金小蜂基因组学研究前沿

金小蜂不仅是重要的昆虫天敌资源,还是理想的模式生物。2010年1月15日,3种金小蜂(丽蝇蛹集金小蜂Nasonia vitripennis、吉氏金小蜂N.giraulti和长角金小蜂N.longicornis)的基因组在《科学》上发表。这一事件标志着金小蜂研究"后基因组时代"的开始。金小蜂基因组测序完成后,科学家们就利用生物信息学、比较基因组学、功能基因组学等方法,基于基因组平台,在进化遗传学、发育生物学、神经生物学、行为学等领域开展了系列研究,取得了重要进展,国际著名杂志《昆虫分子生物学》和《遗传》还以特刊的形式进行刊载。本文就金小蜂基因组学相关研究取得的进展予以扼要概述,并探讨其研究方向和发展前景。     

丽蝇蛹集金小蜂的生物学及其滞育调控机制研究进展

丽蝇蛹集金小蜂可以蛹外寄生多种双翅目蝇科害虫,在防治卫生害虫及牲畜害虫方面效果显著。该蜂是实验室内研究寄生蜂的理想模式昆虫,其发育学、行为学、生态学以及遗传学等的研究已有70多年的历史,近年来,随着金小蜂基因组测序的完成,系统的RNAi方法和CRISPR-Cas9技术在丽蝇蛹集金小蜂中的成功应用,该蜂现已成为优良的新型遗传学研究模式昆虫。丽蝇蛹集金小蜂以末龄幼虫进行兼性滞育,其滞育由母代经历的短光照和低温决定,研究该蜂的滞育,不仅有助于揭示昆虫滞育的分子机制,也可帮助解决有益昆虫实际应用中的贮存、运输和适时防治等问题,为提高天敌昆虫的应用效果提供技术支撑。本文总结了近年来丽蝇蛹集金小蜂的研究文献,主要介绍了该蜂的基本生物学特征,概括了现代分子生物学前沿技术在金小蜂研究中的应用情况,并重点讨论了丽蝇蛹集金小蜂滞育的最新研究进展,以期为深入金小蜂的滞育研究和促进该蜂其他研究领域的发展提供参考。     

去除Wolbachia 对丽蝇蛹集金小蜂繁殖适合度和成蜂寿命的影响

【目的】明确内共生菌 Wolbachia 对丽蝇蛹集金小蜂 Nasonia vitripennis 繁殖适合度和成蜂寿命的影响。【方法】通过给自然感染 Wolbachia 的丽蝇蛹集金小蜂成蜂喂食不同浓度的利福平来消除其体内的 Wolbachia,然后进行10个世代的连续饲养,探究不同浓度利福平对丽蝇蛹集金小蜂体内 Wolbachia 的去除效果和去除 Wolbachia 后对丽蝇蛹集金小蜂繁殖力、性比（雌蜂占子代数量的比值）和成蜂寿命的影响。【结果】低浓度利福平（0.1~0.5 mg/mL）对丽蝇蛹集金小蜂的毒害作用较小,而高浓度利福平（0.7~10.0 mg/mL）对丽蝇蛹集金小蜂的毒害作用较大,但二者均能去除丽蝇蛹集金小蜂体内的 Wolbachia;去除 Wolbachia 后丽蝇蛹集金小蜂的出蜂量显著下降（P <0.01）,子代中性比显著下降（P <0.01）,但寿命无明显差异。【结论】不同浓度利福平均能去除丽蝇蛹集金小蜂体内Wolbachia,但效果不一致;Wolbachia 对丽蝇蛹集金小蜂的出蜂量和子代性比均有显著影响,对成蜂寿命无显著影响。     

Cytonuclear genic incompatibilities cause increased mortality in male F2 hybrids of Nasonia giraulti and N. vitripennis

The haplodiploid wasp genus Nasonia is a promising model for studying the evolution of genic incompatibilities due to the existence of interfertile species and haploid males. The latter allows for significantly reducing the sample size required to detect and map recessive dysfunctional genic interactions. We exploited these features to study the genetics of intrinsic hybrid inviability in male F2 hybrids of Nasonia giraulti and N. vitripennis. Analyzing marker segregation in 225 hybrid embryos, we inferred a linkage map with 38 framework markers. The markers were tested for marker transmission ratio distortion (MTRD) and interchromosomal linkage disequilibrium in populations of embryonic and adult hybrids. We found evidence for four transmission ratio distorting loci (TRDL). Three TRDL showed a deficit of the N. giraulti allele in hybrids with N. vitripennis cytoplasm. A separate TRDL exhibited a deficiency of the N. vitripennis allele in hybrids with N. giraulti cytoplasm. We ascribe the observed MTRD in adult hybrids to cytonuclear genic incompatibilities causing differential mortality during development since hybrid embryos did not show MTRD. The identified cytonuclear genic incompatibilities in F2 hybrids with N. vitripennis cytoplasm account for most of the intrinsic hybrid inviability in this cross. The high mortality rate in F2 hybrids with N. giraulti cytoplasm cannot be explained by the single identified TRDL alone, however.     

A major role for zygotic hunchback in patterning the Nasonia embryo

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.     

Chromosomal anchoring of linkage groups and identification of wing size QTL using markers and FISH probes derived from microdissected chromosomes in Nasonia (Pteromalidae: Hymenoptera)

Nasonia vitripennis is a small parasitic hymenopteran with a 50-year history of genetic work including linkage mapping with mutant and molecular markers. For the first time we are now able to anchor linkage groups to specific chromosomes. Two linkage maps based on a hybrid cross (N. vitripennis x N. longicornis) were constructed using STS, RAPD and microsatellite markers, where 17 of the linked STS markers were developed from single microdissected banded chromosomes. Based on these microdissections we anchored all linkage groups to the five chromosomes of N. vitripennis. We also verified the chromosomal specificity of the microdissection through in situ hybridization and linkage analyses. This information and technique will allow us in the future to locate genes or QTL detected in different mapping populations efficiently and fast on homologous chromosomes or even chromosomal regions. To test this approach we asked whether QTL responsible for the wing size in two different hybrid crosses (N. vitripennis x N. longicornis and N. vitripennis x N.giraulti) map to the same location. One QTL with a major effect was found to map to the centromere region of chromosome 3 in both crosses. This could indicate that indeed the same gene/s is involved in the reduction of wing in N. vitripennis and N. longicornis.     

Water relationships in the ectoparasitoid Nasonia vitripennis during larval diapause

Abstract. Final instar larvae of the ectoparasitoid Nasonia vitripennis overwinter in diapause within the puparium of the host fly. Wasp larvae that are in diapause have a different set of water relations than larvae that are not in diapause. Diapausing larvae contain less water, lose water at a lower rate, and have the capacity to absorb water vapour. Water conservation in larvae of N. vitripennis is also enhanced considerably by the puparium of its fly host.     

Female dispersal and isolation-by-distance of Nasonia vitripennis populations in a local mate competition context

Dispersal behavior directly influences the level of inbreeding, but the effect of inbreeding avoidance on dispersal is less well studied. The parasitoid wasp Nasonia vitripennis (Walker) (Hymenoptera: Chalcidoidea: Pteromalidae) is known to mate exclusively on the natal patch, and females are the only dispersing sex. A previous study has shown that foundresses on a patch are typically unrelated, implying that females disperse for a considerable distance from their natal patch after mating. We investigated dispersal of N. vitripennis on two scales. On a local scale we used a mark-release-recapture experiment, and on the larger scale we investigated isolation by distance using a population genetic approach. We found that N. vitripennis females are long-distance dispersers, capable of covering at least 2 km in 48 h. Populations within a range of 100 km showed no substructure, but larger distances or major geographical barriers restricted gene flow and led to significant population structure. The results provide a basis for future research on dispersal of parasitoids and are discussed in the context of dispersal abilities and inbreeding avoidance in Nasonia .     

No patrigenes required for femaleness in the haplodiploid wasp Nasonia vitripennis

The parasitoid wasp Nasonia vitripennis is an emerging model organism for developmental and behavioral genetics. It reproduces by haplodiploidy; males typically develop parthenogenetically from haploid eggs and females from fertilized diploid eggs. A polyploid mutant strain is available in which females are triploid and lay haploid and diploid eggs that normally develop into males when unfertilized. In contrast to previous reports, approximately 2% of triploid females were found to occasionally produce daughters as well as gynandromorphs from diploid unfertilized eggs. Daughter production increased with age and differed among familial lineages. This is the first report of parthenogenetic female development in Nasonia. The results show that a paternally provided genome is not required for femaleness and call for modifications of existing models of sex determination in Nasonia.     

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila.     

The structure and possible mode of functioning of the female reproductive system in Nasonia vitripennis (Hymenoptera: Pteromalidae)

Associated with the female insect reproductive system are a number of glands. Those present in Nasonia vitripennis are described, their microanatomy examined and where possible this is linked with their function during the process of drilling into the host puparium, feeding on the host fluids and oviposition. The structures dealt with are the ovaries, spermatheca, alkaline gland, colleterial gland and the acid gland with reservoir.     

蝇蛹俑小蜂、丽蝇蛹集金小蜂触角感器的扫描电镜观察

本文利用扫描电镜首次对蝇蛹俑小蜂(Spalangia endius)触角及其感器进行了研究.共观察到七类感器:触觉毛类感器、板状感器、薄壁化学感受器、锥形乳头状感器、鬃形感器、短刺形感器和棒状感器.同时在丽蝇蛹集金小蜂(Nasonia Vitripennis)触角上观察到七种感器.比较研究发现,在两种小蜂间.同种小蜂异性间.感器的种类、数量、排列及分布均有差异,并对此进行了讨论.     

Host genotype determines cytoplasmic incompatibility type in the haplodiploid genus Nasonia

In haplodiploid species, Wolbachia-induced cytoplasmic incompatibility (CI) can be expressed in one of two ways: as a "conversion" of diploid fertilized eggs into haploid males or as embryonic mortality. Here we describe CI-type variation within the parasitic wasp genus Nasonia and genetically analyze the basis of this variation. We reach four main conclusions: (i) CI is expressed primarily as conversion in N. vitripennis, but as embryonic mortality in the sibling species N. giraulti and N. longicornis; (ii) the difference in CI type between N. giraulti (mortality) and N. vitripennis (conversion) is determined by host nuclear genotype rather than by Wolbachia differences; (iii) N. vitripennis "conversion genes" are recessive in hybrid females; and (iv) a difference in CI level between the sibling species N. giraulti and N. longicornis is due to the different Wolbachia infections in the species rather than to the host genotype. These results show that host nuclear genes can influence the type of CI present in a species. On the basis of these findings, we propose a model for how different CI types evolve in haplodiploids due to selection on nuclear genes modifying CI.     

Temperature-related development of the parasitoid wasp Nasonia vitripennis as forensic indicator

Development times of the forensically significant parasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) from oviposition to pupation, and from oviposition to adult emergence, were studied in the laboratory at temperatures of 15-35 degrees C using host pupae of the blowfly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae). Total developmental time of N. vitripennis from oviposition to adult emergence (mean+/-SD) was 43.5+/-2.4, 22.5+/-1.1, 14.8+/-1.7 and 11.3+/-0.9 days when reared at 15, 20, 25 and 30 degrees C, respectively. At 35 degrees C, N. vitripennis did not develop successfully. The rate of total immature development (1/days) increased with temperature. From linear regression of development rates, it was determined that the minimum threshold (tL) for total immature development was 9.8 degrees C (approximately 10 degrees C). Above this threshold, the overall thermal constant (K) for N. vitripennis was found to be 224.3+/-1.7 degree-days.     

管氏肿腿蜂毒液过敏原3基因的克隆及表达分析

为研究管氏肿腿蜂Scleroderma guani毒液过敏原3基因SgA3的功能,通过RT-PCR克隆SgA3基因,采用荧光定量PCR分析其在不同发育阶段和组织中的表达特征,并利用载体pSUMO-Mut对其进行原核表达。克隆获得SgA3基因开放阅读框长699 bp,编码233个氨基酸,其中信号肽位于N端第1~23位氨基酸。多序列比对分析发现,SgA3与丽蝇蛹集金小蜂Nasonia vitripennis、粉蝶盘绒茧蜂Cotesia glomerata、红火蚁Solenopsis invicta和常见黄胡蜂Vespula vulgaris过敏原3的氨基酸序列一致性分别为50.44%、50%、48.89%和47.83%。荧光定量PCR结果表明,SgA3基因在雌成虫中高表达,且在毒液器官中的表达量显著高于雌成虫其他组织和雄成虫中的表达量。利用pSUMO-Mut表达载体,成功表达并纯化得到高纯度的SgA3重组蛋白。该研究结果为进一步研究SgA3基因的功能奠定了基础。     

The quantitative genetic basis of polyandry in the parasitoid wasp, Nasonia vitripennis

Understanding the evolution of female multiple mating (polyandry) is crucial for understanding sexual selection and sexual conflict. Despite this interest, little is known about its genetic basis or whether genetics influences the evolutionary origin or maintenance of polyandry. Here, we explore the quantitative genetic basis of polyandry in the parasitoid wasp Nasonia vitripennis, a species in which female re-mating has been observed to evolve in the laboratory. We performed a quantitative genetic experiment on a recently collected population of wasps. We found low heritabilities of female polyandry (re-mating frequency after 18 h), low heritability of courtship duration and a slightly higher heritability of copulation duration. However, the coefficients of additive genetic variance for these traits were all reasonably large (CV(A)>7.0). We also found considerable dam effects for all traits after controlling for common environment, suggesting either dominance or maternal effects. Our work adds to the evidence that nonadditive genetic effects may influence the evolution of mating behaviour in Nasonia vitripennis, and the evolution of polyandry more generally.     

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae)

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.     

Intraspecific variation in sexual isolation in the jewel wasp Nasonia

Abstract.— Divergence in mate recognition systems can lead to reproductive isolation. In this study, we investigate patterns of intraspecific variation that contribute to premating isolation within and between two haplodiploid species, Nasonia vitripennis and N. longicornis . In a broad-scale survey of 17 North American isofemale lines encompassing the two species, we report strong asymmetric sexual isolation between species and a dramatic level of intraspecific variation for mate discrimination between species. A general lack of incipient speciation was found, with the exception of low levels of interpopulational sexual isolation within N. vitripennis . Regression analysis shows that the degree of intraspecific variation for within-species mating frequency is not associated with the degree for between-species mating frequency. Reinforcement or reproductive character displacement may be involved in some of the variation in inter-species premating isolation.     

How parasitoid females produce sexy sons: a causal link between oviposition preference, dietary lipids and mate choice in Nasonia

Sexual selection theory predicts that phenotypic traits used to choose a mate should reflect honestly the quality of the sender and thus, are often costly. Physiological costs arise if a signal depends on limited nutritional resources. Hence, the nutritional condition of an organism should determine both its quality as a potential mate and its ability to advertise this quality to the choosing sex. In insects, the quality of the offspring's nutrition is often determined by the ovipositing female. A causal connection, however, between the oviposition decisions of the mother and the mating chances of her offspring has never been shown. Here, we demonstrate that females of the parasitic wasp Nasonia vitripennis prefer those hosts for oviposition that have been experimentally enriched in linoleic acid (LA). We show by (13)C-labelling that LA from the host diet is a precursor of the male sex pheromone. Consequently, males from LA-rich hosts produce and release higher amounts of the pheromone and attract more virgin females than males from LA-poor hosts. Finally, males from LA-rich hosts possess three times as many spermatozoa as those from LA-poor hosts. Hence, females making the right oviposition decisions may increase both the fertility and the sexual attractiveness of their sons.