SOME REPRODUCTIVE PATTERNS OF CULTURED MEDITERRANEAN MUSSEL ( Mytilus galloprovincialis Lamarck, 1819) IN BOKA KOTORSKA BAY

SUMMARY Results about annual reproductive cycle of Mediterranean mussel ( Mytilus galloprovincialis Lamarck, 1819) in Boka Kotorska Bay are given in this paper. Sex ratio, gonad developmental stages, oocytes diameter, mean gonad index, condition index as well as correlation to water temperature were analysed. Sex ratio was 1:1. Maturation and spawning occurred over a great part of the year, from October to June, with short resting period during the late summer, when water temperature was above 25°C. Mean oocytes diameter ranged from 56.68 ± 10.52 µm in October 2015 to 66.58 ± 12.09 in December 2015. The highest mean oocytes diameter appeared during period when most of individuals were in ripe stage. Significant negative correlation between mean gonad index and sea water temperature was found, as well as weak negative correlation between condition index and sea water temperature. Positive correlation between condition index and mean gonad index showed that condition index followed the mean gonad index pattern. Obtained qualitative and quantitative data on reproduction cycle of cultured M. galloprovincialis are of great significance for farmers.


INTRODUCTION
Despite great natural potential, marine aquaculture is still poor developed in Montenegro. At this moment marine aquaculture is developed only in Boka Kotorska Bay. Since 2020, with the support of the Montenegrin Ministry of Agriculture, Forestry and Water Management, studies regarding potential locations for marine aquaculture along the open part of the Montenegrin coast have begun .
Mediterranean mussel (Mytilus galloprovincialis Lamarck, 1819) is the main cultured bivalve species in Montenegrin marine aquaculture. Initial exploration on the possibility of mussel farming goes back to the early 1960s, while commercial farming began to develop in the second half of the 1980s (Mandić et al., 2016). Today, there are about twenty mussel farms situated in Boka Kotorska Bay (Gvozdenović et al., 2020;MONSTAT, 2022). On all farms the traditional farming method is implemented (floating parks), and annual mussel production during 2021 was 186 tons (MONSTAT, 2022). Additionally, on mussel farm in settlement Ljuta, raft culture (a method of growing mussels using a floating platform with rope or socking material hanging from its bottom) is implemented. The consumption size of cultured mussels is 5 cm according to the law of Montenegro (Službeni list Crne Gore 65/15, 2015) and mussel reaches that length in the second year of their life.
Mediterranean mussel has fan-shaped, triangular or elongated shells. Shell valves are equal, pointed at the front and rounded at the back. The shell is blackblue on the outside, and nacreous inside. The mussel secrete a byssus, which plays a role in attaching to hard substrates and even in moving. It lives in dense colonies, and the densest settlements are located in the tidal zone. The mussel primary feeds on phytoplankton. Shell length can be up to 15 cm, and weight up to 200 g. The sexes are separate, and mature in its first year of life. As mussels are good examples of bivalves with a flexible reproductive strategy, adjusting their cycle according to prevailing environmental conditions (Gosling, 2003), annual reproductive cycle of cultured mussel is very important for farmers, mainly because of spawning period and subsequently period of young collection from nature environment.
Although, reproductive biology is described in many bivalve species, especially in economically important species, there is a lack of data related reproductive biology of M. galloprovincialis in Montenegro. Stjepčević (1974) gave some results about reproductive biology of cultured M. galloprovincialis in Boka Kotorska Bay, and described prolonged spawning period with spring and autumn peak.
Thus, the aim of this study was to investigate the annual reproductive cycle of cultured M. galloprovincialis in Boka Kotorska Bay, using quantitative and qualitative analysis (gonad stage analysis and oocyte diameter), as well as sex ratio, mean gonad index and condition index.

MATERIAL AND METHODS
Sampling was done monthly on two mussel farms (Orahovac and Sv. Nedjelja) in Boka Kotorska Bay (Figure 1). During each month, starting from February 2015 till January 2016, 15 individuals of M. galloprovincialis were sampled from pergolas at depths 2-3 m. In total, 180 individuals were collected. For each specimens following parameters were measured: shell length (SL), total weight (TW) and wet meat weight (WMW). SL was measured as maximal anterior-posterior axis (Prgić, 2019), using vernier caliper to the nearest 0.1 mm, while weighing was done by balance to the nearest 0.01 g. Condition index (CI) was calculated as ratio between wet meat weight and total weight (Almeida et al., 1999). Sea water temperature was measured each month at depth 2-3 m using the Multiline P4 WTW Probe. For quantitative and qualitative analysis, whole gonads were separated from rest of tissue and fixed in 10% formaldehyde in plastic bottles volume of 15 ml. The gonad tissue was then dehydrated, embedded in paraffin and stained by the haematoxylin and eosin (e.g. Crnčević et al., 2013;Popović et al., 2013). Each histology prepared gonad slide was examined at 100, 200 and 400 magnifications (Zeiss Axio microscope) and development stages were assigned according to Lubet (1959) (Table 1).
A mean gonad index (MGI) was calculated for each month for both sexes together. It was calculated by multiplying the number of individuals from each developmental stage (ni) by the numerical ranking of that stage (Si), and dividing the result by the total number of individuals (N) (e.g. Seed, 1975;Gosling, 2003;Benomar et al., 2006;Bhaby, 2015).

MGI=∑ni*Si/N
Quantitative analysis included oocytes counting and diameter measures. Oocytes with visible nuclei within one visual field at 100 magnification were measured as described by Popović et al. (2013), using AxioCam ICc3 camera and Axio Vision Rel. 4.6 program. Table 1. Gonad development stages in mussels (according Lubet, 1959) For sex ratio, additional 1080 individuals were used, so for sex ratio in total 1260 individuals were processed (180+1080 individuals). The gender for 180 individuals is determined visual (based on color) and also by histology, while for additional 1080 individuals gender is determined only based on color (the mantle containing the gametes is typically orange in females and creamy-white in males) (Dardignac-Corbel, 1990;Gosling, 2003).
To test sex ratio, a chi-square test was used. Differences in shell length with respect to sex were analysed using Mann-Witney U test, as Levene‵s test showed that variance were not homogeneity. Correlation between CI and MGI, CI and temperature, MGI and temperature was determined using Pearson correlation.

RESULTS
There was in total 627 females and 605 males, while for 28 individuals sex could not be determined. There was no statistical differences in sex ratio (chi-square=3.12, p=0.07). Percentage ratio between males and females is presented on Figure 2. Mean shell length of analysed females was 57.7±8.1 mm, while for males was 55.5±5.9 mm.
There was no statistically significant differences in the shell lengths between males and females (U=3178, p= 0.0536).

Figure 2. Sex ratio of Mytilus galloprovincialis
The reproductive cycle of M. galloprovincialis showed prolonged maturation and spawning period, from October to June (Figure 3).   (Figure 6). There was moderate positive correlation between MGI and CI (r=0.5517, p=0.06), significant negative correlation between MGI and sea water temperature (r=-0.7695, p=0.003), and weak negative correlation between CI and sea water temperature (r=-0.0277, p=0.933).
Obtained data showed that M. galloprovincialis is a dioecious species with a sex ratio 1:1. Similar sex ratio was reported by other authors for M. galloprovincialis (Da Ros et al., 1985;Suárez et al., 2005;Bhaby et al., 2014;Bhaby, 2015;Azpeitia et al., 2017), as well as for M. edulis (Sunila 1981;Toro et al., 2002). Contrary to those authors, Chelyadina et al. (2018) in M. galloprovincialis populations from Black Sea found more males during all investigated period (M:F=2.8:1). Sex determination mechanisms in bivalves seems neither purely genetic nor purely environmental (Dalpé, 2020), and sea water temperature is considered as one of the most important environmental factor affecting sex determination in animals (Wedekind, 2017).
In 180 individuals which sex was examined both, by mantle color and by histology, we found a high overlap between these two methods, even in 76% specimen sex was exactly determined. Although in ripe mussels the mantle containing the gametes is typically orange in females and creamy-white in males (Gosling, 2003), sex determination based only on color is considered as not reliable without histology, as it is known that carotenoid pigments, responsible for the orange coloration, are involved in defense against oxidative stress, thus males could also display high concentrations of carotenoids which could lead to gender misidentification (Puljas and Brkić, 2020).
Bivalves are known to have annual reproductive cycle with one or more spawning picks depends on species, geographical location and environmental conditions and mussels are therefore good examples of bivalves with a flexible reproductive strategy, adjusting their cycle according to prevailing environmental conditions (Gosling, 2003). Qualitative results which we obtained in this paper showed that maturation and spawning occurred over a great part of the year, through even nine months while spawning peak was from January to April. Dominant resting period (August 2015) overlap with period when sea water temperature was the highest (> 25°C). Almost the same reproductive pattern in M. galloprovincialis population from Venice Lagoon (north Adriatic Sea) was reported by Da Ros et al. (1985). Da Ros et al. (1985) have also found prolonged spawning period with peak in January and February, as well as reproductive quiescent in July and August. Besides, Hrs-Brenko (1971; and Dardignac-Corbel (1990) reported prolonged spawning in populations across Mediterranean Sea including Adriatic Sea, as well as Stjepčević (1974) in populations from Boka Kotorska Bay and Azpeitia et al. (2017) in populations from Bay of Biscay. Kunduz and Erkan (2008) in populations from Sea of Marmara as well as Bhaby (2015) in populations from Marocco reported spawning during almost all year including summer. Prolonged spawning period of cultured bivalves is of great advantageous for farmers as brood stock may be available over a longer period of the year (Popović et al., 2013).
Food availability and temperature are two main environmental factors affecting reproductive cycle in mussels (Suárez et al., 2005). Boka Kotorska Bay is characterised mainly as mesotrophic to eutrophic area where chlorophyll a reaches maximum values mainly in winter (sometimes > 10 mg m -3 ) and phytoplankton abundance quite often reaches values up to 10 7 cells L -1 (Drakulović et al., 2016), what is very favorable for reproductive activity in cultured M. galloprovincialis. Our results showed significant correlation between MGI and sea water temperature indicating that temperature has strong effect on reproductive cycle as it is reported by other autors (Rand, 1973;Lubet, 1987;Brenko, 1980;Bilecik, 1989;Da Ros et al., 1985;Egzeta-Balić et al., 2020). We also found that most intensive spawning in M. galloprovincialis appear at temperatures between 10 and 15°C, what is in coincides with Lubet (1987) and Hrs-Brenko (1980) who pointed that temperatures below 8°C and above 16°C affect gametogenesis inhibition in mussels.
Quantitative analysis included oocytes counting and diameter measures. Quantitative results were in coincides with qualitative results. The largest oocyte was measured in December 2015 and has diameter 132.63 µm. Additionally, the highest mean values of oocytes were during November and December 2015 (66.31 i 66.58 µm, respectively), indicating that the largest oocytes appear in ripe and partly spawned individuals, as observed in other bivalve species (Gribben et al., 2004;Meneghetti et al., 2004;Peharda et al., 2006;Mladineo et al., 2007;Moura et al., 2009;Crnčević et al., 2013;Popović et al., 2013). Toro et al. (2002) reported mean oocytes diameter from 70 to 75 µm in M. edulis, and 60 to 65 µm in M. trossolus, while Smart et al. (2020) reported values from 28.7 to 35.2 µm in M. galloprovincialis populations from New Zealand.
The CI is highly dependent on reproductive cycle, and was investigated as such in different bivalve species (e.g., Hrs-Brenko, 1973;Peharda et al., 2003;Marušić et al., 2009). In our study, moderate positive correlation between CI and MGI was found, means that CI followed the MGI pattern. Bhaby (2015) also reported that CI follows the changes in the reproductive cycle in M. galloprovincialis as well as other authors for other bivalve species e.g., Venus verrucosa (Popović et al., 2013) and Glycymeris nummaria (Crnčević et al., 2013).
Although obtained results give great insight in reproductive biology of cultured M. galloprovincialis in area of Boka Kotorska Bay, limitations of the study can be related to sample size, so data analysis (e.g., developmental stages, mean gonad index) were done on total sampleboth sexes were analyzed together.

CONCLUSIONS
Obtained data on the reproductive cycle showed that cultured M. galloprovincialis in Boka Kotorska Bay has long spawning period with a short resting period what can be defined as "bradyctic" as described in Da Ros et al. (1985). Sea water temperature has strong influence on the reproduction pattern, thus temperatures below 20°C has positive effect on gametes activity. Obtained qualitative and quantitative data on reproduction cycle of cultured M. galloprovincialis are of great significance for farmers in Boka Kotorska Bay.