Intrahemocoelic Injection Research Articles

Immune modulation by dexketoprofen trometamol, a selective eicosanoid biosynthesis inhibitor of cellular immune response and phenoloxidase reaction in response to viral infection in Pimpla turionellae adults

Nodulation is the first immune defence mechanism related to melanisation in response to microbial infections in insects. Adult parasitoid insects have been hypothesised to produce nodules with melanisation in response to viral infections and, eicosanoids, to mediate nodulation reactions and phenoloxidase (PO) activation in this type of infections. To test this hypothesis, endoparasitoid Pimpla turionellae adults were first inoculated with a novel generation nonsteroidal anti-inflammatory drug (NSAID) dexketoprofen trometamol (DT) (5 μg/adult), which is a selective cyclooxygenase-1 (COX-1) inhibitor. These adults were then immediately injected with intrahaemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1) as a model insect-virus interaction. Additionally, adults were fed on artificial diet with increasing concentrations of DT (0.001, 0.01, or 0.1 g/100 ml diet) per os prior to intrahaemocoelic injection of BHSV-1 (2 × 103 PFU/adult) and nodulation and PO activity were recorded at 2 h post inoculation (PI). BHSV-1-treated newly emerged adults fed with inhibitors showed low levels of nodulation and increased PO enzyme activity. DT-treated Pimpla adults produced significantly fewer nodules (approximately nine nodules/adult), whereas viral infection provoked nodules (approximately 33 nodules/adult) in comparison with needle (vehicle)-treated controls (approximately five nodules/adult). Increasing dietary dexketoprofen trometamol concentrations decreased nodulation (by 12-fold at the highest concentration) and increased PO reactions (by approximately 3-fold at the highest concentration) to BHSV-1 injection. Compared with control adults, adults orally fed on the lowest DT concentration (0.001 %) significantly increased PO activity (1.22 ± 0.23–2.74 ± 0.31 unit/min/mg protein) while nodules significantly decreased (43.19 ± 4.26–17.84 ± 2.19) in response to virus infections. These findings suggest that eicosanoid biosynthesis, at least in the context of prostaglandins (PGs) formed by COX-1, mediates nodulation reactions and PO activation in response to viral infection in adults of this endoparasitoid. This is the first demonstration that the immune response of P. turionellae adults to viral pathogens is modulated by DT, which initiates haemolymph PO activation.

Field isolates of Beauveria bassiana exhibit biological heterogeneity in multitrophic interactions of agricultural importance

Beauveria bassiana (Bb) is a widespread entomopathogenic fungus widely used in agriculture for crop protection. Other than pest control, fungi belonging to the B. bassiana complex represent an important microbial resource in agroecosystems, considering their multiple interactions with other microorganisms as antagonists of phytopathogens, or with plants as endophytic colonizers and growth promoters. Here, we characterised field collected or commercial isolates of B. bassiana relative to the environmental factors that affect their growth. We further compared the metabolome, the entomopathogenic potential and biocontrol activity of the tested isolates respectively on the insect pest Spodoptera littoralis or against the fungal plant pathogen Fusarium oxysporum. Our analysis revealed that the B. bassiana complex is characterised by a high level of inter-isolate heterogeneity in terms of nutritional requirements, establishment of intra- or inter-kingdom interactions, and the nature of metabolites produced. Interestingly, certain B. bassiana isolates demonstrated a preference for low nutrient plant-derived media, which hints at their adaptation towards an endophytic lifestyle over a saprophytic one. In addition, there was a noticeable variation among different B. bassiana isolates in their capacity to kill S. littoralis larvae in a contact infection test, but not in an intrahaemocoelic injection experiment, suggesting a unique level of adaptability specific to the host. On the other hand, most B. bassiana isolates exhibited similar biocontrol efficacy against the soil-dwelling ascomycete F. oxysporum f. sp. lycopersici, a pathogen responsible for vascular wilt disease in tomato plants, effectively averting wilting. Overall, we show that the effectiveness of B. bassiana isolates can greatly vary, emphasising the importance of isolate selection and nutritional adaptability consideration for their use in sustainable agriculture.

The Study of Cell-Penetrating Peptides to Deliver dsRNA and siRNA by Feeding in the Desert Locust, Schistocerca gregaria.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

The diarrhetic shellfish-poisoning toxin, okadaic acid, provokes gastropathy, dysbiosis and susceptibility to bacterial infection in a non-rodent bioassay, Galleria mellonella

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays.

Hemocyte profile, phagocytosis, and antibacterial activity in response to immune challenge of the date fruit stalk borer, Oryctes elegans

The date fruit stalk borer, Oryctes elegans Prell, is a destructive pest on date palms in Saudi Arabia. We evaluated the immune response of the third instars (last instars), by the intrahemocoelic injection of lipopolysaccharide (LPS) (a pathogen-associated molecular peptide, PAMP), which significantly increased the total hemocyte count. Phase-contrast light microscopy revealed the presence of five hemocyte types: prohemocytes, granulocytes, plasmatocytes, oenocytoids, and spherulocytes. Transmission electron microscopy demonstrated that, among these hemocytes, only the granulocytes and plasmatocytes phagocytosed latex beads. Injection with LPS also significantly decreased the number of oenocytoids. The antibacterial activity of plasma proteins of larvae injected with LPS, measured using the agar well diffusion method, against Gram-positive and Gram-negative bacteria varied based on the bacterial strain, the total concentration of plasma protein, and time post-injection with LPS. The results of the current study may be useful in the biological control of O. elegans. Furthermore, new compounds with antibacterial activity that might be useful for the development of innovative drugs of natural origin can be identified in O. elegans.

Indomethacin-induced gut damage in a surrogate insect model, Galleria mellonella

Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased ‘leakiness’ in rat models, and is used routinely as a toxicology assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (0.5–7.5 µg/larva; 2–30 mg/kg) to G. mellonella via intrahaemocoelic injection and gavage (force-feeding) and monitored survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4- to 24 h by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accompanied by significant increases in detoxification-associated activities (superoxide dismutase and glutathione-S-transferase). Herein, we present the first evidence that G. mellonella larvae force-fed indomethacin display broad symptoms of gastric damage similar to their rodent counterparts.

Induction of Antimicrobial Peptides in the Hemolymph of Spodoptera littoralis Larvae Following Treatment with Salmonella typhimurium

This study investigated the efficiency of intrahaemocoelic injection of sublethal concentration (LC20 = 5.7×103 cells/ml) of Salmonella typhimurium in the induction of immune response in 5th larval instar of Spodoptera littoralis. The role of antimicrobial peptides (AMPs) induced in the larval hemolymph was examined as natural antibiotics against different Gram-negative (G-ve) or Gram-positive (G+ve) bacteria. Where the larval plasma injected with Salmonella showed a significant antimicrobial activity against different strains of pathogenic bacteria (Staphylococcus aureus, Salmonella typhimurium, Escherichia coli and Enterococcus faecalis). The biochemical characterization of the immune hemolymph indicated drastic changes in both the total protein content and the protein banding patterns following bacterial injection. The total hemolymph proteins (THPs)decreased significantly 48 h post-Salmonella-injection. The hemolymph proteins profile was also qualitatively changed through the induction of new proteins and the disappearance of others simultaneously, which may be attributed to their participation in the immune reactions. The appearance of six new synthesized protein bands that were found to be specific for Salmonella injection, with a characteristic band of MW ~22 KDa predicting that this protein band may be Attacin-like AMP.

IrC2/Bf – A yeast and Borrelia responsive component of the complement system from the hard tick Ixodes ricinus

Ticks possess components of a primordial complement system that presumably play a role in the interaction of the tick immune system with tick-borne pathogens and affect their transmission. Here we characterized a novel complement component, tagged as IrC2/Bf, from the hard tick Ixodes ricinus, the principal vector of Lyme disease in Europe. IrC2/Bf is a multi-domain molecule composed of 5–7 CCP modules, varied by alternative splicing, followed by a von Willebrand factor A domain and a C-terminal trypsin-like domain. The primary structure and molecular architecture of IrC2/Bf displays the closest homology to the C3-complement component convertases described in horseshoe crabs. The irc2/bf gene is mainly expressed in the tick fat body associated with the trachea and, as determined by western blotting, the protein is present in low amounts in tick hemolymph. Expression of irc2/bf mRNA was significantly up-regulated in response to the intra-hemocoelic injection of the yeast Candida albicans and all tested Borrelia sp. strains (B. burgdorferi NE5264, B. burgdorferi CB26, B. garinii MSLB, B. afzelii CB43), but was not affected by injection of model Gram-negative and Gram-positive bacteria or the aseptic injection control. In-line with these results, RNAi-mediated silencing of irc2/bf inhibited phagocytosis of B. afzelii and C. albicans but not the other bacteria. Tissue expression profiles, specific responses to microbial challenges, and patterns of phagocytic phenotypes upon RNAi silencing observed for IrC2/Bf match well with the previously reported characteristics of I. ricinus C3-related molecule 1 (IrC3-1). Therefore we presume that IrC2/Bf functions as a convertase in the same complement activation pathway protecting ticks against yeast and Borrelia infection.

Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions.

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA1B1 and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of Galleria mellonella larvae was investigated. We found that the protein had haemocoel insecticidal activity against G. mellonella with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA1B1 into G. mellonella resulted in significant decreases in haemocyte number and phagocytic ability. In in vitro experiments, PirA1B1 inhibited the spreading behaviour of the haemocytes of G. mellonella larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA1B1 toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA1B1 toxin disarmed the insect cellular immune system.

The Host Specificities of Baculovirus per os Infectivity Factors.

Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host’s midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses.

The Autographa californica multiple nucleopolyhedrovirus ac110 gene encodes a new per os infectivity factor

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac110 gene is especially conserved in lepidopteran-specific baculoviruses and is uncharacterized. To investigate the role of ac110 in the baculovirus life cycle, an ac110-knockout (vAc110KO) and a repair (vAc110:HA) viruses were constructed in this study. Budded virion production and occlusion body formation were unaffected in vAc110KO-transfected or infected Sf9 cells. Intrahemocoelic injection of budded virions of vAc110KO killed Trichoplusia ni larvae as efficiently as the repair or the wild-type viruses. However, per os inoculation of occlusion bodies of vAc110KO failed to establish infection in T. ni larvae, while the repair virus was as efficient as the wild-type virus. Treatment with calcofluor white, a reagent that damages the peritrophic membrane, did not rescue the oral infectivity of vAc110KO. These results suggested that Ac110 is a new per os infectivity factor that may play a role after occlusion-derived virions pass through the peritrophic membrane during oral infection.

Effect of Staphylococcus aureus Infection on Biochemical and Antioxidant Activities of Fifth Instar Silkworm Larvae (Bombyx mori L.)

In the present study silkworm used as a model animal for evaluating the pathogenicity of Staphylococcus aureus. The fifth instar silkworm larvae was used and infected by intrahaemocoelic injection of bacterial sample. The haemolymph was collected from the infected and control group larvae at 6, 12, 18 and 24 hours of post infection and stored at -4°C in eppendorf tubes to use. Lipid peroxidation, phenol oxidase and acid phosphatase activity were estimated in the haemolymph of control and infected group. This indicated that there was a gradual increase in lipid peroxidation, phenol oxidase and acid phosphatase activities in infected group when compared with control group. The antioxidant enzyme activities were estimated in the haemolymph of control and infected group. We found that the antioxidant enzyme activities were decreased after 24 hours of infection with S. aureus. The silk glands were removed and the wet weight was measured, the wet weight of the silk glands was decreased on the 24 hours of infection when compared with control group.

A Critical Assessment of Bombyx mori Haemolymph Extract on Staphylococcus aureus an In vitro and In silico Approach

The newly reemerging drug-resistant Gram-positive pathogens require the discovery of new drug targets and the development of new therapeutics. We focus on antimicrobial proteins which inhibits growth of Staphylococcus aureus. In the present study fifth instar Bombyx mori larvae was used and infected with S. aureus by intrahaemocoelic injection of bacterial sample. The haemolymph was collected from the healthy and infected larvae after 24 h of infection and stored at -4°C in eppendorf tubes until use. Haemolymph extract was prepared and antimicrobial activity was done by the classical well diffusion method. Haemolymph extract prepared form infected larvae exhibit maximum zone of inhibition on S. aureus when compared with healthy haemolymph extract. Bioinformatics tools were used to find out the molecular interaction and binding mode of antimicrobial peptides from B. mori such as Moricin and Cecropin on three different drug target proteins Glycerol phosphate lipoteichoic acid synthase (PDB: 2W5Q), ABC transporter (PDB: 1P99) and DNA Gyrase (PDB: 2XCO) from S. aureus. Hex Docking software was used for the docking studies. The interaction mode and binding results can be represented in terms of docking energy. The best binding energies were obtained from the docking of DNA gyrase with Moricin and Cecropin (-702.13 Kcal/mol and -639.39 Kcal/mol) respectively.

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection.

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.

Characterization of Melanoplus sanguinipes oviposition stimulating protein expression and re-examination of its potential role in stimulating oviposition

Melanoplus sanguinipes oviposition stimulating protein (MsOSP) was characterized and its role in stimulating oviposition in virgin females was examined. A 967nt MsOSP mRNA sequence with homology to previously characterized N-terminal amino acid sequence data for MsOSP was identified in a RNAseq library generated from an mRNA pool from the long hyaline tubule (LHT) of the male accessory gland complex. This transcript contained a predicted 729nt open reading frame encoding the 242aa putative MsOSP protein and had the second highest read abundance in the library. The MsOSP transcript was detected exclusively in the LHT tissue of adult males and its abundance increased with time until 7days post-eclosion. Western blot analysis using an anti-MsOSP antibody showed high levels of MsOSP protein in the LHT luminal secretions of virgin males and to a lesser degree was associated with the aedeagus and ejaculatory duct. MsOSP was shown to be a major protein component of the spermatophore packet transferred from the male to female during copulation. However, only minor amounts of MsOSP could be detected in the female bursa, spermatheca and oviduct. Intrahemocoelic injection of LHT luminal protein into mature virgin females stimulated oviposition in ∼65% of females. A similar but non-significant trend was observed upon injection of purified recombinant MsOSP protein, and immunoprecipitation of LHT protein with anti-MsOSP antibody led to abrogation of oviposition stimulation upon injection of mature virgin females. Despite the demonstration of stimulation of oviposition upon intrahemocoelic injection of LHT-derived-MsOSP into mature virgin females, the potential mode of action of MsOSP in this process remains to be determined. MsOSP cannot be detected in the tissues other than the bursa, spermatheca and oviduct of female grasshoppers and relatively large quantities of MsOSP are required to stimulate oviposition upon injection.

Intrahaemocoelic infection of Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus does not induce tracheal cell basal lamina remodelling

Infection of the lepidopteran insect Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by the oral route stimulates activation of host matrix metalloproteases (MMP) and effector caspases, a process dependent on expression of the viral fibroblast growth factor (vFGF). This pathway leads to tracheal cell basal lamina remodelling, enabling virus escape from the primary site of infection, the midgut epithelium, and establishment of efficient systemic infection. In this study, we asked whether the MMP-caspase pathway was also activated following infection by intrahaemocoelic injection. We found that intrahaemocoelic infection did not lead to any observable tracheal cell or midgut epithelium basal lamina remodelling. MMP and caspase activities were not significantly stimulated. We conclude that the main role of the AcMNPV vFGF is in facilitating virus midgut escape.

The Baculovirus Core Gene ac83 Is Required for Nucleocapsid Assembly and Per Os Infectivity of Autographa californica Nucleopolyhedrovirus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

Transgenerational transmission of the Glossina pallidipes hytrosavirus depends on the presence of a functional symbiome.

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.

EICOSANOIDS MEDIATE CELLULAR IMMUNE RESPONSE AND PHENOLOXIDASE REACTION TO VIRAL INFECTION IN ADULT Pimpla turionellae

Nodulation is the predominant insect cellular immune response to microbial infections. We posed the hypothesis that parasitoid insects in their adulthood express melanotic nodulation reactions to viral challenge and that eicosanoids mediate nodulation reactions and phenoloxidase (PO) activation in response to viral challenge. To test this idea, we injected Pimpla turionellae adults with indomethacin, a nonsteroidal anti-inflammatory drug, immediately prior to intrahemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1). Treating newly emerged adults of P. turionellae with BHSV-1 induced nodulation reactions, and decreased PO activity at high viral doses. Relative to vehicle-treated controls, indomethacin-treated adults produced significantly reduced numbers of nodules following viral infection (down from approximately 21 nodules per adult to less than six nodules per adult). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 0.1%) were associated with decreasing nodulation (by six-fold) and PO (by about three-fold) reactions to BHSV-1 injection. Wasp adults orally fed with the lowest dietary indomethacin concentration (0.001%) expressed significantly increased PO activity (1.45 unit/min/mg protein) while nodulation reaction was not affected in response to viral challenge compared to control adults. We infer from these findings that cyclooxygenase (COX) products, at least prostaglandins, mediate nodulation response and PO action to viral infection in adults of these highly specialized insects.

Comparative analysis of budded virus infectivity of Bombyx mandarina and B. mori nucleopolyhedroviruses

Bombyx mandarina nucleopolyhedrovirus (BomaNPV) is a variant of Bombyx mori nucleopolyhedrovirus (BmNPV). BomaNPV S1 strain has been reported to be significantly less virulent than the BmNPV T3 strain via the oral infection route in B. mori larvae, but other features of S1 including budded virus (BV) infectivity and virus propagation in cultured cells are still unknown. In this study, we compared BV infectivity of S1 and T3 in B. mori larvae and cultured cells. Larval bioassays by intrahemocoelic BV injection revealed that the median lethal dose of S1's BV was approximately three times lower than that of T3. In addition, S1 produced more BVs and occlusion bodies (OBs) in the hemolymph of B. mori larvae compared with T3. Furthermore, we observed that the locomotion was enhanced earlier and the median lethal time was shorter in S1-infected larvae compared with those in T3-infected larvae. Western blot analysis of S1- and T3-infected BmN cells revealed that expression of late and very late gene products in S1-infected cells was higher than that in T3-infected cells. Collectively, these results clearly show that S1's BV infectivity is higher than that of T3 in both B. mori larvae and cultured cells, although S1's OBs are much less infectious to B. mori larvae than T3's.