Abstract:Chinese horseshoe crab (Tachypleus tridentatus) belongs to the Arthropoda, Chelicerata, and Merostomata taxonomic groups and is, specifically, a member of the horseshoe crab family (Limuroidea). Tachypleus amebocyte lysate (TAL) reagent, prepared from the hemolytic solution of T. tridentatus, can quickly coagulate into a colloid with bacteria; therefore, TAL is a conventional reagent for the detection of endotoxins. Due to habitat destruction and overfishing, T. tridentatus has become an endangered marine animal, with its resources on the verge of depletion. Artificial breeding and release are important ways to increase its population in the wild. Artificial breeding of T. tridentatus has been conducted as early as the 1980 s. However, artificial breeding strategies involve killing horseshoe crabs to obtain eggs, with few reports on larval cultivation; therefore, this breeding process still lacks detailed technical standards and specifications. In recent years, artificial breeding has been adopted to promote natural spawning of broodstock horseshoe crabs under simulated wild conditions to reduce the damage to these rare broodstock crabs; however, their corresponding hatchability and survival rates are relatively low. With limited artificial space indoors, high-density culture leads to an extremely small living space for fertilized eggs and larvae; therefore, these eggs and larvae become susceptible to infection by various bacteria, fungi, viruses, parasites, and other pathogens. Therefore, to improve the hatchability rate of fertilized eggs and the survival rate of larvae for large-scale artificial breeding, it is necessary to address these issues.
In this study, nutrient fortification of the broodstock horseshoe crabs was initially conducted, followed by natural fertilization to obtain fertilized eggs for hatching. Then, the eggs infected with bacteria were collected to study the characteristics of different colored eggs. Next, 16 s rDNA and ITS sequencing technology were used to identify bacteria and fungi on the surface of T. tridentatus fertilized eggs, and the composition of these microbial communities was analyzed. Then, acute toxicity testing of the 1st instar larvae with BCDHM solution was conducted, and disinfection concentrations for fertilized eggs and larvae were determined according to the safe concentration range. The results demonstrated that a total of 16493 eggs were laid by the four pairs of horseshoe crabs, with an average fertilization rate of 66.25%, an average hatchability rate of 52.25%, a bacterial infection rate of 47.75%, and a mortality rate of 100% for fertilized eggs that were infected with pathogens; this resulted in the final production of 5704 1st instar larvae. Compared to previous breeding studies, more horseshoe crab eggs were harvested, which was attributed to the use of nutritional fortification in this study. Nonetheless, both fertilization and hatchability were low, which may be related to the poor physique of the broodstock horseshoe crab and the breeding environment. The dominant bacteria observed were Shewanella, Neptuniibacter, Desulfobacter, Terasakiella, and Halarcobacter, with relative abundances of 23.67%, 7.21%, 6.64%, 5.41%, and 5.16%, respectively. The dominant fungi included an unknown genus of Hypocreaceae and Aspergillus species, with relative abundances of 81.81% and 10.48%, respectively. The LC(50–96 h) of BCDHM for the 1st instar larvae was 945.06 mg/L; consequently, the safe concentration (SC) was determined to be 94.51 mg/L. The 1st instar larvae were observed to possess strong tolerance, but the specific mechanism of this tolerance requires further evaluation. The optimal BCDHM concentration (SC) for egg hatching and breeding of 1st instar larvae was determined to be 30–90 mg/L, which could significantly increase the hatchability of fertilized eggs (P<0.05) and reduce the mortality rate of 1st instar larvae (P<0.05). A recommended concentration of BCDHM for the hatching of fertilized eggs is 30–60 mg/L, with a specific value within that range being determined according to the infection of fertilized eggs; additionally, lower BCDHM concentrations could be used for disinfection when infection is not severe. In this study, eggs were disinfected every two days during incubation at a concentration of 30 mg/L. When infected eggs were observed, they were picked out and changed in time; they were then disinfected with an increased BCDHM concentration of 60 mg/L and daily water changes. Overall, BCDHM can be used to effectively reduce the risk of fertilized eggs and larvae being infected by bacteria, fungi, and other microorganisms, ultimately providing daily disinfection during the artificial breeding of T. tridentatus.
