Winter honeybee (Apis mellifera) populations show greater potential to induce immune responses than summer populations after immune stimuli.

In the temperate climates of central Europe and North America, two distinct honeybee (Apis mellifera) populations are found in colonies: short-living summer bees emerge in spring and survive until summer, whereas long-living winter bees emerge in late August and overwinter. Besides the difference in their life spans, each of these populations fulfils a different role in the colonies and individual bees have distinct physiological and immunological adaptations depending on their roles. For instance, winter worker bees have higher vitellogenin levels and larger reserves of nutrients in the fat body than summer bees. The differences between the immune systems of both populations are well described at the constitutive level; however, our knowledge of its inducibility is still very limited. In this study, we focus on the response of 10-day-old honeybee workers to immune challenges triggered in vivo by injecting heat-killed bacteria, with particular focus on honeybees that emerge and live under hive conditions. Responses to bacterial injections differed between summer and winter bees. Winter bees exhibited a more intense response, including higher expression of antimicrobial genes and antimicrobial activity, as well as a significant decrease in vitellogenin gene expression and its concentration in the hemolymph. The intense immune response observed in winter honeybees may contribute to our understanding of the relationships between colony fitness and infection with pathogens, as well as its association with successful overwintering.

Vigour-related traits and immunity in hybrids of evolutionary divergent cyprinoid species: advantages of hybrid heterosis?

Hybrid advantage, described as the superiority of hybrids in some traits over their parents and termed the "heterosis effect," is widely documented in the case of reciprocal crosses of parental species (i.e., hybrids representing the F1 generation). In fish, high survival, fast growth and better health status have been widely documented in F1 hybrids. Nonetheless, the effects of interspecific hybridization on vigour, physiology and immunity-related traits in fish are largely unknown, especially concerning native systems of coexisting parental and hybrid genomes in the same habitat. The present study examined the potential physiological and immune aspects of hybrid heterosis by comparing condition status (measured especially by indexes), haematological profile, glucose concentration and selected parameters of non-specific and specific immunity between the evolutionarily divergent non-congeneric cyprinoid species Abramis brama and Rutilus rutilus and their hybrids representing the F1 generation, all of them caught in nature. Clear differences were documented for vigour-related, physiological and immune parameters between the two divergent species. Hybrids generally tended to express intermediate characters of the measured traits, likely generated by the evolutionary divergence of the hybridizing species; nonetheless, for some traits, hybrids exhibited a character that was more similar to one parental species than to the other. This was interpreted as the heterozygote advantage for F1 hybrids. It is suggested that a maternally inherited genetic background may potentially influence the expression of some branches of non-specific immunity or other aspects related to the fish health status.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

A Novel Fucose-binding Lectin from Photorhabdus luminescens (PLL) with an Unusual Heptabladed ß-Propeller Tetrameric Structure.

Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed ß-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora.

Seasonal changes in immune parameters of rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis) and brook trout × Arctic charr hybrids (Salvelinus fontinalis × Salvelinus alpinus alpinus).

Despite the high number of studies concerning seasonality of immune response in fish, information for some fish species is still scarce. Here, we assess seasonal changes in leukocyte counts and several immune parameters in three groups of farmed salmonids, i.e. brook trout (Salvelinus fontinalis), brook trout x Arctic charr hybrids (Salvelinus fontinalis x Salvelinus alpinus alpinus) and rainbow trout (Oncorhynchus mykiss) reared under the same conditions and fed with the same feed. Fish were sampled in five periods of the year (late April, early July, late August, early November and early February) and leukocyte counts, respiratory burst of blood phagocytes, lysozyme concentration in skin mucus and total complement activity were measured. Generalized linear models using fish body length as a continuous predictor and sampling period and fish species as categorical predictors, were significant for each of the parameters analysed. The highest seasonal variations in measured parameters were found in rainbow trout and lowest in hybrids. Our results confirm that measures of innate and adaptive immunity are strongly affected by season in all three groups of salmonids. The results will contribute to the improved assessment of immunocompetence in farmed fishes, essential for future sustainable development in aquaculture.

Physiological and condition-related traits in the gynogenetic-sexual Carassius auratus complex: different investments promoting the coexistence of two reproductive forms?

BACKGROUND: Carassius auratus complex is an extraordinary species complex including the diploid and polyploid forms exhibiting asexual and sexual reproduction modes. The coexistence of both forms in the same habitats is currently reported. The stable coexistence of asexual and sexual forms assumes some disadvantages for asexuals that balance the costs of sex. In our study, we hypothesized and tested the differences in physiological (including heamatological and immunological), growth-related, condition-related, and fitness-related traits between gynogenetic females and sexuals. RESULTS: Our results revealed similar growth performance in gynogenetic females and sexuals measured by body size and weight, or expressed by condition factor. The energy allocation in reproduction measured by the relative size of gonads revealed no difference between gynogenetic and sexual females; in addition, both females in spawning expressed the same estradiol levels in blood plasma. We found a gender specific trade-off between investment in reproduction and immunocompetence (measured by the spleen-somatic index). Higher aerobic performance expressed by the heart index and higher oxygen-carrying capacity were found in sexual males, with increasing values before and during spawning. Our study evidenced significantly lower aerobic performance but higher oxygen-carrying capacity per erythrocyte in gynogenetic females when compared to sexuals. IgM production differed between gynogens and sexuals of C. auratus complex. CONCLUSIONS: Our study indicates that a similar amount of energy is invested by both gynogenetic and sexual females of C. auratus complex in reproductive behaviour. We suggest that lower aerobic performance in gynogens may represent their physiological disadvantage balancing the cost of sexual reproduction. A trade-off between the number of erythrocytes and the oxygen-carrying capacity per erythrocyte in sexual males and gynogenetic females may contribute to the coexistence of gynogenetic and sexual forms. In addition, the differences in specific immunity between gynogens and sexuals may also reduce the evolutionary disadvantage of sexual reproduction. In conclusion, we propose that several mechanisms contribute to the coexistence of the gynogenetic-sexual C. auratus complex.

Adaptive phylogeography: functional divergence between haemoglobins derived from different glacial refugia in the bank vole.

Over the years, researchers have used presumptively neutral molecular variation to infer the origins of current species' distributions in northern latitudes (especially Europe). However, several reported examples of genic and chromosomal replacements suggest that end-glacial colonizations of particular northern areas may have involved genetic input from different source populations at different times, coupled with competition and selection. We investigate the functional consequences of differences between two bank vole (Clethrionomys glareolus) haemoglobins deriving from different glacial refugia, one of which partially replaced the other in Britain during end-glacial climate warming. This allows us to examine their adaptive divergence and hence a possible role of selection in the replacement. We determine the amino acid substitution Ser52Cys in the major expressed ß-globin gene as the allelic difference. We use structural modelling to reveal that the protein environment renders the 52Cys thiol a highly reactive functional group and we show its reactivity in vitro. We demonstrate that possessing the reactive thiol in haemoglobin increases the resistance of bank vole erythrocytes to oxidative stress. Our study thus provides striking evidence for physiological differences between products of genic variants that spread at the expense of one another during colonization of an area from different glacial refugia.

Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

The Influence of Selected Insecticides on the Oxidative Response of Atta sexdens (Myrmicinae, Attini) Workers.

Reactive oxygen species (ROS) are generated as products of normal cellular metabolic activities; however, the use of pesticides to control leafcutter ants leads to unbalanced ROS production. We evaluated the effects of two insecticides (fipronil, sulfluramid) and metallic insecticide complex (magnesium complex [Mg(hesp)2(phen)] (1)) on the superoxide dismutase (SOD), glutathione (GSH) and the overall antioxidant capacity using two different methodologies: total radical-trapping potential (TRAP) and oxygen radical absorbance capacity (ORAC). Media workers of Atta sexdens (C. Linnaeus) were exposed to the insecticides for 24 h, 48 h, 72 h and 96 h before their fat bodies were dissected for analysis. The results showed that although the sulfluramid may cause the production of ROS, its slow action in the organism does not lead to oxidative stress. There is a rise in oxidative stress in workers of leafcutter ants treated with fipronil because SOD significantly increased when compared to the control group. On the other hand, Mg1-complex suppressed both GSH and SOD, indicating that the immune system may be affected by Mg1-complex, which has a delayed activity ideal for its use in chemical pest control. Both TRAP and ORAC evaluated total antioxidant capacities; however, ORAC proved to be a more sensitive method. In conclusion, the Mg1-complex is a new compound that should be further investigated as a potential replacement for fipronil and sulfluramid in pest control.

Omics-based analysis of honey bee (Apis mellifera) response to Varroa sp. parasitisation and associated factors reveals changes impairing winter bee generation.

The extensive annual loss of honey bees (Apis mellifera L.) represents a global problem affecting agriculture and biodiversity. The parasitic mite Varroa destructor, associated with viral co-infections, plays a key role in this loss. Despite years of intensive research, the complex mechanisms of Varroa - honey bee interaction are still not fully defined. Therefore, this study employed a unique combination of transcriptomic, proteomic, metabolomic, and functional analyses to reveal new details about the effect of Varroa mites and naturally associated factors, including viruses, on honey bees. We focused on the differences between Varroa parasitised and unparasitised ten-day-old worker bees collected before overwintering from the same set of colonies reared without anti-mite treatment. Supplementary comparison to honey bees collected from colonies with standard anti-Varroa treatment can provide further insights into the effect of a pyrethroid flumethrin. Analysis of the honey bees exposed to mite parasitisation revealed alterations in the transcriptome and proteome related to immunity, oxidative stress, olfactory recognition, metabolism of sphingolipids, and RNA regulatory mechanisms. The immune response and sphingolipid metabolism were strongly activated, whereas olfactory recognition and oxidative stress pathways were inhibited in Varroa parasitised honey bees compared to unparasitised ones. Moreover, metabolomic analysis confirmed the depletion of nutrients and energy stores, resulting in a generally disrupted metabolism in the parasitised workers. The combined omics-based analysis conducted on strictly parasitised bees revealed the key molecular components and mechanisms underlying the detrimental effects of Varroa sp. and its associated pathogens. This study provides the theoretical basis and interlinked datasets for further research on honey bee response to biological threats and the development of efficient control strategies against Varroa mites.

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.

Pathogen entrapment by transglutaminase--a conserved early innate immune mechanism.

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.

1H NMR Profiling of Honey Bee Bodies Revealed Metabolic Differences between Summer and Winter Bees.

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.

Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

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 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.

Assessing relationships between chemical exposure, parasite infection, fish health, and fish ecological status: a case study using chub (Leuciscus cephalus) in the Bílina River, Czech Republic.

Multiple stressor scenarios, as they are relevant in many watersheds, call for approaches extending beyond conventional chemical-focused approaches. The present study, investigated the fish population, represented by chub (Leuciscus cephalus), in the Bílina River (Czech Republic), which is impacted by various pollution sources and might pose a risk on the fish population. To confirm or reject this hypothesis it was examined whether there exists an association between abundance of chub and exposure to toxic chemicals as well as natural stressors, represented by parasites, and whether health-related suborganismal traits, namely, organ indices, tissue histopathology, and immune parameters, would help in revealing relationships between stressor impact and population status. Toxic pressure was assessed by the toxic unit approach, which gives an integrative estimate of toxic effect concentrations and by measuring the biomarkers cytochrome P4501A and vitellogenin, which indicate exposure to bioavailable arylhydrocarbon- or estrogen receptor ligands. Parasite pressure was estimated by determining abundance and species composition of ecto- and endoparasites of chub. Chub abundance was high upstream in the Bílina, low to zero in the middle stretches, and increased again downstream. Toxic pressure increased in the downstream direction, while parasite intensity decreased in this direction. Health status of chub did not differ clearly between up-, middle-, and downstream sites. Thus, it appears that neither toxic pressure nor parasite pressure nor their combination translates into a change of chub health status. By using varied assessment tools, this study provides evidence against a presumed causative role of toxicants impairing the fish ecological status of the Bílina River.

Special Issue: Insects, Nematodes, and Their Symbiotic Bacteria.

This special issue contains articles that add to the ever-expanding toolbox of insect pathogenic nematodes (entomopathogenic nematodes; EPNs) as well articles that provide new insights into the mutualistic interaction between EPNs and their hosts. The study of natural infection models such as EPNs allows detailed insight into micro- and macro-evolutionary dynamics of innate immune reactions, including known but also emerging branches of innate immunity. Additional new insights into the kinetics of EPN infections are gained by increased spatiotemporal resolution of advanced transcriptome studies and live imaging.

Differences in the growth rate and immune strategies of farmed and wild mallard populations.

Individuals reared in captivity are exposed to distinct selection pressures and evolutionary processes causing genetic and phenotypic divergence from wild populations. Consequently, restocking with farmed individuals may represent a considerable risk for the fitness of free-living populations. Supportive breeding on a massive scale has been established in many European countries to increase hunting opportunities for the most common duck species, the mallard (Anas platyrhynchos). It has previously been shown that mallards from breeding facilities differ genetically from wild populations and there is some indication of morphological differences. Using a common-garden experiment, we tested for differences in growth parameters between free-living populations and individuals from breeding facilities during the first 20 days of post-hatching development, a critical phase for survival in free-living populations. In addition, we compared their immune function by assessing two haematological parameters, H/L ratio and immature erythrocyte frequency, and plasma complement activity. Our data show that farmed ducklings exhibit larger morphological parameters, a higher growth rates, and higher complement activity. In haematological parameters, we observed high dynamic changes in duckling ontogeny in relation to their morphological parameters. In conclusion, our data demonstrate pronounced phenotype divergence between farmed and wild mallard populations that can be genetically determined. We argue that this divergence can directly or indirectly affect fitness of farmed individuals introduced to the breeding population as well as fitness of farmed x wild hybrids.

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.

Bioactive Excreted/Secreted Products of Entomopathogenic Nematode Heterorhabditis bacteriophora Inhibit the Phenoloxidase Activity during the Infection.

Entomopathogenic nematodes (EPNs) are efficient insect parasites, that are known for their mutualistic relationship with entomopathogenic bacteria and their use in biocontrol. EPNs produce bioactive molecules referred to as excreted/secreted products (ESPs), which have come to the forefront in recent years because of their role in the process of host invasion and the modulation of its immune response. In the present study, we confirmed the production of ESPs in the EPN Heterorhabditis bacteriophora, and investigated their role in the modulation of the phenoloxidase cascade, one of the key components of the insect immune system. ESPs were isolated from 14- and 21-day-old infective juveniles of H. bacteriophora, which were found to be more virulent than newly emerged nematodes, as was confirmed by mortality assays using Galleria mellonella larvae. The isolated ESPs were further purified and screened for the phenoloxidase-inhibiting activity. In these products, a 38 kDa fraction of peptides was identified as the main candidate source of phenoloxidase-inhibiting compounds. This fraction was further analyzed by mass spectrometry and the de novo sequencing approach. Six peptide sequences were identified in this active ESP fraction, including proteins involved in ubiquitination and the regulation of a Toll pathway, for which a role in the regulation of insect immune response has been proposed in previous studies.