Asian Horseshoe Crabs Research Articles

The Malay Peninsula as a barrier to gene flow in an Asian horseshoe crab species, Carcinoscorpius rotundicauda Latreille

Horseshoe crabs are marine arthropods that are amongst the oldest living creatures that still exist today. Among the four extant species of horseshoe crabs, Carcinoscorpius rotundicauda differs from the other species by having poisonous eggs and lays its eggs in sandy-mud areas near river mouths. With the rapid development of coastal areas worldwide, C. rotundicauda habitats are decreasing. Until now, however, there has not been any study on the species' genetic variation. Simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers were employed to study the genetic variation in five C. rotundicauda populations from the east and west coasts of the Malay Peninsula. Both markers showed differing levels of genetic variation, but concurred on the pattern of genetic structuring among populations of the species. This includes showing that little, although significant, genetic differentiation is present among populations, suggesting a low rate of gene flow among populations. The results also suggested that C. rotundicauda may be subjected to the land barrier effect of the Malay Peninsula, whereby gene flow is limited between populations occurring on both sides of the peninsula, increasing their genetic differentiation through time.

Validation of a New (1 → 3)-β-D-Glucan Test Kit, Fungitec G Test MKⅡ " Nissui "

Using 415 residual blood samples subjected to (1→3) -β-D-glucan assay, we studied the correlation of measurement results between Fungitec G Test MKII "Nissui" (Nissui Pharmaceutical Co., Ltd., Tokyo) and its predecessor, Fungitec G Test MK (Seikagaku Corporation, Tokyo), which is now out of production. Their major difference is that MK II uses reagents derived from blood cells of Limulus polyphemus, the American horseshoe crab, whereas MK used those of Tachypleus tridentatus, an Asian horseshoe crab. Passing-Bablok analysis showed a linear regression with nearly one-to-one correspondence (slope=1.065, intercept=-0.287) between the two test kits over the regular range of measurements (4.0 pg/ml -500 pg/ml ). This was also true when data were subdivided and analyzed in the low to medium (≦150 pg/ml ) and in the low range (≦50 pg/ml ). There were several cases, however, that showed a wide discrepancy between the pair of measurements. This discrepancy is believed to be due in part to the difference between Limulus and Tachypleus in their reactivity to β-glucans with diverse side chains. Despite of this, the even distribution on either side of the regression line of those samples that are associated with deep fungal infection and the abrupt disappearance of such samples below 20 pg/ml attest that MK II "Nissui" is an acceptable successor of MK.

Complete mitochondrial genomes of Carcinoscorpius rotundicauda and Tachypleus tridentatus (Xiphosura, Arthropoda) and implications for chelicerate phylogenetic studies.

Horseshoe crabs (order Xiphosura) are often referred to as an ancient order of marine chelicerates and have been considered as keystone taxa for the understanding of chelicerate evolution. However, the mitochondrial genome of this order is only available from a single species, Limulus polyphemus. In the present study, we analyzed the complete mitochondrial genomes from two Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus tridentatus to offer novel data for the evolutionary relationship within Xiphosura and their position in the chelicerate phylogeny. The mitochondrial genomes of C. rotundicauda (15,033 bp) and T. tridentatus (15,006 bp) encode 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. Overall sequences and genome structure of two Asian species were highly similar to that of Limulus polyphemus, though clear differences among three were found in the stem-loop structure of the putative control region. In the phylogenetic analysis with complete mitochondrial genomes of 43 chelicerate species, C. rotundicauda and T. tridentatus were recovered as a monophyly, while L. polyphemus solely formed an independent clade. Xiphosuran species were placed at the basal root of the tree, and major other chelicerate taxa were clustered in a single monophyly, clearly confirming that horseshoe crabs composed an ancestral taxon among chelicerates. By contrast, the phylogenetic tree without the information of Asian horseshoe crabs did not support monophyletic clustering of other chelicerates. In conclusion, our analyses may provide more robust and reliable perspective on the study of evolutionary history for chelicerates than earlier analyses with a single Atlantic species.

Horseshoe Crab, Tachypleus gigas (Müller, 1785) Spawning Population at Balok Beach, Kuantan, Pahang, Malaysia

Local and regional decline of Asian horseshoe crabs has spurred a study on its spawning population at Balok Beach, Kuantan, Pahang, Malaysia. This location was identified as spawning site due to the occurrence of horseshoe crab spawning pairs and nests. Size-frequency, length-weight relationships, sex ratio and epibiont infestation of Tachypleus gigas were studied. Instar stage was estimated based on prosomal width. Condition of the horseshoe crab carapace was reported. Visual search technique of horseshoe crab was conducted during high tide of new and full moons. Prosomal, opisthosomal and telson length and weight of each horseshoe crab were measured. Largest female was recorded with mean prosomal length and width of 154.4 and 246.9 mm, respectively. About 69.8% of the males belonged to size group of 151-200 mm and 53.3% of females were grouped into 201-250 mm. All individuals were of fourteenth to sixteenth instar stages. Sex ratio varied from 0.313 to 2.5 and attributed to commercial harvest and monsoon season. Sand sediment of study site showed 93% of fine sands with grain size ranged from 120 to 250 microm. Acorn and pedunculate barnacle, conical and flat slipper shells were found on the carapace of the specimens. Most males had damaged eyes and carapaces while females with broken telsons. Body damages of about 19.9% on the specimens were likely due to nearby fishing activities. Lack of satellite male indicated low spawning population. The finding of this study showed that the species is extremely threatened by human activities and coastal development.

Effect of Salinity on the Egg Hatching and Early Larvae of Horseshoe Crab Tachypleus gigas (Muller, 1785) in Laboratory Culture

Tachypleus gigas (Muller) the Southeast Asian horseshoe crab, is found to nest on sandy beaches in Malaysia, thus biological information about survival of this living fossil become crucial. Laboratory experiments were carried out to investigate the effect of salinity on duration to maturation and hatching success of their eggs. The effect on size, morphological changes and growth of the hatched larvae was also investigated. Eggs of T. gigas were collected from the newly nests made on breeding beach of Pekan, Pahang (Lat 3° 56.915’N; Long 103°21.933’ E) in Peninsular Malaysia. Triplicates of five different salinities (15, 20, 25, 30 and 35 ppt) were chosen for the experiments. A vernier caliper was used for size measurement under a stereo microscope. The morphological changes in prosomal width and length, body length, telson length, opisthosoma length and total length were recorded. Salinity range of between 25 and 35 ppt could be an optimal incubation condition. No egg hatched at salinity 15 and 20 ppt. Highest percentage of hatching was found at 30 ppt (91.11±5.57%). Hatching also occurred at 25 and 35 ppt with 65.56±1.73% and 74.4±2.31% success rate, respectively. The shortest molting time occurred at 15 ppt (25 days after hatching). Total size changes in larvae were also highest at 15 ppt but lowest at 35 ppt. There was no mortality in larvae maintained in all treatments. Early larval development would also affected by the salinity stress but its seemed tolerable.

Intraspecific shape variation in horseshoe crabs: The importance of sexual and natural selection for local adaptation

A morphometric analysis of the body shape of three species of horseshoe crabs was undertaken in order to infer the importance of natural and sexual selection. It was expected that natural selection would be most intense, leading to highest regional differentiation, in the American species Limulus polyphemus, which has the largest climatic differences between different populations. Local adaptation driven by sexual selection was expected in males but not females because horseshoe crab mating behaviour leads to competition between males, but not between females. Three hundred fifty-nine horseshoe crabs from nine populations, representing three species, were analyzed using a digitizer to position sixty morphometric landmarks in a three-dimensional space. Discriminant analysis revealed strong regional structuring within a species, which suggests strong philopatry, and showed the existence of geographically-based intraspecific variation. An admixture analysis showed regional intraspecific differentiation for males and females of L. polyphemus and males of the Asian horseshoe crab Carcinoscorpius rotundicauda, but not for females of C. rotundicauda and another Asian horseshoe crab, Tachypleus gigas. Differences in shape variation between sexes were tested with F-tests, which showed lower intrapopulation morphometric variation in males than females. These results indicate a lower degree of local adaptation on body shape in C. rotundicauda and T. gigas than in L. polyphemus and a lower degree of local adaptation in females than in males.

Effect of starvation on the energy budget of two Asian horseshoe crab species: Tachypleus tridentatus and Carcinoscorpius rotundicauda (Chelicerata: Xiphosura)

Energy budget is one of the most studied parameters in aquatic animals under environmental challenge. To examine how prolonged starvation would affect their energy budget, respiration rate (RR), ammonia excretion rate (ER), oxygen consumption to ammonia–nitrogen excretion (O:N) ratio and scope for growth (SfG) representing the balance between energy intake and metabolic output, two Asian horseshoe crab species, Tachypleus tridentatus and Carcinoscorpius rotundicauda, were investigated in two feeding regimes (fed and starved) over a period of 7 weeks. No significant effects of species and time course, as well as their interaction, on absorption efficiency were observed in the fed treatments. For both species, RR and ER of the starved treatments significantly decreased, while their O:N ratio significantly increased during the experiment. However, such values for the fed treatments remained relatively stable over the study period. A rapid reduction in SfG was only apparent in the first week of the starved treatments for both species; thereafter, their SfG remained relatively constant. In the fed treatments, SfG of T. tridentatus was significantly lower than that of C. rotundicauda throughout the experiment. In general, C. rotundicauda showed a greater decrease in SfG under starvation than T. tridentatus, suggesting that they may have a more competitive life-history strategy for adjusting to poor nutritional conditions.

Reproductive behavior in horseshoe crabs: Does density matter?

Abstract While the four species of horseshoe crabs share many common reproductive traits with respect to their reproductive systems, they do differ with respect to their mating behavior (monogamy vs. polygynandry). Past research has attributed these differences to a number of factors including: spawning densities, operational sex ratios (OSR's), male condition (or age), environmental and/or genetic factors, or a combination thereof. Mating behaviors in the three Asian horseshoe crab species (Tachypleus gigas, T. tridentatus, and Carcinoscorpius rotundicauda) with low spawning densities and 1:1 operational sex ratios are typically monogamous. In Limulus polyphemus, mating behavior is more variable ranging from monogamy to polygynandry. Here we provide evidence, through a long term behavioral study, that variation in mating behavior is influenced by population density in L. polyphemus. Our study population on two beaches in Connecticut (Long Island Sound) have a spawning density 400 times less than that found in Delaware Bay (0.002 females/m2 vs. 0.8 females/m2) but similar operational sex ratios. Between 90%-95% of all spawning females in CT were paired with only one male, thus exhibiting monogamous behavior. In contrast, between 30 and 60% of spawning females in Delaware Bay have more than one mate and produce clutches of eggs with multiple paternities. Male condition played no role in mating behavior in CT populations. We also observed that on average 18% of the females on the spawning beaches are single. These results suggest that population density is an important condition that determines mating behavior. Also, low population density may lead to decreased mate finding ability and lost opportunities for spawning.

Effect of prolonged starvation on body weight and blood-chemistry in two horseshoe crab species: Tachypleus tridentatus and Carcinoscorpius rotundicauda (Chelicerata: Xiphosura)

To determine the effect of prolonged starvation on body weight and blood-chemistry of two Asian horseshoe crab species, Tachypleus tridentatus and Carcinoscorpius rotundicauda, the animals were stocked into indoor recirculation systems under two food treatments (fed and starved) for seven weeks. Seven blood-chemistry parameters: alkaline phosphatase, calcium, cholesterol, glucose, lipase, triglyceride and total protein of plasma, were measured. During the experimental period, no significant differences of body weight were detected between the starved group and fed group in the two species. Plasma alkaline phosphatase increased slightly and then decreased. Plasma calcium remained fairly constant during the experimental period. A continuous decrease of plasma glucose in the starved treatment was observed. Additionally, cholesterol, lipase and triglyceride in the plasma increased significantly at later times, while plasma total protein remained fairly constant during the experiment. Significant interactive effects between feeding regime and starved time on some parameters were also evident in the two species. The results suggested that, for adult horseshoe crabs, starvation may stimulate a “hunger response”, which in turn might lead to consumption of some nutrient reserves in the animals. By consuming such nutrient reserves, adult horseshoe crabs can withstand prolonged starvation. The present findings contribute to the understanding of the blood biochemical aspect of the biology of T. tridentatus and C. rotundicauda, when these animals encounter starvation in the wild.

Osmoregulation and acid base regulation of the Asian horseshoe crab Carcinoscorpius rotundicauda

Osmoregulation and acid base regulation of the Asian horseshoe crab Carcinoscorpius rotundicauda

Rotation of the central pair microtubules in eukaryotic flagella.

SUMMARY It has been 20 years since the phenomenon of centralpair rotation in eukaryotic flagella was reported(Omoto, and Kung, 1979). At that time, a model wasproposed in which the central pair functions as adistributor to regulate dynein activity among the outerdoublet microtubules. The video evidence obtainedsince then and gathered together in this essay is con-sistent with this model. Geometric arguments indicatethat there must be circumferential and longitudinalregulation of shear forces to produce effective bendingmotion of an axoneme. Central pair microtubules areideally situated to perform this regulatory function.The regulation of outer doublet sliding by the centralpair, together with the rotation of the latter, where thisoccurs, may explain the wide diversity of two- andthree-dimensional flagellar and ciliary waveforms thatis found in organisms using the same basic 9 1 2structure. ACKNOWLEDGMENTS We thank Mike McLaughlin of Material Media Services, Washing-ton State University for converting 16-mm film to video format, andDenise A. Palmen (Technical Services, Washington State University)for editing the videos, assembling the QuickTime movies, and pro-ducing the jpeg images to use as figures. The research based onvideo images shown in this essay was first published by Kamiya(1982), Omoto and Witman (1981), Gibbons

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 crab's hemocyanin molecules.

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×106 colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 μg/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.

A Case of Orthologous Sequences of Hemocyanin Subunits for an Evolutionary Study of Horseshoe Crabs: Amino Acid Sequence Comparison of Immunologically Identical Subunits of Carcinoscorpius rotundicauda and Tachypleus tridentatus

About 83% of the amino acid sequence of hemocyanin subunit HR6 from the Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda, has been determined. There is a difference of about 43% between HR6 and complete sequences of chelicerate hemocyanin subunits from the American horseshoe crab, Limulus polyphemus, and a tarantula, Eurypelma californicum. However, the immunologically identical subunits HR6 and HT6 from Tachypleus tridentatus (Japanese horseshoe crab) show 2.7% sequence difference. Based on the amino acid sequences of HR6 and HT6, the divergence between C. rotundicauda and T. tridentatus occurred about 9.6 million years ago. In the case of horseshoe crab hemocyanin subunits, it seems that the orthologous homologues in many homologous subunits between species are immunologically detectable.

A lectin from the Asian horseshoe crab Tachypleus tridentatus: purification, specificity and interaction with tumour cells.

A lectin from the haemolymph of the Asian horseshoe crab Tachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-bound N-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues, N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin the N-acetyl group and the glyceryl side chain of N-acetylneuraminic acid are important, while presence of an aglycon, specially an alpha-glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin and Collocalia mucin, all being O-chain glycoproteins but carrying completely different carbohydrate chains. The majority of N-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the 'Tachypleus tridentatus agglutinin-receptor' which is present on red cells and O-chain glycoproteins.

Isolation and characterization of a sialic acid-specific binding lectin from the hemolymph of Asian horseshoe crab, tachypleus tridentatus

A lectin was isolated from the hemolymph of Asian horseshoe crab Tachypleus tridentatus by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The lectin's molecular weight was approx. 533 kDa; being a simple protein comprised of two non-identical subunits with molecular weights fo 30 and 32 kDa. The hemagglutinating activity of the lectin against equine erythrocytes was strongly inhibited by several sialoglycoproteins and weakly inhibited by sialic acid, although not inhibited by neutral sugars, hexosamines, N-acetylhexosamines, glucronic acid, or several asialoglycoproteins. In addition, glycophorin HA was more effective than glycophorin HA digested with trypsin in inhibiting hemagglutination of the lectin. These result suggest that the purified lectin specifically reacts with sialic acids containing glycoprotein.

Isolation and characterization of a sialic acid-specific lectin from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas.

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.

Induction of Secondary Embryos by Intra- and Interspecific Grafts of Center Cells under the blastopore in Horseshoe Crabs: (interspecific graft/secondary embryo/organizer/induction/horseshoe crab).

When the cell mass (center cells) of the early gastrulae in both American and Asian horseshoe crabs was grafted into the embryo of the homologous species, secondary embryos were formed as a result of these grafts. Secondary embryos were also formed in a similar way when the center cells of heterologous embryos were grafted between the American and Asian horseshoe crab embryos. The characteristics of the secondary embryos were similar to the host embryos in both cases, indicating that the center cells played the roles same as those by the amphibian organizer. The homogenate of center cells also induced the formation of secondary embryos. This is the first published study in which secondary embryos of horseshoe crabs have been induced by grafting. The fact may mean that this type of embryonic induction is widespread in the animal kingdom.

Tachyplesins Isolated from Hemocytes of Southeast Asian Horseshoe Crabs (Carcinoscorpius rotundicauda and Tachypleus gigas)

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.

Hemocyanin from Tachypleus gigas. II. Cooperative interactions of the subunits.

Six subunits (I to VI) were isolated from hemocyanin of an Asian horseshoe crab, Tachypleus gigas, by anion exchange chromatography of the dissociated hemocyanin. The subunit preparations were nearly homogeneous as judged by alkaline electrophoresis, but they still showed the presence of isoproteins in isoelectric focusing. The subunits were reassembled (in 10 mM CaCl2 at pH 7.5) and tested for restoration of the cooperativity in O2 binding. The reassembly of the subunits gave equilibrium mixtures of the monomer and hexamer with small amounts of larger molecules. Homogeneous and heterogeneous hexamers were prepared by reassembling a single kind or two kinds of subunits, followed by isolation of the hexamer fraction by gel filtration. Among the homohexamers, only the subunit V hexamer showed cooperativity in O2 binding with the Hill coefficient of 1.6. Among the heterohexamers the subunit I/V hybrid was most noteworthy, showing a Hill coefficient (1.7) higher than that of any other heterohexamer examined. It was concluded that there are specific interactions between the subunits I and V. It is suggested that their interactions are important for the cooperativity in the native hemocyanin.