Morphological,ultrastructural, histochemical and electrophoretic studies on the venom system of Nasonia vitripennis walker (hymenoptera: Pteromalidae)

The venom system of Nasonia vitripennis is well-developed and composed of an unbranched acid gland and associated reservoir. Fine-structural, histochemical and electrophoretic studies indicate that the venom is produced by two protein-secreting epithelia. The bulk of the venom is synthesised in the columnar cells of the acid gland and discharged via “vesicular organelles” and the efferent ductular system into the lumen of the reservoir. The acid gland also contains squamous chitogenous cells, situated either around the central lumen or interposed between the bases of the columnar cells. Once within the reservoir, the venom is probably activated by enzymatic secretions from the reservoir secretory cells. Each of these cells has a “vesicular organelle” but, in contrast to the columnar cells of the acid gland, the cytoplasm contains a preponderance of free ribosomes, and protein segregation apparently occurs outside the Golgi complexes. The venom is expelled through the efferent discharge duct by muscular contractions, which open the duct lumen and bring it into contact with the funnel of the ovipositor. Excessive distortion of the duct is prevented by a massive ventral ligament.     

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.     

Formation of protein yolk in the eggs of a parasitoid hymenopteran,Nasonia vitripennis (Walker) (Pteromalidae: Hym)

Summary Electron micrograph profiles of developing yolk inclusions in the oöcytes and comparative electrophoresis of the haemolymph of males and females and of mature oöcytes, indicates that a fraction is present in the haemolymph of the females, which does not occur in the haemolymph of the males. Changes occur to the protein fractions from the haemolymph which are passed into the egg. This suggests that the egg synthesises some of its own yolk and does not have a synthetically passive role during vitellogenesis.We would like to thank Professor E. W. Knight-Jones in whose Department the work was done.     

Pteromalidae etEulophidae nouveaux d'Europe (Hym., Chalcidoidea)

Summary In the present paper the Author has described three new genera ofPteromalidae, two new species of the genusEnaysma Del. and one of the genusEpilampsis Del. (Euloph. Entedontinae). The first Pteromalid genus,Stenoselma, is closely related to the well knownAnisopteromalus Ruschka; the second new genus,Apelioma, has been proposed forDinotiscus (Dinotus) pteromalinus Thomson; the third,Aggelma, has been described for the new speciesabdominalis, collected in Central Europe onPinus montana. The other new species ofEntedontinae has been bred from leaf-miners (Lepidoptera).      

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.     

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.)     

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.     

Marking Behavior and Discrimination of Concealed Hosts by the Ectoparasitoid, Dinarmus basalis Rond. (Hym. Pteromalidae)

The parasitoid species, Dinarmus basalis, attacks bruchid beetle larvae concealed within hard seeds. Female wasps are able to discriminate the quality of their host but the position in the concealed host system where this discrimination takes place remains unclear. Under laboratory conditions, we investigated whether the cues were detectable: (1) at a medium or short distance from the seed, (2) on the seed, or (3) within the seed, at the point of contact with the host. In particular, we investigated whether internal or external markings are applied by the parasitoid and then recognized in a subsequent encounter or by a conspecific encountering the host. Detailed behavioral observations did not provide evidence of markings being applied onto the seed or used, but showed that host discrimination occurred on the basis of internal cues. The lack of evidence of external markings is unexpected and raises the question as to which circumstances in the parasitoid's ecology and life history could result in such a lack not causing a reduction of fitness.     

Laboratory studies on the effects of fungicides, acaricides and insecticides on the entomopathogenic fungus, Verticillium lecanii

Tests on agar suggested that several fungicides, insecticides and acaricides could be recommended for use with the fungus, Verticillium lecanii (Zimm) Viégas; some chemicals, benodanil, oxycarboxin, iprodione, vinclozolin, dinocap, carbaryl, dicofol, dienochlor, pirimicarb, permethrin and white oil are in theory sufficiently innocuous to V. lecanii for spraying simultaneously with is spores while separate aphlications would be recommended for diflubenzuron which is slightly more toxic. Results of tests on aphids with some of the compounds of greater but intermediate toxicity on agar, suggested that separate applications are advisable with bupirimate, pyrazaphos, triforine, cyhexatin, dioxathion and tetradifon but any use of zineb is best avoided. The most toxic compounds on agar in this study were thiram, captan, maneb, mancozeb, chlorothalonil, dichlofluanid, fenarimol, imazalil, BTS 40542, quinomethionate, fluotrimazole and diazinon. Of these compounds tested with V. lecanii on aphids, captan, chlorothalonil, dichlofluanid, quinomethionate and thiram proved to be incompatible with V. lecanii but fenarimol proved relatively harmless. On the basis of the results of this study, careful selection of pesticides and fungicides would permit the combined use of V. lecanii and chemicals in integrated control programmes.

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Résumé Des tests sur agar ont suggéré que plusieurs fongicides, insecticides et acaracides pouvaient être recommandés pour une utilisation simultanée avec le champignon Verticillium lecanii (Zimm) Viégas; quelques substances chimiques — benodanil, oxycarboxine, iprodione, vinelozoline, dinocap, carbaryl, dicofol, dienochlor, pirimicarb, permethrine et huile blanche — sont en théorie suffisamment innoffensives contre V. lecanii pour être pulvérisées simultanément avec ses spores, tandis que des applications séparées devraient être préconisées pour le diflubenzuron qui est légèrement plus toxique. Les résultats d'essais sur agar, contre des aphides, avec quelques produits de toxicité plus élevée mais cependant moyenne, ont conduit à suggérer des applications séparées pour: bupirimate, pyrazaphos, triforine, cyhexatine, dioxathion et tetradifon, mais d'éviter toute utilisation de zineb. Les produits les plus toxiques sur agar ont été: thiram, captan, maneb, mancozeb, chlorothalonil, dichlofluanide, fenarimol, imazalil, BTS 40542, quinomethionate, fluotrimazole et diazinon. Après essais contre aphides, avec V. lecanii, captan, chlorothalonil, dichlofluanide, quinomethionate et thiram ont montré leur incompatibilité avec V. lecanii, tandis que fenarimol se montrait relativement sans nocivité. Sur la base des résultats de cette étude, une sélection soigneuse des pesticides et des fongicides devrait permettre l'utilisation combinée de V. lecanii et de produits chimiques dans des programmes de lutte intégrée.

     

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.     

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.

     

Host-size-dependent feeding behaviour and progeny sex ratio of Anisopteromalus calandrae (Hym., Pteromalidae)

Host-size related feeding and oviposition behaviour, and allocation of progeny sex by Anisopteromalus calandrae (Howard) were tested on Sitophilus oryzae L. The parasitoid showed a host-size-dependent partition of feeding and oviposition behaviour, preferring small hosts for feeding, but large hosts for oviposition. Neither the mutual interference nor the host density showed any effect on the behaviour of the parasitoid. Allocation of progeny sex by the female parasitoid appeared to be based more likely on absolute than on relative host size encountered. A model for the progeny sex ratio was constructed based on: (1) ovipositional preference of the parasitoid on large hosts; (2) feeding preferentially on small hosts; and (3) host-size-related regulation of progeny sex ratio. The progeny sex ratio of the parasitoid predicted by the model was in close agreement with the observed value.     

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.     

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.).     

Toxicity of some insecticides and acaricides to the predatory bugDicyphus tamaninii (Het.: Miridae)

Several pesticides were tested in the laboratory for their side-effects upon the mirid bugDicyphus tamaninii, a polyphagous predator used for IPM programmes in some vegetable crops. Residual toxicity to 3rd -4th instar nymphs on tomato leaflets was checked 24, 48 hours and seven days after treatment. The acaricides bromopropylate, dicofol+tetradifon and fenpyroximate were harmless to the nymphs. The insect growth regulators azadirachtin, buprofezin, lufenuron and pyriproxyfen were also harmless to nymphs and teflubenzuron was slightly harmful seven days after treatment. Among the conventional insecticides tested, only pirimicarb and tau-fluvalinate were harmless toD. tamaninii nymphs     

Side effects of insecticides on aphidius rhopalosiphi (Hym.: Aphidiidae) In laboratory

The toxicity of bifenthrin, cyfluthrin, λ-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, fluvalinate, phosalon and pirimicarb have been assessed in laboratory on the parasitic hymenoptera Aphidius rhopalosiphi. A strong toxic effect was found with every tested product when adult parasitoids were exposed to freshly applied residues on both glass plates and maize leaves for 24 hours. Only two products, fluvalinate and esfenvalerate, did not kill all the insects. No differences were observed between mortalities on glass plates and leaves. Applied on aphids mummies, cyfluthrin and deltamethrin slightly reduced the emergence of young parasitoids, but not their reproductive performance. The other tested products had no effects on adult emergence. On basis of these results, the insecticides are of comparable toxicity to A. rhopalosiphi in laboratory.     

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.