Data Paper
Print
Data Paper
Seasonal variations of the microflora of wedge clam Donax trunculus (Linnaeus, 1758) from the region of Arkutino (Bulgarian Black Sea aquatory)
expand article infoSevginar Ibryamova, Borislava Pavlova, Elitca Stanachkova, Seniha Salim, Aysel Lyatif, Dimitar Dimitrov, Darina Bachvarova, Nesho Chipev§, Nikolay Natchev, Tsveteslava Ignatova-Ivanova
‡ Shumen University “Konstantin Preslavski”, Shumen, Bulgaria
§ Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Open Access

Abstract

The main goal of the present study was to investigate the impact of the state of the environment on the microbiota of the wedge clam Donax trunculus (Linnaeus, 1758) from the region of Arkutino (Bulgarian Black Sea aquatory). The species Enterococcus hirae was isolated during the summer (from May to August). The species P. mendocina prefers the warmer months and the species P. alcaligenes the colder ones. The temperature followed a course of decrease during the period September 2020 to January 2021, followed by a slow increase from February 2021. Comparing May 2020 with May 2021, it became evident that in 2021 the temperature was 1.5 °C lower. 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 lower optimum temperatures and constant other parameters. For the species Enterococcus hirae such dependence was not observed - the number remained constant in May, but with increasing temperature the number of microorganisms decreased during the summer months. The species seemed to preferably develop at pH 7.78. The species A. gyllenbergii preferably grows at temperatures between 20.3–25.7 °C and the optimal temperature was 25.7 °C. For C. farmeri the optimum conditions were temperature 26.2 °C and 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.

Keywords

Black sea, Donax trunculus, microbial identification, pathogens

Introduction

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).

Materials and methods

Place and duration of the study

The samples were collected from the region of Arkutino (exact field coordinates 42.3341 N, 27.7317 E: Datum WGS 84) from May 2019 until May 2021. The laboratory studies were conducted at the Department of Biology, University of Shumen, Bulgaria.

Sample collection

The wedge clams were harvested from the Bulgarian Black Sea aquatory. After collection of the three subsamples (each of about 1 kg), they were refrigerated (4 °C) and transported to the laboratory for further immediate analysis, without freezing the specimens.

In this study, we examined wedge clams of similar size, weight, and shape to ensure maximal uniformity in the applied methods (Duquesne et al. 2004). The average length of mussels used in the study was 2.2 ± 0.43 cm.

Physico-chemical analysis of the inhabited sea waters

During the mussel sampling, we measured in situ the temperature, total salinity (by using YSI Model 33 salinity meter), and pH (by using ATC Piccolo HI1280 pH-meter).

Microbiological analysis

Three subsamples (each of about 1 kg of wedge clams) were used for the microbiological analyses. The clams were scrubbed free of dirt, washed in hypochlorite solution (20 mg l-1), rinsed with sterile distilled water, and shucked with a sterile knife. Tissue liquor samples (about 100 g) were homogenized (Maffei et al. 2009).

Fecal coliforms (FC) were enumerated through five tubes per dilution most probable number (MPN) series (Ignatova-Ivanova et. al. 2018). After 3 h at 37 °C plus 21 h at 44 °C, gas positive tubes were recorded for FC. From each 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 was 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, surrounded by an opaque area due to the precipitation of bile salts in this environment. Pseudomonas sp. was enumerated by Cetrimide Agar (Merck KGaA, 64271 Darmstadt, Germany).

Microbial identification databases for the “Biolog” systems

The microbial identification was performed by the Biolog Microbial Identification System (VIO45101AM). The isolated strains were screened on BL4021502 Tryptic Soy Agar (TCA), cultured for 24 hours at 37 °C, and then subjected to Gen III plaque identification to identify Gram-positive and Gram-negative aerobic bacteria. The microscopic pictures were made using stereomicroscope OPTIKA (Italy) with a DinoEye, Eyepiece camera with 5 megapixels. The photographs were performed by using a Canon EOS 60D camera. The GEN III MicroPlate test panel provides a standardized 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) is used to identify the bacterium from its phenotypic pattern in the GEN III MicroPlate. The BIOLOGIST 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 information on the properties of the strains, in addition to species-level identification. Biolog’s carbon technology identifies the environment and pathogenic microorganisms by producing a characteristic pattern or “metabolic fingerprint” of discrete test reactions performed in a 96-well microplate. The culture suspensions are tested with a panel of pre-selected assays, then incubated, read and compared with extensive databases. https://www.biolog.com/products-portfolio-overview/microbial-identification/

Results

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.

Table 1.

The physico-chemical parameters of the marine water.

Region Date Depth Temperature pH Salinity Dissolvedo2
M C] [pH] [ppt] [mg/l]
Arkutino 05.2020 2 to 4 24.4 7.78 12.2 7.9
Arkutino 06.2020 2 to 4 25.5 7.32 13.5 7.7
Arkutino 07.2020 2 to 4 27.2 8.26 13.5 7.8
Arkutino 08.2020 2 to 4 27.7 8.36 11.2 8.07
Arkutino 09.2020 2 to 4 26.2 8.20 11.2 8.1
Arkutino 10.2020 2 to 4 25.7 8.13 11.2 8.1
Arkutino 11.2020 2 to 4 22.5 7.2 12.78 6.9
Arkutino 12.2020 2 to 4 19.9 6.5 12.69 6.8
Arkutino 01.2021 2 to 4 19.8 6.5 12.71 6.9
Arkutino 02.2021 2 to 4 20.3 6.9 12.78 7.5
Arkutino 03.2021 2 to 4 22.5 7.3 12.99 7.7
Arkutino 04.2021 2 to 4 23.1 7.4 13.1 7.7
Arkutino 05.2021 2 to 4 22.9 7.70 13.3 7.6

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

Figure 1.

Dynamics in the change of physicochemical parameters of marine water.

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.

Table 2.

The number of bacterial cells in 1 ml on the different media.

Region/clam species Pseudomonas agar Cetrimid agar Chromokult agar MacConkey agar Strain BIOLOG
Arkutino 17.05.2020/ D. trunculus 3.8×104 8.4×104 Enterococcus hirae Pseudomonas mendocina
Arkutino 20.06.2020/ D. trunculus 5.3×104 1.7×104 Enterococcus hirae
Arkutino 25.07.2020/ D. trunculus 1.6×107 Escherichia vulneris
Arkutino/ 25.08.20 D. trunculus 5.8×104 Enterococcus hirae
Arkutino/ 02.09.2020 D. trunculus 3.4×104 Citrobacter farmeri
Arkutino 17.10.2020/ D. trunculus 9.2×106 Acinetobacter gyllenbergii
Arkutino 18.11.2020/ D. trunculus 8.9×106 Pseudomonas alcaligenes
Arkutino 18.12.2020/ D. trunculus 1.34×107 P. Pseudomonas alcaligenes
Arkutino 18.01.2021/ D. trunculus 3.4×104 P. Pseudomonas alcaligenes
Arkutino 18.02.2021/ D. trunculus 4.4×104 Acinetobacter gyllenbergii
Arkutino 18.03.2021/ D. trunculus 8.4×105 Acinetobacter gyllenbergii
Arkutino 18.04.2021/ D. trunculus 3.2×104 P. mendocina
Arkutino 17.05.2021/ D. trunculus 7.4×104 7.3×103 Pseudomonas mendocina Enterococcus hirae -

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.

Figure 2.

Dynamics of microorganism species, isolated from wedge clams for 2020–2021 period.

Discussion

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.

Conclusion

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.

Acknowledgements

This study was financially supported by the National Research Fund of the Bulgarian Ministry of Education and Science (Grant- H31/6 KP-06-H31/6/10.12.19) and by Shumen University, Department of Biology (Grant 08-67 / 25.01.2021).

References

  • Arias CR, Macián MC, Aznar R, Garay E, Pujalte MJ (1999) Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast. Journal of Applied Microbiology 86(1): 125–134. https://doi.org/10.1046/j.1365-2672.1999.00641.x
  • Borrego JJ, Castro D, Luque A, Paillard C, Maes P, García M, Ventosa A (1996) Vibrio tapetis sp. nov., the causative agent of the brown ring disease affecting cultured clams. International Journal of Systematic and Evolutionary Microbiology 46(2): 480–484. https://doi.org/10.1099/00207713-46-2-480
  • Colwell RR, Liston J (1960) Microbiology of shellfish. Bacteriological study of the Nova natural flora of Pacific oysters (Crassostraea gigas). Applied Microbiology 8(2): 104–109. https://doi.org/10.1128/am.8.2.104-109.1960
  • Duquesne S, Liess M, Bird DJ (2004) Sub-lethal effects of metal exposure: Physiological and behavioral responses of the estuarine bivalve Macoma balthica. Marine Environmental Research 58(2–5): 245–250. https://doi.org/10.1016/j.marenvres.2004.03.066
  • Fernández-Pérez J, Froufe E, Nantón A, Gaspar MB, Méndez J (2017) Genetic diversity and population genetic analysis of Donax vittatus (Mollusca: Bivalvia) and phylogeny of the genus with mitochondrial and nuclear markers. Estuarine, Coastal and Shelf Science 197: 126–135. https://doi.org/10.1016/j.ecss.2017.08.032
  • Garnier M, Labreuche Y, García C, Robert M, Nicolas JL (2007) Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas. Microbial Ecology 53(2): 187–196. https://doi.org/10.1007/s00248-006-9061-9
  • Gaspar MB, Chı’charo LM, Vasconcelos P, Garcia A, Santos AR, Monteiro CC (2002) Depth segregation phenomenon in Donax trunculus (Bivalvia: Donacidae) populations of the Algarve coast (southern Portugal). Scientia Marina 66(2): 111–121. https://doi.org/10.3989/scimar.2002.66n2111
  • Ignatova-Ivanova Ts, Ibrjmova S, Stanachkova E, Ivanov R (2018) Microbiological characteristic of microflora of (Mytilus galloprovincialis Lam.) in The Bulgarian Black Sea aquatory. Research Journal of Pharmaceutical, Biological and Chemical Sciences 9(1): 199–205. https://www.rjpbcs.com/pdf/2018_9(1)/[29].pdf
  • Jorquera MA, Silva FR, Riquelme CE (2001) bacteria in the culture of the scallop Argopecten purpuratus (Lamarck, 1819). Aquaculture International 9(4): 285–303. https://doi.org/10.1023/A:1020449324456
  • Maffei M, Vernocchi P, Lanciotti R, Guerzoni ME, Belletti N, Gardini F (2009) Depuration of Striped Venus Clam (Chamelea gallina L.): Effects on Microorganisms. Sand Content. and Mortality. Journal of Food Science 74(1): M1–M7. https://doi.org/10.1111/j.1750-3841.2008.00971.x
  • Park KI, Paillard C, Le Chevalier P, Choi KS (2006) Report on the occurrence of brown ring disease (BRD) in Manila clam, Ruditapes philippinarum, on the west coast of Korea. Aquaculture (Amsterdam, Netherlands) 200(1–4): 610–613. https://doi.org/10.1016/j.aquaculture.2005.12.011
  • Prieur D, Mével G, Nicolas JL, Plusquellec A, Vigneulle M (1990) Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. International Microbiology 2: 259–266.
  • Pujalte MJ, Ortigosa M, Macián MC, Garay E (1999) Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. International Microbiology 2: 259–266.
  • Reid HI, Soudant P, Lambert C, Paillard C, Birkbeck TH (2003) Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis. Diseases of Aquatic Organisms 56: 249–258. https://doi.org/10.3354/dao056249
  • Romalde JL, Diéguez AL, Doce A, Lasa A, Balboa S, López C, Beaz-Hidalgo R (2012) Advances in the knowledge of the microbiota associated with clams from natural beds. Chapter 7, Clam fisheries and aquaculture. Nova publishers, Neu York, 163.
  • Seguineau C, Laschi-Loquerie A, Moal J, Samain JF (1996) Vitamin requirements in great scallop larvae. Aquaculture International 4(4): 315–324. https://doi.org/10.1007/BF00120948
  • Signorelli JH, Raven JGM (2018) Current knowledge of the family Cardiliidae (Bivalvia, Mactroidea). Journal of Paleontology 92(02): 130–145. https://doi.org/10.1017/jpa.2017.86