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Entomopathogenic nematodes (EPNs) are obligate insect parasites symbiotically associated with entomopathogenic bacteria. They can be used as a natural infection model combining bacterial infection with infection by multicellular parasite and as such, can be used in biological control and also offer a powerful tool to study insect immunity. Both mix of non-pathogenic bacteria and even nectar alkaloids were previously hypothesized to positively modulate honey bee health. We used EPNs for evaluating the overall immune resistance of honey bee larvae treated with potentially immuno-modulating substances - a plant alkaloid sanguinarine (extracted from Macleaya cordata) and non-pathogenic strains of Lactobacillus apis, Lactobacillus melliventris and Gilliamella apicola, native to honey bee gut isolates. Honey bee 5th instar larvae (L5) and white pupae were infected with Heterorhabditis bacteriophora or Steinernema feltiae, both carrying their symbiotic bacteria. In comparison to untreated honey bee larvae we observed an increase in survival of 13.5 ± 6.43 or 11.25 ± 5.77% in case of sanguinarine and S. feltiae or H. bacteriophora, respectively. Similarly, mix of above mentioned bacteria inoculated at the same time increased survivorship to 23.25 ± 1.53 or 11.0 ± 6.0% for S. feltiae or H. bacteriophora. This is the first record of use of nematobacterial infection for evaluating the immune status of a beneficial insect. Addition of low doses of both sanguinarine and non-pathogenic strains of selected bacteria had a positive impact on the resistance of honey bee to pathogen. This method can serve as a valuable tool for immunological tests in honey bees. [ABSTRACT FROM AUTHOR]

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or--as previously shown--the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions. [ABSTRACT FROM AUTHOR]

Simple Summary: The European honey bee, Apis mellifera, is well-known to have two distinct populations in temperate climate zone: short-living summer bees and long-living winter bees. Several biological factors related to the different lifespans of the two populations have been studied. However, the link between the metabolic changes and basic physiological features in the bodies of summer bees and winter bees is limited. This study aimed to identify the metabolic fingerprints that characterize summer and winter bees using proton nuclear magnetic resonance (1H NMR) spectroscopy. In total, we found 28 significantly changed metabolites between the two populations. The results suggest that the metabolites detected in honey bee bodies can distinguish the summer and winter bees. Changes in carbohydrates, amino acids, choline-containing compounds, and an unknown compound were noticeable during the transition from summer bees to winter bees. The results from this study give us a broad perspective on honey bee metabolism that will support future research related to honey bee lifespan and overwintering management. In temperate climates, honey bee workers of the species Apis mellifera have different lifespans depending on the seasonal phenotype: summer bees (short lifespan) and winter bees (long lifespan). Many studies have revealed the biochemical parameters involved in the lifespan differentiation of summer and winter bees. However, comprehensive information regarding the metabolic changes occurring in their bodies between the two is limited. This study used proton nuclear magnetic resonance (1H NMR) spectroscopy to analyze the metabolic differences between summer and winter bees of the same age. The multivariate analysis showed that summer and winter bees could be distinguished based on their metabolic profiles. Among the 36 metabolites found, 28 metabolites have displayed significant changes from summer to winter bees. Compared to summer bees, trehalose in winter bees showed 1.9 times higher concentration, and all amino acids except for proline and alanine showed decreased patterns. We have also detected an unknown compound, with a CH3 singlet at 2.83 ppm, which is a potential biomarker that is about 13 times higher in summer bees. Our results show that the metabolites in summer and winter bees have distinctive characteristics; this information could provide new insights and support further studies on honey bee longevity and overwintering. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2013 S. Karger AG, Basel)

We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation., (Copyright © 2010 S. Karger AG, Basel.)

Antioxidant enzymes play a major role in the defense against pro-oxidative effects of xenobiotics and pro-oxidant plant allelochemicals in insects. We posed the hypothesis that eicosanoids also mediate antioxidant enzymatic defense reactions to pro-oxidant challenge. To test this idea, we reared first-instar larvae of Galleria mellonella (L.) with the lypoxygenase inhibitor, esculetin (0.001%), the phospholipase A(2) inhibitor, dexamethasone (0.001%) and the dual inhibitor of cyclooxygenase and lipoxygenase, phenidone (0.1%) to seventh-instars. Newly ecdysed seventh-instars were then fed on artificial diet containing 0.05% xanthotoxin (XA) for 2 days. Treating seventh-instar larvae of G. mellonella with XA induced lipid peroxidation and protein carbonylation as evident from the increased content of malondialdehyde (MDA) and protein carbonyls respectively, and antioxidative enzymatic response in a dose-dependent manner. High dietary XA concentrations (0.005 and 0.1%) were associated with increasing MDA and carbonyl content (by 3-fold) and antioxidant enzyme activities, superoxide dismutase (SOD) (by 3-fold) and catalase (CAT) (by 4-fold), and glutathione-dependent enzymes, glutathione S-transferase (GST) (by 15-fold) and glutathione peroxidase (GPx) (by 7-fold). Relative to control, eicosanoid biosynthesis inhibitors (EBIs) esculetin, dexamethasone and phenidone also resulted in impaired MDA content and antioxidant enzyme activities. However, carbonyl content did not differ between control- and EBIs-feeding larvae. Finally, MDA and carbonyl content, and antioxidant enzymes SOD, GST and GPx activities exhibited an incremental increase while CAT activity was decreased in the experimental larvae that had been reared on media amended with esculetin, dexamethasone and phenidone and then challenged with our standard XA challenge dose. Two of the markers indicated that significantly higher levels of oxidative stress were produced in the hemolymph tissue of larvae fed diets supplemented with EBIs and then challenged with XA. This oxidative stress was associated with elicited antioxidative responses by increasing SOD, GST and GPx and decreasing CAT activities in hemolymph. We infer from these findings that eicosanoids mediate insect antioxidant enzymatic responses to dietary pro-oxidants., (Copyright 2010 Elsevier Inc. All rights reserved.)

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management., ((c) 2007 Wiley-Liss, Inc.)

Nitric oxide production by the hemocytes of the last instar larvae and sessile pharate prepupa of Galleria mellonella (Lepidoptera: Pyralidae) was demonstrated in vitro in response to preparations of bacterial lipopolysaccharide (LPS) from Escherichia coli using the Griess reaction. Augmented, dose dependent nitric oxide production was observed in the pharate prepupal hemocytes compared with larval hemocytes. This was partially reversed in a dose dependent manner with S-methyl thiourea (SMT), a specific inhibitor of inducible nitric oxide synthase (iNOS). A decrease in NO production was also observed when non-selective inhibitors such as N(G)-nitro-L-arginine (L-NAME) and N-omega-nitro-L-arginine (L-NNA) were used, albeit the inhibition was not to the extent of SMT. Challenge with the entomopathogenic Gram-negative bacterium Photorhabdus asymbiotica also enhanced NO production by hemocytes of both stages. SMT, alone or in combination with P. asymbiotica significantly decreased levels of NO production. However, it was observed that phenoloxidase activity (a cascade for innate immune responses) was independent of NO production stimulation. NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and diethylenetriamine NO adduct (DETA/NO) at various concentrations (100-500 microM) resulted in the lysis of hemocytes dose dependently. The nitrite production in these cases was however similar to LPS stimulation (10 microg/mL) and 1.5-3 fold lower than those observed upon P. asymbiotica (2.5 x 10(7) cfu/mL) stimulation. Survival analysis (Kaplan-Meier) following injection of P. asymbiotica alone or in combination with SMT revealed that only 12.5% (median survival 25.5 h) of co-injected larvae of G. mellonella survived in comparison to 28.6% (median survival 29 h) survivors in P. asymbiotica alone-injected groups till the end of the study. In contrast, co-injected pharate prepupa survived longer (median survival 28 h) than the P. asymbiotica alone-injected individuals (median survival 24 h); however, both co-injected and P. asymbiotica-injected groups showed 100% mortality at the end of the study. Based on the above, we propose that although NO production is involved in cellular immune responses of this insect to bacterial infection it does not appear to be a part of the signalling pathway that initiates the prophenoloxidase (PPO) cascade, and the extended NO production/overproduction by pharate prepupal hemocytes could result in cytotoxic rather than cytoprotective effects compared with larval hemocytes.