Diet composition has a differential effect on immune tolerance in insect larvae exposed to Mesorhabditis belari, Enterobacter hormaechei and its metabolites.

In insects, diet plays an important role in growth and development. Insects can vary their diet composition based on their physiological needs. In this study we tested the influence of diet composition involving varying concentrations of macronutrients and zinc on the immune-tolerance following parasite and pathogen exposure in Spodoptera litura larvae. We also tested the insecticidal potential of Mesorhabditis belari, Enterobacter hormaechei and its secondary metabolites on Spodoptera litura larvae. The results shows macronutrient composition does not directly affect the larval tolerance to nematode infection. However, Zinc supplemented diet improved the immune tolerance. While larvae exposed to bacterial infection performed better on carbohydrate rich diet. Secondary metabolites from bacteria produced an immune response in dose dependent mortality. The study shows that the larvae maintained on different diet composition show varied immune tolerance which is based on the type of infection.

Eicosanoids mediate Galleria mellonella immune response to hemocoel injection of entomopathogenic nematode cuticles.

Entomopathogenic nematodes are symbiotically associated with bacteria and widely used in biological control of insect pests. The interference of symbiotic bacteria with insect host immune responses is fairly well documented. However, knowledge of mechanisms regulating parasite­host interactions still remains fragmentary. In this study, we used nematode (Steinernema carpocapsae and Heterorhabditis bacteriophora) cuticles and Galleria mellonella larvae as parasite­host model, focused on the changes of innate immune parameters of the host in the early phase of nematode cuticle infection and investigated the role of eicosanoid biosynthesis pathway in the process. The results showed that injection of either S. carpocapsae or H. bacteriophora cuticles into the larval hemocoel both resulted in significant decreases in the key innate immune parameters (e.g., hemocyte density, microaggregation, phagocytosis and encapsulation abilities of hemocyte, and phenoloxidase and antibacterial activities of the cell-free hemolymph). Our study indicated that the parasite cuticles could actively suppress the innate immune response of the G. mellonella host. We also found that treating G. mellonella larvae with dexamethasone and indomethacin induced similar depression in the key innate immune parameters to the nematode cuticles. However, these effects were reversed when dexamethasone, indomethacin, or nematode cuticles were injected together with arachidonic acid. Additionally, we found that palmitic acid did not reverse the influence of the dexamethasone, indomethacin, or nematode cuticles on the innate immune responses. Therefore, we inferred from our results that both S. carpocapsae and H. bacteriophora cuticles inhibited eicosanoid biosynthesis to induce host immunodepression.

Susceptibility of the Giant African snail (Achatina fulica) exposed to the gastropod parasitic nematode Phasmarhabditis hermaphrodita.

The Giant African snail (Achatina fulica) is a major pest in tropical countries. Current control methods involve the use of slug pellets (metaldehyde) but they are ineffective, therefore new methods of control are needed. We investigated whether A. fulica is susceptible to the gastropod parasitic nematode Phasmarhabditis hermaphrodita, which has been developed as a biological control agent for slugs and snails in northern Europe. We exposed A. fulica to P. hermaphrodita applied at 30 and 150nematodes per cm(2) for 70days and also assessed feeding inhibition and changes in snail weight. We show that unlike the susceptible slug species Deroceras panormitanum, which is killed less than 30days of exposure to P. hermaphrodita, A. fulica is remarkably resistant to the nematode at both doses. Also P. hermaphrodita does not reduce feeding in A. fulica nor did it have any effect on weight gain over 70days. Upon dissection of infected A. fulica we found that hundreds of P. hermaphrodita had been encapsulated, trapped and killed in the snail's shell. We found that A. fulica is able to begin encapsulating P. hermaphrodita after just 3days of exposure and the numbers of nematodes encapsulated increased over time. Taken together, we have shown that A. fulica is highly resistant to P. hermaphrodita, which could be due to an immune response dependent on the snail shell to encapsulate and kill invading parasitic nematodes.

Biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode Steinernema carpocapsae.

A trypsin-like serine protease was purified by gel filtration and anion-exchange chromatography from the excretory-secretory products of parasitic phase Steinernema carpocapsae. The purified protease exhibited a molecular mass of about 29 kDa by SDS-PAGE and displayed a pI of 6.3. This protease exhibited high activity with trypsin-specific substrate N-Ben-Phe-Val-Arg-p-nitroanilide and was highly sensitive to aprotinin and benzamidine. The purified trypsin protease digested the chromogenic substrate N-Ben-Phe-Val-Arg-p-nitroanilide with K(m), V(max) and k(cat) values of 594.2 mum, 0.496 mum/min and 22.8/s, respectively. The optimal pH and temperature for protease activity were 9 and 30 degrees C, respectively. Internal amino acid sequencing yielded 150 amino acids and these were homologous to other trypsin sequences. In vitro investigation was carried out to monitor prophenoloxidase suppression in Galleria mellonella by the purified protease; about 38.9-52.6% suppression of prophenoloxidase was observed. The purified protease affected insect haemocyte spreading, causing cells to become spherical or round. Protease-treated actin filaments were highly disorganized in haemocytes. In vitro, G. mellonella haemocytes recognized infective juveniles of Heterorhabditis bacteriophora; however, S. carpocapsae and Steinernema glaseri were not recognized. We provide experimental evidence that the purified trypsin has the potential to alter host haemocytes, actin filaments and to inhibit host haemolymph melanization.

Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.

SUMMARY: Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.

A nematode symbiont sheds light on invertebrate immunity.

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.

Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State.

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemoless) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.

Encapsulation of the entomopathogenic nematode Steinernema feltiae in Tipula oleracea.

The encapsulation response of Tipula oleracea to the entomopathogenic nematode Steinernema feltiae was investigated by exposing the insects to nematode dauer juveniles (DJs) and by injecting DJs with and without the symbiotic bacteria Xenorhabdus bovienii. The encapsulation response varied considerably between individual insect larvae. The variation could not be attributed to a more or less scattered nematode invasion over time since it was also recorded after simultaneous injection of a fixed DJ dose. The proportion of encapsulated nematodes declined with increasing dose (injected DJs/larva) from approx 80% for 1 DJ/larva to 33-34% for 20 DJ/larva. Tipula oleracea larvae were capable of encapsulating nematodes with and without symbionts inside the hemocoel; however, at doses of 10 and 20 DJ/larva, axenic nematodes were encapsulated less frequently than monoxenic nematodes. Injected axenic nematodes that were not encapsulated did not develop in T. oleracea larvae but disappeared from the insect's hemocoel. Coinjection of symbiotic bacteria increased encapsulation of axenic nematodes, showing that X. bovienii is triggering the encapsulation response of T. oleracea against S. feltiae.

Active resistance of entomophagous rhabditid Heterorhabditis bacteriophora to insect immunity.

A specific extracellular proteinase, degrading selectively the cecropin-based defence system of insects, is secreted into the larval body during parasitism of the greater wax moth by the Heterorhabditis bacteriophora/Photorhabdus luminescens complex and by phase 1 of P. luminescens. The proteolytic digestion of insect inducible cecropin-like immune molecules was demonstrated by the disappearance of the Galleria mellonella cecropins and purified Hyalophora cecropin B peptide PAGE bands upon exposure to infected extracts, and a similar abrogation of antibacterial activity using an agar diffusion assay. Proteolytic activity of infected extracts produced by nematode/bacterial complex and phase 1 variant of P. luminescens was shown to be correlated with cecropin-inhibitory activity, suggesting that this anti-cecropin agent may be responsible for the ability of bacteria to establish infection and the insecticidal nature of H. bacteriophora. Antibacterial activity of Galleria lysozyme and that of chicken egg-white lysozyme to which P. luminescens is insensitive, was unaffected by H. bacteriophora proteinase.