Transmission of the Diachasmimorpha longicaudata rhabdovirus (DlRhV) to wasp offspring: an ultrastructural analysis

During oviposition, the parasitic wasp Diachasmimorpha longicaudata introduces an entomopoxvirus (DlEPV) and a rhabdovirus (DlRhV) into larvae of its tephritid fruit fly host Anastrepha suspensa. DlEPV and DlRhV replicate, respectively, in host hemocytes and epidermal cells. Both viruses, like many beneficial viruses of parasitic wasps, are retained in all wasp generations but their avenue(s) of transmission are unknown. This study tests the hypothesis that DlRhV is transmitted transovarially or through larval feeding on infected host hemolymph. Transmission electron microscopy (TEM) revealed no virions in pre-vitellogenic or vitellogenic ova, or in the lateral oviduct of D. longicaudata females. However, numerous virions occurred in subchorionic regions of 33-36-h-old oviposited eggs. This suggests that DlRhV is introduced into the egg either as (a) intact virions after chorionogenesis but prior to oviposition and/or as (b) unencapsidated RNA molecules, undetectable by TEM in pre-vitellogenic ova, that subsequently replicate and assemble into mature virions. DlRhV particles also occurred in the midgut lumen of 20-24-h-old wasp first instars, suggesting that they were ingested. These virions may have been released from the egg into the hemolymph during hatching or may have come from virions introduced by the female wasp directly into the host, separate from the egg. DlRhV particles were also evident in the intracellular vesicles and intercellular spaces of the larval midgut. Taken together, these data support the hypothesis that DlRhV is transovarially transmitted as virions and/or as unencapsidated RNA. Further studies are needed to determine whether the DlRhV that ultimately resides within the female wasp's accessory gland filaments is the progeny of the virus from the egg and/or larval midgut cells.     

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.     

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.     

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

The enigma of the haemogram "left-shift" in periwinkles infected with trematodes

To make further progress in the understanding of digenean immune evasive tactics in their snail host, we compared the haemopoietic parameters and haemocyte functional potencies in the prosobranch Littorina littorea, which were either healthy or infected with echinostome trematode Himasthla elongata. The haemocyte concentration in the circulation of infected individuals was significantly higher than in uninfected ones, 3300 microl(-1) and 1882 microl(-1), respectively. Intense haemopoiesis in haemolymph of infected snails was evidenced by 4-fold higher BrdU incorporation into nuclei of haemocytes as well as elevated level of cyclin D expression in these cells. Evident skewing of the haemocyte population toward a higher frequency of immature, undifferentiated haemocytes in infected L. littorea was found. Haemocytes in infected snails had a much lower functional potency in production of reactive oxygen intermediates (ROI) and cell-mediated cytotoxicity. Correlation analysis shows that both cytotoxic and ROI generation values were significantly and negatively correlated with proportion of juvenile cells in circulation. Experimental injection of H. elongata excretory/secretory products modulated haemopoiesis toward increasing a juvenile cells proportion by 7th day post-injection. This can be considered a haemogram "left-shift" by analogy to that seen in the human neutrophil compartment, when more immature bandforms are found in blood during acute inflammation or bone marrow disorders. We hypothesize that echinostomatide trematodes may interfere in the normal neuroendocrine management of haemopoiesis in the host and cause a haemopoietic signal to initiate multiplication to near neoplastic levels.     

Toxicity of imidacloprid-treated spheres to Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae) and its parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in the laboratory

No-choice cage tests were used to study the toxicity of imidacloprid-treated spheres to Caribbean fruit fly, Anastrepha suspensa (Loew), and its associated parasitoid, Diachasmimorpha longicaudata (Ashmead), in the laboratory. Three imidacloprid sphere treatments (2, 4, and 8% active ingredient [AI] Provado 1.6 F) and an untreated control sphere (no toxicant) were evaluated against A. suspensa. Throughout the observation period (2-72 h), all concentrations of imidacloprid-treated spheres killed significantly more A. suspensa compared with control spheres. After 4 h of exposure to imidacloprid-treated spheres, significantly more A. suspensa were killed on spheres treated with 8% compared with 2% (AI). At 48 and 72 h, there were no significant differences in the mean number of A. suspensa killed at 2, 4, and 8% (AI), potentially indicating that a period of 24 h was sufficient for flies to ingest a lethal dose of the pesticide. Overall, significantly more A. suspensa males were killed after 72 h of exposure to imidacloprid-treated spheres compared with females. For D. longicaudata, only two imidacloprid sphere treatments, 2 and 4% (AI), and an untreated sphere (control) were evaluated for mortality in cage tests. There were no significant differences in mortality of D. longicaudata between the 2 and 4% (AI) imidacloprid-treated spheres. Both rates killed significantly more D. longicaudata compared with the control. However, after 24, 48, and 72 h of exposure to imidacloprid-treated spheres, significantly more D. longicaudata were killed in cages containing 4% compared with 2% (AI) and untreated control spheres. The study demonstrates the potential use of imidacloprid-treated spheres for control of A. suspensa in areas where it may be difficult to apply broad-spectrum insecticides.     

Spindle bodies of Heliothis armigera entomopoxvirus develop in structures associated with host cell endoplasmic reticulum

Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism.     

Parasitism of Lacanobia oleracea (lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, disrupts the cytoskeleton of host haemocytes and suppresses encapsulation in vivo

Parasitism of Lacanobia oleracea larvae by the ectoparasitic wasp Eulophus pennicornis suppressed host haemocyte-mediated encapsulation of Sephadex DEAE A-25 beads in vivo. Beads dissected out of parasitized larvae had fewer haemocytes associated with them. Moreover, those haemocytes that were associated with the beads tended to retain a rounded configuration and rarely flattened. Similar results were obtained using in vitro encapsulation assays. SDS PAGE indicated that for parasitized and PBS injected larvae, there were some differences in the plasma proteins that bound to Sephadex DEAE A-25 beads, suggesting that parasitism-mediated changes to host plasma proteins might contribute to the differences in the encapsulation response occurring in these larvae. However, in vitro encapsulation assays using beads that had been pre-incubated in plasma from parasitized and unparasitized larvae, demonstrated that major differences in the extent of encapsulation did not occur. These results, plus in vitro haemocyte attachment and spreading assays, suggest that parasitism-mediated suppression of encapsulation is primarily due to reductions in the ability of host haemocytes to attach to (i.e., recognize) and flatten over non-self surfaces and other haemocytes. This proposal is corroborated by staining of actin in the haemocyte cytoskeleton by FITC-labelled phalloidin, which indicated that parasitism disrupts the formation of stress fibers and focal adhesions in plasmatocytes. By contrast, experimental injection of adult female wasp venom into unparasitized L. oleracea larvae had no significant effect on in vivo encapsulation responses or the haemocyte cytoskeleton. Arch. Insect Biochem. Physiol. 49:108-124, 2002. Published 2002 Wiley-Liss, Inc.     

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia

Background

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.

Methodology/Principal Findings

In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10³ Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson''s test = 12 822.98, df = 2, p<0.001).

Conclusions/Significance

The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.     

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia)

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.     

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the Defence Activity of Radix lagotis (Lymnaeidae) Haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.     

Parasitization of Lacanobia oleracea (Lepidoptera: Noctuidae) by the ectoparasitc wasp,Eulophus pennicornis. Effects of parasitization,venom and starvation on host haemocytes

In contrast to the situation with endoparasitic wasps, little is known about the effects of ectoparasitoids and their secretions on the haemocytes of their insect hosts. To address this deficit, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and it's host, the tomato moth, Lacanobia oleracea. Using light microscopy, it was determined that L. oleracea has five main haemocyte types, namely, plasmatocytes, granular cells, spherule cells, oenocytoids and pro-haemocytes, representing 56%, 30%, 10%, 2% and 2% of the population, respectively. Parasitization by E. pennicornis, resulted in an increase in the number of circulating haemocytes up to day three, followed by a decrease towards day eight; the latter being associated with changes to the morphology and viability of the cells. For example, on day five after parasitization, plasmatocytes and granular cells had become more rounded and put out pseudopods less readily compared with those from non-parasitized controls, whilst from day seven onwards there was a significant decrease in haemocyte viability and by day nine, extensive haemocyte damage and disintegration was evident. These changes were not observed when larvae were injected with E. pennicornis venom, or when haemocytes were exposed directly to venom in vitro, neither did they occur in starved larvae. Thus, although the observed effects on L. oleracea haemocytes are definitely associated with parasitization they are not due to wasp venom components, nor are they a non-specific effect resulting from nutritional deprivation. The possibility that the feeding wasp larvae produce factors which perturb host haemocytes in order to help condition the host to ensure that successful parasitization occurs, is discussed.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)

Abstract Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte‐spreading behaviour of P. xylostella is analysed by measuring F‐actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte‐spreading peptide, in a dose‐dependent manner for each calyx component and fail to exhibit haemocyte‐spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte‐spreading could be explained by measuring F‐actin contents, in which parasitization by C. plutellae inhibits F‐actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F‐actin development in the nonparasitized haemocytes. In addition, co‐incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte‐spreading and F‐actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.