Sex inversion in cultivated mussels Mytilus galloprovincialis Lam . ( Crimea , Black Sea ) under influence of external environmental factors

In the last decade, there has been a shift in the sex ratio of the mussel Mytilus galloprovincialis in the Black Sea towards increase of males. In modern literature, focus is mainly on mechanisms of sex inheritance in mussels and hormonal regulation of the reproduction, and there is no information on sex inversion in M. galloprovincialis under the influence of environmental factors. The goal of this work is to establish the fact of sex change in mussels cultivated near the coast of Crimea under the influence of some external environmental factors. We establish that mussels change sex from female to male, but some specimens become hermaphrodites, with their fraction reaching 13%. Under unfavorable environmental conditions, mussel females change sex, and their mortality rises up to 69%. In water areas subject to anthropogenic impact, the proportion of sex inversion in the mollusks may be as high as 58%. The influence of various adverse environmental factors on sex inversion in mussel females is unequal, and its strength decreases in the following order: diesel fuel > hypoxia > anionic detergents > starvation.


Introduction
Мussel Mytilus galloprovincialis Lam. is one of the most abundant species of bivalve mollusks of the Black Sea (Ivanov, 1989;Kholodov et al., 2017), This mollusk is harvested, and it is an important element of the food base of fish and invertebrates.The status of mussel populations can be estimated using certain integral population characteristics that reflect the influence of environmental factors on mollusks in specific habitats (Dekhta & Katalevsky, 2000;Ctadnichenko, 2010;Shurova, 2013).One of such indicators is the sex ratio in populations of mussels from different habitats (Chelyadina, 2014;Shulman & Soldatov, 2014).Until 2000-s, in most cases, researchers observed the balanced sex ratio equal to 1: 1 (female : male) in the mussel M. galloprovincialis near the coast of the Black Sea, with 1-3% of hermaphrodites being present (Kudinsky & Shurova, 1990;Pirkova, 1994).The shifts in the gender composition of artificial and natural mussel populations towards the predominance of males have been shown in the more recent studies (Karavantseva, 2009;Machkevskij et al., 2011;Chelyadina, 2014).One of the reasons for the variations in the gender structure of the mussel population can be related to the sex change in the mollusk in ontogenesis.In modern literature, focus is mainly on mechanisms of sex inheritance in mussels and hormonal regulation of the reproduction (Kenchington et al., 2002;Yusa et al., 2013), and there is no information on sex inversion in M. galloprovincialis under the influence of environmental factors.Therefore, the goal of this work is to establish the fact of sex inversion and the direction of this process under the influence of certain environmental factors during post-spawning period in mussels cultivated near the Crimean coast.

Material and Methods
Mussel Mytilus galloprovincialis with a shell size of 50  1.2 mm was collected during the spring spawning period (April) in 2015 -2017 at the mussel-and-oyster farm in the coastal waters of Sevastopol (44 о 37′13.4″N; 33 о 30′13.6″E).To determine the sex of mussels in the laboratory, temperature stimulation of spawning was performed for each mollusk individually in the range 22-25 о С (Pirkova, 1994).To separate the males and females, the spawned gametes were examined under a Jenaval Carl Zeiss microscope with a magnification of ×200.Only females and males of M. galloprovincialis were used for the experimental work, and hermaphrodites were ignored.Experiments were conducted in natural conditions and laboratories.
To study the direction of sex inversion male and female mussels were placed in two separate cages, 100 specimens of the same sex in each.The cages were hung in the water of a semi-enclosed harbor (44 о 36′56.4″N;33 о 30′10.6″E),which is periodically polluted by domestic sewage (Ignat'eva, 2007).Further in natural conditions experiments, were carried out, with mussel females.Female, were placed, into two separate cages of 100 specimens in each and hung out in different areas: unpolluted water areas at the mussel-and-oyster farm (Kuftarkova et al., 2011;Chelyadina et al., 2017) and semi-enclosed polluted harbor.Six months later of each experiment, mortality of the mussels from the cages, their sex and the stage of maturity of their gonads were determined by visual examination of gonadal smears under the Jenaval microscope (Pirkova, 1994).
After temperature stimulation of spawning of each mussel, females were selected.Then, the effects of starvation, hypoxia, and chemical pollutants on the mussel sex change were investigated in laboratory.As the chemical pollutants synthetic anionic surfactants (detergents) and diesel fuel were used.The influence of each factor was studied on female mussels, which were placed in individual six containers of 20-L volume filled with sea water, 20 specimens in each.In the experiments, the water temperature did not exceed 17.8 -18.5°C, the filtered water was changed daily, and one container with mussels was used as a control.
A mixture of live microalgae consisting of chrysophytes (Isochrysis galbana Parke 1949 and Monochrysis lutheri Droop 1953), chlorophytes (Tetraselmis viridis (Rouchijajnen) R.E.Norris, Hori &Chihara 1980 andDunaliella viridis Teodoresco 1905), and diatoms (Chaetoceros calcitrans (Paulsen) H. Takano 1968 and Phaeodactylum tricornutum Bohlin 1897) was used for feeding mussels.The microalgae were grown in a batch cultivator (Trenkenshu et al., 2017).At the initial stage, microalgae were cultivated in a cumulative mode.After the stationary stage of growth had been achieved, the algae were transferred to a quasi-continuous culture by replacing a portion of the microalgae suspension with an equal volume of a freshly prepared medium.This was necessary to ensure that the culture density and the content of biologically valuable substances in the containers were approximately constant.An algae suspension in a concentration of 4 mg L -1 was used as feed.The average annual abundance of phytoplankton in the farm area is 0.5 mg L -1 (Ivanov, 2007).Therefore, in order to provide the food requirement of the mollusks (the average filtration rate of mussel 50 mm in size is 1-1.2 l H -1 (Pechen-Finenko, 1992) for 8 hours, 80 mg of the microalgae suspension was added to each tank (V= 20 L) thrice daily.Accordingly, in an experiment with a lack of food, 0,4 mg/l of a mixture of algae was added to the tank with mollusks.
The dissolved oxygen concentration in the sea water in the experimental containers was determined by the Winkler method (Metody…, 1988) and was maintained at a level of 5.0 -6.0 mg L -1 by aerators.Under hypoxia, the dissolved oxygen concentration in the sea water did not exceed 2 mg L -1 .The chemical pollutants were added to the tanks daily for the anionic surfactants to reach a concentration of 0.06 -0.10 mg L -1 and the diesel fuel concentration to be at a level of 0.03 mg L -1 .
The concentrations of chemical pollutants do not exceed MPCs (0.03 mg L -1 for diesel fuel and 0.1 mg L -1 for anionic surfactants) and are non-toxic to mollusks with prolonged action (Korshenko et al., 2008;Katunina & Smyrnova, 2015).
The mussels were affected by the factors in question for one month.The mollusks were then placed in separate cages, which were hung in the mussel-and-oyster farm area.After the three-months conditioning in the sea, sex and stage of gonad maturity of the mollusks were determined in the laboratory.The laboratory experiments were carried out in duplicate.In total, 1200 mussels were used in the study.All results are given as the mean +SD.

Results and Discussion
The results of the experiment conducted in 2015 were showed, that after six months of exposure of both females and males of M. galloprovincialis to water polluted with domestic sewage, sex inversion in males was not observed.Among females, 75% of the specimens had changed their sex, and the percentage of hermaphrodites was 4%.High mortality of the mussels (48% of females and 25% of males) was registered.
The mollusks were at stages 3 (active gametogenesis) and 4 (pre-spawning) of gonad maturity.
Since the sex change in males of M. galloprovincialis did not occur, the further study of sex inversion under the influence of environmental factors was carried out only on females.Cages with mussels were hung in the area of the sea farm and in the polluted water area.In 2016, after six months, the mortality of female mussels was 19% in the farm area and 69% in the polluted water.The fraction of hermaphrodites was higher at the sea farm (13%), which may also indicate the sex change.The percentage of survived female mollusks that had changed sex was 38% in the mussel-and-oyster farm and 58% in the polluted region (Fig. 1).The sex inversion in M. galloprovincialis is faster in the polluted water.Earlier it was noted (Chelyadina, 2014) that the shift in the sex ratio towards the increase of males occurred in water areas subject to anthropogenic impact.The number of male mussels cultivated near the Black Sea coasts of Crimea increased, and their fraction depended on location of sea farm.Under adverse environmental conditions, the sex ratio reached 1 : 7 (female : male).
It was shown that both genetic and environmental factors contribute to gender formation in different species of bivalve mollusks (Kenchington et al., 2002;Lee, 2015).The explanations of the genetic mechanisms of this process are contradictory (Zouros et al., 1992;Kenchington et al., 2002;Yusa et al., 2013).
The environmental factors affecting the sex ratio of the mollusks include: unfavorable habitat conditions, anthropogenic impact, food availability, temperature, etc. (Yusa, 2007;Stenyakina et al., 2010;Shurova, 2013;Chelyadina, 2014).Under the influence of pollutants, masculinization of the mollusk population can occur, which is the result of depression of some genes (Ivanov, 1989).
To study the differentiated influence of some external factors on the sex inversion in females, laboratory studies were carried out.Among various environmental factors that can change sex of the mollusks, we selected the anthropogenic pollution of Sevastopol bays by anionic surfactants and diesel fuel,

%
the content of which in bays of the Black Sea reaches 0.06-0.1 mg L -1 and 0.05-0.15mg L -1 , respectively (Korshenko et al., 2008;Katunina & Smyrnova, 2015), hypoxia, and starvation.It is known, that mollusks can change sex during the post-spawning development of gonads, which is controlled by the neuroendocrine mechanism (Dolgov, 1985;Ivanov, 1989;Kudikina, 2013).Therefore, the mussels after artificial spawning were under the influence of each of the factors in the laboratory for one month and then were placed for three months in the water area of the mussel-and-oyster farm.
At the end of the experiment, the mussels were at stages 3 and 4 of the reproductive cycle, their mortality did not exceed 2%, and the fraction of hermaphrodites was below 4%.The percentage of females that changed sex under the influence of the factors in question is shown in Fig. 2. Despite the short period of influence of the studied factors on M. galloprovincialis, the sex inversion in the females increased by a factor of 2-3 in comparison with the control group.
The most significant factor causing the sex inversion in 40% of the mussel females is diesel fuel.Petroleum hydrocarbons cover the tissues of mollusks, disrupting gas exchange and access of oxygen in the tissues (Shulman & Soldatov, 2014).Hypoxia is the second significant factor causing the sex inversion in female mussels.In the hypoxic experimental conditions, about one-third of the mussel females change their sex.In natural conditions, oxygen limitation affects the sex ratio variation in populations of mollusks, as well (Shurova, 2013).In cultivated mussels hypoxia can occur as the consequence of increase of specimen density in the cluster: when mollusks grow in several layers on a collector, mussels in the inner layers suffer from a lack of food and oxygen.
The next group of factors, which cause sex inversion in 25% of female mussels, includes the influence of starvation and anionic surfactants.The mollusks can accumulate anionic surfactants up to 0.5-0.4mg per 100 g of soft tissue (Katunina & Smirnova, 2015).The surfactants negatively affect the life activity of hydrobionts, with activating lipid peroxidation processes in them, disrupting the structure and functions of membranes, and damaging proteins, nucleic acid molecules and cellular and tissue structures (Ostroumov & Kolesnikov, 2003;Shulman & Soldatov, 2014).
Thus, adverse environmental situation induces variations in the biochemical status of mollusks.Changes in the internal environment lead to switching of gene expression, and, often, to an increase in the frequency of mutations (Korochkin, 1999), which can affect the sex change in mussels.In the coastal water area of Sevastopol, sex inversion in the cultivated mussel M. galloprovincialis proceeds only in one direction: from females to males.In this process, some of the individuals become hermaphrodites, which are a transitional form between males and females (Ivanov, 1989).

Conclusions
1.In the coastal waters of Sevastopol bays, the inversion of sex of the cultivated mussel M. galloprovincialis proceeds in one direction from females to males, whereas some specimens become hermaphrodites, whose fraction reaches 13%. 2. Under adverse environmental conditions including anthropogenic impact, the sex inversion in females of M. galloprovincialis rises to 58%. 3. The degree of influence of adverse environmental factors on sex inversion in the mussels M.
galloprovincialis is different and decreases in the following order: diesel fuel → hypoxia → anionic surfactants → starvation.

Figure 1 .
Figure1.Sex distribution after six-month exposure of females of the mussel M. galloprovincialis in 2016 in water of (A) the mussel-and-oyster farm area (81 specimens survived mussels) and (B) polluted area (31 specimens survived mussels).

Figure 2 .
Figure 2. Sex inversion in females of the cultivated mussel M. galloprovincialis after a one-month-long laboratory experiment and three-months-long conditioning at the mussel-and-oyster farm, 2016-2017.