Publication:

Feeding ecology, molecular phylogeny and TAL production from Malaysian horseshore crabs

Present study was aimed at exploring the feeding ecology, molecular phylogeny and TAL production from Malaysian horseshoe crabs (Tachypleus gigas and Carcinoscorpius rotundicauda). The influence of seasons and lunar cycle on the diversity and distribution of major macrobenthic communities was studied in the nesting grounds of horseshoe crabs (Balok and Pekan) by correlating with hydrological parameters and sedimentological nature of the nesting grounds. It was interesting to note that macrobenthic diversity was comparatively lower during full moon (FM) days compared to new moon (NM) days in both the sampling stations. However, the diversity and richness of macrobenthos was higher during peak mating seasons of horseshoe crab (June, 2010) in both stations. Seasonal variation in the macrobenthic diversity showed significant variation in Balok station (P < 0.05) while it was not apparent in Pekan nesting ground. Overall, macrobenthic diversity was lower in both the sampling sites (H‟ = < 1.1) indicating severe habitat degradation in these nesting grounds. Physicochemical parameters showed apparent seasonal fluctuation in the salinity of the surface water in both the sampling stations (P < 0.05) while other parameters such as temperature, pH and dissolved oxygen did not vary significantly. The sedimentological parameters such as mean sediment size and sediment sorting value indicated that the nature of sediments in both stations was moderately well sorted with medium sand particle size helping in creating a well oxygenated micro-environment for the normal development of horseshoe crab eggs and larvae, as well as juveniles. Analysis of selected heavy metals (Fe,Zn,Cu and Cd) in different body parts of horseshoe crab (T. gigas) showed that bioaccumulation of essential metal concentration in all the analyzed body parts were higher than non-essential heavy metals and the order of accumulation of heavy metals was Fe > Zn > Cu > Cd. Statistical predictions revealed that bioaccumulation of metals was not significantly influenced by weight, total length and carapace width of the animal. The heavy metal accumulations in samples were higher than the national and international permissible limits and hence is not suitable for human consumption. Gut content analysis showed monthly and seasonal variations in the food preferences of Tachypleus gigas and Carcinoscorpius rotundicauda. It was observed that T. gigas prefers lesser number of polychaetes than bivalves during non-monsoon while it was the reverse during monsoon. On the other hand C. rotundicauda prefers lesser number of bivalves than polychaetes during non-monsoon period while it was reverse during monsoon. Overall, gut content analysis showed that mollusks were highly preferred by both the crab species more especially bivalves (in case of T. gigas) and gastropods (in case of C. rotundicauda). It was found that horseshoe crabs feed intensely during their peak mating season (May-August) in Pahang coast. Molecular phylogenetic (Distance matrix method: NJ and UPGMA) study and genetic distance (GD) data analysis revealed the distant genetic relatedness of American horseshoe crab (Limulus polyphemus) with Asian conspecifics. More interestingly, the monophyletic origin of Tachypleus gigas and Tachypleus tridentatus was quite evident in the phylogram which other molecular markers failed to address. Close genetic relatedness of horseshoe crabs with insects showed that they might have evolved from ancient aquatic insects. The efficiency of cytochrome oxidase C subunit 1 gene in species level identification among the crab genome was clear in both the phylograms together with the precise identification of the differential developmental stages to the species level. Nucleotide Substitution pattern study portrayed the higher number of transitional mutations (Ti) over transversional mutation (Tv) in the mitochondrial genome. A portable Kit (Endo sensor) was developed to detect the presence of bacterial endotoxin in liquid biological samples using Tachypleus Amebocyte Lysate (TAL) as a source. Sensitivity of the Kit was determined using various concentrations of prepared endotoxin standards and pyrogen free water samples. It was noted that Endo sensor could detect up to 0.1 EU/ml (λ) level of endotoxin in liquid biological samples. Basic gel clotting principle method was utilized for the detection/semi-quantification of bacterial endotoxin in unknown sample. The post bleeding mortality of the Malaysian horseshoe crab (Tachypleus gigas) due to biomedical bleeding process was studied. The mortality rate of T. gigas due to the bleeding process was slightly higher than what has been reported for its conspecific American horseshoe crab (Limulus polyphemus). Overall mortalities of bled horseshoe crabs were 17.8% (Male = 18.75%; Female = 17.2%) while mortality of unbled crabs was 3.5%. The quantification of the mortality due to the extent of bleeding commensurate with the commercial production of LAL/TAL providing a basis for the rational exploitation of T. gigas for LAL/TAL production.