Hagen's glands of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae): ultrastructure and the detection of entomopoxvirus and parasitism-specific proteins

Hagen's glands of males of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae) secrete compounds that are involved in courtship and defense. Like the poison glands of female wasps, the Hagen's glands are secretory, membranous, and of ectodermal origin. The poison glands contain the symbiotic entomopoxvirus, DlEPV and the parasitism-specific protein, PSP 24. DlEPV proteins were detected in homogenates of male wasps. Our goal was to describe the ultrastructure of the Hagen's glands and determine whether they contain DlEPV virions and/or proteins as well as PSP 24. The Hagen's glands are bilateral and each consists of 12-16 tubules arranged in two clusters. In cross-section, a tubule has three zones that enclose a central cuticle-lined lumen. The outermost zone consists of aggregates ('islands') of small vesicles, interconnected by narrow ductules that lead to large cuticle-lined ducts, which transport electron-dense material to the lumen prior to its release from the gland. Large vesicles in Zone 2 and a thick layer of ribosomes and rough endoplasmic reticula in Zone 3 are the likely sites of storage and protein synthesis, respectively. While DlEPV virions were not seen in the Hagen's gland, DlEPV and PSP 24 proteins were present.     

Morphogenesis and cytopathic effects of the Diachasmimorpha longicaudata entomopoxvirus in host haemocytes

The Diachasmimorpha longicaudata entomopoxvirus (DlEPV), the first reported symbiotic entomopoxvirus, occurs in the venom apparatus of D. longicaudata female wasps and is introduced into Anastrepha suspensa larvae during parasitism. The DlEPV 250-300 kb double stranded DNA genome encodes putative proteins having 30 to >60% amino acid identity with poxvirus homologs such as DNA helicase, DNA-dependent RNA polymerase, and the poxvirus-specific rifampicin resistance protein. Although the molecular characterization of DlEPV is progressing, little is known about its morphogenesis in and effects on host haemocytes. This paper describes (1) haemocytes of third instar A. suspensa, (2) DlEPV infection and morphogenesis, and (3) DlEPV-induced changes in haemocytes. A. suspensa third instars have 3-4 haemocyte morphotypes. Dot blots of DNA from infected haemocytes hybridized with a digoxigenin-labeled DlEPV genomic probe as early as 4 h post-parasitism (hpp) and the intensity of the signal increased with time through 40 hpp. Immunofluorescence microscopy localized DlEPV proteins in cytoplasmic (but not nuclear) sites of infected haemocytes, within 24-36 hpp. Electron microscopy confirmed the presence of viral envelopes, immature spheroids with centric nucleoids, budding virus, and extracellular enveloped virus in three haemocyte types, 24-84 hpp and later. Infected haemocytes exhibited blebbing, DNA concatenation, and inability to encapsulate sephadex beads in vitro. These data indicate that DlEPV disrupts the normal function of host haemocytes, thereby insuring the successful development of D. longicaudata offspring and as such should be regarded as a symbiont of the wasp.     

Genomic analysis reveals an exogenous viral symbiont with dual functionality in parasitoid wasps and their hosts

Insects are known to host a wide variety of beneficial microbes that are fundamental to many aspects of their biology and have substantially shaped their evolution. Notably, parasitoid wasps have repeatedly evolved beneficial associations with viruses that enable developing wasps to survive as parasites that feed from other insects. Ongoing genomic sequencing efforts have revealed that most of these virus-derived entities are fully integrated into the genomes of parasitoid wasp lineages, representing endogenous viral elements (EVEs) that retain the ability to produce virus or virus-like particles within wasp reproductive tissues. All documented parasitoid EVEs have undergone similar genomic rearrangements compared to their viral ancestors characterized by viral genes scattered across wasp genomes and specific viral gene losses. The recurrent presence of viral endogenization and genomic reorganization in beneficial virus systems identified to date suggest that these features are crucial to forming heritable alliances between parasitoid wasps and viruses. Here, our genomic characterization of a mutualistic poxvirus associated with the wasp Diachasmimorpha longicaudata, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), has uncovered the first instance of beneficial virus evolution that does not conform to the genomic architecture shared by parasitoid EVEs with which it displays evolutionary convergence. Rather, DlEPV retains the exogenous viral genome of its poxvirus ancestor and the majority of conserved poxvirus core genes. Additional comparative analyses indicate that DlEPV is related to a fly pathogen and contains a novel gene expansion that may be adaptive to its symbiotic role. Finally, differential expression analysis during virus replication in wasps and fly hosts demonstrates a unique mechanism of functional partitioning that allows DlEPV to persist within and provide benefit to its parasitoid wasp host.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

中华花翅跳小蜂生物学特性和种群动态的研究

中华花翅跳小蜂Microterys sinicus Jiang是白蜡虫成虫产卵期的主要天敌。该蜂成虫喜在自然光下活动,取食和交配都在寄主树上进行。中华花翅跳小蜂每雌平均怀卵量34.39粒,产卵前期2～3天。产卵量、产卵率和产卵天数与温度密切相关,27℃时产卵量和产卵率最高。30℃时产卵天数最少。21～27℃卵孵化率达80%以上。幼虫共5龄。每头跳小蜂幼虫平均取食蜡虫卵数分别为：1龄14.6粒,2龄29.8粒,3龄69.4粒,4龄126.9粒。5龄幼虫不取食。全代发育起点温度和有效积温分别为11.84℃和574.32日·度。补充营养能显著延长成虫寿命和产卵天数,提高雌虫产卵量。中华花翅跳小蜂成虫在田间有两个高峰期,分别发生在4月上中旬和5月中下旬。每粒虫囊内跳小蜂幼虫数与白蜡虫健卵百分率呈负指数关系,拟合方程为：Y=96.0829Exp(-0.1872x)。     

The midgut ultrastructure of the endoparasite Xenos vesparum (Rossi) (Insecta, Strepsiptera) during post-embryonic development and stable carbon isotopic analyses of the nutrient uptake

Females of the endoparasite Xenos vesparum (Strepsiptera, Stylopidae) may survive for months inside the host Polistes dominulus (Hymenoptera, Vespidae). The midgut structure and function in larval instars and neotenic females has been studied by light and electron microscope and by stable carbon isotopic technique. The 1st instar larva utilizes the yolk material contained in the gut lumen, whereas the subsequent larval instars are actively involved in nutrient uptake from the wasp hemolymph and storage in the adipocytes. At the end of the 4th instar, the neotenic female extrudes with its anterior region from the host; the midgut progressively degenerates following an autophagic cell death program. First the midgut epithelial cells accumulate lamellar bodies and then expel their nuclei into the gut lumen; the remnant gut consists of a thin epithelium devoid of nuclei but still provided with intercellular junctions. We fed the parasitized wasps with sugar from different sources (beet or cane), characterized by their distinctive carbon isotope compositions, and measured the bulk (13)C/(12)C ratios of both wasps and parasites. Female parasites developing inside the wasp hemocoel are able to absorb nutrients from the host but, after their extrusion, they stop incorporating nutrients and survive thanks to the adipocytes content.     

HaNPV在宿主内的复制及其对宿主蛋白质代谢的影响

对棉铃虫Helicoverpaarmigera的中肠、血淋巴及脂肪体进行SDS-PAGE蛋白质分析表明：感染早期,HaNPV对棉铃虫的蛋白质合成有刺激作用,晚期则表现出抑制作用。感染96 h和120 h脂肪体有较高水平的多角体蛋白合成。电镜观察表明：①HaNPV感染棉铃虫除病毒在中肠复制后经出芽进入血腔感染脂肪体等组织外,还可能存在病毒粒子直接经中肠进入血腔而感染脂肪体这样的途径;②中肠与血腔之间存在屏障结构;③中肠不仅是一次感染时病毒粒子复制的场所,它完全可能被病毒粒子二次感染。在中肠细胞中没有发现多角体的形成。     

Intrinsic, inter-specific competition between egg, egg–larval, and larval parasitoids of plusiine loopers

Abstract.  1. Intrinsic, inter-specific competition between parasitoid wasp species is a key factor in ecological community dynamics and is particularly important for application in biological control. Here three parasitoid wasp species with overlapping host ranges and differing life history strategies were chosen to examine parasitoid–parasitoid interactions: the egg parasitoid Trichogramma pretiosum, the egg–larval, polyembryonic parasitoid wasp Copidosoma floridanum, and the gregarious larval parasitoid Glyptapanteles pallipes , with the plusiine loopers Acanthoplusia agnata and Trichoplusia ni as hosts.
2.  Copidosoma floridanum has been shown to be an intrinsically superior competitor against larval parasitoids because of their production and increased investment in a soldier larval caste during development, but little is known of their interactions with egg parasitoid species. Trichogramma pretiosum completely dominated intrinsic competition with C. floridanum regardless of oviposition order or sex of the C. floridanum egg.
3. Competition between C. floridanum and G. pallipes , however, depended on the host stage at which parasitism occurred, the sex of the C. floridanum egg, and parasitoid development time. Copidosoma floridanum outcompeted G. pallipes overall, despite the fact that G. pallipes injects a polyDNA virus into the host.
4. The sex of the C. floridanum egg was a significant factor in its ability to shift caste ratios to produce more soldiers in response to G. pallipes competition.
5. Only developing female C. floridanum responded to competition with G. pallipes by increasing the ratio of soldier to reproductive larvae, and this happened only when multiparasitism occurred in the host's 1st and 2nd instar.     

Adaptive value of host discrimination in parasitoids: when host defences are very costly

Host acceptance decision in parasitic wasps strongly depends on the parasitism status of the encountered host. In solitary species, a host only allows the development of a single parasitic larva and then, any oviposition in an already parasitised host leads to larval competition and loss of offspring. Females of many parasitoid species are able to discriminate between parasitised hosts and healthy ones. However, the host discrimination process may require more time than oviposition, exposing the wasp to high risks when the host has efficient defences. Consequently, depending on the degree of success of the host defence, the cost of host inspection for discrimination can outweigh the benefit of superparasitism avoidance. In the present paper, a theoretical approach was developed for determining how host defences may affect optimal host acceptance behaviour in parasitoids. The present model compares the lifetime reproductive success over the strategy used, discrimination and no-discrimination: a discriminating wasp sets a relatively greater value in its current oviposition, while a non-discriminating female sets a greater value in its own survival and future reproduction. The model predicts that depending on physiological state variables and environmental state variables, the optimal policy is not discriminating. Our results suggest that the low discriminating ability observed in some parasitic wasps could probably be an evolutionary response to host defences pressure.     

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

Divvying up an incubator: How parasitic and mutualistic fig wasps use space within their nursery microcosm

Differential occupancy of space can lead to species coexistence. The fig–fig wasp pollination system hosts species-specific pollinating and parasitic wasps that develop within galls in a nursery comprising a closed inflorescence, the syconium. This microcosm affords excellent opportunities for investigating spatial partitioning since it harbours a closed community in which all wasp species are dependent on securing safe sites inside the syconium for their developing offspring while differing in life history, egg deposition strategies and oviposition times relative to nursery development. We determined ontogenetic changes in oviposition sites available to the seven-member fig wasp community of Ficus racemosa comprising pollinators, gallers and parasitoids. We used species distribution models (SDMs) for the first time at a microcosm scale to predict patterns of spatial occurrence of nursery occupants. SDMs gave high true-positive and low false-positive site occupancy rates for most occupants indicating species specificity in oviposition sites. The nursery microcosm itself changed with syconium development and sequential egg-laying by different wasp species. The number of sites occupied by offspring of the different wasp species was negatively related to the risk of syconium abortion by the plant host following oviposition. Since unpollinated syconia are usually aborted, parasitic wasps ovipositing into nurseries at the same time as the pollinator targeted many sites, suggesting response to lower risk of syconium abortion owing to reduced risk of pollination failure compared to those species ovipositing before pollination. Wasp life history and oviposition time relative to nursery development contributed to the co-existence of nursery occupants.     

TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.

Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosyl-L-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of Aea-TMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsin-like enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch.     

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.     

In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle – development from egg through the pupal stage

A variety of semi-defined artificial diets were developed and tested for their ability to support the in vitro development of Edovum puttleri. In the most effective diet, 2.6% of E. puttleri pupated. This diet contained high levels of hen egg yolk combined with Manduca sexta larval hemolymph, or with a mixture of M. sexta egg homogenate and larval hemolymph. Egg homogenate alone (without the addition of hemolymph) was not capable of supporting the parasitoid's development. Thus, hemolymph appears to contain unidentified factor(s) important for inducing pupation of the wasp. Addition of M. sexta pupal fat body tissue extract (in place of hemolymph) also promoted pupation of E. puttleri. Gypsy moth (Lymantria dispar) larval hemolymph could not replace M. sexta larval hemolymph. Fractionation irreversibly reduced the growth-promoting effects of M. sexta larval hemolymph. However, the most effective fraction contained components whose molecular weights were 1000 kd. In diets that were devoid of insect materials, the best results were achieved when hen egg yolk, FreAmine, yeast extract, lactalbumin, trehalose, fetal bovine serum and bovine milk were included. This is the first report of an artificial diet for in vitro rearing an eulophid parasitoid from the egg through the pupal stage.     

Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Trichogrammatidae)

The role of sex-controlling behaviour at oviposition in generating primary sex ratios, and the effect of larval competition on secondary sex ratios, were studied in the gregarious endoparasitoid, Trichogramma chilonis. The production of a fertilized (female) egg is indicated by the incorporation of a pause in abdominal movements during oviposition, while the absence of it indicates the production of an unfertilized (male) egg. During each ovipositional bout, the first male egg is deposited at the second oviposition, and thereafter at intervals of about eight eggs. This simple pattern enables the wasps to adjust their progeny sex ratios under local male competition to a wide range of host size. Inexperienced wasps do not distinguish between parasitized and healthy hosts. Immature mortality is not significantly different between the sexes when a host is attacked by a single wasp, while females suffer higher immature mortality than males when superparasitism occurs.     

Protein expression during parasite redirection of host (Trichoplusia ni) biochemistry

Expression of proteins during normal egg and larval development of Trichoplusia ni was compared with that occurring in hosts stung as eggs by the parasitic wasp Chelonus sp. near curvimaculatus. Those stung hosts which produced a parasite (truly parasitized), precociously expressed proteins associated with larval-pupal metamorphosis, as did those stung hosts which did not contain a developing endoparasite (pseudoparasitized). No highly abundant, low-intermediate molecular weight hemolymph proteins were observed in truly or pseudoparasitized larvae which did not also occur at some point in the development of normal larvae. A low abundance, high molecular mass (160,000 Da) protein was observed in the hemolymph of truly parasitized larvae, but not of normal or pseudoparasitized larvae. The protein is glycosylated and very acidic (pI near 4.5). The data show that any parasitization proteins injected or induced by the ovipositing female parasite are in low abundance, in contrast to situations reported for parasitic wasps which sting hosts as larvae.     

Effect of Temperature on Development and Survival of the Mermithid Filipjevimermis leipsandra

The development and survival of egg, juvenile (parasitic), preovipositing, and ovipositing stages of the parasitic mermithid Filipjevimermis leipsandra were determined at eight temperatures. Infection of the host, Diabrotica balteata, peaked at 30 C and fell off sharply at warmer or cooler temperatures. No development, oviposition, or infection occurred at < 20 C. Fecundity was highest at 25 C, and 50% of the eggs were laid in the first 4-7 days of oviposition.