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.

Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

BACKGROUND: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. METHODS AND RESULTS: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. CONCLUSIONS: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

Hemolymph metabolism of black soldier fly (Diptera: Stratiomyidae), response to different supplemental fungi.

The black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), is commonly used for organic waste recycling and animal feed production. However, the often inadequate nutrients in organic waste necessitate nutritional enhancement of black soldier fly larvae, e.g., by fungal supplementation of its diet. We investigated the amino acid composition of two fungi, Candida tropicalis (Castell.) Berkhout (Saccharomycetales: Saccharomycetaceae) and Pichia kudriavzevii Boidin, Pignal & Besson (Saccharomycetales: Pichiaceae), from the black soldier fly gut, and commercial baker's yeast, Saccharomyces cerevisiae Meyen ex E.C. Hansen (Saccharomycetales: Saccharomycetaceae), and their effects on larval growth and hemolymph metabolites in fifth-instar black soldier fly larvae. Liquid chromatography-mass spectrometry was used to study the effect of fungal metabolites on black soldier fly larval metabolism. Amino acid analysis revealed significant variation among the fungi. Fungal supplementation led to increased larval body mass and differential metabolite accumulation. The three fungal species caused distinct metabolic changes, with each over-accumulating and down-accumulating various metabolites. We identified significant alteration of histidine metabolism, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism in BSF larvae treated with C. tropicalis. Treatment with P. kudriavzevii affected histidine metabolism and citrate cycle metabolites, while both P. kudriavzevii and S. cerevisiae treatments impacted tyrosine metabolism. Treatment with S. cerevisiae resulted in down-accumulation of metabolites related to glycine, serine, and threonine metabolism. This study suggests that adding fungi to the larval diet significantly affects black soldier fly larval metabolomics. Further research is needed to understand how individual amino acids and their metabolites contributed by fungi affect black soldier fly larval physiology, growth, and development, to elucidate the interaction between fungal nutrients and black soldier fly physiology.

Toxic effects of acaricide fenazaquin on development, hemolymph metabolome, and gut microbiome of honeybee (Apis mellifera) larvae.

Fenazaquin, a potent insecticide widely used to control phytophagous mites, has recently emerged as a potential solution for managing Varroa destructor mites in honeybees. However, the comprehensive impact of fenazaquin on honeybee health remains insufficiently understood. Our current study investigated the acute and chronic toxicity of fenazaquin to honeybee larvae, along with its influence on larval hemolymph metabolism and gut microbiota. Results showed that the acute median lethal dose (LD50) of fenazaquin for honeybee larvae was 1.786 µg/larva, and the chronic LD50 was 1.213 µg/larva. Although chronic exposure to low doses of fenazaquin exhibited no significant effect on larval development, increasing doses of fenazaquin resulted in significant increases in larval mortality, developmental time, and deformity rates. At the metabolic level, high doses of fenazaquin inhibited nucleotide, purine, and lipid metabolism pathways in the larval hemolymph, leading to energy metabolism disorders and physiological dysfunction. Furthermore, high doses of fenazaquin reduced gut microbial diversity and abundance, characterized by decreased relative abundance of functional gut bacterium Lactobacillus kunkeei and increased pathogenic bacterium Melissococcus plutonius. The disrupted gut microbiota, combined with the observed gut tissue damage, could potentially impair food digestion and nutrient absorption in the larvae. Our results provide valuable insights into the complex and diverse effects of fenazaquin on honeybee larvae, establishing an important theoretical basis for applying fenazaquin in beekeeping.

Acid-treated Staphylococcus aureus induces acute silkworm hemolymph melanization.

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis. Staphylococcus aureus and Cutibacterium acnes are pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized that S. aureus and C. acnes interact to increase the innate immunity-activating properties of S. aureus. In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization by S. aureus. Autoclaved-killed S. aureus (S. aureus [AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applying S. aureus [AC] treated with C. acnes culture supernatant increased the silkworm hemolymph melanization. Adding C. acnes culture supernatant to the medium decreased the pH. S. aureus [AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreated S. aureus [AC]. S. aureus [AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity of S. aureus [AC] treated with hydrochloric acid was inhibited by protease treatment of S. aureus [AC]. These results suggest that acid treatment of S. aureus induces innate immune activation in silkworms and that S. aureus proteins are involved in the induction of innate immunity in silkworms.

Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Refinement of a technique for collecting and evaluating the osmolality of haemolymph from Drosophila larvae.

Ex vivo physiological experiments using small insect models such as Drosophila larvae have become increasingly useful to address fundamental biological questions. To perform such experiments, various artificial saline solutions have been developed, but their osmolality varies significantly from one to the next. Such a variation of osmolality stems, in part, from the difficulty of determining the true value of haemolymph osmolality in Drosophila larvae. Thus, there is a pressing need to refine protocols for collecting and measuring the osmolality of the larval haemolymph. Two major obstacles are thought to impede the accurate analysis of haemolymph collected from small insects: melanin formation and gut-derived contamination. Here, we greatly refined existing haemolymph collection methods, evaluated the purity of the collected haemolymph under melanin-free conditions, and concluded that the true value of haemolymph osmolality is close to 306.0 mOsm kg-1 in Drosophila larvae.

Management of inorganic elements by overwintering physiology of cold hardy larvae of European corn borer (Ostrinia nubilalis, Hbn.).

The European corn borer (Ostrinia nubilalis, Hbn.), enters diapause, a strategy characterized by arrest of development and reproduction, reduction of metabolic rate and the emergence of increased resistance to challenging seasonal conditions as low sub-zero winter temperatures. The aim of this study was to investigate the potential role of inorganic elements in the ecophysiology of O. nubilalis, analysing their content in the whole body, hemolymph and fat body, both metabolically active, non-diapausing and overwintering diapausing larvae by ICP-OES spectrometer following the US EPA method 200.7:2001. O nubilalis as many phytophagous lepidopteran species maintain a very low extracellular sodium concentration and has potassium as dominant cation in hemolymph of their larvae. Changes in hemolymph and the whole body sodium content occur already at the onset of diapause (when the mean environmental temperatures are still high above 0 ºC) and remain stable during the time course of diapause when larvae of this species cope with sub-zero temperatures, it seems that sodium content regulation is rather a part of diapausing program than the direct effect of exposure to low temperatures. Compared to non-diapausing O. nubilalis larvae, potassium levels are much higher in the whole body and fat body of diapausing larvae and substantially increase approaching the end of diapause. The concentration of Ca, Mg, P and S differed in the whole body, hemolymph and fat body between non-diapausing and diapausing larvae without a unique trend during diapause, except an increase in their contents at the end of diapause.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.

Quantification of proteomic profile changes in the hemolymph of crayfish during in vitro coagulation.

Hemolymph is the circulatory fluid that fills the body cavity of crustaceans, analogous to blood in vertebrates. Hemolymph coagulation, similar to blood clotting in vertebrates, plays a crucial role in wound healing and innate immune responses. Despite extensive studies on the clotting process in crustaceans, no comparative quantitative analysis of the protein composition of non-clotted and clotted hemolymph in any decapod has been reported. In this study, we used label-free protein quantification with high-resolution mass spectrometry to identify the proteomic profile of hemolymph in crayfish and quantify significant changes in protein abundances between non-clotted and clotted hemolymph. Our analysis identified a total of two-hundred and nineteen proteins in both hemolymph groups. Furthermore, we discussed the potential functions of the top most high and low-abundant proteins in hemolymph proteomic profile. The quantity of most of the proteins was not significantly changed during coagulation between non-clotted and clotted hemolymph, which may indicate that clotting proteins are likely pre-synthesized, allowing for a swift coagulation response to injury. Four proteins still showed abundance differences (p < 0.05, fold change>2), including C-type lectin domain-containing proteins, Laminin A chain, Tropomyosin, and Reverse transcriptase domain-containing proteins. While the first three proteins were down-regulated, the last one was up-regulated. The down-regulation of structural and cytoskeletal proteins may affect the process of hemocyte degranulation needed for coagulation, while the up-regulation of an immune-related protein might be attributed to the phagocytosis ability of viable hemocytes during coagulation.

SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application.

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

Survival of Salmonella Typhimurium in the hemolymph of the German cockroach vector is limited by both humoral immune factors and hemocytes but not by trehalose metabolism.

The German cockroach (Blattella germanica) has been linked to transmission of Salmonella enterica serovar Typhimurium (S. Typhimurium), but infection dynamics within this vector are poorly characterized. Our recent work has focused on S. Typhimurium infection in the cockroach gut. However, microbial dissemination to the hemolymph is an essential aspect of many vector-borne pathogen transmission cycles and could potentially contribute to S. Typhimurium colonization of cockroaches. Therefore, the goal of this study was to examine the ability of S. Typhimurium to disseminate, survive, and proliferate in the hemolymph of cockroaches after oral infection. We detected only low numbers of bacteria in the hemolymph of a minority of insects (~26%) after oral infection. Further, S. Typhimurium was unable to survive overnight in cell-free hemolymph. Several hypotheses to explain the inability of S. Typhimurium to colonize hemolymph were tested. First, we investigated the ability of S. Typhimurium to metabolize trehalose, the primary sugar in hemolymph. S. Typhimurium grew efficiently in vitro using trehalose as a sole carbon source and mutant strains lacking trehalose metabolism genes exhibited no growth deficiencies in media mimicking the composition of hemolymph, suggesting that trehalose metabolism ability is not a factor involved in restricting survival in hemolymph. On the other hand, heat-inactivated cell-free hemolymph was permissive of S. Typhimurium growth, demonstrating that survival in hemolymph is limited specifically by heat-labile humoral factors. The involvement of cellular immune responses was also investigated and cockroach hemocytes in culture were observed to internalize S. Typhimurium within 1 h of exposure. Most hemocytes harbored few to no bacteria after 24 h, indicating that hemocyte responses are additionally involved in clearing infection from the hemolymph. However, dense intracellular clusters of S. Typhimurium were observed sporadically, suggesting a small subset of hemocytes may serve as reservoirs for bacterial replication. Together, our results reveal that a minute proportion of ingested S. Typhimurium is able to escape the cockroach gut and enter the hemolymph, but this systemic population is limited by both humoral effectors and hemocytes. Thus, we conclude that invasion of the hemolymph appears minimally important for colonization of the cockroach vector and that colonization of the gut is the main driver of vector-borne transmission. Our insight into the antimicrobial mechanisms of cockroach hemolymph also highlights the strong ability of these prevalent pests/vectors to cope with frequent infectious challenges in septic habitats.

Es-ß-CATENIN affects the hemolymph-testes barrier in Eriocheir sinensis by disrupting cell junctions and cytoskeleton.

ß-CATENIN is an evolutionarily conserved multifunctional molecule that maintains cell adhesion as a cell junction protein to safeguard the integrity of the mammalian blood-testes barrier, and also regulates cell proliferation and apoptosis as a key signaling molecule in the WNT/ß-CATENIN signaling pathway. In the crustacean Eriocheir sinensis, Es-ß-CATENIN has been shown to be involved in spermatogenesis, but the testes of E. sinensis have large and well-defined structural differences from those of mammals, and the impact of Es-ß-CATENIN in them is still unknown. In the present study, we found that Es-ß-CATENIN, Es-α-CATENIN and Es-ZO-1 interact differently in the testes of the crab compared to mammals. In addition, defective Es-ß-CATENIN resulted in increased Es-α-CATENIN protein expression levels, distorted and deformed F-ACTIN, and disturbed localization of Es-α-CATENIN and Es-ZO-1, leading to loss of hemolymph-testes barrier integrity and impaired sperm release. In addition to this, we also performed the first molecular cloning and bioinformatics analysis of Es-AXIN in the WNT/ß-CATENIN pathway to exclude the effect of the WNT/ß-CATENIN pathway on the cytoskeleton. In conclusion, Es-ß-CATENIN participates in maintaining the hemolymph-testes barrier in the spermatogenesis of E. sinensis.

Oxidative and genotoxic effect of piperazine on Galleria mellonella (Lepidoptera: Pyralidae) hemolymph.

Recently, there are many studies suggesting antibacterial, antifungal, and anthelmintic agents as alternative chemicals to insecticides. In this study, the oxidative and genotoxic effect of Piperazine, a clinically important hexahydropyrazine anthelmintic, on Galleria mellonella L. hemolymph tissue by adding artificial diet were investigated. Galleria mellonella larvae were reared until 7th larval stage in artificial diet containing 0.001, 0.01, 0.1, and 1 g piperazine per 100 g of diet. Using hemolymph collected from 7th-instar larvae, the amount of lipid peroxidation final product malondialdehyde (MDA), protein oxidation product protein carbonyl (PCO), and detoxification enzymes glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cyt P450) activity, comet assay were measured. According to the results obtained, when the piperazine high concentrations tested with the control group were compared, statistically significant differences were found in MDA, PCO content, cyt P450, GST activity, and comet assay in the hemolymph of the insect. While MDA content was 0.01 ± 0.0021 nmol/mg protein in the control group, this amount increased approximately 2-fold at the highest concentration (0.0231 ± 0.0050 nmol/mg protein). On the other hand, when the control group and the highest piperazine concentration were compared in the GST and cyt P450 activity, it was determined that there was a statistically significant increase. We obtained similar results in comet assay and micronucleus formation data. This study showed that the tested piperazine concentrations caused significant changes in the detoxification capacity, oxidative stress, and genotoxic markers in the insect's hemolymph tissue.

Metabolomics analysis of dietary restriction results in a longer lifespan due to alters of amino acid levels in larval hemolymph of Bombyx mori.

Dietary restriction (DR) has been a very important discovery in modern aging biology research. Its remarkable anti-aging effect has been proved in a variety of organisms, including members of Lepidoptera, but mechanisms by which DR increases longevity are not fully understood. By using the silkworm (Bombyx mori), a model of lepidopteran insect, we established a DR model, isolated hemolymph from fifth instar larvae and employed LC-MS/MS metabolomics to analyze the effect of DR on the endogenous metabolites of silkworm, and tried to clarify the mechanism of DR to prolong lifespan. We identified the potential biomarkers by analyzing the metabolites of the DR and control groups. Then, we constructed relevant metabolic pathways and networks with MetaboAnalyst. DR significantly prolonged the lifespan of silkworm. The differential metabolites between the DR and control groups were mainly organic acids (including amino acid), and amines. These metabolites are involved in metabolic pathways such as amino acid metabolism. Further analysis showed that, the levels of 17 amino acids were significantly changed in the DR group, indicating that the prolonged lifespan was mainly due to changes in amino acid metabolism. Furthermore, we identified 41 and 28 unique differential metabolites in males and females, respectively, demonstrating sex differences in biological responses to DR. The DR group showed higher antioxidant capacity and lower lipid peroxidation and inflammatory precursors, with differences between the sexes. These results provide evidence for various DR anti-aging mechanisms at the metabolic level and novel reference for the future development of DR-simulating drugs or foods.

Bombyx mori as a model for Niallia circulans pathogenicity.

Increasing incidences of resistance to antibiotics by pathogenic bacteria is a worldwide concern and isolation of antibiotic-resistant strains of Niallia circulans (formerly known as Bacillus circulans), an opportunistic human pathogen, has been reported. Due to their lack of ethical constraints as well as their cost-effective rearing, invertebrates have been commonly used to study infection by bacteria pathogenic to humans. In this study, we demonstrate that a foodborne strain of N. circulans kills larvae of the silkworm, Bombyx mori within 48 h after hemolymph injection. The infected larvae turned black with an increase in the phenoloxidase (PO) activity in the hemolymph. Midgut injection of N. circulans resulted in the killing of larvae within 96 h. A significant increase in bacterial load was observed in the hemolymph 12 h after infection. The viable hemocyte number decreased to 48% within 12 h of injection. RT-qPCR analysis revealed that upon hemolymph infection with N. circulans the expression of the antimicrobial peptide (AMP) genes, Bmdefensin-B and Bmgloverin-3, were upregulated 2.5- and 1.8-fold, respectively, whereas 1.6-fold upregulation was observed for BmToll-2 in the larval fat body. Therapeutic effects of antibiotics like tetracycline, imipenem, ceftriaxone, ampicillin, and clindamycin were observed against N. circulans in the Bombyx larvae with varying efficacies. Results from this study suggest that larvae of B. mori can be used as infection models for screening therapeutics that are effective against N. circulans.

Purification and characterization of a hemocyanin with lectin-like activity isolated from the hemolymph of speckled shrimp, Metapenaeusmonoceros.

Lectins or agglutinins are mainly proteins or glycoproteins, reported to uphold an ability to agglutinate the red blood cells (RBCs) with a known sugar specificity in a diverse group of organisms. In the present study, we purified a hemocyanin (named as MmHc) from a shrimp, Metapenaeus monoceros by size-exclusion chromatography. Further characterization revealed that the purified MmHc showed hemagglutination activity that was found to be specifically inhibited by Lewis B and Lewis Y tetrasaccharides. The MmHc displayed two oligomers of molecular weight approximately ∼78 and ∼85 kDa in SDS-PAGE. The native molecular mass of MmHc was found to be ∼457 kDa as determined by size-exclusion chromatography which indicated that the purified MmHc is an oligomeric protein. MmHc showed a maximum activity within pH 7.0-8.0, while a wide range of temperature stability was observed between 4 to 55 °C, however, it did not show any dependency on metal ions for binding. Subsequently, the analysis of the peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) identified the purified MmHc as shrimp hemocyanin showing significant similarity to the hemocyanin of Penaeus vannamei. The results of multiple sequence alignment and detailed analysis of the molecular interactions predicted by AutoDock suggested that besides the oxygen carrier function, this MmHc may have multiple roles and can interact well with the Lewis Y antigen through a typical sugar binding motif containing the similar hydrophilic amino acids as the conserved residues.

Spätzle1 is a constituent of the extracellular signaling pathway that promotes nodule formation, a cell-mediated immune response, in caterpillar hemolymph.

The existence of an extracellular signaling pathway that mediates nodule formation, a cell-mediated immune response, has been reported in Bombyx mori larvae. In this pathway, C-type lectins and the hemolymph serine proteinase BmHP-8 function in pathogen associated molecular pattern (PAMPs) recognition and signaling transduction. However, which molecule elicits the cellular response at the end of the pathway is unknown. In this study, the Toll ligand Bombyx mori Spätzel1 was shown to be involved in the pathway by applying anit-Spätzel1 antiserum in an in vitro nodule-like aggregate formation assay and an in vivo nodule formation assay.