Impact of exposure length and pupal source on Muscidifurax raptorellus and Nasonia vitripennis (Hymenoptera: Pteromalidae) parasitism in a New York poultry facility

Commercially obtained Nasonia vitripennis Walker and Muscidifurax raptorellus Kogan & Legner were released weekly for 12 wk into a high-rise, caged-layer poultry house. After the release period, parasitoids were sampled using sentinel house fly (Musca domestica L.) pupae that were either laboratory-reared or field-collected as larvae and exposed for 2, 4, 7, and 14 d. Parasitoid-induced mortality was observed in 31% of laboratory colony pupae and in 26% of field-collected pupae, whereas successful parasitism rates of 48 and 51% were observed from these pupal sources, respectively. Parasitism was primarily by M. raptorellus (88%), and Muscidifurax raptor Girault & Sanders (11%), while N. vitripennis accounted for <1%. Percent female progeny ranged from 43%, in M. raptorellus to 76% in N. vitripennis. Parasitoid emergence from 2-d exposed sentinel pupae was the lowest of all treatments. Parasitoid emergence from 7-d exposed sentinel pupae was the highest of all treatments. We found no differences between pupal source, suggesting that when sampling for M. raptor, M. raptorellus, and N. vitripennis, in poultry facilities, pupal source is not a confounding factor.     

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.     

Competition between the filth fly parasitoids Muscidifurax raptor and M. raptorellus (Hymenoptera: Pteromalidae)

Competition bioassays were conducted with the filth fly pupal parasitoids Muscidurax raptor (Girault & Sanders) and M. raptorellus (Kogan & Legner) (Hymenoptera: Pteromalidae) using house fly Musca domestica L. (Diptera: Muscidae) hosts at different host densities. Muscidifurax raptor had a significant impact on M. raptorellus when hosts were limiting in sequential parasitism tests. Fewer than six M. raptorellus adult progeny emerged from groups of 50 fly pupae that were parasitized by M. raptor at the same time or when M. raptor parasitism preceded M. raptorellus by 48 h, respectively, compared with 42–55 M. raptorellus progeny produced when this species was tested alone. Production of M. raptor was significantly lower when parasitism by this species was preceded by M. raptorellus (25) than when M. raptor was tested alone (43). When the two species parasitized hosts at the same time in different proportions at low host:parasitoid densities (5:1), M. raptorellus produced 13 progeny per parent female when it was the sole species present and fewer than two when M. raptor was present. No negative impact of M. raptorellus on M. raptor was observed. Neither species had a substantial effect on the success of the other at higher host:parasitoid densities.     

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.     

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.     

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.     

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.)     

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.

     

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.     

Diploidy restoration in Wolbachia-infected Muscidifurax uniraptor (Hymenoptera: Pteromalidae)

Thelytokous reproduction, where females produce diploid female offspring without fertilization, can be found in many insects. In some Hymenoptera species, thelytoky is induced by Wolbachia, a group of cytoplasmically inherited bacteria. We compare and contrast early embryonic development in the thelytokous parthenogenetic species Muscidifurax uniraptor with the development of unfertilized eggs of the closely related arrhenotokous species, Muscidifurax raptorellus. In the Wolbachia-infected parasitic wasp M. uniraptor, meiosis and the first mitotic division occur normally. Diploidy restoration is achieved following the completion of the first mitosis. This pattern differs in the timing of diploidy restoration from previously described cases of Wolbachia-associated thelytoky. Results presented here suggest that different cytogenetic mechanisms of diploidy restoration may occur in different species with Wolbachia-induced thelytoky.     

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.     

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.).     

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。     

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.     

Bacterial infections associated with the son-killer trait in the parasitoid wasp Nasonia (= Mormoniella) vitripennis (Hymenoptera: Pteromalidae)

The son-killer (sk) trait in the parasitoid wasp, Nasonia vitripennis, causes the production of very female-biased sex ratios through the mortality of males. Some 80% of all male eggs fail to hatch. The trait is both maternally and contagiously transmitted. Here evidence is presented that the trait is associated with systemic and chronic bacterial infections in adult wasps. Infections develop trans-stadially, originating in the midgut of larvae and subsequently spreading to other tissues, including the brain, fat body, muscles, eyes, and hemocytes. Female reproductive tracts often show infections but infections are absent in male reproductive organs; other sex differences occur as well. The bacteria involved are pleomorphic, with straight rods being the most common form.     

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.).     

Development of microsporidia-infected Muscidifurax raptor (Hymenoptera: Pteromalidae) at different temperatures

Muscididfurax raptor, a pupal parasitoid of house flies and other filth flies, is commonly infected with the microsporidium Nosema muscidifuracis. To determine the effects of infection on developmental time, uninfected and infected adult M. raptor were allowed to parasitize pupae of the house fly (Musca domestica) for 24 h. Exposed pupae of the two groups (infected and uninfected) were held at 15, 20, 25, 30, 32, and 34 °C with 75–80% relative humidity. Development of infected M. raptor was significantly longer at all temperatures than that of uninfected parasitoids, resulting in approximately 7% extensions of developmental times. Uninfected females completed development in 14.6, 19.6, and 30.4 days at 30, 25, and 20 °C, respectively, compared with 15.8, 20.7, and 32.3 days for infected females at these temperatures. The differences in developmental times provided narrow windows for isolating large proportions of uninfected M. raptor females for disease management programs. This window was greatest at 20 °C; 61% of the uninfected females emerged by day 30, at which time only 10% of the infected females had emerged.     

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.     

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。