Distinguishing wild from released juvenile 

Sparus macrocephalus with otolith chemical signatures 

Abstract: A central goal of stock enhancement is to increase species abundance and potential harvest by supplementing recruitment of wild populations. The black sea bream Sparus macrocephalus represents very important recreational fishery along China’s coastline and is stocked in coastal bays to enhance natural fish abundance.To distinguish aquacultured fish from wild population is essential for evaluating the potential stock enhancement. Otolith chemistry signatures are regarded as natural tags to distinguish wild from released Sparus macrocephalus.

From May to June 2019, before the enhancement and releasing of Sparus macrocephalus in Xiangshan Bay, juvenile samples were collected with gill nets, and water temperature and salinity of sampling sites were recorded. The body length of collected Sparus macrocephalus was 3.053.89 cm with a sample size of 20. The samples captured were identified to be wild population according to sampling time and body length. During the rearing of Sparus macrocephalus, fresh water was pumped into the seawater every day to adjust the salinity of cultured seawater, and the salinity of cultured seawater was kept at a suitable range of 1825 for at least 30 days. Thirty cultured fish were collected. In August 2020, adult Sparus macrocephalus samples were collected from Xiangshan Bay, with a body length of 10.619.5 cm, and the sample size was 20. Samples of natural seawater, fresh water and cultured seawater were collected randomly for 5 days, filtered by microporous filter membrane (0.45 μm) to remove impurities, collected and stored in plastic bottles, and brought back to the laboratory for detection of Sr and Ba concentrations. From each individual, sagittal otolith pairs were extracted from the fish, cleaned with H2O2, rinsed with 18.2 MΩ MQ water and dried. Sagittae were embedded in araldite resin, ground on a sagittal plane to expose their primordium and polished with diamond papers of decreasing grain diameter. Finally, the otoliths were double rinsed with 18.2 MΩ MQ water, dried prior to LAICPMS analysis. A continuous linear transect was taken across the otoliths passing through the core to the edge at a speed of 2 μm·s-1. Five elements (Sr,Ba,Mn,Mg,Ca) were initially monitored.

The salinity of natural seawater in Xiangshan Bay was 2527, and the surface water temperature was 1822 ℃. The salinity of cultured sea waterwas about 2 degrees lower than that of natural seawater after pumping fresh water. The Sr concentrations of natural seawater and cultured seawater were (5 428.67±72.93) μg·L-1 and (4 377.59±52.85) μg·L-1, respectively. Ba concentration was (27.33±1.39) μg·L-1, (41.97±1.62) μg·L-1; Ca concentration was (342.6±2.57) mg·L-1, (289.2±2.78) mg·L-1. The Sr/Ca ratios of natural seawater and cultured seawater were (7.2±0.09), (6.96±0.08) mmol·mol-1, and the Ba/Ca ratios were (23.3±1.2), (42.3±1.6) μmol·mol-1, respectively. The Sr/Ca ratio and Ba/Ca ratio of cultured seawater were about 0.96 and 1.82 times of those of natural seawater. The concentration of Ba in cultured seawater was significantly higher than that in natural seawater (P<0.05).

The Sr/Ca and Ba/Ca ratios of otoliths of cultured and wild Sparus macrocephalus were measured from the core to the edge. The Sr/Ca ratios of cultured and wild Sparus macrocephalus were 1.52.0 mmol·mol-1, and the Sr/Ca ratios had little fluctuation. The Ba/Ca ratio of cultured Sparus macrocephalus was 1025 μmol·mol-1, and the Ba/Ca ratio of wild Sparus macrocephalus was 38 μmol·mol-1. The fluctuation of Ba/Ca ratio in cultured Sparus macrocephalus otolith was obvious, but the fluctuation of Ba/Ca ratio in wild Sparus macrocephalus otolith was relatively small. The results showed that there was no significant difference in Sr/Ca ratio between cultured and wild Sparus macrocephalus, but Ba/Ca ratio in cultured Sparus macrocephalus was significantly higher than that in wild. The otolith radius of cultured Sparus macrocephalus was about 400 μm. 

The Sr/Ca ratio near the otolith core (CSr/Ca) of cultured and wild Sparus macrocephalus was (1.96±0.12) mmol·mol-1, (1.97±0.06) mmol·mol-1, respectively, and there was no significant difference between them (P>0.05). The Ba/Ca ratio of the otolith core (CBa/Ca) in cultured and wild Sparus macrocephalus was (16.09±6.23) μmol·mol-1 and (3.05±0.58) μmol·mol-1, respectively. CBa/Ca of cultured Sparus macrocephalus was significantly higher than that of wild(P<0.05). The mean Sr/Ca ratios(XSr/Ca) of whole otoliths of cultured and wild Sparus macrocephalus were (1.77±0.06),(1.82±0.07) mmol·mol-1, respectively, and there was no significant difference between them (P>0.05). The mean Ba/Ca ratios value (XBa/Ca) of cultured and wild Sparus macrocephalus were (16.96±2.02) μmol·mol-1 and (4.08±0.32) μmol·mol-1, respectively. The mean otolith Ba/Ca ratio (XBa/Ca) of cultured Sparus macrocephalus was significantly higher than that of wild(P<0.05).  The coefficients of variation of Sr/Ca ratio (CVISr/Ca) of cultured and wild Sparus macrocephalus were 0.14±0.01 and 0.13±0.01, respectively, and there was no significant difference between them (P>0.05). The coefficients of variation of Ba/Ca ratio element (CVIBa/Ca) of cultured and wild Sparus macrocephalus were 1.26±0.3 and 0.69±0.15, respectively. The coefficient of variation of Ba/Ca ratio (CVIBa/Ca) of cultured Sparus macrocephalus was significantly higher than that of wild(P<0.05). CBa/Ca, XBa/Ca and CVIBa/Ca of the otolith core of cultured Sparus macrocephalus were significantly higher than those of wild(P<0.05). 

According to the characteristics that cultured Sparus macrocephalus’s CBa/Ca, XBa/Ca and CVIBa/Ca were significantly higher than those of wild population, otolith Ba/Ca ratio can be used as a microchemical marker for the identification of released and wild Sparus macrocephalus. Original source discriminant analysis was conducted on 20 adult Sparus macrocephalus samples captured in Xiangshan Bay. The CBa/Ca near otolith cores of two Sparus macrocephalus were 15.56,10.33μmol·mol-1, respectively. And XBa/Ca within 400 μm from the core were 16.08,9.23 μmol·mol-1, and CVIBa/Ca were 1.96 and 1.62, respectively. CBa/Ca, XBa/Ca and CVIBa/Ca at the juvenile stage of two Sparus macrocephalus were significantly higher than those of the other samples. According to Ba/Ca marker, the discrimination of 20 adult Sparus macrocephalus caught in Xiangshan Bay showed that two Sparus macrocephalus were released.

Keywords: Sparus macrocephalus；otolith；Ba；microchemical marker；element analysis