Effects of 5-HT and thermal stimulation on artificial induction

 of spawning of Ruditapes philippinarum 

ZHOU Liqing1,2, WU Lei1,2,3, DONG Zhiguo3, ZHANG Tianshi1,2, 

LIU Zhihong1,2, WU Biao1,2, WANG Songlin1,2,3, SUN Xiujun1,2

(1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, 

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao Shandong266071, 

China; 2.  Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for 

Marine Science and Technology (Qingdao), Qingdao Shandong266071, China; 3. College of Marine Science 

and Fisheries, Jiangsu Ocean University, Lianyungang Jiangsu222005, China)

Abstract: Manila clam Ruditapes philippinarum belongs to veneridae, which is a mudflat shellfish widely cultivated in the north and south coast of China. In recent years, artificial culture of R. philippinarum has gradually increased year by year, with huge economic benefits. In 2020, the total production of clams in China reached 4.21 million tons, of which R. philippinarum accounted for more than 80%. However, the clam breeding industry is faced with many problems, such as lack of improved varieties and low growth rates, which are coupled with the excessive development and utilization of offshore waters and environmental degradation in the north and south coast of China. Efficient family construction is one of the important methods to evaluate the heritability of shellfish and breeding of new varieties during clam genetics and breeding. In clam hatcheries, sperms and eggs from mature gametes can usually be obtained by artificially induction of male and female parents. However, it is very difficult to obtain highquality sperms and eggs, which are generally essential to be naturally released by artificial induction. Recently, the traditional artificial induction has now faced the problems of unsynchronized, unstable and low emission rate of gametes in the clam industry. The combination of thermal stimulation and 5HT injection may have the potential to improve the induction rates of gametes, which may further improve the synchronization of parental spawning of sperms and eggs. In this study, the clams were treated with three induction methods: 5HT injection combined with thermal stimulation, single thermal stimulation, and single 5HT injection. R.philippinarum in the control group were injected with clean and filtered seawater. After clam shells opened, we placed tweezers between upper and lower shells for the injection. The mature gonads of R. philippinarum were injected with 0.30.4 ml of 2 mmol·L-1 5HT solution using a disposable sterile syringe (1 ml). For thermal stimulation, temperature of seawater rapidly increased from 20 ℃ to 29 ℃. After the treatment, the time for releasing of sperms and eggs was recorded. The measured data were analyzed by oneway ANOVA on SPSS 26 software, which was followed by Duncan multiple comparison method to compare the differences among groups (P<0.05). 

In the combined treatment group, the number of eggs released within 10 minutes after injection accounted for 57.45% of the total clams. The number of spawning clams released eggs after 2060 minutes accounted for 19.15% of the total clams. As indicated, the releasing of eggs for clams mainly appeared within 20 minutes in the combined induction group. For single 5HT injection group, 42.86% of the total clams released eggs in 1020 minutes, and the total spawning period lasted for 80 minutes after injection. In the heat stimulation group, only one female clam spawned in 3040 minutes, while no spawning occurred in other time periods. For male clams, the number of spermiation within 1020 minutes accounted for 67.39%91.3% of the total clams in the combined induction group. In the combined induction group, the induction effects on male clams mainly occurred within 20 minutes. In single 5HT injection group, the spermiation rate of male clams after injection of 5HT accounted for 78.57% within 40 minutes, and the spermiation lasted for 80 minutes after injection. In the heat stimulation induction group, there was one male clam spermiation in 2030 minutes after injection, without spermiation in the rest of time. In the control group, one male clam released sperms within 3040 minutes, but there was no spermiation in the rest of time. One way ANOVA showed that there were significant differences in spawning rates among different groups (P<0.01). The maximum proportion of gametes released by the clams treated with 5HT injection and thermal stimulation reached 75.00%. The total spawning ratios of the heat stimulation group and the control group were both less than 5%. As indicated by oneway ANOVA, a significant difference in the spawning rates of males and females was found between the combined induction group and 5HT injection group (P<0.01). The clams from the combined treatment had spawning rates of 50% in males and females. In contrast, 66.67% and 33.33% of clams injected with 5HT alone released sperms and eggs, respectively. 

As a result, when 5HT was injected with a concentration of 2 mmol·L-1 into the gonads of clams, the spawning rates of female and male parents were calculated to be 52.4% and 50.0%, respectively. The spawning time after the induction was more than 60 minutes. In contrast, the spawning rates of clams were 80.9% in females and 91.3 % in males within 20 min, which were induced by the combined effects of 5HT injection and thermal stimulation. However, thermal stimulation alone had little induction effects on spawning rates. There was a significant difference in spawning rates among different groups (P<0.01). After the whole experiments, the highest induction rate (85.19%) was found in the combined induction of 5HT injection and thermal stimulation. For 5HT injection or thermal stimulation alone, the induction rate of 5HT injection was 75.0%, while the induction rate of thermal stimulation alone was less than 5.0%. A significant difference was found in the induction rates between the combined treatment and single injection of 5HT (P<0.01). Therefore, our present findings reveal that the combined treatment of thermal stimulation and 5HT can effectively induce spawning of clams. Thermal stimulation can effectively shorten the induction time of 5HT, which has the advantages of high efficiency and strong synchronization of artificial induction. For male and female parents, 5HT injection induces a relatively high spawning rates of eggs from female parents than those of sperms from male parents. The combined treatment of 5HT injection and thermal stimulation is a good strategy for the rapid and efficient method for clam spawning, which will provide foundational basis and technical support for spawning induction and genetic improvement of clams and other shellfish.

Keywords: Ruditapes philippinarum; spawning induction; thermal stimulation; 5hydroxytryptamine