Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

Investigating bacterial infections in Galleria mellonella larvae: Insights into pathogen dissemination and behavior.

The insect Galleria mellonella is an alternative animal model widely used for studying bacterial infections. It presents a wide range of advantages, including its low cost, easy maintenance and lack of ethical constraints. Among other features, their innate immune system is very similar to that of mammals. In this study, we dissected several larvae infected with important human pathogens: Mycobacterium abscessus, Staphylococcus aureus and Pseudomonas aeruginosa. By observing the fat body, gut, trachea, and hemolymph under the microscope, we were able to describe where bacteria tend to disseminate. We also quantified the number of bacteria in the hemolymph throughout the infection course and found significant differences between the different pathogens. With this work, we aimed to better understand the behavior and dissemination of bacteria in the infected larvae.

Acute melanization of silkworm hemolymph by peptidoglycans of the human commensal bacterium Cutibacterium acnes.

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.

Host-pathogen interactions upon Candida auris infection: fungal behaviour and immune response in Galleria mellonella.

Candida auris has globally emerged as a multidrug-resistant fungus linked to healthcare-associated outbreaks. There is still limited evidence on its virulence, pathogenicity determinants, and complex host-pathogen interactions. This study analyzes the in vivo fungal behaviour, immune response, and host-pathogen interactions upon C. auris infection compared to C. albicans and C. parapsilosis in G. mellonella. This was performed by immunolabelling fungal structures and larval plasmatocytes and using a quantitative approach incorporating bioinformatic morphometric techniques into the study of microbial pathogenesis. C. auris presents a remarkably higher immunogenic activity than expected at its moderate degree of tissue invasion. It induces a greater inflammatory response than C. albicans and C. parapsilosis at the expense of plasmatocyte nodule formation, especially in non-aggregative strains. It specifically invades the larval respiratory system, in a pattern not previously observed in other Candida species, and presents inter-phenotypic tissue tropism differences. C. auris filaments in vivo less frequently than C. albicans or C. parapsilosis mostly through pseudohyphal growth. Filamentation might not be a major pathogenic determinant in C. auris, as less virulent aggregative phenotypes form pseudohyphae to a greater extent. C. auris has important both interspecific and intraspecific virulence and phenotype heterogeneity, with aggregative phenotypes of C. auris sharing characteristics with low pathogenic species such as C. parapsilosis. Our work suggests that C. auris owns an important morphogenetic plasticity that distinguishes it from other yeasts of the genus. Routine phenotypic identification of aggregative or non-aggregative phenotypes should be performed in the clinical setting as it may impact patient management.

The pathological effects of a Nosema ceranae infection in the giant honey bee, Apis dorsata Fabricius, 1793.

Nosema ceranae is an intracellular microsporidian pathogen that lives in the midgut ventricular cells of all known honey bee Apis species. We suspect that N. ceranae may also cause energetic stress in the giant honey bee because this parasite is known to disrupt nutrient absorption resulting in energetic stress in the honey bee species Apis mellifera. To understand how N. ceranae impacts the energetic stress of the giant honey bee, A. dorsata, we measured the hemolymph trehalose levels of experimentally infected giant honey bees on days three, five, seven, and fourteen post infection (p.i.). We also measured the hypopharyngeal gland protein content, the total midgut proteolytic enzyme activity, honey bee survival, infection ratio, and spore loads comparing infected and uninfected honey bees across the same time frame. Nosema ceranae-infected honey bees had significantly lowered survival, trehalose levels, hypopharyngeal gland protein content, and midgut proteolytic enzyme activity. We found an increasing level of parasitic loads and infection ratio of N. ceranae-infected bees after inoculation. Collectively, our results suggest that the giant honey bee suffers from energetic stress and limited nutrient absorption from a N. ceranae infection, which results in lowered survival in comparison to uninfected honey bees. Our findings highlight that other honey bee species besides A. mellifera are susceptible to microsporidian pathogens that they harbor, which results in negative effects on health and survival. Therefore, these pathogens might be transmitted at a community level, in the natural environment, resulting in negative health effects of multiple honey bee species.

Microbiomes of an oyster are shaped by metabolism and environment.

Microbiomes can both influence and be influenced by metabolism, but this relationship remains unexplored for invertebrates. We examined the relationship between microbiome and metabolism in response to climate change using oysters as a model marine invertebrate. Oysters form economies and ecosystems across the globe, yet are vulnerable to climate change. Nine genetic lineages of the oyster Saccostrea glomerata were exposed to ambient and elevated temperature and PCO2 treatments. The metabolic rate (MR) and metabolic by-products of extracellular pH and CO2 were measured. The oyster-associated bacterial community in haemolymph was characterised using 16 s rRNA gene sequencing. We found a significant negative relationship between MR and bacterial richness. Bacterial community composition was also significantly influenced by MR, extracellular CO2 and extracellular pH. The effects of extracellular CO2 depended on genotype, and the effects of extracellular pH depended on CO2 and temperature treatments. Changes in MR aligned with a shift in the relative abundance of 152 Amplicon Sequencing Variants (ASVs), with 113 negatively correlated with MR. Some spirochaete ASVs showed positive relationships with MR. We have identified a clear relationship between host metabolism and the microbiome in oysters. Altering this relationship will likely have consequences for the 12 billion USD oyster economy.

Microbiome of the Successful Freshwater Invader, the Signal Crayfish, and Its Changes along the Invasion Range.

Increasing evidence denotes the role of the microbiome in biological invasions, since it is known that microbes can affect the fitness of the host. Here, we demonstrate differences in the composition of an invader's microbiome along the invasion range, suggesting that its microbial communities may affect and be affected by range expansion. Using a 16S rRNA gene amplicon sequencing approach, we (i) analyzed the microbiomes of different tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) of a successful freshwater invader, the signal crayfish, (ii) compared them to the surrounding water and sediment, and (iii) explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations. This indicates that they may be partly determined by population density, which was higher in the invasion core than in the invasion front. The highly diverse microbiome of exoskeletal biofilm was partly shaped by the environment (due to the similarity with the sediment microbiome) and partly by intrinsic crayfish parameters (due to the high proportion of exoskeleton-unique amplicon sequence variants [ASVs]), including the differences in invasion core and front population structure. Hemolymph had the most distinct microbiome compared to other tissues and differed between upstream (rural) and downstream (urban) river sections, indicating that its microbiome is potentially more driven by the effects of the abiotic environment. Our findings offer an insight into microbiome changes during dispersal of a successful invader and present a baseline for assessment of their contribution to an invader's overall health and its further invasion success. IMPORTANCE Invasive species are among the major drivers of biodiversity loss and impairment of ecosystem services worldwide, but our understanding of their invasion success and dynamics still has many gaps. For instance, although it is known that host-associated microbial communities may significantly affect an individual's health and fitness, the current studies on invasive species are mainly focused on pathogenic microbes, while the effects of the remaining majority of microbial communities on the invasion process are almost completely unexplored. We have analyzed the microbiome of one of the most successful crayfish invaders in Europe, the signal crayfish, and explored its changes along the signal crayfish invasion range in the Korana River, Croatia. Our study sets the perspective for future research required to assess the contribution of these changes to an individual's overall health status and resilience of dispersing populations and their impact on invasion success.

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean.

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.

Etiological characteristics of "tail blister disease" of Australian redclaw crayfish (Cherax quadricarinatus).

In November 2019, an acute disease outbreak in Australian redclaw crayfish (Cherax quadricarinatus) occurred in a farm in Hubei, China, with a cumulative mortality rate of over 80%. One of the characteristic symptoms of the disease was blisters on the tail. This symptom is also common in diseased Procambarus clarkii every year in this country, but the causative agent has not been determined. This study analyzed the etiological characteristics of this disease. Bacterial isolation and identification combined with high-throughput sequencing analysis were conducted to obtain the microbiota characteristics in the hemolymph, hepatopancreas, and intestines. Results showed that this outbreak was caused by infection from Aeromonas hydrophila and Aeromonas veronii. The underlying cause was stress imposed on crayfish during transferring from outdoor pond to indoor pond because of temperature drops. Aeromonas infection caused remarkable changes in the structure of the microbial composition in the hemolymph, hepatopancreas, and intestines of the crayfish. The abundance of Aeromonas in the hemolymph of the sick crayfish was as high as 99.33%. In particular, KEGG metabolic pathway analysis showed that some antibiotic synthesis, enterobactin biosynthesis, and myo-inositol degradation pathways were abundant in healthy crayfish hemolymphs, which may be the mechanism of maintaining crayfish health. Conversely, inhibition of these pathways led to the disorder of microbiota structure, finally leading to the occurrence of diseases. To the knowledge of the authors, this study was the first to use high-throughput amplicon sequencing targeting the 16S rRNA gene to find the causative bacteria in aquatic animals. This protocol can provide more comprehensive and reliable evidence for pathogen identification, even if the pathogenic bacteria are anaerobes or other hard-to-culture bacteria.

The Enhanced Immune Protection in Small Abalone Haliotis diversicolor Against a Secondary Infection With Vibrio harveyi.

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

Identification and analysis of the biological activity of the new strain of Pseudoalteromonas piscicida isolated from the hemal fluid of the bivalve Modiolus kurilensis (F. R. Bernard, 1983).

A cultivated form of bacteria (strain 2202) was isolated from the hemal fluid of the bivalve mollusk Modiolus kurilensis. Based on the set of data collected by genetic and physiological/biochemical analyses, the strain was identified as the species Pseudoalteromonas piscicida. Strain 2202 exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans, and Bacillus subtilis but not against Escherichia coli and Pseudomonas aeruginosa. These activities characterize the behavior of strain 2202 as predator-like and classify it as a facultative predator. Being part of the normal microflora in the hemolymph of M. kurilensis, when external conditions change, strain 2202 shows features of opportunistic microflora. The strain 2202 exhibits selective toxicity towards larvae of various invertebrates: it impairs the early development of Mytilus edulis, but not of Strongylocentrotus nudus. Thus, the selective manner in which P. piscicida strains interact with various species of microorganisms and eukaryotes should be taken into consideration when using their biotechnological potential as a probiotic in aquaculture, source of antimicrobial substances, and factors that prevent fouling.

Cellular Immune Response of an Endemic Lake Baikal Amphipod to Indigenous Pseudomonas sp.

Studies of invertebrates have shown that the internal environment of crustaceans is not always sterile in normal conditions, and in many species, it can be populated by microorganisms even in the absence of any visible pathological processes in the body. This observation raises the question of whether genetically modified indigenous hemolymph microorganisms can be used for biotechnological purposes inside the crustacean either as local producers of some compounds or as sensors to physiological parameters. In this study, we tested the ability of the bacteria isolated from the hemolymph of the amphipod Eulimnogammarus verrucosus to hide from the cellular immune response of the host as the most important feature for their potential long-term application in vivo. 16S rDNA amplicon sequencing revealed five common bacterial genera in all analyzed samples of the amphipod hemolymph, among which Pseudomonas is most easily subjected to genome modification and, thus, the most prospective for biotechnological application. Cultivation of Pseudomonas gave us a number of strains undoubtedly derived from the amphipod hemolymph, and one of them (belonging to the Pseudomonas fluorescens group) was chosen for further tests. The primary culture of amphipod hemocytes was used to analyze the immunogenicity of the strain and showed a pronounced reaction of the immune cells to a high amount of the bacteria within six hours. This result indicates that modulation of cellular immune response to metabolically active bacterial cells is not mandatory for the survival and wide distribution of these microorganisms in the hemolymph of numerous amphipod individuals.

First record of a new microsporidium pathogenic to Gonipterus platensis in Brazil.

Microsporidia are naturally occurring fungal-related parasites that can infect nearly all animal hosts, but their biocontrol potential of insect pests is routinely overlooked in agriculture and forestry. This research brings the first report describing the natural occurrence of a microsporidium causing disease in field-collected populations of the invasive eucalyptus snout beetle, Gonipterus platensis (Coleoptera: Curculionidae), a major destructive pest of eucalyptus plantations in Brazil. Adult beetles were collected during field surveys in commercial eucalyptus plantations in southern Brazil to be examined and dissected with typical symptoms to verify presence of microsporidian spores in haemolymph. From 14 plantations in different sites, the natural infection occurrence in these populations ranged from 0 to 65%, while a lab colony exhibited an infection incidence of 70%. Spore density in haemolymph of symptomatic insects averaged 2.1 (± 0.4) × 107 spores/beetle. Symptoms in infected adults were identified by an abnormal abdomen with malformation of the second pair of wings, impairing their flight activity. Electron transmission microscopy of the pathogen showed morphological features similar to species belonging to the genus Nosema or Vairimorpha. Phylogenetic analysis of the full-length small subunit ribosomal RNA gene suggests this pathogen's placement in the genus Vairimorpha, but with a sequence identity of ~ 94% with the nearest neighbours. The low level of sequence identity suggests this pathogen may represent a novel taxon in the genus and further requires whole genome sequencing for definitive taxonomic resolution. These findings provide insights on the natural occurrence of this novel pathogen of this invasive pest in Eucalyptus plantations in Brazil. Further studies are needed to determine potential of this microsporidium in the design of conservative or augmentative biological control programs for this invasive pest.

Proteomic Analysis of the Hemolymph After Metschnikowia bicuspidata Infection in the Chinese Mitten Crab Eriocheir sinensis.

The "milky disease" of the Chinese mitten crab, Eriocheir sinensis, is a highly lethal fungal disease caused by Metschnikowia bicuspidata infection. To elucidate the immune responses of the hemolymph of E. sinensis to M. bicuspidata infection, a comparative analysis of the hemolymph of E. sinensis infected with M. bicuspidata and that treated with phosphate buffered saline was performed using label-free quantitative proteomics. A total of 429 proteins were identified. Using a 1.5-fold change in expression as a physiologically significant benchmark, 62 differentially expressed proteins were identified, of which 38 were significantly upregulated and 24 were significantly downregulated. The upregulated proteins mainly included cytoskeleton-related proteins (myosin regulatory light chain 2, myosin light chain alkali, tubulin α-2 chain, and tubulin ß-1 chain), serine protease and serine protease inhibitor (clip domain-containing serine protease, leukocyte elastase inhibitor, serine protein inhibitor 42Dd), catalase, transferrin, and heat shock protein 70. Upregulation of these proteins indicated that phenoloxidase system, phagocytosis and the ROS systems were induced by M. bicuspidata. The downregulated proteins were mainly organ and tissue regeneration proteins (PDGF/VEGF-related factor protein, integrin-linked protein kinase homing pat-4 gene) and hemagglutination-associated proteins (hemolymph clottable protein, hemocyte protein-glutamine gamma-glutamyltransferase). Downregulation of these proteins indicated that M. bicuspidata inhibited hemocyte regeneration and hemolymph agglutination. Fifteen differentially expressed proteins related to immunity were verified using a parallel reaction monitoring method. The expression trend of these proteins was similar to that of the proteome. To the best of our knowledge, this is the first report on the proteome of E. sinensis in response to M. bicuspidata infection. These results not only provide new and important information on the immune response of crustaceans to yeast infection but also provide a basis for further understanding the molecular mechanism of complex host pathogen interactions between crustaceans and fungi.

Dual proteomics of Drosophila melanogaster hemolymph infected with the heritable endosymbiont Spiroplasma poulsonii.

Insects are frequently infected with heritable bacterial endosymbionts. Endosymbionts have a dramatic impact on their host physiology and evolution. Their tissue distribution is variable with some species being housed intracellularly, some extracellularly and some having a mixed lifestyle. The impact of extracellular endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however difficult to appreciate because biofluid composition can depend on the contribution of numerous tissues. Here we investigate Drosophila hemolymph proteome changes in response to the infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly hemolymph and gets vertically transmitted over generations by hijacking the oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered a weak, chronic activation of the Toll immune pathway by S. poulsonii that was previously undetected by transcriptomics-based approaches. Using Drosophila genetics, we also identified candidate proteins putatively involved in controlling S. poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in combination with previously published transcriptomics data, improves our understanding of the post-transcriptional regulations operating in this bacterium.

Pathogenicity of Metarhizium rileyi against Spodoptera litura larvae: Appressorium differentiation, proliferation in hemolymph, immune interaction, and reemergence of mycelium.

The pathogenicity of Metarhizium rileyi is a multi-faceted process that depends on many factors. This study attempts to decipher those factors of M. rileyi by investigating its pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae) larvae. Through morphogenesis analysis, we for the first time demonstrated the infection structure, appressorium, of M. rileyi that can generate a more than 4 MPa turgor pressure. The Mrpmk1 gene was found to be essential for appressorium differentiation and mycelium reemerging, ΔMrpmk1 mutant exhibited no pathogenicity towards S. litura by natural infection process. Delayed appressorium formation time, decreased appressorium formation rate and turgor pressure of ΔMrpbs2 mutant manifested itself in postponed death time and lower mortality against S. litura. Following invasion into the larval hemocoel, M. rileyi cells transformed into blastospores, which may be conducive to dispersal and propagation, moreover, the blastospore form M. rileyi may subverted phagocytic defenses. Then M. rileyi cells morphed into extended hyphal body to cope with elongated hemocytes that participated in encapsulation. In the end, M. rileyi mycelia reemerged from the larval cadaver evenly to form muscardine cadaver. Eventually, conidia were produced to complete the infection cycle. During the infection, M. rileyi triggered both cellular and humoral immunity of S. litura. Besides morphological changes, stage-specifically produced oxalic acid and F-actin arrangement may play roles in nutrient acquisition and mycelium reemerging, respectively.

Analysis of nonribosomal peptide synthetase genes in haemolymph microbes of marine crabs.

Nonribosomal peptide synthetases (NRPS) are multi-domain enzymes that have innumerably beneficial health applications. Realizing the significance of marine microorganisms in search for NRPS sequences, study was conducted for analysis of NRPS gene sequences of marine crab haemolymph bacteria for the first time. Strains belonging to five different species were found to have NRPS genes. The study generated NRPS sequences from four bacterial species, for which NRPS gene information was not available earlier. Two new putative adenylation domain signatures were identified from phylum Firmicutes. In silico analysis of amino acid sequences from four species showed less identity (42-50%) to the characterized NRPS compounds that integrate serine residue in active site, suggesting the novelty or uncharacterized nature. Altogether, the study warrants future research exploiting marine crab haemolymph bacteria, an unexplored niche of microbial genetic wealth to discover microbial novel NRPS genes and natural products using emerging tools and technologies.

Alterins Produced by Oyster-Associated Pseudoalteromonas Are Antibacterial Cyclolipopeptides with LPS-Binding Activity.

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins' mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.