Some biological parameters of Patella caerulea from the Black Sea

Limpets are common inhabitants of the midlittoral and upper infralittoral zones and play an important ecological role in the coastal ecosystem. The study aimed to assess growth, meat yield, morphological aspects, condition and reproduction features of Patella caerulea. About 58% of collected Mediterranean limpets were concentrated in the 25.0-34.9 mm shell length group. The average meat yield ratio was calculated as 39.34%. Mean growth increments for shell length (SL) and total weight (TW) were 23.99% and 97.99%, respectively. Results exhibited relatively high correlation coefficients among variables. Mean condition factor value was calculated as 14.2. Spawning occurs over a short period with ovigerous females observed on two month periods. Mean fecundity was calculated as 90,983±28,675 eggs/g whereas mean egg diameter was estimated as 160.6 μm. This study presents first baseline information about biological and morphological of Mediterranean Limpets population in Black Sea. Please cite this paper as follows: Aydın, M., Şahin, A. E., & Karadurmuş, U. (2021). Some biological parameters of Patella caerulea from the Black Sea. Marine Science and Technology Bulletin, In press. https://doi.org/10.33714/masteb.906225


Introduction
Marine gastropods are situated at several levels in the food chain, which increases their determining role in the functioning of marine ecosystems (Hakenkamp & Morin, 2000). Limpets of the genus Patella are grazing gastropods, common inhabitants of the hard substrate communities in the mid-littoral and upper infralittoral zones of the East Atlantic and Mediterranean coasts in temperate latitudes (Vafidis et al., 2020). Limpets are considered the "keystone" species of the mid-littoral zone and The Mediterranean limpet Patella caerulea Linnaeus, 1758 is among the most common species of rocky shores in the infralittoral and midlittoral, especially Mediterranean (Küçükdermenci et al., 2017). P. caerulea is considered endemic to the Mediterranean Sea (Christiaens, 1973). It is very prevalent in Turkish seawater (Öztürk & Ergen, 1996). This species primarily feed by scraping algae off rocks and plants such as Cyanophyceae, Ulva lactuca, Corallina elongate (Silva et al., 2008;Ayas, 2010). P. caerulea is a hermaphrodite protandric species and may reach a length of 70 mm (Kastanevakis et al., 2008). Mediterranean limpet fulfills the criterion an ideal bioindicator for heavy metal contamination is extensively used in marine monitoring programs (Storelli & Marcotrigiano, 2005).
Various studies have been conducted on the morphology, biology, ecology, and distribution of Patella species, especially in the Mediterranean Sea (Navarro et al., 2005;Espinosa et al., 2006;Prusina et al., 2014;Prusina et al., 2015;Bouzaza & Mezali, 2018). In Turkey, generally, morphometric, taxonomic, distribution, and heavy metals content studies on P. caerulea have been conducted (Bat et al., 1999;Akşit & Falakalı Mutaf, 2007;Ayas, 2010;Çulha & Bat, 2010). Although P. caerulea is the most dominant intertidal gastropod on the rocky shore of the Black Sea in Turkey, there is no information about these species in the region yet. The present study aims to make an extensive assessment of population structure and characteristics, growth, meat yield and reproductive status of P.
caerulea in the Black Sea. To our knowledge, this is the first study in Turkey of the species, on the Black Sea.

Study Area and Sampling
This study was conducted along the Turkish coast of the Black Sea (between 41.149538° N, 37.273525° E−40.971953° N, 38.059734° E). In this study, P. caerulea were collected from the intertidal zone of the coastline monthly from April 2018 to March 2019. Limpets were collected by hand with a penknife, and injured limpets (broken, shattered, severed) were not included in the sampling. Collected specimens were put into drums filled with seawater and then transferred to the laboratory. Collected samples were identified on the basis of form and external sculpture of the shell, the color of the pallial tentacles and the color of the foot (Christiaens, 1973).

Growth and Morphological Analyses
Shell length (SL − the longest distance between the anterior and posterior of limpet), shell width (SWi − the longest distance perpendicular to the anterior-posterior axis), and shell height (SH − the longest vertical distance from the apex to the shell base) of samples were measured using a digital caliper to the nearest 0.01 mm. Total wet weight (TW), meat weight (MW), and shell weight (SWe) was measured with an electronic balance of 0.001 g accuracy. The growth performance of samples according to SL and TW was determined with the formula below: where n is the number of size classes (Ricker, 1975). The SL, SWi and SH relationships with each one were determined using the equation (3).

Reproduction, Fecundity, and Egg Diameter
Sex was determined using macroscopic methods. The spawning period was determined by analyzing the monthly variation in the gonad maturity as well as the gonadosomatic index (GSI). In this study, 191 females were used to calculate the gonadosomatic index as described by (Bagenal, 1978) (Equation 5): The limpet eggs were removed from 26 ovigerous females in the spawning period (in October and November), and the total weight of eggs was measured using a balance with a sensitivity of 0.0005 g. Fifty eggs were taken from different regions of an ovary, and egg diameters were measured with an calibrated ocular micrometer. Eggs were counted with an ocular micrometer as (Equation 6): where W0 denotes the weight of gonad and n represents the number of eggs in the subsample, X stands for subsample weight (g), F represents the number of eggs (Bagenal, 1978;Kwei, 1978;Jones et al., 1990).

Data Analysis
Statistics and data analyses were performed by statistical software SPSS v26.0. The normality of the data was checked using the Kolmogorov-Smirnov test, depending on the sample size. In the former analysis, homogeneity of variance was tested using Levene's test. Since the data in this study were not normally distributed, monthly morphological values were compared using the Mann-Whitney U-test. Monthly and combined variables were analyzed with the Pearson correlation and regression analysis to investigate the relationships among morphological characters (Sokal & Rohlf, 1969;Düzgüneş et al., 1983). Hierarchical Ward cluster analysis after Z score correction was used to determine closely related variables (Lopez et al., 2004). The sex ratio of samples, the expected 1:1 male to female ratio was assessed using the chi-square (χ²) test (Düzgüneş et al., 1983).

Size Distribution and Meat Yield
In the present study, 1830 P. caerulea individuals were sampled during the study period. Shell length distribution of collected samples showed that 57.98% of Mediterranean limpets were in a range of 25 to 34.9 mm. It was observed that 77.40% of the samples collected were smaller than 5 g, according to the TW ( Figure 1). Ayas et al. (2008) reported the SL, SWi, and SH in a range of 19-39 mm, 15-33 mm and 5−11 mm from samples caught in the Mersin Bay (Mediterranean). Küçükdermenci et al. (2017) reported the minimum and maximum SL as 24.4 and 30.7 mm in Izmir Bay (Aegean Sea), respectively. Vafidis et al. (2020), who conducted a study in the Pagasitikos Bay (Aegean Sea), determined the average SL, SWi, and SH as 23.36, 18.96, and 6.35 mm, respectively. In our study, biometric parameters were more variable compared to similar studies, possibly attributed to variation in the number of samples or sampling regions. The environmental and ecological factors (food availability, water parameters, and meteorological factors) may lead to morphological variations in limpets of the same species that live in different ecosystems (Crothers, 1983). Monthly biometric measurements and statistical differences of P. caerulea are presented in Table 1. A closer look at the monthly SL reveals that the maximum value was 31.70 mm in December and the minimum value of 25.80 mm in May. Minimum SL, SWi, SH, TW, and MW were determined in May and July. High SL, SWe, TW, and SW values were obtained in April and December. Average MW of P. caerulea was calculated as 1.49±0.02 g with a rate of 39.34% of TW. In other words, 1 kg of edible meat can be obtained from approximate the 2.5 kg live weight of P.
caerulea. It was determined that the minimum and maximum meat yield ratio was 35.32% in November and 43.68% in August, respectively. A weak relationship was found between the SL and MW. However, the maximum ratio of meat weight (41.56%) was observed in the 25.0-29.9 mm shell length classes ( Figure 2). The mean meat weight of samples was significantly different by month (χ²= 99.851; df= 11; .001). Similarly, Küçükdermenci et al. (2017) determined the percentage of meat yield as 37% in Izmir (Turkey) for P. caerulea. Hamad et al. (2019) who conducted a study in Libya, determined the average meat yield as 1.09 g. The present study's findings concerning meat yield are in agreement with the findings reported by Küçükdermenci et al. (2017) and Hamad et al. (2019). Water conditions, especially temperature, may also affect the meat yield of Patella species (Fretter & Graham, 1976). Thus, it is usual to observe differences in meat yield. Note: Superscript and capital letters denote comparisons between two months (a capital and a superscript). There is no statistical difference between a capital letter and each superscript letter (P < 0.05). A -November, B -December, C -January, D -February, E -March, F -April, G -May, H -June, I -July, J -August, K -September, L -October.

Biometric Relationships and Growth
According to the proximity matrix, close relationships were exhibited between SWe and MW (3.39), TW and SWe (62.59),and TW and MW (79.88). For instance, the relationships of SL with SWe and MW were highly distant. However, there appeared to be weak relationships of SL and SWi with other variables, a tendency similar to that presented by the correlation analysis (Table 2, Figure 3). The mean growth increment value was calculated as 23.99% and 97.99% according to the SL and TW, respectively.
According to SL and TW growth increments, the highest growth (51.18% and 264.85%, respectively) occurred in 10.0−14.9 mm size class individuals (Table 3). Both growth types inversely correlated with the size classes but seemed to be close relations. Furthermore, the growth of individuals decreased with the increase of SL size classes and almost plateaued after 50 mm. The most remarkable difference between SL and TW increments concerning the models is that the TW growth increment increases after 40 g weight classes (Figure 4).
In this study, the finding showed that the correlation coefficients among variables were quite high (Table 4). According to Pearson correlation values, the strongest and weakest correlations were determined between SL and SWi (R 2 = 0.982) and between ST and MW (R 2 = 0.868), respectively. A linear relationship and strong interaction (R 2 = 0.95) were reported between SL and SWi of P. caerulea in the Mediterranean was found by Bouzaza and Mezali (2018). These findings are consistent with our results. Unlike these findings, Belkhodja and Romdhane (2012) reported that curvilinear regression equations between morphometric measures for Mediterranean limpets in Tunisia. Compared with our findings, weaker correlation coefficients (R 2 = 0.962 and R 2 = 0.908 for sheltered and exposed, respectively) were obtained of morphometric measurements of P. caerulea by Vafidis et al. (2020). Patella species may show significant morphological variability depending on environmental variation (Mauro et al., 2003). Correlations among the morphometric parameters are of particular significance in terms of understanding of smoothness of organism shell structure. It should be underlined, however, that big sample sizes could make a slight difference among the correlations that are significant (Aydın et al., 2014). The relationship of SL with TW and the linear relationship of SL with SWi and SH are presented in Figure 5. However, the b value of SL and TW of individuals was found as 3.09 (positive allometry). Considering the growth value (b > 3), it can be said that the sampled limpets have well enough environmental conditions, and the total growth of the individuals is provided as required. The b value was reported as 2.89 between SL and TW in the exposed site in Pagasitikos Gulf. Isometric growth type for SL and TW was reported by Vafidis et al. (2020). This value may vary depending on many conditions such as the number of samples, catch season, characteristics of the aquatic ecosystem, gonadosomatic index, and nutrition (Bagenal, 1978).

Condition Factor (K)
The mean condition factor (K) value was calculated as 14.2 for sampled individuals from the study area. The maximum K value occurred in June with 15.47, whereas the minimum K value occurred in December with 12.79. A significant increase in the condition of the individuals was observed in the summer with 15.03 ( Figure 6). The mean K value of individuals was significantly different by monthly (χ²= 164.21; df= 11; .001). The highest condition factor can signify the period before spawning in gastropods; water temperature may also affect both the index and reproduction of Patella species (Fretter and Graham, 1976;Vafidis et al., 2020). Based on this statement, it was seen that the increase in the K value before the ovulation period was significant. Thus, it was determined that the spawning season started in October and ended in November.
The maximum and minimum K value of P. caerulea was reported as 41. 20±1.18 in winter and as 30.46±1.54 in autumn, respectively, by Küçükdermenci et al. (2017). Vafidis et al. (2020) conducted that the maximum K value occurred in April (32.762±7.38), whereas the minimum K value occurred in February (26.58±4.47). The findings of our study were lower than the findings in other studies. Factors, such as different areas, spawning periods, and water parameters, may cause monthly changes in the condition of the limpets (Fretter & Graham, 1976;Belkhodja et al., 2011). caerulea. Horizontal lines in the boxes represent the mean values, and whiskers represent the standard deviations. Error bars represent 95% confidence intervals.

Reproductive Characteristics
Ovigerous females were observed for only two months (October and November), implying that spawning takes place for quite a short period for P. caerulea. The overall sex ratio of all individuals was calculated as 1:1.93 (M/F), with a significant deviation from the expected 1:1 ratio (χ²= 29.19; df= 1; .05) in the spawning period. The GSI values of P. caerulea ranged from 1.41 to 12.32%, with a mean value of 5.69%. It has been reported that the Mediterranean limpets spawn period is in winter in south Eastern Australia (Parry, 1982), whereas in Eastern Tunisian coasts was between February and May (Gharred et al., 2019). These values are different from those obtained in the present study, suggesting that the regional differences may play a significant role in the spawning period. The fecundity of 26 ovigerous females ranged from 39,773 to 127,273 with an average value of 90,983±28,675 eggs/g. The egg diameter of samples ranged between 115.0 μm and 242.5 μm (mean 160.6 μm). Gharred et al. (2019) reported the mean egg diameters for three sites on the eastern Tunisian coast as 56.93, 41.22 and 40.71 µm, respectively. The egg diameters of Mediterranean limpets are unequivocally higher than those obtained in the present study. The present monitoring study suggests that reproduction parameters of Mediterranean limpets may exhibit variations depending on the regions.

Conclusion
In conclusion, due to its ever-changing spatiotemporal structure, the Black Sea should be closely monitored in terms of biodiversity, food chains and ecosystems. Thus, it is essential and valuable to know the current status, growth, and reproductive performance of available populations. This study presents baseline information on the Mediterranean Limpets population in this area, where no studies have been carried out. This study assessed growth, morphometric parameters and population aspects of the Mediterranean limpet, contributing valuable new data on P. caerulea along the Black Sea coastal zones.