BioRisk 17: 253-262 (2022) . Ce anne ge ee a Se ls
doi: 10.3897/biorisk.| 7.77097 eit seals & B lO R IS kK

https://biorisk.pensoft.net

Seasonal variations of the microflora of wedge clam
Donax trunculus (Linnaeus, |758) from the region of
Arkutino (Bulgarian Black Sea aquatory)

Sevginar Ibryamova', Borislava Pavlova', Elitca Stanachkova', Seniha Salim’,
Aysel Lyatif', Dimitar Dimitrov', Darina Bachvarova', Nesho Chipev’,
Nikolay Natchev', Tsveteslava Ignatova-Ivanova'

| Department of Biology, Shumen University, Shumen, Bulgaria, Shumen University “Konstantin Preslavski”
Department of Biology 115 Universitetska Str, Shumen, Bulgaria 2. Laboratory of Free Radical Processes, In-
stitute of Neurobiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 23, Sofia, Bulgaria

Corresponding author: Tsveteslava Ignatova-Ivanova (ts.ignatovaivanova@shu.bg)

Academic editor: Galina Radeva | Received 26 October 2021 | Accepted 11 December 2021 | Published 21 April 2022

Citation: [bryamova S, Pavlova B, Stanachkova E, Salim S, Lyatif A, Dimitrov D, Bachvarova D, Chipev N, Natchev N,
Ignatova-Ivanova T (2022) Seasonal variations of the microflora of wedge clam Donax trunculus (Linnaeus, 1758) from
the region of Arkutino (Bulgarian Black Sea aquatory). In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K,
Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 253-262. https://doi.org/10.3897/biorisk.17.77097

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 dur-
ing 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.

Copyright Sevginar Ibryamova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

254 Sevginar Ibryamova et al. / BioRisk 17: 253-262 (2022)

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 high-
est 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 (Fernandez-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 ris-
ing 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 (Fernandez-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, ie. 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 stud-

ies were conducted at the Department of Biology, University of Shumen, Bulgaria.

Investigation on the impact of the white mussel 259

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 trans-
ported 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 en-
sure 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 us-

ing 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 microbiologi-
cal analyses. The clams were scrubbed free of dirt, washed in hypochlorite solution (20
mg |"), 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 prob-
able 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. £. 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 enumer-

ated 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 Sys-
tem (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 mi-
croscopic pictures were made using stereomicroscope OPTIKA (Italy) with a Dino-
Eye, Eyepiece camera with 5 megapixels. The photographs were performed by using
a Canon EOS 60D camera. The GEN HI MicroPlate test panel provides a standard-
ized 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 bac-

256 Sevginar Ibryamova et al. / BioRisk 17: 253-262 (2022)

Table |. The physico-chemical parameters of the marine water.

Region Date Depth Temperature pH Salinity Dissolved. ,
M xe [pH [ppd [mg/l]
Arkutino 05.2020 2to4 24.4 7.78 12.2 7.9
Arkutino 06.2020 2to4 25:5 7.32 13.5 7.7
Arkutino 07.2020 2to4 2/2 8.26 13.5 7.8
Arkutino 08.2020 2 to4 Dif 8.36 11.2 8.07
Arkutino 09.2020 2to4 26.2 8.20 11.2 8.1
Arkutino 10.2020 2to4 25.7 8.13 11.2 8.1
Arkutino 11.2020 2to4 22.5 72 12.78 6.9
Arkutino 12.2020 2to4 19.9 6.5 12.69 6.8
Arkutino 01.2021 2to4 19.8 6.5 12.71 6.9
Arkutino 02.2021 2to4 20.3 6.9 12.78 ap)
Arkutino 03.2021 2to4 22.5 7.3 12.99 7.7
Arkutino 04.2021 2to4 23.1 74 13.1 Leh
Arkutino 05.2021 2to4 22.9 7.70 13.3 7.6

teria, 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 microorgan-
isms 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 data-
bases. 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 physicochemi-
cal 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 pa-
rameters. 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 wa-
ter temperature but also to the anthropogenic factor, as it was found only in July. The

Investigation on the impact of the white mussel 257

Temperature [* C] * pH bh
30 9
5
as
20 6
F ; |
vis z
4 q
to |
a
1
a 0
£ a as 4 a
re) oh ot age a ian”, a Pu OL oF Ee
Salinity [ppt] Pr Dissolved oxygen

Pa

e
e

2 2

Figure |. Dynamics in the change of physicochemical parameters of marine water.

Table 2. The number of bacterial cells in 1 ml on the different media.

Region/clam species Pseudomonas Cetrimid Chromokult MacConkey Strain BIOLOG
agar agar agar agar
Arkutino 17.05.2020/ D. trunculus 3.8x104 8.4x104 Enterococcus hirae Pseudomonas mendocina
Arkutino 20.06.2020/ D. trunculus 5.3x104 1.7x10* Enterococcus hirae
Arkutino 25.07.2020/ D. trunculus 1.6x 107 Escherichia vulneris
Arkutino/ 25.08.20 D. trunculus 5.8x104 Enterococcus hirae
Arkutino/ 02.09.2020 D. trunculus 3.4x104 Citrobacter farmer
Arkutino 17.10.2020/ D. trunculus 9.2x10° Acinetobacter gyllenbergii
Arkutino 18.11.2020/ D. trunculus 8.9x10° Pseudomonas alcaligenes
Arkutino 18.12.2020/ D. trunculus 1.34107 P. Pseudomonas alcaligenes
Arkutino 18.01.2021/ D. trunculus 3.4x104 P. Pseudomonas alcaligenes
Arkutino 18.02.2021/ D. trunculus 4.4x104 Acinetobacter gyllenbergii
Arkutino 18.03.2021/ D. trunculus 8.4x10° Acinetobacter gyllenbergii
Arkutino 18.04.2021/ D. trunculus 3.2104 P. mendocina
Arkutino 17.05.2021/ D. trunculus 7.4x104 7.3x10° Pseudomonas mendocina
Enterococcus hirae -

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.

258 Sevginar Ibryamova et al. / BioRisk 17: 253-262 (2022)

10000

x 10000

1000

100
10

E. hiare P.mendocina = E.vulneris Cjarmeri A. gvilenbegii P. alcaligenes

5/17/2020 = 6/20/2020 @ 7/25/2020 = 8/25/2020 9/2/2020 10/17/2020 m 11/18/2020
@ 12/18/2020 m 1/18/2021 m 2/18/2021 3/18/2021 m 4/18/2021 5/17/2021

Number of cells in 1 ml

Figure 2. Dynamics of microorganism species, isolated from wedge clams for 2020-2021 period.

Fecal coliforms (FC) were represented by the species Escherichia vulneris, which
was isolated only in July in a significant number of 1.6x10’ (Table 2; Fig. 2). The spe-
cies 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 (iso-
lated in April and May) and P alcaligenes (isolated in November, December and Janu-
ary). Ihe species Citrobacter farmeri was isolated in September, and the species Acine-
tobacter 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.

Discussion

The microbiota found in marine organisms, and mussels in particular, can be consid-
ered in two respects - the so-called ‘resident’ microbiota, which is stable and unaffected
by the environment, and the ‘transitional’, which depends on the environmental con-
ditions. Specifically, our studies show that fecal coliforms represented by E. vulneris,
which was isolated only in July, Citrobacter farmeri, which was isolated only in Sep-
tember, most likely belong to the transitional species. On the opposite, the species of
Pseudomonas sp., Enterococcus sp. and Acinetobacter gyllenbergii are resident for the in-

Investigation on the impact of the white mussel 259)

vestigated 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 vari-
ous 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, Morax-
ella, 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 gen-
eral, the detected bacteria can be considered as typical psychrophilic marine bacteria,
physiologically adapted to survive within the bivalve. In the literature, studies analyz-
ing 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 affect-
ing 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). Envi-
ronmental parameters, such as variations in temperature and salinity, can affect the di-
versity 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 cor-
relates 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 patho-
genic 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

260 Sevginar Ibryamova et al. / BioRisk 17: 253-262 (2022)

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 demon-
strated the presence of bacterial species of genera Pseudomonas, Enterococcus, Escheri-
chia, Citrobacter, and Acinetobacer in wedge clams Donax trunculus (Linnaeus, 1758).
We found that the concentrations of Escherichia vulneris exceed 190 times the maxi-
mum available values according to Ordinance No. 4/20.10.2000. Inflated concentra-
tions 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, Maciadn MC, Aznar R, Garay E, Pujalte MJ (1999) Low incidence of Vibrio vul-
nificus 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, Garcia M, Ventosa A (1996) Vibrio tapetis

sp. nov., the causative agent of the brown ring disease affecting cultured clams. Interna-

Investigation on the impact of the white mussel 261

tional Journal of Systematic and Evolutionary Microbiology 46(2): 480-484. https://doi.
org/10.1099/002077 13-46-2-480

Colwell RR, Liston J (1960) Microbiology of shellfish. Bacteriological study of the Nova natu-
ral flora of Pacific oysters (Crassostraea gigas). Applied Microbiology 8(2): 104-109. https://
doi.org/10.1128/am.8.2.104-109.1960

Deval MC (2009) Growth and reproduction of the wedge clam (Donax trunculus) in the
Sea of Marmara, Turkey. Journal of Applied Ichthyology 25(5): 551-558. https://doi.
org/10.1111/j.1439-0426.2009.01258.x

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 Re-
search 58(2—5): 245-250. https://doi.org/10.1016/j.marenvres.2004.03.066

FAO The State of World Fisheries and Aquaculture (2016) Contributing to Food Security and
Nutrition for All. Rome, 220 pp. http://www.fao.org/3/a-i5555e/pdf

Fernandez-Pérez J, Froufe E, Nantén 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, Garcia C, Robert M, Nicolas JL (2007) Evidence for the involvement
of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas. Micro-
bial Ecology 53(2): 187-196. https://doi.org/10.1007/s00248-006-9061-9

Gaspar MB, Chr charo LM, Vasconcelos P, Garcia A, Santos AR, Monteiro CC (2002) Depth
segregation phenomenon in Donax trunculus (Bivalvia: Donacidae) populations of the Al-
garve 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 characteris-
tic 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 Argopect-
en 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-
3841.2008.0097 1.x

Maugeri TL, Caccamo D, Gugliandolo C (2000) Potentially pathogenic vibrios in brack-
ish waters and mussels. Journal of Applied Microbiology 89(2): 261-266. https://doi.
org/10.1046/}.1365-2672.2000.01096.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

262 Sevginar Ibryamova et al. / BioRisk 17: 253-262 (2022)

Prieur D, Mével G, Nicolas JL, Plusquellec A, Vigneulle M (1990) Aerobic and facultative
anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater. Inter-
national Microbiology 2: 259-266.

Pujalte MJ, Ortigosa M, Macidn MC, Garay E (1999) Aerobic and facultative anaerobic het-
erotrophic bacteria associated to Mediterranean oysters and seawater. International Micro-
biology 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/da0056249

Romalde JL, Diéguez AL, Doce A, Lasa A, Balboa S, Lopez C, Beaz-Hidalgo R (2012) Advanc-
es 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, Mac-
troidea). Journal of Paleontology 92(02): 130-145. https://doi.org/10.1017/jpa.2017.86