Mortality of the mussel Mytilus galloprovincialis (Lamark, 1819) depending on sex.pdf Introduction The mortality rate is one of the most important population parameters. It is necessary for assessing the state of various types of hydrobionts, as it largely determines the features of the size-age structure of a population, the production properties of organisms, and the lifetime and survival of hydrobionts [1-3]. The mussel M. galloprovincialis is one of the most important marine resources for aquaculture in boreal ocean waters [4]. It is one of the most abundant species of the Black Sea [5-6]. Previous research has primarily focused on the study of the mortality of molluscs based on age and size, as well as the issues of mollusc survival in various habitats [1, 7-10]. However, there are limited studies examining the mortality of M. galloprovincialis depending on sex. The relationship between the sex ratio of a population and the survival rate of the different sexes is an interesting yet insufficiently studied phenomenon. Until the 2000s, most researchers observed a balanced sex ratio equal to 1:1 ($:$) in M. galloprovincialis settlements in the Black Sea [5, 11-12]. However, over the last decades, the ecosystem of the Black Sea has undergone considerable natural and anthropogenic changes [13-14]. This has led to a transformation of the sexual structures in both natural and artificial settlements of M. galloprovincialis, trending towards an increase in the number of males [15-16]. In unfavourable environmental conditions, the sex ratio can reach 1:7 ($:^) [15, 17]. The sex-dependent mortality of mussels can influence the shift in the sex ratio of M. galloprovincialis populations. This can lead to negative consequences for populations of M. galloprovincialis in the Black Sea. In recent years, there has been a decrease in the number of M. galloprovincialis in the northern Black Sea, as well another species of mussel, Mytilaster lineatus, in natural settlements [18]. The current study suggests that the unbalanced number of male individuals in the coastal waters of Crimea may be due to the increased mortality of M. galloprovincialis females. Thus, the main aim of the current study was to assess the sex-dependent mortality ofM. galloprovincialis in natural and laboratory conditions. Materials and methods Material sampling area and research objects The mortality of M. galloprovincialis was studied in natural and laboratory experiments in 2017 and 2018, respectively. Mussels with shell sizes of 55.7 ± 2.9 and 30 ± 1.6 mm were used, as it enabled any variability along the length of the shell to be excluded. Furthermore, the choice of these sizes is due to molluscs with a shell length of 30 mm being able to reproduce and, therefore, able to have their sex determined. 55 mm molluscs are of commercial size and have a practical interest to farmers. For the current study, mussels from a mussel-oyster farm located at the outer roadstead of Sevastopol were collected from a depth of 3 - 4 metres. The natural experiment took place at the mussel-oyster farm Natalya S. Chelyadina, Mark A. Popov 168 (44°37’13.4’’N; 33°30’13.6’’E), in a semi-enclosed harbour (44°36’56.4’’N; 33°30’10.6’’Е; Fig. 1). Fig- 1- The map of the study area Thewaters at the mussel-and-oyster farwiss classified asmesotrophic [19]. Feed resourcor rt tire cwndns fermorofavo urable for the growth and development of melluscs [20]. The water area sf fhe adjacent semi-enciosed hatbour -s occastonally polluteW by hc^r^s ohoSd wastewater-resulting id idcreaser in woter Srophicity. The moximom niState concentiatlon was recwrded as (rgL4, WSo phosphate ooncentiotlon es -00 dr Sr h fiss aohform imtex ss 1000 L-1, and the total bacterial count (TBC) as9l0 mL"1 [21]. The concentration of the nutrients was 8 times higher than in the conditionalls aSoas coostal icrenrof ihr sew. anC orgonio pollution indicrtors sxceeded Sha tSrr^slfold limit vaTues bySl-lO times [22]. The eoncentration ohShr nutriente war determine1 by soandarh .ychoc.octioal metisads [23. Edpwhsmentps musA the itt wcillopscvincialis population was samyled during the mnsr spring spawning period. CiWificial rpnwmng wes hurried oui tn determine the ueo nf Ide mnssuls.The spawning was trmperaSure-stimwlated us laboralorycondilioni dor each uadinitliflmussd to discriminate tiieti sex [e].To stirndate spawnm9, each moltuocwes placed in a seporetu 250 mL container. Wild wader in tdecowtemer was heo)ed uplu brtwoen 2VX - )t40C withthc temperaeure meosnrod usiwst ss metenrolngieal 0hermomuterTW-10 fTeunopribwa. Ktlip Russia). The tex o, raid mofiwsc wardetsnnined efter apaouting wsmg a tenoval mtesosco.e (Css9 Zeiss Juno, Gem-esty). Hrrmadhsodhes were Ciscarded [2d]. Mortality of the mussel Mytilus galloprovincialis 169 For individual identification of molluscs, the marking method was used. A marking denoting whether a mollusc was female or male was engraved on each mollusc shell using a Sturm GM2316 handheld electric engraver. This method reduced the laboriousness of the labelling process and increased the reliability of the research, as mollusc identification is not affected by prolonged exposure in the marine environment. In the natural experiment, 100 individual females and 100 individual males of each size (30 mm or 55 mm) were put in four separate Ostriga-5 cages (Isti-tuto Delta Ecologia Applicata, Italy) and placed in a semi-closed polluted harbour (Fig. 1). At the same time, the same number of cages and mussels were placed in relatively clean water on the mussel-oyster farm. The duration of sea exposure was six months for each cage of mussels (from April to October). After 6 months, the number of dead mussels was counted. The laboratory experiments examining the mortality of mussels were carried out in triplicate. The experiment was conducted in six aquariums, with each aquarium considered an individual experiment. Three aquariums contained 30 mm mussels, and three aquariums contained 55 mm mussels. Oxygen content in the aquariums was measured monthly using the Winkler method [23]. The oxygen content for the studied period was in the range of 4 - 4.5 ^g-L-1. In each aquarium, there were 15 males and 15 females, labelled according to their sex. The molluscs were placed in separate 20 L aquariums filled with seawater. The water was taken from the mussel-oyster farm. Water in the aquarium was replaced after the death of any individual mollusc, and there was no feeding or aeration. Each experiment lasted until 50% of the molluscs in the aquarium died. The duration of the experiments ranged from 1 - 6 months. The aquariums were in a semi-basement unheated laboratory room in order to prevent rapid death of the mussels due to any rise in temperature, and the water temperature was measured daily. The mortality of mussels was calculated as the fraction of dead molluscs compared to the total number of molluscs. The individual age of the mussels was determined using the method of sclerochronology [25]. This method is based on the calculation of the seasonal growth layers in a shell when it is cut. The cut shows alternating light and dark layers of calcium carbonate. Summer layers are dark, and winter layers are light. A pair of stripes (dark and light) is formed in one year. This method makes it possible to determine age with accuracy within six months. The age of mussels with a shell size of 30 mm was in the range of 6 months, whereas the age range of the 55 mm mussels was 1 - 1.5 years. Data analysis 580 samples of mussels were investigated in total. In the natural experiment, the results were processed by a pseudo-random number sampling method [26]. Up to 33 molluscs were randomly sampled from each cage, and each sample was analysed individually. The results are presented as mean (M), standard deviation (S), and confidence interval (Ax). An unpaired Student’s ?-test was used for statistical evaluation (P < 0.05). Natalya S. Chelyadina, Mark A. Popov 170 Results of the research Mortality of mussels in a natural experiment High mortality of M. galloprovincialis was observed in the natural experiment in both harbour cages, regardless of mussel size. The mortality of females compared to males was 23% higher in the 55 mm size group, while in the 30 mm size group, it was 18% higher. There was no significant difference in mortality of mussels based on the size of the mussels (Fig. 2). Fig. 2. Mortality of M. galloprovincialis depending on sex and size in the natural experiment, 2017. Data are presented as means (M) In the clear water area at the mussel-oyster farm, mussel mortality was low. In the 55 mm size group, 4 females died. In the 30 mm size group, 2 males and 4 females died. Mortality of mussels in the laboratory experiment In the laboratory experiment, the mortality rate of females of both sizes was 16% higher than males (P < 0.01). Additionally, females began to die first. There was no significant difference in the mortality of mussels depending on size (Fig. 3). The mortality of mussels in each aquarium varied. The onset of death depended on the viability of the molluscs and the water temperature. Molluscs lived up to 6 months under laboratory conditions. However, weakened mussels that died in the aquariums poisoned the water with the decay products from their dead tissues after 1-4 days [27]. Mussels in the experimental aquariums began to die gradually (within two to three weeks). The mortality of mussels increased sharply if the water temperature in the aquariums exceeded 22°C. Figure 4 shows the mortality of mussels with a shell size of 55 mm in the longest experiment. Mortality of the mussel Mytilus galloprovincialis 171 10 9 8 7 7 6 5 4 Sin 3 І 2 3 w 1 0 □ 55 mm Vk 30 mm Ma1 es Females Fig, 3. Mortality of the mussel M. galloprovinaalisdependmg on sex and size in the laboratory experiment, 2018 Fig. 4. Monthly mortality ofM galloprosiyclaSis with a sheSl size of 55 mm in the laboratory experiment, 2018. Dishuisioh of thereseatoh The suodvai of molluacs ls influence, by such factoai such ao ags, cize, the density of aggoegations, temperatei1gahmty, aud waterpoflntionlH 9, y8s30l. T^li^ high mortality of mussels (315%) in fUe haobour (Curing ahe natural exptri-myni wse associated wsth the mWavaurnfi e oonditions in OUis ares sust aa iimifed water exohange md wwstewnsee dsaoharga. fUigli mortahty wndor trsinflaonneoa untreated mnnicipsd wastewater wni also ebcenreF l-s fres^ioi)^^yr mussrtf Fsii-blsma pllcafa (Says l^llh) amd Casbloula Wgmiwna )MulSeOi 1074a of ths Colo- Natalya S. Chelyadina, Mark A. Popov 172 rado River located in eastern Travis County, Texas [9]. In the clear waters of the mussel-oyster farm, the mortality of mussels was 4% - 6%. The conditions for growing mussels at a mussel-oyster farm were favourable for the development of suspended conchioculture. In the laboratory experiment, environmental conditions influenced the mortality of mussels. The oxygen content in the aquariums was at the lower limit of the total limit value [31] and had no effect on mussel mortality. Therefore, the biggest impact may have been a lack of nutrition. During the experiment, mussels lived off their internal body resources for an extended period of time. It is known, with prolonged exposure to unfavourable factors, the mechanisms of anaerobic resynthesis of adenosine triphosphate, using carbohydrate and protein substrates, is activated in mussels [32]. Over time, the body’s resources, therefore, become depleted. In the last two months of the experiment, molluscs that were still alive were not attached to the walls of the aquarium or to each other by a byssus. Some mussel shells were opened slightly. After the death of a mussel, their tissue was observed to be thin and had a cadaverous smell. It is well-known that as molluscs increase in age (size), so too does their individual mortality [8]. This phenomenon has been described in populations of Mytilus edulis (Linnaeus, 1758) off the coast of Canada [33] and inMacoma balthica (Linnaeus, 1758) in the northern Baltic Sea and Hudson Bay [34]. A significant difference in the mortality of M. galloprovin-cialis depending on age (size) was not observed in the laboratory experiment (p > 0.01). In the current study, a slight increase in the rate of elimination in larger mussels was observed. This could be due to a small difference in the age of the mussels studied. The influence that the density of mussel aggregations and water salinity has on mortality has been noted in previous literature. However, these factors can be disregarded in the current study, as the experiments were conducted in aquariums and cages, where mussels did not experience dense aggregations. The salinity of water in the experiments also did not affect mortality, as the salinity of the mussel-oyster farm from 2001 - 2018 was in the range of 17.25% -18.40% [35]. The optimal salinity range for M. galloprovincialis is 12% - 25% [5]. Sex ratios of mollusc populations are often presented as the proportion of males and females among adult individuals. Typically, these ratios do not account for mortality, size, or other features that could potentially affect the ratio. Therefore, disregarded differential sex-dependent mortality can cause misrepresentation of the sex ratio in adult molluscs [36]. In the current study, significant differences in the mortality of mussels depending on sex were observed, with the mortality of females found to be higher. The uneven death and survival between sexes are common among animals. However, females are usually more viable. One of the popular explanations for the difference in life expectancy between the sexes is that the heterogametic sex lives less than the homogametic one due to the recessive X-linked deleterious mutations negatively affecting the lifetime of the heterogametic sex [37]. However, the sex of mussels is not genetically determined [38]. Thus, the differences in the sur- Mortality of the mussel Mytilus galloprovincialis 173 vival rates of male and female mussels is thought to be associated with the high energy expenditures needing for oogenesis [30, 39]. Myrand et al. [40] noted that after spawning, the mussel M. edulis, which is found in the southern bay of the Magdalena Islands, had low post-spawning glycogen content, which weakened their vitality. Similar results were obtained experimentally in the freshwater bivalve mollusc of the genus Unio [41]. In the current study, it was demonstrated that female mussels are more sensitive to unfavourable environmental conditions, with the mortality of M. galloprovincialis females from natural Black Sea settlements being higher than that of males during prolonged anoxia [1]. Furthermore, a lower female survival rate was previously noted in the White Sea Hydrobia ulvae mollusc populations (Pennant 1777) due to a decrease in water salinity and temperature [30]. A shift in the sexual structure, trending towards an increase in the number of males, has also been observed in Macoma calcarea populations (Gmelin, 1790) from the Barents and Pechora seas due to the high mortality rate of females [42]. Conclusion In the natural experiment of the current study, the mortality of females compared to males in the polluted harbour was 23% higher in the 55 mm group and 18% higher in the 3o mm group. However, both males and females experienced high mortality rates of up to 35% in the harbour, which was associated with the unfavourable conditions of the area. In the conditionally clear water area of the mussel-oyster farm, the mussel mortality was comparable lower, at 4% - 6%. In the laboratory experiment, the mortality of females of both sizes was 16% higher than that of males. It was also observed that females began to die first in the laboratory experiment. Therefore, in both the natural and laboratory experiments, the mortality of females was significantly higher than that of males. Differential mortality is important for studying the state of dynamic equilibrium in the population of M. galloprovincialis. These studies can be useful and applicable to other species of bivalve molluscs. One of the reasons for the increase in the number of males in the settlement of the mussel M. galloprovincialis of the Black Sea is the high mortality of females compared to males. However, the study of sex-dependent mortality requires further, more detailed research.

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