Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

Eicosanoids mediate nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya megacephala

Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.     

Changes in immune effort of male field crickets infested with mobile parasitoid larvae

Insect immune defenses include encapsulation and the production of lysozymes and phenoloxidase. However, the highly mobile larvae of parasitoid Ormiine flies (Ormia ochracea) can evade initial encapsulation, and instead co-opt host immune responses to form a critical respiratory funnel connecting them to outside oxygen. Here we ask how field crickets (Teleogryllus oceanicus) respond immunologically to O. ochracea infestation. Host encapsulation and phenoloxidase play important roles in the formation of the respiratory funnel, so we hypothesized that decreases in these immune parameters during infestation may interfere with respiratory funnel formation and increase the likelihood of larval death. Encapsulation ability decreased after infestation with O. ochracea larvae, but phenoloxidase activity increased in both infested crickets and controls, whereas lysozyme activity decreased in infested crickets but remained constant in controls. Hosts with fewer established larvae showed greater decreases in encapsulation, and phenoloxidase activity was positively associated with the degree of larval respiratory funnel melanization. Differences between phenoloxidase and lysozyme activity in infested crickets are consistent with a trade-off within the immune system of hosts, and our results demonstrate the effects of a prior immune challenge on the ability to mount a subsequent response.     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Effect of temperature and relative humidity on the cellular defense response of Ephestia kuehniella larvae fed Bacillus thuringiensis

While maintained under all combinations of three temperatures and two RH, fifth instar larvae of the Mediterranean flour moth, Ephestia kuehniella were fed wheat treated with spores and crystals of Bacillus thuringiensis var. kurstaki. Larvae that had fed on wheat with the bacterial preparation contained higher concentrations of nodules in their haemocoel than did larvae fed on wheat without bacteria. Nodule concentrations in larvae fed untreated wheat were unaffected by temperature or relative humidity. Larvae fed treated wheat had higher nodule concentrations at 32 degrees C than at 15 and 23 degrees C, and higher nodule concentrations at a relative humidity of 85% than at 43%. The percentage of larvae that pupated was lower when larvae were fed the bacterial preparation, and was significantly higher at 23 degrees C than at 15 and 32 degrees C, regardless of whether larvae were fed bacteria or not. Less time was required for larvae to develop to pupation at higher temperatures and at higher humidity. Mean time to pupation was lower for bacteria-fed larvae than for those fed untreated wheat, and this appeared to be a result of truncation of the distribution of times to pupation because only rapidly developing larvae survived to pupation.     

Regulation of CD8 T cell responses to infection with parasitic protozoa

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8⁺ T cell response. This review will highlight some of the key studies that established that CD8⁺ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8⁺ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8⁺ T cell responses are initiated and new insights into their phenotypic plasticity     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Active suppression of D. melanogaster immune response by long gland products of the parasitic wasp Leptopilina boulardi

To develop inside their insect hosts, endoparasitoid wasps must either evade or overcome the host's immune system. Several ichneumonid and braconid wasps inject polydnaviruses that display well-studied immune suppressive effects. However, little is known about the strategies of immunoevasion used by other parasitoid families, such as figitid wasps. The present study provides experimental evidence, based on superparasitism and injection experiments, that the figitid species Leptopilina boulardi uses an active mechanism to suppress the Drosophila melanogaster host immune response, i.e. the encapsulation of the parasitoid eggs. The immune suppressive factors are localised in the long gland and reservoir of the female genital tractus, where virus-like particles (VLPs) have been observed. Parasitism experiments using a host tumorous strain indicate that these factors do not destroy host lamellocytes but that they impair the melanisation pathway. Interestingly, they are not susceptible to heating and are not depleted with prolonged oviposition experience, in contrast to observations reported for L. heterotoma, another figitid species. The mechanisms that prevent encapsulation of eggs from L. boulardi and L. heterotoma differ in several respects, suggesting that different physiological strategies of immunosuppression might be used by specialised and generalist parasitoids.     

Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: Examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against vibriosis

Three putative beneficial unicellular organisms, the marine bacterium Roseobacter sp, the yeast Saccharomyces cerevisiae mnn9 strain (SC-mnn9) and the microalga Tetraselmis suecica were cooked and offered separately as diets to developing germ-free (GF) Artemia larvae, in order to analyze their alimentary and protective effects. GF Artemia larvae were able to grow with cooked Tetraselmis and SC-mnn9 but failed with cooked Roseobacter. In spite of its high dietary quality, Tetraselmis failed to provide protection against Vibrio proteolyticus infection, while Roseobacter failed as food as well to provide protection. Cooked SC-mnn9 appeared to possess both values, dietary for growth and protective against Vibrio infection. GF Artemia larvae were apparently rapid adapted to dietary swaps; from yeast to algal and from algal to yeast. While the diets swap from algal or yeast, to bacterial diet appeared to be detrimental. Phenoloxidase-L (PO-L) and trypsin-L were used as biochemical indicators of defense and digestive functions, respectively. Developmental trypsin-L patterns were similar when fed on yeast and microalgae diets, suggesting a good digestive adaptation to plant or fungal substrates at early stages. On the contrary, diets swap or Vibrio infection affected PO-L and trypsin-L suggesting a sort of ‘alteration’ of digestive and defense functions.     

Eicosanoids mediate nodulation reactions to bacterial Escherichia coli K 12 infections in larvae of the oriental blowfly, Chrysomya megacephala

Abstract  Nodulation is the predominant cellular defense reaction to bacterial challenges in insects. In this study, third instar larvae of Chrysomya megacephala were injected with bacteria, Escherichia coli K 12 (10⁶ CFU/mL, 2 μL), immediately prior to injection of inhibitors of eicosanoid biosynthesis, which sharply reduced nodulation response. Test larvae were treated with specific inhibitors of phospholipase A₂ (dexamethasone), cyclo-oxygenase (indomethacin, ibuprofen and piroxicam), dual cyclo-oxygenase/lipoxygenase (phenidone) and lipoxygenase (esculetin) and these reduced nodulation except esculetin. The influence of bacteria was obvious within 2 h of injection (5 nodules/larva), and increased to a maximum after 8 h (with 15 nodules/larva), and then significantly reduced over 24 h (9 nodules/larva). The inhibitory influence of dexamethasone was apparent within 2 h of injection (4 vs. 5 nodules/larva), and nodulation was significantly reduced, compared to control, over 24 h (5 vs. 8 nodules/larva). Increased dosages of ibuprofen, indomethacin, piroxicam and phenidone led to decreased numbers of nodules. Nodules continued to exist during the pupal stage. However, the effects of dexamethasone were reversed by treating bacteria-injected insects with an eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. These findings approved our view that eicosanoid can mediate cellular defense mechanisms in response to bacterial infections in another Dipteran insect C. megacephala .     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera,Scarabaeidae): involvement of both granulocytes and oenocytoids in in vivo phagocytosis

In the context of comparative studies on immunity defence mechanisms of adults and larvae of the coleopteran Cetonischema aeruginosa (Drury, 1770) the ultrastructure of the circulating hemocytes of the third instar larval stage has been investigated by means of light and transmission electron microscopy (TEM). Six types of hemocytes were found in the hemolymph of C. aeruginosa and they were identified as prohemocytes, granulocytes, plasmatocytes, coagulocytes, oenocytoids and spherule cells. In order to identify the "professional" phagocyte cell, phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads. It was demonstrated that the granulocytes and the oenocytoids of C. aeruginosa were the only hemocyte types involved in this cellular response.     

The effect of dietary nickel on the immune responses of Spodoptera litura Fabricius larvae

By exposing Spodoptera litura Fabricius larvae to nickel (Ni) in artificial diets for successive three generations, we investigated the impacts of the dietary Ni on growth and immune response of the fifth and sixth instar larvae at 24 h intervals. The time of newly moulted fifth instar larvae was labelled as 0 h. After exposure to 5 mg/kg Ni for two generations, Ni exposure significantly improved larval phenoloxidase activity and encapsulation grade in fifth instar larvae when compared to controls, except for encapsulation grade at 72-120 h in the second generation. However, higher concentrations of Ni (≥10 mg/kg) only significantly reduced encapsulation grade at 72-120 h. In the third generation, insects given higher dietary levels of Ni (≥10 mg/kg) showed lower immune responses and retarded relative growth rate (RGR) compared to controls, but those exposed to lower Ni levels (≤5 mg/kg) had a significantly improved encapsulation grade at 24-72 h. Larvae at lower Ni level (≤5 mg/kg) treatments had significantly higher RGR in comparison with that in controls. There was no significant difference in food relative consumption rate (RCR) and RGR among any treatment of the fifth instar larvae in three successive generations. These results indicated that the type and extent of effects on growth and immune responses of S. litura varied with the Ni concentrations and exposure periods.     

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.     

Effects of parasitism by Asobara tabida (Hymenoptera: Braconidae) on the development,survival and activity of Drosophila melanogaster larvae

The impact of parasitism by Asobara tabida on Drosophila melanogaster larval development, survival features and larval activity has been investigated using two strains of the parasitoid. The successful parasitism rate of the A1 strain was four times greater than that of the WOPV strain. Both strains induced equivalent mortality rates but hosts parasitized by A1 predominantly died as pupae. The time necessary for the host pupariation and emergence, and the larval weight at 72, 96 and 120 h post-parasitization were measured. Parasitized larvae exhibited longer periods of development and lower weights than controls, especially when parasitized by A1. These results suggest that hosts underwent physiological costs varying with respect to the outcome of the parasitic relationship. Of the parasitoid factors possibly responsible for these costs, we examined venoms for their impact on host mortality. Artificial injections of WOPV venoms induced higher mortality rates than did A1 venoms. Venoms were also found responsible for the induction of a transient paralysis, naturally occuring after parasitization. Again, the strongest effect was observed after parasitization by WOPV or injections of its venoms. This study gives new insights into the intriguing features of A. tabida and constitutes the first report of the paralysing properties of the venoms.     

Eicosanoids mediate control of thermoregulatory sweating in the cicada,Tibicen dealbatus (Insecta: Homoptera)

Cicadas prevent body temperature from exceeding tolerable levels by a combination of behavioral responses and sweating. Sweating is activated when body temperature reaches a critical set-point temperature. We investigated control of sweating in the cicada, Tibicen dealbatus, by chemically manipulating biosynthesis of prostaglandins and other eicosanoids. Injecting prostaglandins in amounts equal to those that induce behavioral fever in scorpions and crustaceans resulted in only a small increase in set-point temperature. Blocking prostaglandin biosynthesis with cyclo-oxygenase inhibitors such as aspirin produced significant changes in set-point temperature, confirming that prostaglandins are involved in control of sweating. However, the effect of cyclo-oxygenase inhibitors was not the opposite of the effect of prostaglandins. Instead, the effect of cyclo-oxygenase inhibitors depended strongly on the value of setpoint temperature prior to treatment. Results of biochemical manipulations of other steps in eicosanoid biosynthetic pathways corroborated the results of cyclo-oxygenase inhibition and indicated that eicosanoids other than prostaglandins may be involved in control of body temperature in normothermic T. dealbatus. The effect of cyclo-oxygenase inhibitors on a given set-point temperature depended on the ambient temperature experienced by cicadas during the experiment. Surprisingly, cicadas exposed to ambient temperatures 40°C delayed activation of sweating until body temperature exceeded values normally recorded from T. dealbatus in the field. Control of body temperature in normothermic cicadas is thus complex, involving inputs from body temperature sensors, ambient temperature sensors, and at least two cyclo-oxygenase-dependent regulatory pathways.Abbreviations PUFA polyunsatured fatty acid(s) - T _a ambient temperature - T _b body temperature - T _set set-point of body temperature for activation of sweating     

Tolerance to Bacillus thuringiensis endotoxin in immune-suppressed larvae of the flour moth Ephestia kuehniella

Tolerance to Bacillus thuringiensis crystal endotoxins (Bt-toxins) is correlated with an elevated immune status in larvae of the flour moth Ephestia kuehniella. To gain more specific information about the effector pathways involved in the protection against the toxin, we studied the effects of Bt-toxin formulations in susceptible (non-induced) and tolerant (immune-induced) larvae after natural (parasitism-mediated) and chemical (tropolone-mediated) suppression of defence reactions. Although melanization in hemolymph was significantly reduced, there was no significant effect on susceptibility to the toxin in parasitised or tropolone-treated larvae. This suggests that melanization of hemolymph is correlated with an elevated immune status but not responsible for the observed tolerance to Bt-toxin. To examine whether hemolymph proteins exist in the gut lumen and function as pro-coagulants, we compared gut and plasma proteins of immune-induced with those of non-induced larvae. Here we show that the lipid carrier lipophorin represents a major component in the gut lumen and interacts with mature Bt-toxin to form a complex.     

Haemocyte changes in D. Melanogaster in response to long gland components of the parasitoid wasp Leptopilina boulardi: a Rho-GAP protein as an important factor

The hymenopteran wasp Leptopilina boulardi (Figitidae) is a larval solitary parasitoid of Drosophila larvae of the melanogaster sub-group. The factors used by parasitoid females to prevent encapsulation of their eggs by the host are localized in the female long gland and reservoir. We report here the physiological effects of these factors on host haemocytes using in vivo injection experiments. The total number of haemocytes, the number of plasmatocytes and the number of crystal cells were not modified by injection of long gland extracts. In contrast, long gland extracts either from virulent or avirulent strains had a significant effect on the lamellocyte number. Compared to the Ringer control, the avirulent long gland products induced an increase of the lamellocyte number while virulent extracts induced a drastic decrease together with an alteration of the morphology of these cells. Interestingly, changes in the lamellocyte morphology were also observed following injection of the P4 protein, a major component of L. boulardi female long glands that displays a strong immune suppressive effect on Drosophila larvae. The implication of the P4 protein in suppressing the host cellular immunity is discussed in correlation with its predicted molecular function as a Rho-GAP protein.