Microbial symbioses are common across invertebrate taxa, in part because the suspension-feeding strategy of many species exposes them to free-living and particle-associated microbes. Most research on bivalve microbiomes has involved marine species, but less focus has been on freshwater bivalves. Marine bivalves show a resident and transient framework associated with digestive tissues, with some microbes likely forming persistent, residential associations and others exhibiting transitory associations. The purpose of this study was to characterize the resident and transient microbial communities of the gut and gill from the quagga mussel, Dreissena bugensis, and eastern elliptio, Elliptio complanata. Mussels were collected from natural populations and dissected to isolate tissues immediately or after 24 h of depuration. Environmental water samples and faeces deposited during depuration were collected, and all samples underwent 16S SSU rRNA marker gene sequencing. Organs from both mussel species displayed distinct microbial communities from their source water. Allowing animals to egest faeces, or depurate, affected tissue microbial communities, indicating that the resident and transient framework is prevalent in freshwater bivalve species. Taxonomic differences between quagga mussels and elliptios may indicate that phylogenetic history, habitat, and gill structure play a role in determining the composition of host-associated microbial communities in these species.

Microplastics (MP) have repeatedly been found in commercially cultured species of bivalves. There are concerns regarding the amount of MP released into the environment by aquaculture activities, and questions regarding possible higher MP loads in farm -grown shellfish compared to levels in shellfish collected from recreational beds. To explore this concept, seawater, aquaculture gear, and eastern oysters ( Crassostrea virginica ) were sampled from an aquaculture site in Niantic Bay, CT, USA, and a 2 -week transplantation experiment was performed in which oysters were transplanted between the aquaculture site and a plastic -free cage off the dock at the University of Connecticut -Avery Point campus. The digestive gland -stomach complex (gut) was dissected from the oysters and MP were extracted from the adjacent seawater and oyster gut samples using previously validated extraction methods. Extensive quality assurance and control measures were taken to reduce MP contamination. Particles in all samples were isolated, imaged under a stereomicroscope, and characterized (size, shape, polymer) using ImageJ software and microFourier transform infrared spectroscopy. Water samples contained 0 -0.3 MP/L and oyster gut samples contained 0 -1.3 MP/g wet weight indicating very low concentrations of MP at the farm (0 -2 MP/individual) or away from the farm (0 -3 MP/individual). Aquaculture gear in this area is not contributing to MP ingestion in farmed oysters or elevated MP levels in the surrounding water.

Suspension-feeding bivalve molluscs are dominant benthic fauna in many near-shore environments, with phytoplankton often being their main prey type. Feeding, which involves hydrosol filtration and mucociliary processes to capture particles and process them for digestion, occurs at low Reynolds numbers. Changes in water temperature have been shown to affect feeding processes of bivalves as a result of altered physiological processes or temperature-dependent changes in kinematic viscosity of water. Most studies, however, have focused on feeding rates and have manipulated temperature under laboratory conditions. In this study, experiments were conducted using ambient seawater and acclimatized blue mussels, Mytilus edulis, to examine particle capture efficiency over a 1-year period. During this period, water temperature decreased from similar to 18 degrees C to 5 degrees C with a concomitant increase in viscosity of similar to 41%. The capture of a wide variety of bacteria and phytoeukaryotes (0.8-8 mu m), as well as two sizes of polystyrene microspheres (1 and 6 mu m), was quantified to calculate capture efficiency. Data demonstrate that temperature, and the concomitant change in water viscosity, had no significant effect on capture efficiency. Cyanobacteria were captured at significantly higher rates than other bacteria of similar size. These results suggest that the laterofrontal cirri of blue mussels act as paddles rather than sieves because capture efficiency was constant over a range of viscosities and Reynolds numbers. Additionally, the efficient capture of some bacteria and smaller phytoeukaryotes by the mussels suggests that these plankters could play a larger role in the diet of M. edulis than previously considered. The ecological impact of these findings, especially regarding differences in capture efficiency of different bacterial cells, warrants further study.

Plastic pollution in the marine environment is a persistent and ubiquitous issue. Biodegradable and compostable plastics may present a positive alternative to traditional non-biodegradable polymers for marine applications such as aquaculture. Mater-Bi (MB) is a starch-based polymer used in compostable films and plastic carrier bags and has been proven to completely biodegrade in a variety of laboratory conditions. However, degradation rates in the natural environment differ from static laboratory tests and depend on the specific physicochemical and environmental conditions of a particular location. This study examined the degradation of Mater-Bi in coastal marine conditions in a flow-through mesocosm system, in comparison to polyhydroxybutyrate (PHB), a well-known biopolymer, and high-density polyethylene (HDPE), a traditional plastic polymer. Mass and area loss were both used as proxies for disintegration across a nine-month time span. Results indicate that both Mater-Bi and polyhydroxybutyrate disintegrate when exposed to shallow water column and sediment conditions, losing on average 25 to 47

Metatranscriptomic methods involving RNA sequencing (RNA-seq) are powerful tools for evaluating the gene expression profiles of transcriptionally-active microbes that inhabit the tissues of animals. Bivalve molluscs, like all invertebrates, are holobionts and sites of interactions between host animals and both prokaryotic and eukaryotic symbionts. The present communication describes the metatranscriptomic profile of the resident microorganisms that inhabit the gut of blue mussels, Mytilus edulis, under standard laboratory conditions. Each of the eight mussels described herein were housed in isolated microcosms with routine husbandry for 14 days before their gut tissues were sampled and subjected to RNA-seq. Subsequent mRNA reads that aligned to the mussel genome were removed, and the non-host reads were annotated for function and pathway analyses. Under laboratory conditions, the resident gut microbiota expressed genes associated mostly with aerobic energy processes, with other notable contributions from metabolism and protein processing genes. At the pathway level, the most abundant complete pathways expressed in the resident gut microbial communities were related to aerobic cellular respiration, nucleotide biosynthesis, and catabolism. These data represent novel, baseline microbial gene expression information from the gut of mussels, which are crucial for future research examining the mussel holobiont and bivalve microbial ecology. Public retrieval and secondary analyses of these metatranscriptomic profiles are highly encouraged.

The blue mussel (Mytilus edulis) is a suspension feeder which has been used in gut-microbiome surveys. Although raw 16S sequence data are often publicly available, unifying secondary analyses are lacking. The present work analysed raw data from seven projects conducted by one group over 7 years. Although each project had different motivations, experimental designs and conclusions, all selected samples were from the guts of M. edulis collected from a single location in Long Island Sound. The goal of this analysis was to determine which independent factors (e.g., collection date, depuration status) were responsible for governing composition and diversity in the gut microbiomes. Results indicated that whether mussels had undergone depuration, defined here as voidance of faeces in a controlled, no-food period, was the primary factor that governed gut microbiome composition. Gut microbiomes from non-depurated mussels were mixtures of resident and transient communities and were influenced by temporal factors. Resident communities from depurated mussels were influenced by the final food source and length of time host mussels were held under laboratory conditions. These findings reinforce the paradigm that gut microbiota are divided into resident and transient components and suggest that depuration status should be taken into consideration when designing and interpreting future experiments.

Ingestion of microplastics (MP) by suspension-feeding bivalves has been well-documented. However, it is unclear whether exposure to MP could damage the stomach and digestive gland (gut) of these animals, causing ramifications for organism and ecosystem health. Here, we show no apparent effects of nylon microfiber (MF) ingestion on the gut microbiome or digestive tissues of the blue mussel, Mytilus edulis. We exposed mussels to two low concentrations (50 and 100 particles/L) of either nylon MF or Spartina spp. particles (dried, ground marsh grass), ca. 250-500 μm in length, or a no particle control laboratory treatment for 21 days. Results showed that nylon MF, when aged in coarsely filtered seawater, developed a different microbial community than Spartina spp. particles and seawater, however, even after exposure to this different community, mussel gut microbial communities resisted disturbance from nylon MF. The microbial communities of experimental mussels clustered together in ordination and were similar in taxonomic composition and measures of alpha diversity. Additionally, there was no evidence of damage to gut tissues after ingestion of nylon MF or Spartina spp. Post-ingestive particle processing likely mediated a short gut retention time of these relatively large particles, contributing to the negligible treatment effects.

Microplastics (MP, <5 mm) are found in coastal waters across various environmental compartments (biota, water, marine snow, sediment). The eastern oyster (Crassostrea virginica) is a commercially important species that ingests MP; however, oysters are discriminant suspension feeders that do not consume all particles to which they are exposed. This study explored the relationship between MP in oysters on a recreational oyster bed and the surrounding environmental compartments in Long Island Sound (LIS; USA). The quantity and types of MP in oysters, water, marine snow, and sediment samples were determined. Precautions were taken to minimize and monitor MP contamination in the field and laboratory to improve the quality of data collected. Microplastics were isolated from samples via chemical digestion, and any suspected particles were identified using micro -Fourier transform infrared spectroscopy. A total of 86 MP were identified out of 885 suspected particles across environmental media. The highest MP count in an individual oyster was nine, indicating low concen-trations of MP in oysters and the surrounding environment. Few polymers, except polyethylene terephthalate, were shared between oysters and the surrounding environmental compartments. Sediments contained the highest number of MP across all environmental compartments (42 total). These data aid in determining the types of MP (polymer composition, shape, size) to which oysters are exposed and identified those ingested. The low numbers of MP recorded, coupled with the lack of alignment of polymers between oysters and their surrounding environment, demonstrates further that oysters are a poor bioindicator species for MP pollution.

Introduction Bivalve molluscs are among the most prominent coastal benthic-suspension-feeders and their farming is the largest and fastest-growing sector of aquaculture. More than a century of intensive laboratory studies (but surprisingly few in-situ studies) has yielded the consensus view that bivalves mainly capture particles >4µm. Nonetheless, bivalves thrive throughout the world’s oceans that are mostly oligotrophic, characterized by low food concentration and dominated by minute autotrophic picoplankton (<2 µm). Method We measured, in situ , the capture efficiency of naturally occurring planktonic cells by five suspension-feeding bivalve species from four families and three orders, residing in two oligotrophic basins: the Red Sea and the East Mediterranean Sea. Results Three species captured micron and submicron autotrophic cells with high efficiency (60-90%), suggesting a wider trophic niche than hitherto believed. In contrast, two sympatric species captured mainly particles >10 µm. Discussion These results suggest that the same basic anatomical tool kit, variably modulated according to taxa, habitat, or life history traits, enables the remarkable evolutionary and ecological success of bivalves in trophically-diverse habitats.

Microplastics (MP) are a contaminant of emerging concern and, as such, there has been a rush to action and publication. Over the past two decades, this haste has resulted in a chaotic and cluttered literature rife with inappropriate methodologies, poor experimental protocols, misinterpreted results, overstated significance, and subsequent damaging media stories. This review provides a critical assessment of the current scientific literature on interactions between particle-feeding molluscs and MP and their purported impacts (>750 publications), and recommendations for future efforts. Experimental studies were critically assessed and assigned scores ranging from 0 to 2 as indicators of their veracity. The mean ratio for the 84 papers included in this analysis was 0.9, indicating that most publications contained too many flaws. It is not surprising that MP have been noted in shellfish guts globally. What is surprising is the extremely low level of particles routinely recorded (see Table 1 and references therein). The presence of MP in molluscs has been shown repeatedly, with little regard for quality assurance and control measures. The inconsistencies across studies and lack of proper sampling design have inundated the literature with incomparable studies and inappropriate claims. Common mistakes in field studies from collection through digestion and MP characterization are discussed and identified in 128 studies. Suggestions are made to improve field studies at every stage. The data to date clearly demonstrate extremely low numbers (<10 per individual) of MP in filter-feeding bivalve molluscs globally. There are no data demonstrating presence of MP in these molluscs is a serious risk to human health, and few data to demonstrate negative impacts on the shellfish at environmentally relevant concentrations. Many of the studies on suspension-feeding bivalve molluscs and other invertebrates are weak or fatally flawed. There is a recurring presence in the published literature of misunderstanding of the feeding processes, capabilities for particle selection and rejection, and species-specific differences that all lead to misinformation, misinterpretation, and incorrect assumptions regarding potential impacts. There are major shortcomings to many laboratory studies that examined uptake and accumulation of MP by bivalves and their subsequent effects. The shortcomings have led to a seriously flawed literature on purported interactions and impacts of MP on these animals. If potential investigators do not possess the necessary knowledge and skills to carry out the study, they should engage a collaborator that has the requisite expertise. Bivalves and other particle-feeding molluscs are complex living organisms with extraordinary capabilities for the control of selective capture, ingestion, and egestion of particulate material. They should be recognized and treated as such in any attempt to describe impacts of stressors, including different particle types, on their feeding and ability to accumulate materials. Any future experimental studies need to be focused carefully, based upon clear questions, use standardized analytical procedures, demonstrate a knowledge of the animals being studied, and an understanding of the literature extant. The hype needs to be curtailed and scientists should not imply impacts or potential impacts when there are no data to support the suppositions at environmentally relevant concentrations of MP. The case is further strengthened to stop advocating for the use of bivalve molluscs as reliable indicators of MP in the environment. Recommendations are offered for future efforts including harmonization of methodologies. Finally, a plea is made for editors of scientific journals to make a stronger effort to engage qualified peer-reviewers and stop the flow of poorly done studies and superficial reviews that do nothing more than confuse the literature and reinforce inadequate studies and prior reviews. This review is presented from the viewpoint and consideration of experts in shellfish physiology, and represents the opinions of, and assessments made by, the authors.

Suspension feeding bivalve molluscs interact with different types of microplastics (MP) suspended in the water column. Most bivalves are selective suspension feeders and, thus, do not consume all particles to which they are exposed. Selection depends upon the physicochemical properties and size of the particle. Recent work has provided evidence that blue mussels, Mytilus edulis, and eastern oysters, Crassostrea virginica, ingest and egest microspheres (polystyrene) and microfibers (nylon) differently, but whether other factors, such as polymer type and shape, mediate selection have not been explored. To investigate these factors, mussels and oysters were offered similar sized nylon (Ny) and polyester (PES) microfibers or polyethylene (PE) and polystyrene (PS) microspheres, or different sized PES microfibers during a 2 h exposure. Feces and pseudofeces were collected separately and analyzed for MPs, and the data were used to develop a linear regression model for selection. Results demonstrated clear species-specific differences in the efficiency of particle selection. Both mussels and oysters, however, exhibited size-based rejection of PES microfibers, ingesting a higher proportion of shorter fibers than longer fibers. Polymer type did not impact selection of fibers or spheres. The relative size of particles (area and perimeter) was found to be the most important factor in predicting whether a MP will be rejected or ingested.

ABSTRACT Suspension-feeding bivalve molluscs have evolved a highly effective mechanism for particle discrimination, which allows them to process efficiently the material to which they are exposed. The mechanisms controlling this preingestive sorting process have been described as either passive or active. Evidence of a passive selection mechanism has been demonstrated in several species of suspension-feeding bivalves. In contrast, to date there has been no evidence that active selection mechanisms underlay particle selection in bivalves, although the possibility of such mechanisms has been hypothesized numerous times. The present study was designed to examine active selection on the gill of the eastern oyster Crassostrea virginica. The gill was chosen for study because in oysters this organ is involved in particle selection. Two in vivo assays were designed in which the gill was exposed to dissolved cell exudate or extracts from the microalga Tetraselmis chuii, and the transport of polystyrene microspheres (25 µm) by the frontal cilia of the ordinary filament quantified by means of video endoscopy. Results demonstrated that the addition of exudates or extracts of T. chuii cells had no significant effect on the percentage of particles being transported dorsally (likely ingested) or ventrally (more likely rejected), and no differences in the number of particles in the ventral groove between control and experimental treatments. Results of follow-up experiments using covalently bound neoglycoproteins commonly found on cell surfaces of microalgal species demonstrated differences in transport of captured microspheres (10 µm, carboxylated) depending on the sugar type. Although chemoreception cannot be completely ruled out, these findings further indicate that physicochemical properties of particles, and not an active behavioral or physiological response (i.e., chemoreception of dissolved metabolites) by the animal, mediate particle selection in oysters.

Suspension-feeding bivalves are critical members of aquatic ecosystems worldwide, which is why research into their host-associated microbiota is growing. Experiments that artificially diminish the native microbial communities of bivalves in vivo will be increasingly necessary to evaluate the functional role of microbes within their hosts. Previous methods to manipulate the microbiome of bivalves lack standardization and, often, verification of successful disturbance. The goal of this study was to evaluate antibiotic administration as a method for perturbing the gut microbiome of bivalves in two separate, but related, experiments. In the first, a mixture of antibiotics was delivered to eastern oysters for 4 days to probe effects on gut microbial carbon usage, diversity, and taxonomic composition. In the second, the same antibiotic mixture was administered to blue mussels for 21 days to probe effects on microbial abundance, diversity, and taxonomic composition. In both experiments, animals were administered antibiotics in isolation, and stringent sterilization methods were employed, which included sterilized seawater and microalgal food. The results of the oyster experiment revealed that antibiotics substantially reduced microbial carbon usage and perturbed community composition. In the mussel experiment, antibiotics lowered microbial abundance and species richness and significantly altered community composition. Taken together, results from the two experiments demonstrate that antibiotics can be used to effectively alter the function and composition of the gut microbial community of bivalves. Future research that aims to perturb the microbiomes of suspension-feeding animals should incorporate aspects similar to the protocols described herein. Additionally, future studies must include verification, ideally high-throughput DNA sequencing coupled with microbial quantification, that the antibiotic perturbation was successful.

Direct measurements of the capture efficiency of planktonic cells by seven solitary ascidians were made in situ and in the laboratory and compared with the capture efficiency of polystyrene microspheres. The capture efficiency of the microspheres was significantly higher than that of planktonic cells over the entire tested size range (0.3-15 mu m). Submicron polystyrene spheres with a surface modification consisting of an adsorbed layer of a nonionic, long-chain surfactant were removed at lower efficiencies than uncoated particles whereas for larger microspheres (1-3 mu m), the coating had no effect. Our findings strengthen the concept that some planktonic cells evade capture by mucus-based suspension feeders, and that evasion happens throughout the pico- and nanoplankton size range. Thus, the common assumption that particles larger than similar to 1 mu m are always captured at a 100% efficiency by ascidians should be reconsidered. Some large microalgae cells (> 3-12 mu m) were captured at a lower efficiency than the largest microspheres used (3 mu m) suggesting that other factors, such as surface interactions and particle shape, play an important role in capture throughout the tested size range. Furthermore, given the lack of a known active selection mechanism in ascidians, we propose that some plankton possess traits that allow them to evade predation by mucus-based suspension feeders.

Suspension-feeding bivalve molluscs perform important ecological roles by coupling pelagic and benthic systems during their feeding activities. Particle capture, and thus feeding, is dependent on particle encounter and retention on the gill filaments, with several factors influencing this process. Over the past 30 yr, different types of synthetic microspheres have been used to examine aspects of particle capture and ingestion by bivalves. Critics of this work have posited that manufactured particles may contain surfactants, chemicals commonly used in manufacturing to reduce surface tension, that could produce spurious capture and ingestion rates. The goal of this work was to experimentally assess whether the presence of different types of surfactants on manufactured polystyrene particles can result in instantaneous effects on particle capture by the blue mussel Mytilus edulis. The effects of 3 different types of common surfactants (sodium dodecyl sulfate, benzalkonium chloride, Triton-X) on clearance rates (CR) and capture efficiencies (CE) were tested. Results indicated that none of the surfactant treatments had an effect on CR. Treatment with one of the surfactants (Triton-X) significantly lowered CE for 3 µm sized spheres compared to the control spheres (Milli-Q treated). None of the other tested surfactants significantly affected CE when compared to the control treatment. These data add to an understanding of particle handling by bivalves, and suggest that concentrations of surfactants found on commercially available microspheres used for experiments or found in the environment have little immediate effect on feeding processes.

Titanium dioxide nanoparticles (n-TiO2) are emerging contaminants and the ecological impact of these materials to the nearshore environment is largely unknown. The reactivity of n-TiO2 increases with light exposure, and the photocatalytic effects have been shown on cultures of bacteria and microalgae in the laboratory. The purpose of this study was to assess the response of natural bacterial and microalgal communities associated with marine aggregates to n-TiO2 under conditions similar to those found in the photic zone of nearshore waters. Nano and bulk TiO2 particles were incorporated into marine aggregates over 4 days under two light conditions: 6:18 and 0:24 (hours light:dark). The abundance and metabolic response of heterotrophic bacteria and viability of microalgae associated with aggregates were assessed. Although the proportion of living microalgae was unchanged, the abundance, total metabolic activity and functional diversity of heterotrophic bacteria were significantly altered by irradiated n-TiO2.