Among marine bivalve species, the wedge clam Donax trunculus (Linnaeus, 1758) has one of the highest yields in the world. The habitat of this species is close to open sandy beaches, where it forms thick beds. It is found along the Atlantic-Mediterranean coast with the highest density at a depth from 0 to 3 m (Gaspar et al. 2002). The highest population density is reached at the surf. The species was found in the Mediterranean and the Black Sea from Senegal to the North Atlantic coast of France (Deval 2009). According to The State of World Fisheries and Aquaculture (2016) the annual production of Donax in Europe reaches 970–1353 tons. Both Donax species - D. trunculus and D. variegatus can be found along the Black Sea coast, but the population of D. trunculus (Fernández-Pérez et al. 2017) has a higher density. D. trunculus is commercially important in many countries like France, Italy, Turkey, Portugal and Spain as a food resource. In Galicia, D. trunculus is the bivalve with rising commercial value in the markets during recent years (42.37 €/kg in the year 2017), and its value has increased with the consequent increase in its fishing pressure (Fernández-Pérez et al. 2017). In Recent years have registered an increase in sites on the southern Black Sea coast where D. trunculus has been extracted; it is then mainly exported abroad on account of its high prices. Due to excessive collecting in some spots (including Bulgaria), the wild stocks are drastically depleted. Shellfish are exposed to diseases caused by various bacteria, which can also cause a mass extinction of species along the coast. It was detected, that the cause of outbreaks of diseases in bivalves is related to conditional pathogens, i.e. free-living pathogenic bacteria which, under favorable conditions, can cause diseases. This poses a serious risk to humans as consumers of clam species. Pathogenic bacteria can enter into the clams from seawater, from the microalgae they feed on, and as a result of anthropogenic pollution of the environment. The species D. trunculus is most often used as a bioindicator, as it is very sensitive to changes in the environment (Signorelli and Raven 2018). We have found no scientific publications on the microbiological status of wedge clams D. trunculus from the Bulgarian Black Sea aquatory and the impact of the state of the environment on it.

The main goal of the present study was to investigate the impact of the state of the marine environment on the microbiota of the wedge clams D. trunculus from the region of Arkutino (Bulgarian Black Sea aquatory).

We conducted a physicochemical analysis of the sea waters. The results are summarized in Table 1. The data represents results from the measurement of 4 basic physicochemical parameters.

The dynamics of changes in the physicochemical parameter values are shown in Fig. 1.

From the data presented in Fig. 1, it is visible that the temperature follows a course of decrease from September 2020 to January 2021, followed by a slow increase from February. If we compare May 2020 with May 2021, is evident that the temperature in 2021 was 1.5 °C colder (Fig. 1a). Comparing the results in Fig. 1 we can say that the number of the species P. alcaligenes was twice as high in May 2021. It is likely that this species preferred to grow at lower optimum temperatures and constant other parameters. For the species, Enterococcus hirae such dependence was not observed - the number of this strain remained constant in May, but with increasing temperature, the number of microorganisms decreased during the summer months. Only Enterococcus clarify preferably developed at pH 7.78 (Fig. 1b). For the species A. gyllenbergii, we can say that it preferably grew at temperatures between 20.3–25.7°C, and the optimal temperature was 25.7 °C. For C. farmeri the optimum temperature was 26.2 °C and the pH 7.3. The species E. vulneris was probably related not only to the increase in water temperature but also to the anthropogenic factor, as it was found only in July. The lowest amount of dissolved oxygen was from November to January, when the species P. alcaligenes preferably develops (Fig. 1c).

The microorganisms isolated from D. trunculus were determined by the Biolog Microbial Identification System (Biologist VIO45101AM) to species level. After 24 h of cultivation on different media, the number of cells in 1 ml were obtained - data represented in Table 2 and Fig. 2.

Fecal coliforms (FC) were represented by the species Escherichia vulneris, which was isolated only in July in a significant number of 1.6×10⁷ (Table 2; Fig. 2). The species Enterococcus hirae was isolated during the period from May to August (Table 2; Fig. 2) . From genus, Pseudomonas (Table 2; Fig. 2) were detected P. mendocina (isolated in April and May) and P. alcaligenes (isolated in November, December and January). The species Citrobacter farmeri was isolated in September, and the species Acinetobacter gyllenbergii in February-March. The distribution dynamics of different types of microorganisms isolated from the wedge clam D. trunculus are represented in Fig. 2. Fig. 1 shows that the species Pseudomonas preferably developed in the cooler months, although there was a variation within the genus, as well. The species P. mendocina preferred the warmer months, while the species P. alcaligenes preferred the colder ones.

The microbiota found in marine organisms, and mussels in particular, can be considered in two respects - the so-called ‘resident’ microbiota, which is stable and unaffected by the environment, and the ‘transitional’, which depends on the environmental conditions. Specifically, our studies show that fecal coliforms represented by E. vulneris, which was isolated only in July, Citrobacter farmeri, which was isolated only in September, most likely belong to the transitional species. On the opposite, the species of Pseudomonas sp., Enterococcus sp. and Acinetobacter gyllenbergii are resident for the investigated mussels. Our results demonstrate an increase in the quantity of the coliforms in the region of Arkutino in July, when the quantity of the fecal coliforms is 190 times over the norms prescribed in Ordinance No. 4/20.10.2000 for the quality of fisheries water and the breeding of shellfish (the number of fecal coliforms in the inter-shell content should be less than 300 NVB). This can be harmful to human health following the consumption of mussels. Jorquera et al. (2001) suggested that the bivalve mollusks only present “transition” microbiota. In general, the resident microbiota performs various functions in mussels - it serves as food, a source of vitamins and growth factors, plays also a role in the defense mechanisms to prevent the colonization of bacterial pathogens or eliminate toxic substances (Prieur et al. 1990; Seguineau et al. 1996). On the other hand, the microbiota can also enter the mussels as a result of environmental pollution. According to data from the literature, many microorganisms belonging to different species – such as Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus, and Bacillus (which are part of the bacterial population in the aquatic environment) may enter the mussels as a result of the diet by filtration. The first study analyzing the normal microbiota of a bivalve species was performed by Colwell and Liston (1960) with the Pacific oyster (Crassostrea gigas). The authors found a high proportion of Gram-negative bacteria (> 80%), with a predominance of the genera Pseudomonas and Vibrio, but also of Flavobacterium and Achromobacter. In general, the detected bacteria can be considered as typical psychrophilic marine bacteria, physiologically adapted to survive within the bivalve. In the literature, studies analyzing the diversity, distribution, and density of marine bacteria associated with bivalve mollusks are scarce. In fact, most studies have focused on characterizing bacteria with pathogenic potential for mollusks, or specifically for mussels. Thus, the genus Vibrio is widely studied, as it is known as one of the most important bacterial genera affecting the culture of bivalves. For example, V. tapetis has received special attention since it caused Brown Ring Disease (BRD), the bacterial etiology of which is described in adult clams. In addition, the disease caused by it is considered one of the main limiting factors for the culture of Manila clams (Venerupis philippinarum) (Europe Borrego et al. 1996), and was also detected in cultured clams in Korea (Park et al. 2006). Environmental parameters, such as variations in temperature and salinity, can affect the diversity of microorganisms and the environment as a physiological state of bivalves and its susceptibility to bacterial infections (Arias et al. 1999; Pujalte et al. 1999; Maugeri et al. 2000;Garnier et al. 2007). Evidence was found, that at lower water salinity BRD disease in mussels is much more severe (Reid et al. 2003). This assumption also correlates with our results. In the months of August, September, and October the lowest salinity of the seawater was reported in the region of Arkutino 11.2 ppt, which is also associated with the emergence of transitional species of Citrobacter farmeri and pathogenic coliforms (Table 2; Fig. 1). Romalde et al. (2012), found a wide variety of species of Pseudomonas sp, which make up about 52.8% of the microbiota in bivalves. These results completely correlate with the results obtained by us and are represented in Table 2. This fact shows that the genus Pseudomonas is one of the main groups of microbiota in mussels, although some seasonal variations can be observed, which are related to the physicochemical parameters of the environment - temperature, pH and salinity. Our results indicate the need for deeper research on the microbiota of mollusks and on the pathogenic potential of marine bacteria, using both culture-dependent and molecular methods. To our knowledge, these are the first data of cultivated bacterial species in mussels from the Black Sea.

When studying the microbiota of populations of different species of bivalves, it is very important to know their sanitary status, as well as to determine the pathobiological basis of periodic outbreaks of diseases affecting these populations. Our results demonstrated the presence of bacterial species of genera Pseudomonas, Enterococcus, Escherichia, Citrobacter, and Acinetobacer in wedge clams Donax trunculus (Linnaeus, 1758). We found that the concentrations of Escherichia vulneris exceed 190 times the maximum available values according to Ordinance No. 4/20.10.2000. Inflated concentrations of coliforms in the summer attracted very special attention, indicating a seasonal worsening of the conditions of the seawater as a consequence of anthropogenic activity. We supposed that the pollution was very serious bearing in mind that the habitat of the wedge clams is in depth within the sand. The other important conclusion is the dominance of Pseudomonas, found in the mussels, which correspond to some seasonal variations related to the physicochemical parameters of the environment, such as - temperature, pH and salinity. With the worldwide increase in bivalve consumption, we would like to point out the possibility of the emergence of new diseases due to the interaction between the pathogen, the host, and the environment.