Microorganisms are widespread and essential components in marine ecosystems, establishing diverse and often intimate associations with a wide range of animals (Fraune and Bosch, 2010; Krishnaveni et al., 2025). Marine benthic invertebrates, like all multicellular organisms, engage in complex interactions with a diverse array of microbes, including bacteria (Knowlton and Rohwer, 2003; Li et al., 2023a, b), and these associations can take the form of mutualism, commensalism, or parasitism. Bacteia colonizing marine invertebrates play crucial roles in maintaining host health, supplying essential nutrients, and offering protection against pathogens. Moreover, they contribute significantly to the overall functioning and adaptive responses of hosts to environmental change (Bourne et al., 2016; Pita et al., 2018). Hosts may possess species-specific bacterial communities distinct from their surrounding environment, which can be acquired vertically, from conspecifics, or from the environment (Koskella et al., 2017). The degree of specificity of these communities varies among host species, with some exhibiting only a few highly specific symbionts, whereas others share bacteria with the surrounding environment (Lemieux-Labonté et al., 2016; Lemay et al., 2018; Roth‐Schulze et al., 2018).

Bivalves harbor a wide variety of microorganisms, including protozoa, fungi, viruses, and bacteria (Rey-Campos et al., 2022), with bacteria being the most extensively studied group (Masanja et al., 2023). The microbiome of bivalves plays a critical role in the host health and disease, with the bacterial component involved in processes such as digestion, nutrient cycling, and immune defense (Pierce and Ward, 2018; Timmins-Schiffman et al., 2021). Certain bivalve-colonizing bacteria have been found to produce antimicrobial compounds that help protect the host from pathogens (Destoumieux-Garzón et al., 2020; Balbi et al., 2021). In addition, some species belonging to the families Phyllobacteriaceae and Hyphomicrobiaceae (Pseudomonadota) and Flavobacteriaceae (Bacteroidota) are associated with enhanced disease resistance in bivalves (Dubé et al., 2019). These microorganisms produce enzymes that break down complex carbohydrates and contribute to the immune response of their host (Moruf et al., 2020).

The structure of microbial communities colonizing marine invertebrates, including bivalves, is highly variable, with different body regions harboring distinct microbial communities (Masanja et al., 2023). For instance, the bacterial composition of Eriocheir sinensis (Chinese crab) was dominated by Tenericutes and Pseudomonadota in the gut, whereas Actinobacteria, Pseudomonadota, and Bacteroidetes predominated in the gill samples (Zhang et al., 2016). Similarly, Mytilus galloprovincialis possesses a microbiota with tissue-specific variations: where the digestive gland is characterized by Ruminococcaceae and Lachnospiraceae, the stomach is dominated by Flavobacteriaceae, and the foot by Spirochaetaceae representatives (Musella et al., 2020).

The Boreal-Atlantic bivalve Mya arenaria Linnaeus, 1758, is native to the North Atlantic, off the coasts of America and Canada (Strasser, 1998). Invasive in Europe, it has been reported in the Barents Sea (Galkin, 1998), White (Russanova, 1963), Baltic (Kube, 1996), North (Strasser, 1998), Wadden (Smidt, 1951), Mediterranean seas (Stora et al., 1995), and along the European coasts of the Atlantic Ocean (Conde et al., 2011). In the Black Sea, it was first recorded in 1966 near Odessa (Beshevli and Kolyagin, 1967; Gomoiu and Porumb, 1969), later identified near Berezan Island (Zambriborshch et al., 1968), and within a relatively short time became a common species in the northwestern part of the basin. Considering the age of the identified specimens, as well as the time required for larval development and reaching sexual maturity, Zambriborshch et al. (1968) suggested that the species entered the Black Sea in the early 50s. In the Romanian Black Sea region, this species was first reported in the late 1960s, with a limited number of specimens identified near Zaton and Tomis Harbor (Constanţa) (Gomoiu and Porumb, 1969), and was later observed on the Romanian continental shelf at depths of up to 35 m (Skolka and Gomoiu, 2004).

This bivalve thrives in the organic-rich muds of the Black Sea, particularly in sedimentary habitats influenced by inputs from the Danube River, which carry high bacterial loads, including potential pathogens (Ene et al., 2025). Although not currently consumed in Romania, M. arenaria is recognized for its edible potential and is successfully harvested and farmed in other regions of the world (Wheaton et al., 2008). In the Black Sea, it serves as prey for several commercially important benthic fish species, potentially raising public health concerns. Despite exposure to bacterial pathogens, the species appears to remain unaffected, likely due to immune adaptations that enable tolerance to challenging environmental conditions such as hypoxia, rapid fluctuations and high organic pollution. Some studies have shown that the soft-shell clam hosts a distinct microbiome, suggesting a possible symbiotic relationship between the clam and its associated microorganisms (Cabelli and Heffernan, 1970; Liu et al., 2020).

While extensive research has been conducted on bacterial communities colonizing benthic organisms in marine environments (Gilbertson et al., 2012; Fuirst et al., 2021; Li et al., 2023b; Liu et al., 2023), including bivalves (Musella et al., 2020; Akter et al., 2023), only a few studies have addressed the microbiome of these invertebrates from the Black Sea, notably in the bivalves Mytilaster lineatus (Onishchenko and Kiprianova, 2006) and Donax trunculus (Ibryamova et al., 2022), as well as the tube-dwelling polychaete Melinna palmata (Menabit et al., 2024).

Microbial ecology studies carried out in the Black Sea ecosystem have predominantly focused on bacterial communities from seawater (Jørgensen et al., 1991; Sorokin et al., 1995; Glaubitz et al., 2010; Bryukhanov et al., 2015; Ruginescu et al., 2022) and sediments (Schulz et al., 1999; Thamdrup et al., 2000; Leloup et al., 2007; Schäfer et al., 2007; Coolen and Shtereva, 2009; Schippers et al., 2012), with limited comparison between their diversity and that of invertebrate-associated communities (Onishchenko and Kiprianova, 2006; Menabit et al., 2024).

In this context, the present study aims to assess the structural and functional bacterial diversity associated with the marine bivalve M. arenaria, focusing on organ-specific (siphon, gills, and stomach) microbiomes and their distinctions from surrounding sediment communities using a high-throughput Illumina 16S rRNA gene sequencing approach. The study was based on the hypothesis that M. arenaria harbors a microbiome that facilitates its adaptation to stressful estuarine environments and organic pollution, including bacterial contaminants. Rather than using classical microbiological methods, we applied molecular techniques that enabled the identification of bacterial genes potentially involved in diverse metabolic functions, including antibiotic resistance. This investigation represents the first characterization of microbiome variability across different organs of the bivalve M. arenaria.

This study aimed to characterize the bacterial communities of the bivalve M. arenaria collected from the Romanian part of the Black Sea, and is the first to examine the microbiome of different organs (siphon, gills, and stomach) of this marine invertebrate, as well as the associated sediments.

Previous research on M. arenaria-associated bacteria has primarily focused on microbial contamination, including studies on the kinetics of coliform bacteria uptake and removal by this bivalve (Cabelli and Heffernan, 1970), and, more recently, reports of significant contamination with pathogens such as Arcobacter, Cryptosporidium parvum, Giardia, and Salmonella in clams collected from the north shore of the St. Lawrence River (Quebec, Canada) (Lévesque et al., 2006). The present study applied culture-independent Illumina sequencing of the 16S rRNA gene to characterize the taxonomic and functional diversity of bacterial communities colonizing the siphon, gills, and stomach of M. arenaria, as well as associated sediments from two locations in the Black Sea. This represents a more comprehensive and targeted molecular characterization compared to a previous NGS study of M. arenaria microbiome, which only characterized the bacterial diversity of the whole organisms collected from two locations on the east coast of Canada (Liu et al., 2020). Moreover, this study investigated the hypothesis that M. arenaria hosts a microbiome that may help it cope with stressful estuarine conditions and organic pollution, including bacterial contaminants. This molecular approach also enabled the identification of bacterial genes potentially involved in diverse metabolic functions, including antibiotic resistance, providing insight into functional traits that allow the clam to cope with environmental stressors.

Our data showed that the bivalve microbiota was well differentiated from that of its physical habitat, and exhibited compositional variations depending on the organ, with greater taxonomic diversity (19 phyla) in the sediments as compared to the bivalve organs (14–15 phyla), independent of water depth. The phylum Verrucomicrobiota dominated the siphon and gill samples, Spirochaetota was the most abundant in the stomach, and the sediment communities were mainly composed of Desulfobacterota.

No significant differences in bacterial diversity were observed between samples from different depths, likely reflecting similar environmental conditions across sites. All samples were collected from circalittoral sandy mud habitats, which are characterized by benthic communities dominated by detritivores and suspension feeders such as oligochaetes, polychaetes, mollusks, and bacterivorous nematodes (Nenciu et al., 2023; Mureşan et al., 2019; Teacă et al., 2019, 2020). These results align with other studies showing that microbial communities in similar habitats are comparable, even across different geographical regions (Lauber et al., 2009).

Recent investigations in the Black Sea revealed the dominance of Actinobacteriota and Chloroflexi in sediments from areas with physico-chemical water characteristics and substrate composition comparable to those observed in our research (Menabit et al., 2024). While these bacteria were also detected, the most abundant taxa colonizing the sediments in this study were assigned to Pseudomonadota and Desulfobacterota. Shifts in microbial composition might be attributed to the distinct habitat types (Teacă et al., 2020), as such changes in the structure of the zoobenthic community could influence microbial populations. While prior research (Reveillaud et al., 2014) has demonstrated that microbial communities often reflect host and environmental differences across large spatial scales, our study found relatively minor differences in bacterial composition between the two sampling sites, suggesting limited spatial variation in this context.

Bivalves, like many other organisms, host microbial communities that are specific to various organs and tissues. Previous data have reported the digestive system as containing the highest concentrations of bacteria, particularly the stomach, gastric juice, crystalline style and digestive diverticula (Kueh and Chan, 1985). The results obtained in this survey indicated a specific microbiota in this bivalve, distinct from its physical habitat, with compositional variations depending on the organ. Thus, representatives of the order Spirochaetales showed the highest abundances in siphon and gill samples, with the Verrucomicrobiales group being better represented in gills. Taxa belonging to Spirochaetales have also been identified in the digestive tract of invertebrates such as arthropods (Berlanga et al., 2007) and mollusks (Romero and Espejo, 2001), as well as in bivalve gills (Duperron et al., 2007). A recent study in New Zealand showed these bacteria to be abundant in the siphon and digestive glands of the bivalve Paphies australis (Biessy et al., 2020), consistent with our data, which revealed a high representation of Spirochaetales taxa in the siphon samples. Bacteria classified in the order Verrucomicrobiales have been identified in various environments such as the water column (Yoon et al., 2007), sediments (Yoon et al., 2008), in association with marine invertebrates, such as the polychaete Periserrula leucophryna (Yang et al., 2007) and different organs (gills, stomach, foot) of the bivalve M. galloprovincialis (Musella et al., 2020). The results also indicated a statistical correlation between the phylum Planctomycetales and stomach samples, consistent with data on the stomach microbiome of C. gigas, which showed a high abundance of taxa belonging to this group (Offret et al., 2020).

The present data revealed distinct, organ-specific distributions of bacterial taxa within M. arenaria. Members of the order Bacteroidales were predominantly associated with the siphon, while bacteria from Campylobacterales and Synechococcales exhibited higher relative abundances in the stomach samples. The enrichment of Bacteroidales is consistent with the high representation of this phylum in other marine invertebrates, including the crab Callinectes sapidus (Givens et al., 2013), the gastropod Haliotis diversicolor (Zhao et al., 2018), and the bivalve M. galloprovincialis (Musella et al., 2020). Similarly, Campylobacterales that prevailed in the stomach of M. arenaria have been reported in the digestive tracts of various marine organisms, such as the sea urchin Lytechinus variegatus (Hakim et al., 2015) and the ascidian Ciona intestinalis (Dishaw et al., 2014), suggesting a possible functional role in digestion. The presence of Synechococcales in stomach tissues may be linked to the dietary habits of M. arenaria, as these cyanobacteria are known to constitute a significant nutritional component for other marine taxa, including sipunculid worms (Li et al., 2023a; Liu et al., 2023) and bivalves (Qiao et al., 2022).

Despite variations in composition, our research showed that the Verrucomicrobiota and Spirochaetota phyla exhibited high abundances in all analyzed organs. In addition, a notable proportion of sequences belonging to Planctomycetota was recovered from the stomach samples. A study conducted at two locations on the East Coast of Canada demonstrated that soft-shell clams from Nova Scotia had a higher abundances of Pseudomonadota and Acidobacteria, but a lower abundance of Actinobacteria than those from Quebec. Furthermore, samples from Nova Scotia exhibited greater microbial diversity than those from Quebec (Liu et al., 2020). These findings suggest that environmental factors may shape the microbial communities of this bivalve, influencing both composition and diversity.

The genus Persicirhabdus encompasses nitrate-reducing bacteria (Yoon et al., 2008), which, under certain conditions, such as low oxygen levels, can reduce nitrates to nitric oxide (Strahl and Abele, 2020). Studies have shown that the clam Arctica islandica may harbor nitrate-reducing bacteria, and that nitric oxide could play a crucial role in reducing the metabolic activity of A. islandica during self-burial and shell closure. Under these conditions, nitric oxide contributes to the initiation of metabolic depression, which may help conserve energy during hypoxia or anoxia and protect against oxidative stress (Strahl and Abele, 2020). Accordingly, the high abundance of these bacteria in the analyzed organs of M. arenaria could support the survival and adaptation of this bivalve in the low-oxygen conditions of the investigated area.

The genus Sulfurimonas is characterized as sulfur-oxidizing bacteria, capable of growing chemoautotrophically with reduced sulfur compounds, such as sulfide, thiosulfate and elemental sulfur (Sievert et al., 2008), and has been isolated from various environments, including coastal marine sediments (Hoor, 1975; Cai et al., 2014; Wang et al., 2020), polychaete nests (Inagaki et al., 2003; Takai et al., 2006), and deep-sea hydrothermal vents (Hu et al., 2021). While sulfur-oxidizing bacteria are typically found in the gills of marine bivalves (Dando et al., 1985; Caro et al., 2007), recent investigations have revealed a persistent population of these microbes in the digestive tract of adult hosts lucinid clam Loripes orbiculatus, which contribute to the host’s nutrition by fixing carbon from sulfide oxidation (Alcaraz et al., 2024). These findings are consistent with our investigations, where we detected higher abundances of Sulfurimonas colonizing the stomach samples, further suggesting a role of these bacteria in digestion or other processes that contribute to the host’s nutrition.

Bacteroides, identified in all analyzed organs of M. arenaria, was previously documented in the digestive tract of the mussel M. edulis and the oyster C. virginica, and plays an important role in digestion, including the breakdown of complex carbohydrates and the production of essential amino acids (Pierce and Ward, 2019).

Poseidonibacter is considered a synonym or subgroup within the genus Arcobacter (On et al., 2021), members of which are known pathogens linked to human and animal diseases, previously detected in various water bodies, including wastewater, seawater, and freshwater systems, often associated with fecal contamination (Ghaju Shrestha et al., 2022), as well as colonizing soft-shell clams collected from the north shore of the St. Lawrence River, and other bivalves such as M. galloprovincialis (Ottaviani et al., 2016). In our study, we observed high abundances of these bacteria in the gills of M. arenaria specimens collected from the station located in the Danube influence area, which was not surprising given that the river is permanently subject to anthropogenic pressures such as industrial activities, intensive farming, and agricultural runoff (Gasparotti, 2014). Moreover, the Escherichia/Shigella genera were identified in all analyzed organs. Escherichia includes both commensal and pathogenic species (Braz et al., 2020), whereas Shigella is highly infectious and responsible for significant global morbidity and mortality (Schroeder and Hilbi, 2008). Bivalves, as filter-feeding organisms, concentrate bacteria from the environment and serve as suitable bioindicators of water contamination. Consequently, the presence of these genera in bivalve tissues may pose serious public health risks through seafood consumption and highlight underlying threats to ecosystem health, including microbial pollution and reduced water quality.

Unlike the microorganisms associated with M. arenaria, those colonizing the sediments were mainly represented by taxa belonging to the orders Desulfobulbales, Steroidobacterales, and Flavobacteriales orders. The high abundance of the Desulfobulbales group, mainly composed of sulfur-reducing bacteria thriving in anoxic environments (Ward et al., 2021), could be correlated with the low dissolved oxygen concentrations characterizing the Romanian part of the Black Sea. These particularities could also explain the presence of taxa belonging to Steroidobacterales in sediments, which are known for their ability to oxidize sulfur (Mußmann et al., 2017). Previous studies have demonstrated that Chloroflexi species account for 25.5% to 41.3% of total 16S rRNA sequences in marine sediments globally (Hoshino et al., 2020). Some representatives of this phylum are also known for decomposing organic matter (Landry et al., 2017). In this study, the prevalence of this group in sediment samples could be associated with the marine habitat, characterized by high organic matter content, from which the samples were collected (Bucşe et al., 2020; Teacă et al., 2020). Moreover, the genus Woeseia was consistently detected across all sediment samples. This group comprises halotolerant bacteria capable of sulfur oxidation and denitrification (Du et al., 2016; Mußmann et al., 2017) and has previously been isolated from both coastal and deep-sea surface sediments (Mußmann et al., 2017; Hoffmann et al., 2020).

A preliminary assessment of the functional profile of the microbiome of the bivalve M. arenaria and adjacent sediments was performed by determining the functional diversity of the bacterial communities based on16S rRNA gene sequencing data, using the Tax4Fun platform (Langille et al., 2013). While this tool cannot replace the assessment of gene functionality through metagenomic analysis, studies have shown that it can provide insight into the functions of microorganisms in diverse habitats such as biofilms (Koo et al., 2017), the water column (Bowman and Ducklow, 2015), and sediments (Su et al., 2018). However, the effectiveness of this method depends on the genomic information available in databases, which in some cases does not reflect the functional diversity of the microorganisms in the investigated ecosystem (Wemheuer et al., 2020).

The data obtained in this study on the functional diversity of the bacterial communities associated with the bivalve M. arenaria and surrounding sediments are consistent with previous investigations emphasizing the role of marine bacteria in carbohydrate metabolism (Gavriilidou et al., 2020). Thus, studies have shown that bacteria associated with some bivalves produce enzymes that break down complex carbohydrates, and contribute to the immune response of these organisms (Dubé et al., 2019). In addition, representatives of the orders Verrucomicrobiales and Bacteroidales orders, mainly identified in M. arenaria organs, are involved in the hydrolysis of various polysaccharides (Martinez-Garcia et al., 2012). Likewise, Dunkai et al. (2023) demonstrated that bacterial strains isolated from the digestive system of the bivalve Crenomytilus grayanus can degrade various nutrient substrates (sugars, amino acids, and polysaccharides) as well as xenobiotic substances (petroleum hydrocarbons, bisphenol A, and atrazine). Therefore, the presence of genes involved in xenobiotic metabolism in the microbiota of M. arenaria could play an important role in maintaining the health and adaptability of this species in diverse environments, including polluted areas. Several studies have highlighted the presence and potential of microbial communities colonizing marine sediments to degrade aromatic hydrocarbons, including toluene, ethylbenzene, xylene, chlorocyclohexane, and chlorobenzene (Moore et al., 2020; Suarez-Moo et al., 2020; Nikolova et al., 2021). Similar degradation pathways which have also been identified in the current study, highlighting the potential of these bacteria as valuable resources for bioremediation and the pharmaceutical industry. Moreover, research undertaken by Šyvokienė and Butrimavičienė (2013) demonstrated that bacteria associated with the digestive systems of swollen river mussels (Unio tumidus) and zebra mussels (Dreissena polymorpha) from the Curonian Lagoon (Winters et al., 2011) are involved in the biodegradation of petroleum hydrocarbons. Mollusks, including bivalves, are emerging as a promising source of novel marine bioactive compounds, with certain species containing substances that could have diverse applications across industries (Sousa and Hinzmann, 2020). The current investigation demonstrates that bacteria associated with both the organs of M. arenaria and the surrounding sediments harbor genes involved in antibiotic synthesis. This finding aligns with other research, such as studies from the Red Sea, where the detection of biosynthetic gene clusters polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) in marine invertebrate-associated bacteria indicates their potential to produce bioactive compounds, including antibiotics (El Samak et al., 2018).

This study revealed significant differences in bacterial community diversity between the organs of the bivalve M. arenaria and the adjacent sediments, with greater diversity in the sediments. The bivalve harbors a distinct microbiota that varies by organ and differs clearly from that of its physical habitat. Notably, the phylum Verrucomicrobiota predominated in the siphon and gills, while Spirochaetota were more abundant in the stomach. In contrast, Desulfobacterota dominated the sediment-associated microbial communities. Persicirhabdus bacteria were consistently abundant across all examined organs and may contribute to the clam’s metabolic depression and adaptation to low-oxygen conditions. Additionally, sulfur-oxidizing Sulfurimonas species in the stomach likely contribute to the host’s nutrition. Sediments, in contrast, were primarily colonized by Woeseia species, which are key players in the degradation of organic matter. Functional diversity analyses suggested that bacteria inhabiting both bivalve tissues and adjacent sediments participate in diverse metabolic processes, with a particular emphasis on carbohydrate, amino acid, and energy metabolism. Several pathways were associated with xenobiotic degradation and antibiotic biosynthesis, highlighting the biotechnological potential of these microbial communities for applications such as bioremediation and pharmaceutical development.

This survey, providing the first organ-specific characterization of the microbiome associated with M. arenaria revealed clear structural differences among the siphon, gills, and stomach, as well as a distinct separation from the surrounding sediment microbial communities. The observed compositional divergence highlighted the presence of a specialized and compartmentalized microbiome within the bivalve, likely shaped by organ-specific functions and external environmental exposures. Notably, the internal microbial communities do not mirror the environmental microbiota, suggesting a non-random, potentially symbiotic assemblage with functional relevance to host biology. These results offer a foundation for future investigations into the ecological and physiological roles of the bivalve microbiome, particularly in the context of environmental stress and host adaptation. Further studies employing metagenomic and metatranscriptomic approaches will be essential to elucidate the mechanistic interactions between M. arenaria, its microbiota, and the surrounding habitat. Nevertheless, the small number of specimens analyzed could potentially bias interpretations of the structural and functional diversity within the bivalve microbiome.