Induction, Isolation and Biological function of Excreted/Secreted Products released by entomopathogenic nematode Heterorhabditis bacteriophora.

Entomopathogenic nematodes (EPNs) are a type of parasitic nematode characterized by the development in insect hosts and symbiotic relationship with specific bacterial species. The EPN Heterorhabditis bacteriophora has a symbiotic association with bacterium Photorhabdus luminesces. During the infection of the host, the EPNs produce a variety of molecules known as Excreted/Secreted Products (ESPs) that have been described with immunomodulatory functions. Although EPNs only infect insects, humans and insects share analogous immune reactions of the innate immune system. Characterized ESPs from H. bacteriophora have the capacity to down-regulate the production of antimicrobial peptides (AMPs), induced by insect host hemolymph during infection. In addition, P. luminesces produces lectins that decrease the production of reactive oxygen species (ROS) in human blood phagocytes. In this study, we optimize the production process of H. bacteriophora ESPs in infective juveniles (IJs), by getting rid of contamination and using a variety of materials that induce their production. The different activating materials used come from a variety of compounds from insect host Galleria mellonella, which we name as homogenate. Furthermore, we isolated these ESPs and analyzed their immunomodulatory functions in insects and humans. We confirmed that the contamination found in the production of ESPs was due to Gram-positive and Gram-negative bacteria, and that this contamination could be eliminated with kanamycin (50 mg/ml) and streptomycin/penicillin (10,000 U/ml). Moreover, the use of these antibiotics showed an increase in the protein concentration of ESPs. We did not detect any increase in the protein concentration of ESPs, by the addition or removal of 0,01 % NaClO, nor with activation or secretion times selected in this study. An increase in the protein concentration of ESPs was observed when using as activating material concentrated heat-inactivated homogenate. We analysed the effect of ESPs over the antimicrobial activity of hemolymph from G. mellonella, by viability of bioluminescent Escherichia coli. We could not conclude if ESPs impede the antimicrobial activity of hemolymph or served as nutrients for the bacteria, due to lack of statistical significance. Lastly, we decided to measure the effect of ESPs over the production of ROS in human blood, concluding that collected ESPs do not significantly affect this type of immune response. This study benefits the ongoing research of bioactive ESPs over the areas of pharmacology, biocontrol and human medicine.pre-print873 K

Plant-based and immunostimulant-enhanced diets modulate oxidative stress, immune and haematological indices in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to investigate the effects of three commercial diets, standard, immunostimulant-supplemented (β-glucan, vitamins C and E) and plant-based, on the degree of oxidative stress in tissues of rainbow trout (Oncorhynchus mykiss). Selected immune and haematological indices were measured and challenge with Aeromonas salmonicida was conducted. The plant-based diet systemically modulated all oxidative stress biomarkers (thiobarbituric acid reactive substances; reduced and oxidised glutathione and their ratio) in the intestine. The ratio was elevated in all organs (liver, kidney, muscle and intestine) and an enhancement of respiratory burst and complement activity was observed even in the control fish. With the standard diet, an elevation of thiobarbituric acid reactive substances in the intestine and a decrease in some immune indices appeared after challenge. Less distinctive changes and the lowest mortality rate (the highest being with the plant-based feed) were obtained with immunostimulants. Increased total immunoglobulin, relative lymphocytosis and a decrease in the phagocyte count were observed. This study contributes especially by a thorough examination of oxidative stress indices in different tissues. The exact composition of these commercial diets is a trade secret; however, knowledge of their effects is extremely important for fish farmers using them; therefore, this study has a great practical impact

Clinical and Laboratory Parameters of Carp Edema Virus Disease: A Case Report

In the present study, we describe a natural outbreak of carp edema virus disease (CEVD) in koi carp, concentrating on clinical manifestation, gross and microscopic pathology, immunological parameters, viral diagnostics, and phylogenetic analysis. Examination of white blood cell parameters showed increased monocyte and decreased lymphocyte counts in CEV-affected fish compared to healthy control fish. Regarding immune system functioning, the present work shows, for the first time, enhanced phagocytic activity in CEV-affected fish. Respiratory burst of phagocytes was strongly increased in diseased fish, the increase being attributed to an increased phagocyte count rather than enhancement of their metabolic activity. The present work also newly shows histopathological changes in the pancreatic tissue of diseased koi.O

Boric acid-induced effects on protein profiles of Galleria mellonella hemolymph and fat body

The dietary effects of boric acid (BA) on the protein profiles of greater wax moth, Galleria mellonella (L.), were investigated in hemolymph and fat body of final instar (VIIth) and pupae. The insects were reared from first-instar larvae on an artificial diets containing 156, 620, 1250 or 2500 ppm of BA. We detected many undetermined protein fractions (6.5-260 kDa) in addition to well-defined protein fractions such as lipophorins and storage proteins in the tissues by using sodium dodecyl-sulphate polyacrylamide gradient gel electrophoresis. A marked quantitative change in the 45 kDa protein fraction of the hemolymph was observed in the VIIth instar larvae reared on 2500 ppm dietary BA. © 2008 Akadémiai Kiadó

Eicosanoids mediate hemolymph oxidative and antioxidative response in larvae of Galleria mellonella L.

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 A2 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. © 2010 Elsevier Inc. All rights reserved

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

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. © 2007 Wiley-Liss, Inc

Bioluminescent assay for evaluating antimicrobial activity in insect haemolymph

We describe an antibacterial assay based on bioluminescence of two Gram negative bacteria, Photorhabdus luminescens and transformed Escherichia coli, which can be used as a real-time measurement of antibacterial activity in insect haemolymph. This method is based on the production of the bioluminescence signal depending on the viability of bacterial cells. We observed a significant rapid dose-dependent decrease in bioluminescence using both bacterial species, and Bombyx mori or Galleria mellonella haemolymph, which was confirmed by the decrease in bacterial viability determined by plating. The humoral origin of the antibacterial activity observed in whole haemolymph was confirmed for haemolymph plasma without haemocytes. Antibacterial activity directed against Gram negative bacteria was recorded in unaffected insect larvae as well as after septic injury; increased antibacterial activity of haemolymph was detected in the latter case confirming the inducibility of antimicrobial agents. We think it is likely that this method could be widely used for determining antibacterial activity in insects and other invertebrates

The Effect of Foraging on Bumble Bees, Bombus terrestris, Reared under Laboratory Conditions

Bumble bees are important pollinators broadly used by farmers in greenhouses and under conditions in which honeybee pollination is limited. As such, bumble bees are increasingly being reared for commercial purposes, which brings into question whether individuals reared under laboratory conditions are fully capable of physiological adaptation to field conditions. To understand the changes in bumble bee organism caused by foraging, we compared the fundamental physiological and immunological parameters of Bombus terrestris workers reared under constant optimal laboratory conditions with workers from sister colonies that were allowed to forage for two weeks in the field. Nutritional status and immune response were further determined in wild foragers of B. terrestris that lived under the constant influence of natural stressors. Both wild and laboratory-reared workers subjected to the field conditions had a lower protein concentration in the hemolymph and increased antimicrobial activity, the detection of which was limited in the non-foragers. However, in most of the tested parameters, specifically the level of carbohydrates, antioxidants, total hemocyte concentration in the hemolymph and melanization response, we did not observe any significant differences between bumble bee workers produced in the laboratory and wild animals, nor between foragers and non-foragers. Our results show that bumble bees reared under laboratory conditions can mount a sufficient immune response to potential pathogens and cope with differential food availability in the field, similarly to the wild bumble bee workers

US presidential candidates and energy

With one of the most contested presidential elections under way in the United States in decades, the impact on the energy industry is a particularly relevant issue. On the one hand, all three remaining contenders are likely to be much less favourably inclined to the energy industry than the current Administration; on the other hand, there are serious nuances between the