@ BioRisk BioRisk 21: 29-40 (2023) DOI: 10.3897/biorisk.21.111253 Research Article Study of the dynamics of the microbial communities in the wedge clam Donax trunculus (Linnaeus, 1758) from the Bulgarian aquatory of the Black Sea Sevginar F. lbryamova', Stephany Toschkova', Darina Ch. Bachvarova’', Elitca Stanachkova’, Radoslav I. lvanov', Nikolay D. Natchev'?, Tsveteslava V. Ignatova-lvanova' 1 Department of Biology, Shumen University “Konstantin Preslavski", Department of Biology 115 Universitetska Str., Shumen, Bulgaria 2 Unit for Integrative Zoology, Department of Evolutionary Biology, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria Corresponding author: Tsveteslava V. Ignatova-lvanova (ts.ignatovaivanova@shu.bg) OPEN Qaceess Academic editor: Pavel Stoev Received: 16 August 2023 Accepted: 13 October 2023 Published: 5 December 2023 Citation: lbryamova SF, Toschkova S, Bachvarova DC, Stanachkova E, lvanov RI, Natchev ND, Ignatova-lvanova TV (2023) Study of the dynamics of the microbial communities in the wedge clam Donax trunculus (Linnaeus, 1758) from the Bulgarian aquatory of the Black Sea. BioRisk 21: 29-40. https://doi.org/10.3897/ biorisk.21.111253 Copyright: © Sevginar F. lbryamova et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract In the present work, we investigated the dynamics of the microbial communities in the wedge clam Donax trunculus (Linnaeus, 1758) from the Bulgarian coastal waters of the Black Sea. The samples were collected in the period of January 2020 until December 2022 from Arkutino, Ahtopol, Obzor and Tsarevo. The BIOLOG system was used for mi- crobiological determination. In our investigation were isolated the following microorgan- isms: Enterococcus cancerogenus, Enterococcus hirae, Escherichia vulneris, Citrobacter farmeri, Acinetobacter gyllenbergii, Enterococcus hirae, Escherichia vulneris, Enterobacter cloacae, Escherichia hermannii, Pseudomonas mendocina, Pseudomonas fulva, Pseudo- monas alcaligenes, Pseudomonas putida, Acinetobacter johnsonii, Acinetobacter gyllen- bergii, Enterococcus hirae, Escherichia vulneris, Enterococcus gallinarum, Citrobacter sedakii, Pseudomonas putida, Streptococcus lugdunensis, Enterococcus casseliflavus, Vibrio cincinnatiensis, Vibrio alginolyticus, Vibrio parahaemolyticus, Enterococcus hirae, Streptococcus aureus, Staphylococcus lugdunensis and Enterococcus casseliflavus. During the winter period, we detected the presence of Pseudomonas sp. — P. alcaligenes, P. putida, and A. gyllenbergii. In the autumn months we isolated C. sedakii, C. farmeri, A. gyllenbergii, A. johnsonii, P. fulva and E. casseliflavus. In the spring, E. cancerogenus, E. hirae and Pseudomonas mendocina were found. During the summer, the highest bio- diversity of microorganisms - E. hirae, E. vulneris, E. cloacae, E. gallinarum, P. putida, V. cincinnatiensis, V. alginolyticus, V. parahaemolyticus, S. aureus, E. hermannii and S. lug- dunensis were registered. Although our three-year research showed that some species are permanent and others are transient, we tend to accept the conclusion that there is only a transient microbiota in mussels and it changes depending on environmental con- ditions or is a result of pollution of the Black Sea. Key words: Bivalves, hydrobiology, microbial identification, molluscs, pathogens, pollutions Introduction The wedge clam Donax trunculus (Linnaeus, 1758) is found in the entire region of the Black Sea coast, but according to Fernandez-Pérez et al. (2017) its pop- ulation is extremely high on the territory of the Bulgarian Black Sea aquatory. 29 Sevginar F. Ilbryamova et al.: Study of the dynamics of the wedge clam Donax trunculus The species occurs in the Mediterranean and the Black Seas, and in the Atlantic Ocean from Senegal to the North Atlantic coast of France (Deval 2009). The species inhabits open sandy beaches where it forms thick beds, as the highest density are at depths from 0 to 3 m. It is considered a warm-water temperate species (Bayed and Guillou 1985; Lamine et al. 2020a; Chahouri et al. 2022). The shellfish are exposed to diseases caused by various bacteria, which can Cause a mass extinction of the species. It was detected that the cause of out- breaks of diseases in bivalves is related to conditional pathogens, i.e. free-living pathogenic bacteria which, under favourable conditions, can cause diseases. This poses a serious risk to humans as consumers of bivalve species. Pathogen- ic bacteria can enter into the clams directly from seawater, from the microalgae they feed on, but also as a result of anthropogenic pollution of the environment. For D. trunculus, various studies indicate it importance for the assessment and monitoring of the ecological conditions of the sandy beaches (Moukrim et al. 2004; Idardare et al. 2008; Nadir et al. 2015; Lamine et al. 2020b). D. trunculus can be used as an indicator species to understand population dynamics and to interpret variation in various biological parameters used as biomarkers of pollu- tion. The dynamics of the microbial population of D. trunculus in the Black Sea Region have not been studied and there are no data concerning the microbial communities inhabiting the bivalves. Our study contributes to the study of micro- biota of the Black Sea. It should be linked to additional long-term studies to pro- vide basic information for the development of strategies for the protection and monitoring of the Black Sea coastal ecosystem. In a previous study, the microbial variation in the Arkutino Region was described (Ibryamova et al. 2022a). Our re- sults demonstrated the presence of bacterial species of genera Pseudomonas, Enterococcus, Escherichia, Citrobacter and Acinetobacer in wedge clams Donax trunculus (Linnaeus, 1758). In the present study, only the species D. trunculus was investigated, as it was the only one found in the surveyed area. We found that the concentrations of E. vulneris exceed by 190 times the maximum avail- able values according to Ordinance No. 4/20.10.2000. Increased concentrations of coliforms in the summer indicate a seasonal worsening of the conditions of the seawater as a consequence of anthropogenic activity. The main goal of the present work was to investigate the dynamics of microbial communities in the wedge clam D. trunculus (Linnaeus, 1758) from the Bulgarian Black Sea aquatory. Materials and methods Place and duration of the study The study was conducted at the Department of Biology, University of Shumen, Bulgaria, from January 2020 until December 2022. The samples were collected from the regions of Mussel farms — Arkutino, Ahtopol, Obzor and Tsarevo (Fig. 1). Collection of samples After collection of three subsamples (each of about 1 kg), the mussels were refrigerated (4 °C) and transported to the laboratory for further immediate anal- ysis, without freezing the specimens. In this study, we examined wedge clams of similar size, weight and shape to ensure maximal uniformity in the applied BioRisk 21: 29-40 (2023), DOI: 10.3897/biorisk.21.111253 30 Sevginar F. lbryamova et al.: Study of the dynamics of the wedge clam Donax trunculus , Obzor Bulgaria Black sea Figure 1. Sampling locations at the Black Sea coast. methods (Duquesne et al. 2004). The average length of mussels used in the study was 2.2 + 0.43 cm. The mussels used for the analysis were collected monthly, throughout the three-year period. The collection of samples was car- ried out by trawling by JSC “Black Sea Fishing”, Burgas. Microbiological analyses The mussels were scrubbed free of dirt, washed in hypochlorite solution (20 mg I"), rinsed with sterile distilled water and shucked with a sterile knife. The whole soft tissues of the mussel’s liquor samples (about 100 g) were homogenised. Faecal coliforms (FC) were enumerated through five tubes per dilution most probable number (MPN) series (Ignatova-lvanova et al. 2018). After 3 h at 37 °C plus 21 h at 44 °C, gas positive tubes were recorded for FC. From each of the FC gas positive tubes, 0.1 ml were transferred in tubes with 10 ml of Tryptone Water (Oxoid, Basingstoke, UK) and then incubated for 24 h at 44 °C. E. coli were enumerated by MacConkey agar (Merck, Darmstadt, Germany). The plates were incubated aerobically at 35-37 °C for 18-24 hours. E. coli grows matte dark pink to tile-red colonies, surrounded by an opaque area due to the precipitation of salts in this environment. Pseudomonas sp. were enumerated by Cetrimide Agar (Merck KGaA, 64271 Darmstadt, Germany). Lactic acid bac- teria (LAB) were isolated in media of MRS (de Mann Rogosa Sharpe, Biolife 272-20128, Milano, Italia). The strains were cultured overnight (16-18 h) on MRS at 37 °C and in limited oxygen (tubes or Petri dishes with the strains were incubated in plastic bags, which limited the oxygen content). When determining the number of isolated species of microorganisms, the number of cells in 1 ml of sample was calculated, after which the percentage of the total number of microorganisms in the sample was calculated for each isolated species. Microbial identification databases for the “BIOLOG” system The microbial identification was performed by the BIOLOG Microbial Identifica- tion System VIO45101AM. The isolated strains were screened on BL4021502 BioRisk 21: 29-40 (2023), DOI: 10.3897/biorisk.21.111253 31 Sevginar F. Ilbryamova et al.: Study of the dynamics of the wedge clam Donax trunculus Tryptic Soy Agar (TCA), cultured for 24 hours at 37 °C and then subjected to Gen Ill plaque identification to identify Gram positive and Gram negative aerobic bacteria. The microscopic pictures were performed using stereomicroscope OPTIKA (Italy) with a DinoEye, Eyepiece camera with 5 megapixels. The photo- graphs were performed by using a Canon EOS 60D camera. The GEN III Micro- Plate test panel provides a standardised micromethod using 94 biochemical tests to profile and identify a broad range of Gram-negative and Gram-positive bacteria. BIOLOG’s Microbial Identification Systems software (e.g. OmniLog Data Collection) was used to identify the bacterium from its phenotypic pattern in the GEN III MicroPlate. The BIOLOG system allows to quickly and accurately identify more than 2900 species of aerobic and anaerobic bacteria, yeasts and fungi. BIOLOG’s advanced phenotypic technology provides valuable informa- tion for the properties of the strains, in addition to species-level identification. BIOLOG’s carbon technology identifies the environment and pathogenic micro- organisms by producing a characteristic pattern or “metabolic fingerprint” of discrete test reactions performed in a 96-well microplate. The culture suspen- sions are tested with a panel of pre-selected assays, then incubated, read and compared with extensive data-bases (https://www.biolog.com/products-port- folio-overview/microbial-identification). Results The isolated species of microorganisms by year and percentage are presented in Figs 2-5. The species E. cancerogenus, E. hirae, E. vulneris, C. farmeri and A. gyllenbergii were isolated from the Arkutino Region (Fig. 2). The species E. cancerogenus was isolated only in 2020 in the month of May. The species EF. hirae was isolated in all three years, with the highest percentage recorded in 2021 - 60.71% and the lowest in 2020 - 21.25%. This species was isolated during the months of May, June and August. The species E. vulneris was iso- lated in all three years, with the highest percentage reported in 2022 - 20.15% and, in 2020 and 2021, the percentage was 11.69%. The species was isolated Arkutino, 2020 -2022 2022, A. gyllenberqii 3) 92%, 2022, £. vulneris es 20) 15% 2022, £. hirce 6 93°, 2021, A. gyllenbergii 27 60% 2021,E.vulneris MEE 11,69% 207, AISI —— EEE 60), 1 2020, A. gyllenbergii 32,05% 2020, C. farmeri 11,85% 2020, E. vulneris m™ 11,69% 2020, E. hirae 21,25% 2020, E. cancerogenus 29,27% 0,00% 10,00% 20,00% — 30,00% 40,00% 50,00% — 60,00% 70,00% Figure 2. Microbial population dynamics in the Arcutino Region. BioRisk 21: 29-40 (2023), DOI: 10.3897/biorisk.21.111253 a9 Sevginar F. Ilbryamova et al.: Study of the dynamics of the wedge clam Donax trunculus Ahtopol, 2020-2022 2022, P. putida 21,74% 44,87% 2022, E. cloacae 13,30% —a 3,05% 2022, E. hice —_—_<