析表明, 蝶蛹金小蜂寄生能抑制肌动蛋白、肌动蛋白解聚因子及微管蛋白基因在菜粉蝶蛹血细胞中的转录水平。由此推断蝶蛹金小蜂调控寄主血细胞骨架相关蛋白基因的转录是该蜂抑制寄主血细胞免疫反应的分子机制之一。     

Flight activity in the parasitoid waspNasonia vitripennis (Hymenoptera: Pteromalidae)

Flight activity in females of the parasitoid wasp Nasonia vitripennis(Walker) was examined by measuring still-air tethered flight. There was a large amount of variation among females in flight duration. The longest single flight (with no pauses of more than 5 s) was more than 2 h long. Mating status had a significant and large effect on flight: mated females flew twice as long as virgin females. There also was a slight but significant effect of age on flight, with 3-day-old females being less likely to fly than 1-day-old females. Flight duration was not affected by prior exposure to other females, to honey, or to a low or a high host density.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by a pupal endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae): effects of parasitization and venom on host hemocytes

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.     

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 microg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

Oviposition strategy of the parasitic waspDinarmus basalis (Hymenoptera,Pteromalidae)

Summary Host type choice and sex allocation were examined using the solitary parasitic waspDinarmus basalis (Pteromalidae, Hymenoptera) parasitizing larvae or pupae of the bean weevilCallosobruchus chinensis (Bruchidae, Coleoptera) within azuki beans (Vigna angularis). The wasps were offered two types of host; one was hard for the mother to lay eggs in, but was more beneficial for the offspring; the other was easy for the mother to lay eggs in, but was less beneficial for the offspring. The two types of host were one large host (17-day old host) in one bean and 6 small hosts (12-, or 13-day old hosts) in one bean. The same number of each host was presented at the same time to female wasps. The wasps accepted more 17-day old hosts than 12-day old hosts, and more 13-day old hosts than 17-day old hosts in each pair-wise choice experiment. The proportions of accepted host types were different from the proportions predicted by optimization models of random prey encounter with known or unknown prey densities. The wasps showed partial preference of host types. Incomplete information about prey densities, and about the costs and benefits of the two types of host may have generated the partial preference. Two predictions of host sizemodels, that (1) there should be a negative relationship between host size and offspring sex ratio (proportion of male offsprings), and (2) the sex ratio in each size host changes with the relative frequency of each size host utilized, were qualitatively supported.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

The structure of the rectal papilla in a parasitoid hymenopteran Nasonia vitripennis (Walker) (Hymenoptera Pteromalidae)

Summary The ultrastructure of the rectal papillae of the parasitoid hymenopteran, Nasonia vitripennis (Walk), is described. These organs in this insect consist of four distinct cell types arranged as a closed, hollow cone. The majority of the cells are present in the raised cone, and are characterised by large numbers of mitochondria arranged in a membranous labyrinth. A series of cells form a collar around the base of the cone. Junction cells have been identified which are present at the point of insertion of the cone into the rectal epithelium. The base of the cone consists of cells with elaborately folded plasma membranes facing both the central cavity of the cone, and the haemolymph. The structure of this rectal papilla is compared with those found in other insects.We are indebted to Professor E. W. Knight-Jones in whose department the work was carried out, and to the Science Research Council for support for one of us (I.D.).