Captive breeding and rearing of Tachypleus gigas for its biomedical utilization and conservation

Abstract

Horseshoe crab (HSC) has been exploited by several industries such as agriculture (fertilizer) and as livestock feed, yet, the bigger threats come from fisheries, biomedical industries and habitat destruction. Hence, this present study aimed at investigating various biological parameters, artificial breeding and rearing conditions to be used for horseshoe crab’s conservation. The first stage of this study was aimed at studying the effect of captivity on hemolymph quality for the purpose of LAL production, and sperm traits of T. gigas for captive spawning and rearing studies. The second stage of this study focused on captive spawning and rearing of T. gigas for the restocking/repopulation purpose. The third stage aimed at identifying pathogens that caused mortality during captive breeding and rearing studies. Effect of captivity was assessed in wild and captive horseshoe crabs (5 month captivity) collected from Balok beach, Kuantan, Pahang. The blood was collected in 6 different anticoagulant formulations (A, B, C, D, E and F) and amoebocyte cells’ density, viability and morphology were investigated. While the sperm was collected from wild and captive horseshoe crabs by massaging the gonads and sperm density and viability were studied. Optimum captive spawning and rearing of T. gigas conditions were then investigated. Importance and type of sand substrates (no substrate, coarse sand and fine sand) as well as the addition of fish oil (CLO) to T. gigas’s feed were studied to improve captive spawning. While differential microalgae feeds (Isochrysis, Spirulina and Isochrysis +Spirulina) were studied to induce molting and improve survival of T. gigas instars. Pathogens that infected T. gigas’s adults and eggs were isolated and cultured in various media and culture conditions. Genomic DNA extraction, PCR amplification and DNA sequencing were carried out. Molecular identification of the pathogens was carried out using partial sequencing of 16S rRNA gene for bacteria and ITS gene for fungi. Captivity was found to have profound negative effect on blood and sperm parameters. However, sperm traits showed that it is encouraging for restocking and sea ranching approach. T. gigas preferred spawning in moderately well sorted (Óø = 0.52) sand substrate with mean size (Xø) of 2.57. Captive spawning was then further improved by 9 fold using fish oil (CLO). While Isochrysis at concentration of 7.5 million cells/L induced and accelerated molting in T. gigas instar stages. Aeromonas caviae strains were isolated from the gill fluid, while Lysinibacillus fusiformis strains were isolated from gill tissues of adult T. gigas. Aspergillus aculeatus strains were isolated from infected eggs. It can be concluded that LAL kits can’t be developed and prepared by bleeding captive T. gigas. However, captive spawning and rearing could be a better approach and the most viable option for the restocking/repopulation of T. gigas. The diversity of the pathogens and their varying targets indicated the need for more studies on the design, feed and aquarium parameters used for maintaining horseshoe crabs in laboratory settings.