The effect of age on the ultrastructure of the mid-gut cells of the hymenopteran Nasonia vitripennis (Walk.) (Hymenoptera,Pteromalidae)

Summary The ultrastructure of the mid-gut cells of aged female Nasonia vitripennis is described. The mid-gut is a shrunken and distorted organ in the aged animal. The individual cells are highly disorganised and the organelle components are altered. The small lipid droplets formed in the apical cell region do not coalesce to form the large central lipid inclusions characteristic of the young animal. The rough endoplasmic reticulum is reduced and some of the mitochondria enlarge. The mid- and apical cell regions also contain large numbers of cytolysosomes. The basal cell region is essentially unchanged, but the channels formed by the infolded basal plasma membranes are dilated. The changes observed are discussed in relation to previous observations on other insect species.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.)     

The effect of diet on the ultrastructure of the mid-gut cells of Nasonia vitripennis (Walk.) (Insecta: Hymenoptera) at various ages

Summary The ultrastructure of mid-gut cells of female Nasonia fed on a diet of 10% sterile sucrose is described. There are extensive alterations in cell organelles, particularly the mitochondria, rough endoplasmic reticulum (R.E.R.) and lipid inclusions, when compared to similar insects fed a normal diet of dipteran pupae. A proportion of the mitochondria found in the apical cell region are enlarged in size and contain electron-dense granules. The remaining mitochondria are smaller, but also contain electron-dense granules. Cytochrome-c oxidase activity appears to be absent from the enlarged mitochondria. The R.E.R. appears reduced in many cells of the 1 day old, sugar-fed insects, however, this component fluctuates throughout the remaining life span. The lipid inclusions prominent in the 1 day old, pupae-fed insects are not present in sucrose-fed females of the same age, but lipid deposition was recorded later in the life span. There are many large residual bodies and cytolysosomes present in the old, sugar-fed insects. These changes in ultrastructure are discussed in relation to diet and longevity.     

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.     

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.).     

Effect of host density on offspring sex ratios and behavioral interactions between females in the parasitoid waspNasonia vitripennis (Hymenoptera: Pteromalidae)

Pairs of females of the parasitoid waspNasonia vitripennis were videotaped with one or two hosts. The presence of an additional host decreased the number of interactions between females but had no measured effect on the nature of the interactions, i.e., on whether the interaction involved physical contact or occurred while one of the females was parasitizing a host. The number of hosts did not itself affect offspring sex ratios but did influence which other factors were correlated with sex ratio. When there was one host, the proportion of sons was more positively correlated with utilization of previously drilled holes than with female-female interactions, whereas when there were two hosts, the reverse was true. Parasitizing an already parasitized host appeared to affect a female's sex ratio beyond any effects of the physical presence of another female: When two hosts were present, the proportion of sons was greater from hosts parasitized by both females than from hosts parasitized by only one female. The observation that parasitizations in previously drilled holes and female-female interactions are correlated with sex ratios is consistent with previous studies; however, that these relationships are host density dependent is a new result and remains unexplained.     

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.     

Associative Learning of Color by Males of the Parasitoid Wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

Males of the parasitoid wasp Nasonia vitripennis showed no innate preference for blue versus yellow or for green versus brown. They learned to associate color with mates, but their ability to do so depended on the color used and the strength of the reward. Specifically, males learned to associate brown or green with a reward of many virgin females. With fewer females, fewer training periods, or mated females as the reward, males still learned a preference for green but not for brown. Males did not learn to associate color with rewards of honey or water. Previous studies of color preference and associative learning in parasitoid wasps have focused almost entirely on females. This is the first demonstration of associative learning in response to visual cues by male parasitoid wasps.     

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.     

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.     

The effect of host spatial distribution on patterns of parasitism by Nasonia vitripennis

Patterns of parasitism of Calliphora sp. pupae by Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) are explored under various conditions of host aggregation. Increasing the hosts' spatial aggregation decreased the number of hosts actually parasitised but had no significant effect on the total number of hosts attacked. Increasing host aggregation also resulted in a smaller parasitoid population with a more male biased sex ratio.

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Résumé Nous avons examiné les effets de différents niveaux d'agrégation des hôtes (pupes de Calliphora) sur l'efficacité parasitaire de Nasonia vitripennis. Une augmentation de l'agrégation des pupes a diminué le nombre d'hôtes parasités bien que le nombre de pupes attaquées ait été peu modifié. Cette différence a été attribuée à un accroissement des interactions comportementales entre les parasitoïdes et à une tendance au superparasitisme plus élevée lors des distributions les plus agrégatives. Plus les hôtes étaient concentrés, moins l'effectif de parasitoïdes achevant leur développement et émergeant était important. La fréquence des males a augmenté avec la concentration des pupes. Nous avons estimé que les facteurs déterminants ont été les interactions comportementales entre femelles et leur aptitude à modifier le taux sexuel de leur descendance immédiate.Nous en avons conclu que N. vitripennis est peut-être mieux adaptée aux populations d'hôtes distribuées au hasard, type de distribution le plus vraisemblable dans les populations naturelles.

     

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.     

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。     

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.     

Some aspects of the biology and population dynamics of Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Some aspects of the biology and population dynamics of the chalcid Nasonia vitripennis (Walker) are described.The reproduction capacity and the influence of size and age of the females have been studied, using Calliphora erythrocephala Meig. as the host. The females lay a maximum number of about 30 eggs into one host puparium. Fully parasitized puparia are recognized by females as such. This seems to be the major factor in the determination of the area searched for hosts.Changes in sex ratio of the offspring, in relation to the age and the density of the females are described. Also an influence of the age of the females on the number of offspring entering diapause is reported.

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Zusammenfassung Die beschriebenen Experimente zeigen, dass die Eiproduktion von Nasonia vitripennis in grossem Ausmasse durch das Alter des Muttertieres bedingt ist. Insbesondere während der ersten 4 Tage nach dem Schlüpfen steigt die Produktion schnell von sehr wenig bis zu etwa 100 Eiern pro Tag an (Wirt: Calliphora erythrocephala Meig.). Diese Produktion bleibt einige Tage konstant und nimmt dann langsam ab. Obwohl die individuelle Produktion sehr variabel ist, konnte eine positive Korrelation zwischen der Grösse des Tieres und der Anzahl seiner Nachkommen nachgewiesen werden.Wenn ein Teil der vorhandenen Wirte durch Austrocknen unbrauchbar geworden ist, tritt eine Reduktion der Eiablage auf. Diese Reduktion ist nicht eine Folge von Zeitmangel (verursacht durch das Inspizieren unbrauchbare Wirte), sondern entsteht durch die beschränkte Eiablage-Möglichkeit in einen Wirt. Die Weibchen passen ihre Eiablage der Anzahl der verfügbaren Wirte an. Im Mittel werden die wirte mit nicht mehr als rund 30 Eiern belegt. Eine Reduktion der Nachkommenschaft durch Futterkonkurrenz zwischen den Larven findet nicht statt.Ein Einfluss des Alters der Weibchen auf das Verhältnis der Geschlechter ihrer Nachkommen wird nachgewiesen. Das gefundene Verhältnis (10–15% Männchen) entspricht nicht dem Mechanismus, der von King (1961) für die Berfruchtung vorgeschlagen wird.Durch Mangel an Wirten wird die Anzahl abzulegender Eier reduziert. Eiresorption und damit Steigerung des Anteils der Männchen in der Nachkommenschaft ist die Folge; die ersten Resorptionsstadien werden bei der Eiablage nicht befruchtet, wodurch Männchen entstehen. Die Dichte der Wirte hat also einen Einfluss auf das Geschlechtsverhältnis.Ein dritter Einfluss des Alters der Weibchen besteht in einer Zunahme des Prozentsatzes von Diapauselarven. Bei älteren Weibchen wird eine rasche Änderung von normaler Nachkommenschaft in eine fast nur Diapauselarven umfassende nachgewiesen. Diese Änderung ist nicht die Folge von Futtermangel oder Abkühlung.Die Suchaktivität des Parasiten wird zum grössten Teil durch die Wirtsdichte bedingt. Nasonia-Weibchen bleiben in der Nähe eines Wirtes, bis dieser fast vollständig ausgenutzt ist. Die Weibchen können parasitierte und nichtparasitierte Wirte voneinander unterscheiden und nehmen bei ihrer Suche den ersten freien Wirt an, den sie finden. Dadurch wird die Grösse ihres Wandergebietes durch die Populationsdichte des Wirtes bedingt. Eine zwangsläufige Regulation der Dichte von Wirt und Parasit ist damit aber nicht ausgeschlossen.

     

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.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

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.     

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。