Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Scallop-bacteria symbiosis from the deep sea reveals strong genomic coupling in the absence of cellular integration.

Previous studies have revealed tight metabolic complementarity between bivalves and their endosymbiotic chemosynthetic bacteria, but little is known about their interactions with ectosymbionts. Our analysis of the ectosymbiosis between a deep-sea scallop (Catillopecten margaritatus) and a gammaproteobacterium showed that bivalves could be highly interdependent with their ectosymbionts as well. Our microscopic observation revealed abundant sulfur-oxidizing bacteria (SOB) on the surfaces of the gill epithelial cells. Microbial 16S rRNA gene amplicon sequencing of the gill tissues showed the dominance of the SOB. An analysis of the SOB genome showed that it is substantially smaller than its free-living relatives and has lost cellular components required for free-living. Genomic and transcriptomic analyses showed that this ectosymbiont relies on rhodanese-like proteins and SOX multienzyme complex for energy generation, mainly on the Calvin-Benson-Bassham (CBB) cycle and peripherally on a phosphoenolpyruvate carboxylase for carbon assimilation. Besides, the symbiont encodes an incomplete tricarboxylic acid (TCA) cycle. Observation of the scallop's digestive gland and its nitrogen metabolism pathways indicates it does not fully rely on the ectosymbiont for nutrition. Analysis of the host's gene expression provided evidence that it could offer intermediates for the ectosymbiont to complete its TCA cycle and some amino acid synthesis pathways using exosomes, and its phagosomes, endosomes, and lysosomes might be involved in harvesting nutrients from the symbionts. Overall, our study prompts us to rethink the intimacy between the hosts and ectosymbionts in Bivalvia and the evolution of chemosymbiosis in general.

Comprehensive Multi-omics Approaches Provide Insights to Summer Mortality in the Clam Meretrix petechialis.

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding.

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

IMPORTANCE: This study highlights diversity in iron acquisition and regulation in bacteria. The mechanisms of iron acquisition and its regulation in Teredinibacter turnerae, as well as its connection to cellulose utilization, a hallmark phenotype of T. turnerae, expand the paradigm of bacterial iron acquisition. Two of the four TonB genes identified in T. turnerae exhibit functional redundancy and play a crucial role in siderophore-mediated iron transport. Unlike typical TonB genes in bacteria, none of the TonB genes in T. turnerae are clearly iron regulated. This unusual regulation could be explained by another important finding in this study, namely, that the two TonB genes involved in iron transport are also essential for cellulose utilization as a carbon source, leading to the expression of TonB genes even under iron-rich conditions.

Sanctuaries are not inviolable: Haplosporidium pinnae as responsible for the collapse of the Pinna nobilis population in Lake Faro (central Mediterranean).

The rapid spread of the protozoan Haplosporidium pinnae is having a strong negative effect on Pinna nobilis populations. A case study on a residual population in Lake Faro (Sicily, Central Mediterranean), whose long-term monitoring has revealed a dramatic decline following the 2018-2020 mass mortality event, is presented. In the framework of such monitoring, we performed tissue sampling on nine living P. nobilis, detecting the pathogen in seven of them. In contrast, other pathogens associated with P. nobilis disease in other areas, i.e., Mycobacterium spp. and Vibrio mediterranei, were not recorded. The surviving individuals (approximately twenty) showed that brackish areas only weakly mitigate the effects of H. pinnae disease and might not be resolutive. Nevertheless, the results show that Lake Faro may constitute one of the last Mediterranean P. nobilis sanctuaries.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Fate of microbial contamination in a South European Coastal Lagoon (Ria Formosa) under the influence of treated effluents dispersal.

AIM: Assessment of the fate of microbial contamination driven from treated wastewater disposal at a highly productive zone on a South European coastal lagoon (Ria Formosa). METHODS AND RESULTS: Microbial indicators of contamination (Total coliforms, Escherichia coli, and Enterococci) were evaluated monthly during September 2018-September 2020 at three study areas (Faro, Olhão, and Tavira) under different wastewater discharge flows and hydrodynamic conditions. Additional data on E. coli monitoring in bivalves, available from the national institution responsible for their surveillance was also considered. The maximum microbial contamination was found at Faro, the highest-load and less-flushed study area, contrasting the lowest contamination at Olhão, a lower-load and strongly flushed area. The wastewater impact decreased along the spatial dispersal gradients and during high water, particularly at Faro and Tavira study areas, due to a considerable dilution effect. Microbial contamination at Olhão increased during the summer, while at the other study areas seasonal evidence was not clear. Data also indicate that E. coli in bivalves from bivalve production zones next to the three study areas reflected the differentiated impact of the wastewater treatment plants effluents on the water quality of those areas. CONCLUSIONS: Effluent loads together with local hydrodynamics, water temperature, solar radiation, precipitation, and land runoff as well as seabirds populations and environmentally adapted faecal or renaturelized bacterial communities, contributed to microbial contamination of the study areas.

A review on microbial contamination cases in Tunisian coastal marine areas.

Microbial pollution in marine environments is one of the critical issues with regard to the sanitary status of recreational activities and seafood harvesting due to a potential contamination by pathogenic microorganisms. This review's objectives were to identify instances of bacterial, viral and protozoan parasite pollution in the Tunisian coastal region and to make recommendations for further research. Fecal indicators such as Escherichia coli and Salmonella spp. were detected in samples of clams and mussels. Vibrionaceae species were also recorded in seawater, sediment, fish and clams in different sites from north to south with the dominance of Vibrio alginolyticus. Bivalve mollusks collected from the Tunisian coast have been revealed to harbor viruses as well as protozoan parasites. Furthermore, the isolation of multidrug-resistant bacterial strains from Tunisian coastlines proves the significant spread and circulation of antibiotic resistance caused by the massive use of antibiotics. In conclusion, we suggest intensive monitoring and cutting-edge wastewater treatment technologies to enhance seawater quality and preserve the biodiversity of aquatic life. Rapid detection techniques for the most important pathogenic microorganisms in seafood and seawater must be also developed to reduce human health risk.

Two common nanoparticles exert immunostimulatory and protective effects in Tegillarca granosa against Vibrio parahaemolyticus.

There are many studies revealed that metal-based nanoparticles (NPs) possess excellent bactericidal effect on multitudinous bacteria and fungi. However, the control effect of NPs as antimicrobial agents to against Vibrio parahaemolyticus infection remain in poorly understood for blood clam, Tegillarca granosa. In order to evaluate the effect, the changes in six physiological parameters and the immune-related genes expression of clams exposed to V. parahaemolyticus alone or along with NPs (nZnO or nCuO) were investigated in present study. Results showed that both tested NPs exerted prominent redemptive or mitigative effect in an inverse dose-dependent way on physiological indexes of clam, especially in the total counts, phagocytosis and the cell viability of haemocytes, as well as the concentration and activity of lysozymes, when co-exposed with Vibrio. Gene expression analysis showed NPs at a concentration of 0.1 mg/L generally mitigated the downregulation of immune-related genes after clam exposure to V. parahaemolyticus. The combination of 0.1 mg/mL nZnO and nCuO additives has been shown to significantly enhance the humoral immunity of blood clam, suggesting its potential as a protective measure against V. parahaemolyticus infection in T. granosa.

Investigation of the highly endangered Pinna nobilis' mass mortalities: Seasonal and temperature patterns of health status, antioxidant and heat stress responses.

Recently, P. nobilis populations have suffered a tremendous reduction, with pathogens potentially playing a crucial role. Considering its highly endangered status, mechanisms leading to mass mortalities were examined in one or multiple pathogens infected populations. Thus, seasonal antioxidant enzymatic activities, hsp70 and catalase mRNA levels, were investigated in two different Greek populations, during mass mortality events in summer of 2020. Samples were collected from Fthiotis and Lesvos during February (ToC 14 ± 1.2 and 15 ± 1 respectively), April (ToC 18 ± 1.2 and 17 ± 1.3 respectively), and June (ToC 24.5 ± 1.5 and 21.5 ± 1.5 respectively) 2020. In July of the same year (ToC 26.5 ± 1.7 in Fthiotis and 24.5 ± 1.7 in Lesvos), no live specimens were found. All biochemical parameters and phylogenetic analysis suggest that pathogen infection increases P. nobilis sensitivity to water temperature, subsequently leading to mass mortality. The latter was obvious in Fthiotis individuals, in which Haplosporidium pinnae was also observed with Mycobacterium spp., compared to Lesvos individuals.

Microbiological Quality of Oysters and Mussels Along Its Market Supply Chain.

Oysters and mussels are known vectors of foodborne pathogens because of their immobile and filter-feeding nature leading to the accumulation of biological particles in their tissues. Accumulated bacteria which comes from the culture environment and unsanitary handling can cause food poisoning if these shellfish are consumed raw or partially processed. This study determined the incidence of bacterial pathogen contamination along the different channels of the oyster and mussel supply chain through a time-distribution simulation analysis. First, the route of the fresh bivalve products from a local farm to its market was established through interviews. From the data gathered, a simulation experiment was conducted following the observed time-temperature conditions and the actual bulk packaging material used by the traders. The presence of target pathogens Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae were detected using standard conventional culture techniques. Initial E. coli counts in both mussels and oysters were higher than the safety limit of 330 MPN in 100 g tissue. Interestingly, E. coli counts in mussels decreased after 6 h and maintained low numbers after more than 24 h postharvest. Counts in oysters however increased to 1000 MPN in 100 g tissue. V. parahaemolyticus in mussels and oysters showed a gradual increase in counts with increasing holding time albeit in numbers that are lower than the safety limit of 1000 cfu g-1 tissue. Qualitative detection of Salmonella and V. cholerae showed the presence of both pathogens in all the sampling points. All four pathogens were also detected in the culture waters and in the sediment. Results of the study showed that the culture environment and the handling practices contribute greatly to the pathogen contamination in oysters and mussels.

A lanthanide-based high-sensitivity fluorescence method for the on-site rapid detection of thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Development of the phage lysin-loaded liposomes as preservatives for live clams.

Exogenous applications of phage lysins against Vibrio parahaemolyticus (V. parahaemolyticus) are a challenge due to the gram-negative bacteria outer membrane barrier. This study aimed to improve the antibacterial effect of V. parahaemolyticus phage lysin Lysqdvp001 (Lys), the best-characterized lysin with lytic activity against multiple species of Vibrios, by using liposome delivery. Various kinds of Lys-loaded liposome (Lys-lip) systems were designed and tested. The antibacterial activities of cationic guar gum (CGG) containing liposomes were much higher than the other liposomes, causing >5 log10CFU/mL of reductions of V. parahaemolyticus in buffer and severely damaging the bacterial cell structure. Moreover, some CGG liposome formulations retained high antibacterial effect after both 60-80 °C heat treatments and freeze-drying. Besides, the most stable liposome formulation killed 99 % of V. parahaemolyticus in the seawater with live clams, and its depuration rate against the bacterial contaminated clams also reached 99 %.

El mejillón Semimytilus patagonicus (Mytilida: Mytilidae) y el gusano Pseudonereis gallapagensis (Phyllodocida: Nereididae) como herramientas ecotoxicológica para la contaminación por detergentes en ambientes marinos / The mussel Semimytilus patagonicus (Mytilida: Mytilidae) and the worm Pseudonereis gallapagensis (Phyllodocida: Nereididae) as ecotoxicological tools for detergent pollution in marine environments

Introduction: The bivalve Semimytilus patagonicus is a potentially useful bioindicator because of its feeding mechanism, and the worm Pseudonereis gallapagensis is also interesting as a bioindicator because it is benthonic, abundant, and a food source for the squid Doryteuthis gahi. However, their sensitivity to contaminants has not been sufficiently studied. Objective: To test the usefulness of the mussel Semimytilus patagonicus and the polychaete Pseudonereis gallapagensis as ecotoxicological tools for detergents in the marine environment. Methods: We used 120 individuals of S. patagonicus from Miraflores and 120 of P. gallapagensis from Barranco (both near the city of Lima, Peru). For the bioassays, we used two anionic detergents (active ingredient, ai, Sodium Dodecylbenzene Sulfonate). For S. patagonicus, with an average valve length of 32.3 ± 6.4 mm, we tested "Double power Ariel®" (90 %) at concentrations of 17.5, 35, 70 and 140 mg ai l-1, evaluated after 48 and 72 h of exposure; and for P. gallapagensis, with a total body length of 20.4 ± 8.8 mm, we tested "Caricia®" at 62.5, 125, 250, 500 and 1 000 mg of ai l-1 at 24, 48 and 72 h of exposure. Results: The LC50 values (Mean Lethal Concentration) were 34.95 mg ia l-1 for S. patagonicus and 102.48 mg ia l-1 for P. gallapagensis at 72 h of exposure. The detergents were toxic for S. patagonicus and slightly toxic for P. gallapagensis. The risk classification for S. patagonicus is "harmful" and for P. gallapagensis "not classifiable". Conclusions: These two bioindicators allow evaluating the acute toxicity of SDBS-based commercial detergents in the marine aquatic environment.

Introducción: El bivalvo Semimytilus patagonicus es un bioindicador potencialmente útil por su mecanismo de alimentación, y el gusano Pseudonereis gallapagensis también es interesante como bioindicador por ser bentónico, abundante y fuente de alimento para el calamar Doryteuthis gahi. Sin embargo, su sensibilidad a los contaminantes no ha sido suficientemente estudiada. Objetivo: Probar la utilidad del mejillón S. patagonicus y el poliqueto P. gallapagensis como herramientas ecotoxicológicas para detergentes en el medio marino. Métodos: Se utilizaron 120 individuos de S. patagonicus de Miraflores y 120 de P. gallapagensis de Barranco (ambos cerca de la ciudad de Lima, Perú). Para los bioensayos se utilizaron dos detergentes aniónicos (ingrediente activo, ia, dodecilbenceno sulfonato de sodio). Para S. patagonicus, con una longitud valver promedio de 32.3 ± 6.4 mm, probamos Ariel Doble Poder® (90 %) a concentraciones de 17.5, 35, 70 y 140 mg·ia·l-1, evaluadas a las 48 y 72 h de exposición; y para P. gallapagensis, con una longitud corporal total de 20.4 ± 8.8 mm, probamos Caricia® a 62.5, 125, 250, 500 y 1 000 mg·ia·l-1 a las 24, 48 y 72 h de exposición. Resultados: Los valores de CL50 (Concentración Letal Media) fueron de 34.95 mg·ia·l-1 para S. patagonicus y 102.48 mg·ia·l-1 para P. gallapagensis a las 72 h de exposición. Los detergentes fueron tóxicos para S. patagonicus y levemente tóxicos para P. gallapagensis. La clasificación de riesgo para S. patagonicus es "nocivo" y para P. gallapagensis "no clasificable". Conclusiones: Estos dos bioindicadores permiten evaluar la toxicidad aguda del detergente comercial a base de SDBS en el ambiente acuático marino.

Transport of symbiont-encoded cellulases from the gill to the gut of shipworms via the enigmatic ducts of Deshayes: a 174-year mystery solved.

Shipworms (Bivalvia, Teredinidae) are the principal consumers of wood in marine environments. Like most wood-eating organisms, they digest wood with the aid of cellulolytic enzymes supplied by symbiotic bacteria. However, in shipworms the symbiotic bacteria are not found in the digestive system. Instead, they are located intracellularly in the gland of Deshayes, a specialized tissue found within the gills. It has been independently demonstrated that symbiont-encoded cellulolytic enzymes are present in the digestive systems and gills of two shipworm species, Bankia setacea and Lyrodus pedicellatus, confirming that these enzymes are transported from the gills to the lumen of the gut. However, the mechanism of enzyme transport from gill to gut remains incompletely understood. Recently, a mechanism was proposed by which enzymes are transported within bacterial cells that are expelled from the gill and transported to the mouth by ciliary action of the branchial or food grooves. Here we use in situ immunohistochemical methods to provide evidence for a different mechanism in the shipworm B. setacea, in which cellulolytic enzymes are transported via the ducts of Deshayes, enigmatic structures first described 174 years ago, but whose function have remained unexplained.

Bacteriology & bivalves: Assessing diagnostic tools for geographically remote bivalve populations.

Two sampling approaches for the growth of common or dominant bacteria from bivalve haemolymph were compared: (1) samples processed in the field immediately after collection (field samples), and (2) samples processed in the laboratory at least 24 h after collection (laboratory samples). The sampling approaches were compared on 210 marine bivalve molluscs Paphies subtriangulata and P. australis from two shallow intertidal sites in North Island New Zealand. The approaches were evaluated for the amount of bacterial growth, type of growth, and diversity of growth. Differences in amount and type of growth between the two sampling approaches were observed. Samples processed in the field from P. subtriangulata had significantly more bacterial growth, and a higher diversity of bacteria, including more common or dominant bacterial species. Laboratory samples had a higher proportion of samples with no growth, however common or dominant bacteria were still isolated from these samples. For P. australis, field samples more often had no bacterial growth and laboratory samples had a significantly higher number of common or dominant growth present. Field samples did however contain a higher diversity of bacteria. By conducting bacteriology on bivalves in either the field or the laboratory only, there may be limitations to determining the significance of a bacterial agent isolated. Sampling of both field and laboratory samples should be carried out where possible to optimise detection of important bacteria.

Molecular Mechanisms Underlying Vibrio Tolerance in Ruditapes philippinarum Revealed by Comparative Transcriptome Profiling.

The clam Ruditapes philippinarum is an important species in the marine aquaculture industry in China. However, in recent years, the aquaculture of R. philippinarum has been negatively impacted by various bacterial pathogens. In this study, the transcriptome libraries of R. philippinarum showing different levels of resistance to challenge with Vibrio anguillarum were constructed and RNA-seq was performed using the Illumina sequencing platform. Host immune factors were identified that responded to V. anguillarum infection, including C-type lectin domain, glutathione S-transferase 9, lysozyme, methyltransferase FkbM domain, heat shock 70 kDa protein, Ras-like GTP-binding protein RHO, C1q, F-box and BTB/POZ domain protein zf-C2H2. Ten genes were selected and verified by RT-qPCR, and nine of the gene expression results were consistent with those of RNA-seq. The lectin gene in the phagosome pathway was expressed at a significantly higher level after V. anguillarum infection, which might indicate the role of lectin in the immune response to V. anguillarum. Comparing the results from R. philippinarum resistant and nonresistant to V. anguillarum increases our understanding of the resistant genes and key pathways related to Vibrio challenge in this species. The results obtained here provide a reference for future immunological research focusing on the response of R. philippinarum to V. anguillarum infection.

A Conserved Biosynthetic Gene Cluster Is Regulated by Quorum Sensing in a Shipworm Symbiont.

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites. We determined that 2052S produces the signal N-decanoyl-l-homoserine lactone (C10-HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms. IMPORTANCE Bacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step toward linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

Ranking the biases: The choice of OTUs vs. ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold.

Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline's effect, OTU threshold and rarefaction had a minimal impact on all measurements.