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。     

Adaptive latitudinal cline of photoperiodic diapause induction in the parasitoid Nasonia vitripennis in Europe

Living in seasonally changing environments requires adaptation to seasonal cycles. Many insects use the change in day length as a reliable cue for upcoming winter and respond to shortened photoperiod through diapause. In this study, we report the clinal variation in photoperiodic diapause induction in populations of the parasitoid wasp Nasonia vitripennis collected along a latitudinal gradient in Europe. In this species, diapause occurs in the larval stage and is maternally induced. Adult Nasonia females were exposed to different photoperiodic cycles and lifetime production of diapausing offspring was scored. Females switched to the production of diapausing offspring after exposure to a threshold number of photoperiodic cycles. A latitudinal cline was found in the proportion of diapausing offspring, the switch point for diapause induction measured as the maternal age at which the female starts to produce diapausing larvae, and the critical photoperiod for diapause induction. Populations at northern latitudes show an earlier switch point, higher proportions of diapausing individuals and longer critical photoperiods. Since the photoperiodic response was measured under the same laboratory conditions, the observed differences between populations most likely reflect genetic differences in sensitivity to photoperiodic cues, resulting from local adaptation to environmental cycles. The observed variability in diapause response combined with the availability of genomic tools for N. vitripennis represent a good opportunity to further investigate the genetic basis of this adaptive trait.     

Effects of starvation on the vitellogenesis,ovarian development and fecundity in the ectoparasitoid,Nasonia vitripennis (Hymenoptera: Pteromalidae)

A female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.     

QTL analysis of the photoperiodic response and clinal distribution of period alleles in Nasonia vitripennis

In seasonal environments, organisms synchronize their life cycle with the annual cycle of environmental factors. In many insect species, this includes a diapause response: a timed dormant stage that allows to survive harsh winter conditions. Previously, we have shown that larval diapause in the parasitic wasp Nasonia vitripennis is induced by the mother upon exposure to a threshold number of short photoperiods (named switch point) and diapause response follows a latitudinal cline in natural populations. Here, we present a QTL analysis using two lines derived from the extremes of this clinal distribution: a northern line from Oulu, Finland and a southern line from Corsica, France. A genomic region on chromosome 1 and one on chromosome 5 were found to be associated with photoperiodic diapause induction. Interestingly, these regions contain the putative clock genes period, cycle (chromosome 1) and cryptochrome (chromosome 5). An analysis of period polymorphisms in seven European populations showed a clinal distribution of two main haplotypes that correlate with the latitudinal cline for diapause induction.     

Effects of starvation on the vitellogenesis,ovarian development and fecundity in the ectoparasitoid,Nasonia vitripennis （Hymenoptera： Pteromalidae）

A female-specific protein, vitellogenin （Vg）, and its corresponding egg vitellin （Vt） are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. An indirect sandwich enzyme-linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.     

丽蝇蛹集金小蜂几丁质酶基因鉴定与表达分析

几丁质酶是降解几丁质的糖苷水解酶,参与昆虫蜕皮、器官发育、免疫等重要生理过程。目前,寄生蜂等膜翅目昆虫中几丁质酶的鉴定以及功能研究仍较少。本研究基于生物信息学分析,在丽蝇蛹集金小蜂Nasonia vitripennis基因组中鉴定到14个几丁质酶基因,氨基酸个数介于312~2 682之间。系统进化分析表明丽蝇蛹集金小蜂几丁质酶分为9个亚家族,其中Group Ⅳ、Ⅶ亚家族可能通过基因串联复制而发生基因家族扩增。qRT-PCR分析结果表明几丁质酶基因的表达具有多样性,其中NvCht1、NvCht5、NvCht6、NvCht7 4个基因在1.5 d幼虫表达量最高,NvCht3在5 d幼虫表达量最高,NvCht8在幼虫期高表达。此外,幼虫组织中的表达分析结果表明NvCht4、NvCht5、NvCht10、NvCht12在表皮中高表达,NvCht7、NvCht8、NvCht13在肠道中高表达,NvCht9在脂肪体和表皮中高表达,NvCht11在唾液腺中高表达。本研究为寄生蜂几丁质酶的进化分析以及功能研究提供理论基础。     

Shifts in metabolomic profiles of the parasitoid Nasonia vitripennis associated with elevated cold tolerance induced by the parasitoid's diapause,host diapause and host diet augmented with proline

The ectoparasitoid wasp, Nasonia vitripennis can enhance its cold tolerance by exploiting a maternally-induced larval diapause. A simple manipulation of the fly host diapause status and supplementation of the host diet with proline also dramatically increase cold tolerance in the parasitoid. In this study, we used a metabolomics approach to define alterations in metabolite profiles of N. vitripennis caused by diapause in the parasitoid, diapause of the host, and augmentation of the host's diet with proline. Metabolic profiles of diapausing and nondiapausing parasitoid were significantly differentiated, with pronounced distinctions in levels of multiple cryoprotectants, amino acids, and carbohydrates. The dynamic nature of diapause was underscored by a shift in the wasp's metabolomic profile as the duration of diapause increased, a feature especially evident for increased concentrations of a suite of cryoprotectants. Metabolic pathways involved in amino acid and carbohydrate metabolism were distinctly enriched during diapause in the parasitoid. Host diapause status also elicited a pronounced effect on metabolic signatures of the parasitoid, noted by higher cryoprotectants and elevated compounds derived from glycolysis. Proline supplementation of the host diet did not translate directly into elevated proline in the parasitoid but resulted in an alteration in the abundance of many other metabolites, including elevated concentrations of essential amino acids, and reduction in metabolites linked to energy utilization, lipid and amino acid metabolism. Thus, the enhanced cold tolerance of N. vitripennis associated with proline augmentation of the host diet appears to be an indirect effect caused by the metabolic perturbations associated with diet supplementation.     

Puparial wax restricts passive movement of water during diapause in the flesh fly

Abstract. To determine whether the capacity of diapausing pupae of the flesh fly ( Sarcophaga crassipalpis Macquart) to absorb water from drier air than non-diapausing pupae is due to a simple passive process, we examined water flux in empty puparia. Empty puparia from both types of fly absorb and lose water passively, but those from diapause pupae do so to a lesser degree. The impervious nature of such puparia is attributed to an extra layer of cuticular wax not found in the non-diapause group.     

Regulation and function of tailless in the long germ wasp Nasonia vitripennis

In the long germ insect Drosophila, the gene tailless acts to pattern the terminal regions of the embryo. Loss of function of this gene results in the deletion of the anterior and posterior terminal structures and the eighth abdominal segment. Drosophila tailless is activated by the maternal terminal system through Torso signaling at both poles of the embryo, with additional activation by Bicoid at the anterior. Here, we describe the expression and function of tailless in a long germ Hymenoptera, the wasp Nasonia vitripennis. Despite the morphological similarities in the mode of development of these two insects, we find major differences in the regulation and function of tailless between Nasonia and Drosophila. In contrast to the fly, Nasonia tll appears to rely on otd for its activation at both poles. In addition, the anterior domain of Nasonia tll appears to have little or no segmental patterning function, while the posterior tll domain has a much more extensive patterning role than its Drosophila counterpart.     

The paternal sex ratio chromosome induces chromosome loss independently of Wolbachia in the wasp Nasonia vitripennis

 The paternal sex ratio chromosome (PSR) is a paternally-inherited supernumerary chromosome found in some males of Nasonia vitripennis. PSR induces the loss of N. vitripennis’s paternal autosomes in early fertilized embryos. Previous examinations have not directly addressed the complication of PSR’s co-occurrence with Wolbachia. Wolbachia is the name assigned to a group of cytoplasmic bacteria which induce numerous reproductive alterations in their hosts. In Nasonia, Wolbachia cause cytoplasmic incompatibility (CI) which also results in paternal chromosome loss. Here we address the question of whether PSR’s function (i.e. PSR’s transmission and/or ability to induce chromosome loss) depends upon or interacts with Wolbachia. A strain of PSR males is artificially cleared of Wolbachia. Test crosses and cytological observations of this strain demonstrate that PSR’s transmission and ability to induce chromosome loss is not dependent upon Wolbachia. Comparisons suggest an absence of interactions between PSR and Wolbachia when they co-occur. Fluorescent and confocal microscopy are used to examine and compare early embryos. Observations demonstrate that microtubule interactions with chromatin do not appear to cause the initial loss of the paternal chromosomes. Cytological observations presented here also differ from previous reports of PSR- and Wolbachia-induced chromosome loss. Received: 3 May 1996 / Accepted: 24 June 1996     

Reproductive strategies under multiparasitism in natural populations of the parasitoid wasp Nasonia (Hymenoptera)

Parasitoid Nasonia wasps adjust their progeny sex ratio to the presence of conspecifics to optimize their fitness. Another trait under female control is the induction of offspring diapause. We analysed progeny sex ratios and the proportion of diapausing offspring of individual Nasonia females in host patches parasitized by two species, Nasonia vitripennis and Nasonia giraulti, in North American field populations using microsatellite fingerprinting. Both Nasonia species produced similar sex ratios on hosts that were co‐parasitized by their own species as by the other species, indicating that females do not distinguish between con‐ and heterospecific clutches. The sex ratios of the diapause and adult fractions of mixed broods from single females were not correlated. We found further indications that N. vitripennis females take the emergence time of the offspring into account in their sex allocation. The reproductive strategies of Nasonia under multiparasitism are largely adaptive, but also partially constrained by information.     

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 .     

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.     

Cowpox virus infection in natural field vole Microtus agrestis populations: delayed density dependence and individual risk

1. Little is known about the dynamics of pathogen (microparasite) infection in wildlife populations, and less still about sources of variation in the risk of infection. Here we present the first detailed analysis of such variation. 2. Cowpox virus is an endemic sublethal pathogen circulating in populations of wild rodents. Cowpox prevalence was monitored longitudinally for 2 years, in populations of field voles exhibiting multiannual cycles of density in Kielder Forest, UK. 3. The probability that available susceptible animals seroconverted in a given trap session was significantly positively related to host density with a 3-month time lag. 4. Males were significantly more likely to seroconvert than females. 5. Despite most infection being found in young animals (because transmission rates were generally high) mature individuals were more likely to seroconvert than immature ones, suggesting that behavioural or physiological changes associated with maturity contribute to variation in infection risk. 6. Hence, these analyses confirm that there is a delayed numerical response of cowpox infection to vole density, supporting the hypothesis that endemic pathogens may play some part in shaping vole cycles.     

Juvenile hormone and regulation of dichotomous spermatogenesis during the larval diapause of the codling moth

Spermatogenesis ceases during diapause and resumes after diapause. A possible causal relationship between the regulation of these phenomena and the fluctuations of the juvenile hormone titre connected with the diapause was examined in the codling moth. Spermatocyte lysis and spermatogenesis arrest are directly and reversibly related to these fluctuations. They are induced in the last-larval instar by sole application of the juvenoid Altosid to the penultimate-instar larvae predetermined not to enter diapause. Conversely, allatectomy of early last-instar larvae predetermined to diapause induces renewal of spermatogenesis and disruption of diapause. However, the continuation of the arrest of the spermatogenesis during diapause is not directly related to the presence of a high titre of juvenile hormone. The arrest extends throughout diapause which may continue for months, although no juvenile hormone could be detected in the species after 35 days of larval diapause.     

The ultrastructure of the mid-gut cells of Nasonia vitripennis (Walker) (Hymenoptera,Pteromalidae)

Summary The ultrastructure of the mid-gut cells of female Nasonia vitripennis is described. The mid-gut consists of a uniform, single-cell epithelium. The cells of different gut regions were analysed using morphometric techniques in order to determine any differences in the components. The structure of the cells is described in the unfed animal, and after varying periods of feeding on host body-fluids. Tissues were sampled after 12 h and 24 h of feeding on host body-fluids and after 24 h feeding/24 h starvation. The cells were found to be complex and contain an organelle component that allows solute-transport and extensive lipid synthesis. A limited cytochemical analysis involving the lysosomal marker enzyme-acid phosphatase — and the respiratory enzyme — cytochrome oxidase was carried out.We are indebted to Professor E.W. Knight-Jones, in whose Department this work was carried out, and to the Science Research Council for financial support to one of us (I.D.)     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

Effects of host (Boettcherisca peregrina) copper exposure on development, reproduction and vitellogenesis of the ectoparasitic wasp, Nasonia vitripennis

Effects of copper (Cu) accumulation by the flesh fly Boettcherisca peregrina (R.-D.) (Diptera: Sarcophagidae) on the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) were investigated experimentally by exposing host larvae to contaminated diets with final Cu concentrations of 400 μg/g and 800 μg/g diet fresh weight (DFW), respectively. Results showed that Cu can be transferred along food chains to secondary consumers (parasitoids) in small amounts, resulting in negative effects on parasitoid growth and development (body weight and developmental duration) as well as fecundity (number of offspring per female). Copper exposure also inhibited vitellogenesis of parasitoids from Cu-contaminated host pupae. It is suggested that the decreased fecundity and inhibition of vitellogenesis of N. vitripennis resulted from poor host nutritional state rather than from direct effects of Cu stress.