In order to better protect the horseshoe crabs and develop concepts for a sustainable management it is crucial to investigate their biology and distribution. With this project we will uncover the population genetic structure and evolutionary relationships of the four species of horseshoe crabs using molecular genetic methods. This study will enable us to pinpoint high priority areas for conservation of these species in order to ensure their existence in the future. Physiological examination of the animals will give us a much better picture of how environmental changes (natural and human mediated) effect the distribution and survival of horseshoe crabs.
The ability of horseshoe crabs to adjust to changes in the environment and resist pathogens is highly dependent on their genetic variation. However, genetic variation declines rapidly when populations shrink leading to predominance of inbreeding which can result in the extermination of the species. The chances for a species to survive over long terms rise with its genetic variation. This diversity is likewise important for the medical applicability of an organism as different strains, populations or subspecies can posses various forms of a relevant substance, e.g. genetic traits and immune responses. Therefore it is essential to investigate the three Asian horseshoe crab species with regard to their entire distribution range, which is at this point still unclear.
Little is known on the physiology of Asian horseshoe crabs with regard to the circulation system, respiration, metabolism, as well as acid-base and osmoregulation. This implies that it is nearly impossible at this point to predict how the animals can cope with recent and future environmental changes. Hence it is not possible to anticipate how precisely changes in e.g. water temperature, oxygen content, salinity, influence the animals. An increased insight into the physiological function of Asian horseshoe crabs will give us the possibility for a detailed comparison with the physiology of spiders and scorpions herby unraveling very basic parts of the natural history of chelicerate early evolution in form and function.
Research methodology and techniques
1. Specimen sampling and treatment of animals. Horseshoe crabs will be collected directly by our group or obtained indirectly from locals (e.g. collaborators or fishermen). The animals will be determined for species and sex, weighted, photographed and measured externally. A small tissue sample (0.1mg) and blood sample (0.2 ml) will be taken from every individual and stored in 95% alcohol (this will not harm the animal). The animals will be released after this treatment. A few embryonic, juvenile, and adult individuals will be preserved for anatomical studies.
2. Sequencing and genetic analysis. We will apply one the the most widely used genetic marker for population genetic studies – the mitochondrial cyctochrome c oxidase subunit I (COI) which we have experience with. Alternatively we will be able to use microsatellites for the same purpose. The DNA sequences will be analyzed for each species separately and we will be able to fit the genetic variation within a geographical framework and indicate the presence of isolated populations or subspecies. This information will be compared among the four species and used to construct a phylogeny (evolutionary history). Constructing an evolutionary tree of these four species will help us to determine the relative age of each species and reconstruct speciation events in the past.
3. Cross fertilization tests. We will quantify the degree of reproductive isolation among the three Asian species as well as within each species and among geographical distant populations. These experiments will be conducted by blending eggs of one species/population with sperms of another. The number of viable larvae produced from each fertilization trial will be compared to those obtained by blending eggs and sperms from the same species/population under the same conditions.
4. On site measurements and laboratory work. The ecophysiological studies will begin on site with the measuring of the physical-chemical factors (e.g. temperature, salinity, etc.) at the sampling site where animals are collected. For this purpose we will use portable apparatuses. All other physiological studies (see below) will be performed at Phuket Marine Biological Laboratory by keeping the animals in aquaria and using our portable apparatuses.
5. Osmoregulation. The fact that Asian horseshoe crabs are common in the nearby of river outlets suggests that they are able to tolerate large oscillations in salinity. We will characterize the chemical composition of the hemolymph (blood) and cytoplasm (intracellular fluid) before, during and after changes in the salinity of the surrounding water and simultaneously measure the netto water flux in and out of the body. Using the same setup we will also measure the acid-base regulation in the body facing various salinities.