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The pacific oyster Crassostrea gigas and the Mediterranean mussel Mytilus galloprovincialis are two widely farmed bivalve species which show contrasting behaviour in relation to microbial diseases, with C. gigas being more susceptible and M. galloprovincialis being generally resistant. In a recent study, we showed that different susceptibility to infection exhibited by these two bivalve species may depend on their different capability to kill invading pathogens (e.g., Vibrio spp.) through the action of haemolymph components. Specific microbial-host interactions may also impact bivalve microbiome structure and further influence susceptibility/resistance to microbial diseases. To further investigate this concept, a comparative study of haemolymph and digestive gland 16SrDNA gene-based bacterial microbiota profiles in C. gigas and M. galloprovincialis co-cultivated at the same aquaculture site was carried out using pyrosequencing. Bacterial communities associated with bivalve tissues (hemolymph and digestive gland) were significantly different from those of seawater, and were dominated by relatively few genera such as Vibrio and Pseudoalteromonas. In general, Vibrio accounted for a larger fraction of the microbiota in C. gigas (on average 1.7-fold in the haemolymph) compared to M. galloprovincialis, suggesting that C. gigas may provide better conditions for survival for these bacteria, including potential pathogenic species such as V. aestuarianus. Vibrios appeared to be important members of C. gigas and M. galloprovincialis microbiota and might play a contrasting role in health and disease of bivalve species. Accordingly, microbiome analyses performed on bivalve specimens subjected to commercial depuration highlighted the ineffectiveness of such practice in removing Vibrio species from bivalve tissues.

Recently, it has become apparent that multiple factors are responsible for honey bee decline, including climate change, pests and pathogens, pesticides, and loss of foraging habitat. Of the large number of pathogens known to infect honey bees, very few are bacteria. Because adult workers abandon hives when diseased, many of their pathogens may go unnoticed. Here we characterized the virulence of Serratia marcescens strains isolated from honey bee guts and hemolymph. Our results indicate that S. marcescens , an opportunistic pathogen of many plants and animals, including humans, is a virulent opportunistic pathogen of honey bees, which could contribute to bee decline. Aside from the implications for honey bee health, the discovery of pathogenic S. marcescens strains in honey bees presents an opportunity to better understand how opportunistic pathogens infect and invade hosts. Although few honey bee diseases are known to be caused by bacteria, pathogens of adult worker bees may be underrecognized due to social immunity mechanisms. Specifically, infected adult bees typically abandon the hive or are removed by guards. Serratia marcescens , an opportunistic pathogen of many plants and animals, is often present at low abundance in the guts of honey bee workers and has recently been isolated from Varroa mites and from the hemolymph of dead and dying honey bees. However, the severity and prevalence of S. marcescens pathogenicity in honey bees have not been fully investigated. Here we characterized three S. marcescens strains isolated from the guts of honey bees and one previously isolated from hemolymph. In vivo tests confirmed that S. marcescens is pathogenic in workers. All strains caused mortality when a few cells were injected into the hemocoel, and the gut-isolated strains caused mortality when administered orally. In vitro assays and comparative genomics identified possible mechanisms of virulence of gut-associated strains. Expression of antimicrobial peptide and phenoloxidase genes was not elevated following infection, suggesting that these S. marcescens strains derived from honey bees can evade the immune response in their hosts. Finally, surveys from four locations in the United States indicated the presence of S. marcescens in the guts of over 60% of the worker bees evaluated. Taken together, these results suggest that S. marcescens is a widespread opportunistic pathogen of adult honey bees and that it may be highly virulent under some conditions such as perturbation of the normal gut microbiota or the presence of Varroa mites that puncture the integument, thereby enabling entry of bacterial cells. IMPORTANCE Recently, it has become apparent that multiple factors are responsible for honey bee decline, including climate change, pests and pathogens, pesticides, and loss of foraging habitat. Of the large number of pathogens known to infect honey bees, very few are bacteria. Because adult workers abandon hives when diseased, many of their pathogens may go unnoticed. Here we characterized the virulence of Serratia marcescens strains isolated from honey bee guts and hemolymph. Our results indicate that S. marcescens , an opportunistic pathogen of many plants and animals, including humans, is a virulent opportunistic pathogen of honey bees, which could contribute to bee decline. Aside from the implications for honey bee health, the discovery of pathogenic S. marcescens strains in honey bees presents an opportunity to better understand how opportunistic pathogens infect and invade hosts.

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

Microbiota provide their hosts with a range of beneficial services, including defense from external pathogens. However, host-associated microbial communities themselves can act as a source of opportunistic pathogens depending on the environment. Marine poikilotherms and their microbiota are strongly influenced by temperature, but experimental studies exploring how temperature affects the interactions between both parties are rare. To assess the effects of temperature, temperature stress and infection on diversity, composition and dynamics of the hemolymph microbiota of Pacific oysters ( Crassostrea gigas ), we conducted an experiment in a fully-crossed, three-factorial design, in which the temperature acclimated oysters (8 or 22 °C) were exposed to temperature stress and to experimental challenge with a virulent Vibrio sp. strain. We monitored oyster survival and repeatedly collected hemolymph of dead and alive animals to determine the microbiome composition by 16s rRNA gene amplicon pyrosequencing. We found that the microbial dynamics and composition of communities in healthy animals (including infection survivors) were significantly affected by temperature and temperature stress, but not by infection. The response was mediated by changes in the incidence and abundance of operational taxonomic units (OTUs) and accompanied by little change at higher taxonomic levels, indicating dynamic stability of the hemolymph microbiome. Dead and moribund oysters, on the contrary, displayed signs of community structure disruption, characterized by very low diversity and proliferation of few OTUs. We can therefore link short-term responses of host-associated microbial communities to abiotic and biotic factors and assess the potential feedback between microbiota dynamics and host survival during disease.

Metarhizium anisopliae infects mosquitoes through the cuticle and proliferates in the hemolymph. To allow M. anisopliae to combat malaria in mosquitoes with advanced malaria infections, we produced recombinant strains expressing molecules that target sporozoites as they travel through the hemolymph to the salivary glands. Eleven days after a Plasmodium-infected blood meal, mosquitoes were treated with M. anisopliae expressing salivary gland and midgut peptide 1 (SM1), which blocks attachment of sporozoites to salivary glands; a single-chain antibody that agglutinates sporozoites; or scorpine, which is an antimicrobial toxin. These reduced sporozoite counts by 71%, 85%, and 90%, respectively. M. anisopliae expressing scorpine and an [SM1]₈:scorpine fusion protein reduced sporozoite counts by 98%, suggesting that Metarhizium-mediated inhibition of Plasmodium development could be a powerful weapon for combating malaria.

Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.

Background The entomopathogenic fungus (EPF) Ophiocordyceps sinensis has a long-term coexistence with its host insect, Thitarodes xiaojinensis , making it a unique model for host–pathogen interactions. Hemolymph, a critical component in insects, plays an essential role in maintaining both nutritional and immune homeostasis. However, the mechanism of the host’s immune response remains unclear when O. sinensis proliferates in the hemolymph. Results O. sinensis caused damage to the insect’s intestinal barrier, facilitating the translocation of gut bacteria into the hemocoel. Subsequently, the presence of O. sinensis and opportunistic pathogenic bacteria from the gut disrupted the homeostasis of the hemolymph microbiota, resulting in an increase in bacterial diversity. This disruption triggered a series of physiological responses in the host, including elevated levels of endocrine hormones specifically 20-hydroxyecdysone (20E) and juvenile hormone 3 (JH3). Additionally, there was an enhancement of antioxidant capacity, as indicated by increased total antioxidant capacity and glutathione S-transferase activity, along with the production of antimicrobial peptides (AMPs) as part of the immune defense. Notably, the rise in 20E levels during O. sinensis infection might have significantly contributed to the increased production of AMPs. Conclusions O. sinensis infection significantly alters T. xiaojinensis physiology. Humoral immunity in infected hosts is primarily in response to hemolymph microbial homeostasis due to intestinal translocation. Among them, 20E upregulates AMP-related genes, suggesting a key immune strategy for managing microbial imbalances while tolerating fungal pathogens.

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.

Microplastic (MP) contamination in the aquatic environment is a cause of concern worldwide since MP can be taken up by different organisms, altering different biological functions. In particular, evidence is accumulating that MP can affect the relationship between the host and its associated microbial communities (the microbiome), with potentially negative health consequences. Synthetic microfibers (MFs) represent one of the main MPs in the marine environment, which can be accumulated by filter-feeding invertebrates, such as bivalves, with consequent negative effects and transfer through the food chain. In the mussel Mytilus galloprovincialis, polyethylene terephthalate (PET) MFs, with a size distribution resembling that of an MF released from textile washing, have been previously shown to induce multiple stress responses. In this work, in the same experimental conditions, the effects of exposure to PET-MF (96 h, 10, and 100 μg/L) on mussel hemolymph microbiome were evaluated by 16S rRNA gene amplification and sequencing. The results show that PET-MF affects the composition of bacterial communities at the phylum, family and genus level, with stronger effects at the lowest concentration tested. The relationship between MF-induced changes in hemolymph microbial communities and responses observed at the whole organism level are discussed.

are widespread in coastal environments, where they are being increasingly associated with mortality episodes of farmed bivalves. This is the case for strain r172 isolated from a mortality event of adult mussels in Spain in 2022. In this study, the infection dynamics and immune responses of adult challenged with r172 were investigated using different experimental approaches. First, a virulence assay by injection (1 x 108 CFU/100 µL) was performed at different temperatures (12, 18 and 24 °C). The results showed that mussel mortality (about 50 % within 8 days) was independent of increasing temperatures. Subsequently, an injection/cohabitation experiment was carried out placing together in the same tank -injected (Donors, 108 CFU/mL) with un-injected mussels (Recipients). The time course of infection was then followed by evaluating positivity to by PCR, responses of haemolymph components (haemocyte lysosomal membrane stability and serum lysozyme activity) and tissue histopathology (gills and digestive gland). The results showed a partial horizontal transfer of from infected to uninfected mussels, with transient effects on haemolymph responses and histopathological lesions in both groups. Finally, in order to mimic more realistic environmental conditions, a bath infection experiment was carried out, exposing mussels to in seawater (105 CFU/mL). This condition resulted in lower stress in haemocytes; moreover, no lysozyme release or histopathological alterations were observed. Overall, the results show that is able to cope with challenge with , indicating that this Vibrio species is moderately pathogenic to adult mussels under the established experimental conditions.