Hiroaki Sugita

Post-Embryonic Development of the Horseshoe Crab

Individuals of Limulus polyphemus and Ta- chypleus tridentatus reached, respectively, the fourteen- th-instar and tenth-instar stages during our nine-year rearing experiment. Body sizes were measured using exu viae and body specimens. The results made their growth steps clear, and body sizes of the juveniles at further growth stages could be estimated. We conclude from these data that L. polyphemus males generally molt 16 times to reach maturity in the ninth year; females molt 17 times to reach maturity in the tenth year. Similarly, we conclude from the presumptive numbers of growth stages that T. tridentatus males generally molt 15 times to reach maturity in the thirteenth year; females molt 16 times to reach maturity in the fourteenth year. Although we have few data on growth stages of Tachypleus gigas and Carcinoscorpius rotundicauda, it is thought that T. gigas males molt 12 times to reach adulthood, females molt 13 times, and that C. rotundicauda reaches matu rity after the thirteenth molt in both males and females.

Purification, Characterization, and Amino Acid Sequence of an Embryonic Lectin in Perivitelline Fluid of the Horseshoe Crab

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins orN-acetylhexosamines.

AMPLEXUS AND MATING BEHAVIOR IN THE JAPANESE HORSESHOE CRAB, TACHYPLEUS TRIDENTATUS

Abstract Amplexus and mating behavior in the horseshoe crab, Tachypleus tridentatus, were studied at imari Bay and Kitsuki Bay, Kyushu, Japan, during Summer, 1994. The pairwise size distribution of mated pairs (n =28) showed a lack of size-assortative mating. Long-term amplexus is primarily maintained by the males pair of posterior claspers, which is significantly larger than the anterior claspers. The posterior claspers always attach directly to the females opisthosoma, just lateral to the terminal spines, but the anterior claspers attach in various anteriorward positions on the lateral edges of the females opisthosoma. We conclude that the mating system of T. tridentatus is fundamentally similar to the American horseshoe crab (Limulus polyphemus), despite the >100 million years of isolation between the two groups. T. tridentatus morphologies, however, show more adaptations to long-term amplexus than those of L. polyphemus.

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.

Evolution of Hemocyanin Subunits in Mygalomorph Spiders: Distribution of Hemocyanin Subunits and Higher Classification of the Mygalomorphae

Abstract Mygalomorph spiders hemocyanins examined contain monomer subunits which are the component parts of hemocyanin hexamers and dimer subunits which bond two hexamers together. We determined the N-terminal amino acid sequences of twenty-two monomer subunits and ten constituent monomers of the dimer subunits prepared from hemolymph of seven mygalomorph spiders. We classified these subunits into eight groups based on the sequence comparison. From the distribution of monomer subunits in mygalomorph spiders, it is evident that duplications and losses of monomer subunits occurred frequently. This is the reason why we cannot detect an orthologous monomer subunit derived from a common ancestral sequence in the seven spiders. These monomer subunits were found in either Atypoidea or Avicularioidea. On the other hand, the dimer subunits having similar N-terminal sequences were found widely in both mygalomorph spider groups, showing that the dimer subunits are more conservative than monomer subunits for their function different from monomer subunits.

N-Terminal Amino Acid Sequence Comparison of Asian Horseshoe Crab Hemocyanins: Immunologically Identical Hemocyanin Subunits Are Orthologous in Asian Horseshoe Crabs

Abstract Each hemocyanin of the 3 Asian horseshoe crabs is composed of 6 immunologically different subunits, each subunit of which is immunologically identical with the comparable subunits of 2 other species. These immunologically identical subunits show 0-15% N-terminal sequence differences between Carcinoscorpius rotundicauda and Tachypleus gigas, while the immunologically different subunits show 25-74% sequence differences within species and 25-72% between C. rotundicauda and T. gigas. From the N-terminal sequence comparison and immunological comparison of hemocyanin subunits, it is evident that Asian horseshoe crabs share 6 orthologous hemocyanin subunits which are immunologically identical. We may produce 6 phylogenetic trees of Asian horseshoe crabs using the 6 sets of orthologous hemocyanin subunits and an evolutionary tree of horseshoe crabs hemocyanin molecules.

Hemocyanin Subunits of a Whipscorpion, Typopeltis crucifer, and a Primitive Spider, Heptathela kimurai: Orthologous Hemocyanin Subunits in Arachnids

Abstract The presence of monomer and dimer subunits was revealed, by means of native polyacryl-amide gel electrophoresis (PAGE), in examined arachnid hemocyanins. We determined the N-terminal amino acid sequences of nine monomer subunits prepared from hemolymph of a whipscorpion, Typopeltis crucifer, and a primitive spider, Heptathela kimurai, and two constituent monomers of a dimer subunit from the whipscorpion. Based on a comparison of the sequences, we confirmed that the orthologous hemocyanin subunits are shared between the whipscorpion and the scorpion, Liocheles australasiae, between the primitive spider and the scorpion, and among the whipscorpion, the scorpion, and mygalomorph spiders. This study is the first to demonstrate the presence of orthologus hemocyanin subunits in different orders. Furthermore, it is evident that one of the constituent monomers of the hemocyanin dimer from the whipscorpion is orthologous to the constituent monomers (the group G subunits) of the hemocyanin dimers in mygalomorph spiders and to the subunit LA8 (a constituent monomer of a dimer subunit) of the scorpion, suggesting that these constituent monomers of arachnid hemocyanin dimers originated from a common ancestral gene which existed in a common ancestor of these arachnids.

THE DEVELOPMENTAL APPEARANCE OF PATERNAL FORMS OF LACTATE DEHYDROGENASE AND MALATE DEHYDROGENASE IN HYBRID HORSESHOE CRABS

Differences in electrophoretic mobilities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) existed between three Asian horseshoe crabs, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda, used for interspecific hybridization. After electrophoresis of extracts of hybrid horseshoe crab embryos on starch gels, the paternal, maternal, and hybrid forms of the LDH and MDH were detected with specific enzyme staining. In viable hybrids the paternal form of the LDH was detected at stage 17 (immediately before the 1st embryonic molt). Similarly, evidence of gene expression for mitochondrial MDH was seen at stage 14 (stage of appearance of rudimental appendages). Gene expression for supernatant MDH was seen at stage 17 (immediately before the 1st embryonic molt). Regarding the onset of genome control in embryogenesis, it was suggested that prior to the activation of the maternal gene of the LDH, the paternal gene of the LDH was activated in horseshoe crab hybrids. Furthermore, there was evidence that the maternal effects on early embryogenesis were due to enzymes present in the egg prior to fertilization, not to continued synthesis directed by stable messenger RNA.

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.