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.     

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.     

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。     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

The effects of host deprivation on the calyx cells of Nasonia vitripennis (walker) (hymenoptera: Pteromalidae)

Summary 1. The changes in the ultrastructure of the cells of the calyx in the female reproductive system of Nasonia vitripennis are described during a period extending from a condition when host puparia are readily available to a condition of prolonged host deprivation.2. In conditions when host puparia are available, the calyx cells resemble typical secretory cells with rough endoplasmic reticulum, Golgi complex and mitochondria. After periods of host deprivation the calyx cells increase in size, the organelles change and become reorientated and cytolysomes appear producing a configuration of cells undergoing autophagy.3. When host puparia become available again, the cells show an ability to recover and recommence production of secretory droplets.     

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。     

丽蝇蛹集金小蜂Pacifastin蛋白酶抑制剂基因nvpp-1和nvpp-2的功能研究

Pacifastin蛋白酶抑制剂在昆虫免疫与发育中起着重要作用。为了明确其在寄生蜂中的相关功能, 本研究分别克隆获得编码丽蝇蛹集金小蜂Pacifastin蛋白酶抑制剂开放阅读框的cDNA序列nvpp-1和nvpp-2, 序列长度分别为723和888 bp, 分别编码240和295个氨基酸残基。预测结果表明, nvpp-1和nvpp-2推导氨基酸序列N端均含一个长度为17个氨基酸残基的信号肽序列。序列分析和进化树构建结果表明, NVPP-1和NVPP-2分别含有5个和4个典型的Pacifastin保守结构域, 并与疑黑瘤姬蜂Pimpla hypochondriaca毒液蛋白CVP4 聚为一类。实时荧光定量RT-PCR结果表明, nvpp-1和nvpp-2于该蜂雌蜂各组织中均发生转录, 且在胸、 腹部残体（解剖后腹部剩余部分）和毒器官中的转录水平较高; 于毒器官中, 其在羽化初期（0和1 d）转录水平较高, 其转录水平显著降低。Western blot结果表明, NVPP-1和NVPP-2均只在毒液中被大量检出, 在其他待测组织中均未被检出, 而刚羽化时（0 d）其在毒液中含量较低。利用pET-28a (+) 载体分别对nvpp-1和nvpp-2进行了原核表达, 并对重组表达产物进行纯化。分别测定重组NVPP-1和NVPP-2对4种不同丝氨酸蛋白酶（胰蛋白酶、 糜蛋白酶、 蛋白酶K和弹性蛋白酶）的抑制效果, 结果表明, 重组NVPP-1和NVPP-2分别能显著抑制糜蛋白酶和胰蛋白酶活性。同时还分别测定了两种重组蛋白对寄主家蝇蛹血淋巴自身的酚氧化酶活性及原酚氧化酶激活反应的影响, 结果表明, 重组蛋白对家蝇蛹血淋巴原酚氧化酶激活反应亦有抑制效果, 但其均不能显著影响血淋巴自身的酚氧化酶活性。综上所述, 丽蝇蛹集金小蜂毒液中含有Pacifastin蛋白酶抑制剂NVPP-1和NVPP-2, 分别为糜蛋白酶抑制剂和胰蛋白酶抑制剂家族成员, 均能显著影响寄主家蝇蛹血淋巴原酚氧化酶激活反应, 从而削弱寄主体液免疫水平。本研究所获结果加深了我们对昆虫尤其是寄生蜂Pacifastin蛋白酶抑制剂作用的认识。     

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。     

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.     

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。     

Oösorption in Nasonia vitripennis (Hymenoptera: Pteromalidae)

The process of oösorption is described. Leucine amino peptidase and esterase produced by the follicle cells remove the chorion and vitelline membrane. The oölemma grows into the oöcyte and islands of degeneration are formed. The follicle is the entity of oösorption and is isolated from adjoining follicles.
Under conditions of host deprivation oösorption begins earlier in older individuals than younger ones whose fat body is still large before egg production reaches its peak. The time of onset may be correlated with the reduction in the size of the fat body following the peak of egg production. This suggestion is supported by the reduced longevity of starved older individuals compared with younger ones.
Age has no effect on the rate of oösorption.     

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.     

Eleven microsatellite markers in Nasonia,Ashmead 1904 (Hymenoptera; Pteromalidae)

We designed primer sequences for 11 microsatellite markers in the jewel wasp Nasonia vitripennis. Most loci could be cross‐amplified in Nasonia longicornis and Nasonia giraulti, which make them amenable for linkage analysis in hybrid crosses. Eight loci were assigned to specific chromosomes. Additionally, 10 loci showed allelic variation in a Nasonia vitripennis field population. The observed number of alleles in this population ranged from two to seven, with observed heterozygosities from 0.0750 to 0.4750.     

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.     

Variation in Propensity to Exhibit Thanatosis in Nasonia vitripennis (Hymenoptera: Pteromalidae)

Thanatosis (death-feigning) has rarely been documented for Hymenoptera but occurs in the parasitoid wasp Nasonia vitripennis. The propensity to exhibit thanatosis did not differ with age, sex, or food deprivation. Squeezing a female’s abdomen and contacting her antennae were equally likely to trigger thanatosis. Dropping an object next to a female in order to cause substrate vibrations never triggered thanatosis, and dropping a female from a test tube rarely triggered thanatosis. Thanatosis was not seen during interactions between females. There was some tendency for females to exhibit fewer thanatosis responses on white than on colored backgrounds. Females that were least active had the greatest tendency to exhibit thanatosis.     

Ultrastructural changes in the mitochondria of the acid gland of Nasonia vitripennis (Walker) (Pteromalidae: Hymenoptera) induced by starvation

Summary The formation of mitochondrial-cytoplasmic complexes and their transformation into lipid droplets in the acid gland of Nasonia vitripennis is described. Electron microscopy and histochemistry show that lipid droplets are absent from acid glands in newly emerged, fed and re-fed insects. The droplets develop in the cytoplasm after varying periods of starvation and are not associated with acid phosphatase activity.The mature lipid droplets are rarely associated with intact mitochondria and are probably the residual end-product of the mitochondrial-cytoplasmic associations. The possible role of the associations in the maintenance of mitochondrial function and structure is discussed.We are indebted to Professor E. W. Knight-Jones in whose Department the work was carried out and to the Scientific Research Council for financial assistance to one of us (N.A.R.).     

Temporal events of gypsy moth vitellogenesis and ovarian development

Abstract The vitellogenic period of gypsy moth ovarian development starts on day 3 of the pupal stage and continues through adulthood. During this period, rapid increases occur in follicle size, protein content, and wet weight of the ovary. Patency is observed on day 3 of the pupal stage.
Pre-vitellogenic follicles are formed in the last larval stadium. Newly formed follicles detach from the germarium on day 4, and increase rapidly to 140 per ovariole at the end of the last larval stadium. The pre-vitellogenic follicles are uniformly around 50 um in diameter. No vitellogenin is incorporated into the oocytes until the pupal stage.
Polyacrylamide gel electrophosesis (PAGE) in the presence of sodium dodecylsulphate (SDS) analysis of male and female haemolymph samples and vitellogenic ovaries demonstrates the presence of two female-specific subunits of vitellogenin of 180 kD and 160 kD. These proteins are detected only in haemolymph and ovarian extracts of vitellogenic females. The molecular weight of the native protein determined by size exclusion chromatography is approximately 400–420 kD.
A highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to monitor the temporal changes in vitellogenin titre in haemolymph. Vitellogenin production starts on day 2 of the last larval stadium, reaching a maximum level by day 6 of the last larval stadium, and decreasing in the late pupal stage as vitellogenin was internalized into the oocytes. This is the first report of vitellogenin production occurring in the larval stage of a holometabolous insect. The fact that vitellogenin production and uptake occur during different stages of development in the gypsy moth, opens up some interesting questions concerning the underlying regulatory mechanisms controlling each process.     

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     

Associative Learning in Response to Color in the Parasitoid Wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

A parasitoid that can learn cues associated with the host microenvironment should have an increased chance of future host location and thereby increase its reproductive success. This study examines associative learning in response to simultaneous exposure to the colors yellow and blue in mated females of the parasitoid wasp Nasonia vitripennis. Preference was measured as the proportion of time spent on a color. When trained with one color rewarded with hosts and honey and the other unrewarded, females showed an increase in preference for the rewarded color with increasing number of training days (1, 3, and 7 days). Hosts and honey together produced a slightly greater preference toward the rewarded color than just hosts, which produced a greater preference than just honey. When trained with a variable reward on one color and a constant reward on the other, females preferred the color associated with the variable reward when it was yellow, but not when it was blue. Thus, relative to no reward, the presence of a variable reward decreased the strength of preference toward the constantly rewarded color. Finally, females trained with regular hosts on one color and used hosts on the other preferred the color associated with the regular hosts when that color was blue but showed no preference in the reverse situation. The presence of used hosts instead of no reward did not increase the strength of preference for the color associated with the regular hosts.