Koichi Sekiguchi

Limits on the Global Distribution of Horseshoe Crabs (Limulacea): Lessons Learned from Two Lifetimes of Observations: Asia and America

This paper is based on the premise that understanding the natural history and ecology of the horseshoe crabs (Limulacea) is paramount to conservation of the species. Our objective has been to select those large-scale, global parameters that help to define the opportunities for, and the limits on, their distribution. Essentially, we have selected the big-picture type of conditions pertinent to considerations of the conservation of the species. This has led to an examination of potential biotic and environmental parameters. Of these we have selected eight, four in each category. The biotic aspects are: who they are (taxonomically), whether they are living fossils, what they are (ecologically), and the significance of discrete populations in conservation of the species. Four large-scale environmental parameters that limit their global distribution are geologic (estuarine environments, continental shelves as avenues of distribution, ocean deeps as deterrents), temperature which sets boundaries, tidal types that define predominant potential spawning sites, and the influence of benthic currents.

Basic Habitat Requirements of the Extant Species of Horseshoe Crabs (Limulacea)

Because horseshoe crabs (Limulacea) are ecological generalists that exist in more-or-less discrete populations within a variety of habitats throughout their ranges, we cannot explore all the possibilities within this short chapter. We have concentrated, therefore, on the two major habitats that have supported large population of horseshoe crabs (the Seto Inland Sea, Japan, and Delaware Bay, USA) and the species with which we are most familiar (the Japanese horseshoe crab Tachypleus tridentatus and the American species Limulus polyphemus), including differences between the populations, their habitats, and their distribution, including how the last Great Ice Age probably affected that distribution.

Protein components in the perivitelline fluid of the embryo of the horseshoe crab,Tachypleus tridentatus

Protein components in the perivitelline fluid of the embryo of the horseshoe crab, Tachypleus tridentatus , have been studied during the development of the embryo. The components were classified into two proteins and two protein groups according to the results obtained by electrophoretic and immunological analyses and periodic acid-Schiff test. One group was identified as hemocyanin (H proteins). The others could not be identified and were named B-1 protein, B-2 protein, and the “rest proteins.” These components showed remarkable transition patterns in quantity during development. Tachypleus embryo started to synthesize hemocyanin after the first embryonic molting and secreted it into the perivitelline fluid before the third embryonic molting. The amount of hemocyanin continued to increase until the seventh day after the third embryonic molting and afterward it began to decrease gradually until the fourth embryonic molting. It disappeared completely from the fluid after the fourth embryonic molting. The B-1 protein and the “rest proteins” were found in the perivitelline fluid at all stages of development examined. Roughly speaking, the amount of B-1 protein increased during development. The amount of the “rest proteins” stayed almost constant until the fourth embryonic molting when it suddenly increased about threefold. The B-2 protein was found in the perivitelline fluid only after the fourth embryonic molting and remained constant. Some of these components are considered to be more or less useful for the remarkable swelling of the inner egg membrane of the embryo.

Induction of multiple embryos with NaHCO3 or calcium free sea water in the horseshoe crab

SummaryWhen horseshoe crab embryos were treated with NaHCO3 at the developmental stage when the germ disc appears, multiple embryos were formed. NaHCO3 may effect the formation of multiple embryos by binding Ca2+ ions of the embryo since multiple embryos were also formed by treatment with Ca2+ free sea water.The treatment caused the blastoderm layer to tear. When the embryos were returned to normal sea water after the treatment, the blastoderm recovered. Some cell masses, probably derived from the germ disc or its prospective cells, formed during the process of the recovery. Each cell mass developed into an embryo.

EMBRYO AND ORGAN CULTURES OF THE HORSESHOE CRAB, TACHYPLEUS TRIDENTATUS*

This paper deals with some preliminary technical experiments on cultures of the embryos and organs of Tachypleus tridentatus. In embryo culture, using the embryos at various stages of development, embryonic areas with hardly any yolk remaining in them were cultured in various media and by various methods (Series A); hemispheres of embryos containing the embryonic area (Series B) and whole embryos without egg membranes (Series C) were cultured in sterilized sea water. The suitability of the culture media and methods is described; the medium containing calf serum or 199, and the hanging‐drop method were found to be the most appropriate of those used in respective series of experiments. The changes in external and internal structures of the explants from embryos at various developmental stages cultured in various media and by various methods are described; dorsal closure, secretion of inner egg membrane, thickening of the cell layer and the elongation of limb buds were all observed in the Series A experiments. In Series B and C, coelom formation and also normal development of the embryos cultured after the 2nd embryonic moulting were observed. In organ culture, fragments of the embryonic heart were cultured in various media and by various methods. The hanging‐drop method resulted in longer survival than the method using agar media.

Morphological Characterization of First Instar Larvae of Asian Horseshoe Crabs and their Hybrids

The morphological characters of first instar larvae of three Asian horseshoe crabs and their hybrids were examined. For this purpose, the length of six parts on the larvae, the pattern of pigmentation, the structure of the margin of the opisthosoma, and the distribution and shape of spines on the extracardiac region of the prosoma were recorded. In general, the phenotypes of the hybrids were intermediate between those of the parents, suggesting that both male and female genomes contribute to development of the hybrids. However, there were some exceptional characters and phenotypic variations. Some characters were inherited from only one parent. This was suggested to imply that if the maternal species did not have the gene regulating expression of a given paternal character, the paternal gene did not express this character.