Morphometrics Allometry of the Endangered Tri-spine Horseshoe Crab, Tachypleus tridentatus Leach, 1819 (Xiphosura: Limulidae), and Fishers’ Perceptions from the Philippines

Changes in condition and in carapace length and width of the Chinese mitten crab (Eriocheir sinensis H. Milne Edwards, 1853) harvested in the Odra River estuary in 1999-2007 Carapace length and width as well as condition were analysed in 2336 Chinese mitten crabs, Eriocheir sinensis, caught in 1999-2007 in the Odra River estuary (Poland). Their condition was assessed using the Fulton coefficient and the carapace width-individual weight relationship. September through November catches netted the highest number of crabs (83.60% of the total sample); males accounted for 53.56% of the crabs caught at that time. Males showed a significantly higher mean individual weight, significantly higher carapace width and length, and a higher Fulton coefficient than the females. The lowest mean individual weight, carapace width and length, and the highest condition coefficients were recorded in those crabs caught in 1999-2000. Later on, the mean carapace width and individual weight increased, and the increase was accompanied by a reduction in condition of the crabs. Causes of the differences are being sought in environmental changes taking place in the Odra estuary.

Biology.- Limits on the Global Distribution of Horseshoe Crabs (Limulacea): Lessons Learned from Two Lifetimes of Observations: Asia and America.- Horseshoe Crabs - An Ancient Ancestry Revealed.- The Ecological Importance of Horseshoe Crabs in Estuarine and Coastal Communities: A Review and Speculative Summary.- Relationships Between Sandpipers and Horseshoe Crab in Delaware Bay: A Synthesis.- Horseshoe Crabs, Their Eco-biological Status Along the Northeast Coast of India and the Necessity for Ecological Conservation.- American Horseshoe Crabs, Limulus polyphemus, in Mexico: Open Possibilities.- Basic Habitat Requirements of the Extant Species of Horseshoe Crabs (Limulacea).- The Relationship Between Small- and Large-Scale Movements of Horseshoe Crabs in the Great Bay Estuary and Limulus Behavior in the Laboratory.- Ecology of Horseshoe Crabs in Microtidal Lagoons.- Phylogeography, Demographic History, and Reserves Network of Horseshoe Crab, Tachypleus tridentatus, in the South and East China Seaboards.- Genetic Structure of Japanese Populations of Tachypleus tridentatus by mtDNA AT-Rich Region Sequence Analysis.- Reproductive Competition and Sexual Selection in Horseshoe Crabs.- Vision in Horseshoe Crabs.- Sperm Attachment on the Egg of Malaysian King Crab, Carcinoscorpius rotundicauda.- Distribution and Development of Limulus Egg Clusters on Intertidal Beaches in Delaware Bay.- Comparisons in Prosomal Width and Body Weight Among Early Instar Stages of Malaysian Horseshoe Crabs, Carcinoscorpius rotundicauda and Tachypleus gigas in the Laboratory.- Emergence Behavior of Juvenile Tachypleus tridentatus Under Simulated Tidal Conditions in the Laboratory and at Two Different Sediment Temperatures.- Distribution of Juvenile Horseshoe Crabs in Subtidal Habitats of Delaware Bay Using a Suction-Dredge Sampling Device.- Conservation.- History of Horseshoe Crab Harvest on Delaware Bay.- Biomedical Applications of Limulus Amebocyte Lysate.- The Effect of Hemolymph Extraction Volume and Handling Stress on Horseshoe Crab Mortality.- Horseshoe Crabs in Hong Kong: Current Population Status and Human Exploitation.- Comparative Status and Assessment of Limulus polyphemus with Emphasis on the New England and Delaware Bay Populations.- An Integrative Approach to Horseshoe Crab Multiple Use and Sustainability.- Strategies to Conserve and Enhance Sandy Barrier Habitat for Horseshoe Crabs (Limulus polyphemus) on Developed Shorelines in Delaware Bay, United States.- Conservation Program for the Asian Horseshoe Crab Tachypleus tridentatus in Taiwan: Characterizing the Microhabitat of Nursery Grounds and Restoring Spawning Grounds.- The Effects of Water Quality on Horseshoe Crab Embryos and Larvae.- Heavy Metal Concentration in Horseshoe Crab (Carcinoscorpius rotundicauda and Tachypleus gigas) Eggs from Malaysian Coastline.- A Discussion of Horseshoe Crab Management in Five Countries: Taiwan, India, China, United States, and Mexico.- Clinical Evaluation, Common Diseases, and Veterinary Care of the Horseshoe Crab, Limulus polyphemus.- Aquaculture Methods and Early Growth of Juvenile Horseshoe Crabs (Limulus polyphemus).- Larval Culture of Tachypleus gigas and Its Molting Behavior Under Laboratory Conditions.- Diet Composition of Juvenile Horseshoe Crabs: Implications for Growth and Survival of Natural and Cultured Stocks.- Effect of Sediment Type on Growth and Survival of Juvenile Horseshoe Crabs (Tachypleus tridentatus).- The Conservation Network of Horseshoe Crab Tachypleus tridentatus in Taiwan.- The History of Horseshoe Crab Research and Conservation in Japan.- Public Awareness and Community-Based Conservation for the Horseshoe Crab at Saikai National Park in Nagasaki Prefecture, Japan.- Public Participation in Studies on Horseshoe Crab Populations.- Green Eggs and Sand: A Collaborative Effort of Scientists, Teachers, Resource Managers, and Stakeholders in Educating About Limulus polyphemus.- Community Building: An Integrated Approach to Horseshoe Crab Conservation.

Chinese horseshoe crab ( Tachypleus tridentatus ) is a Xiphosura animal of significant commercial importance and in danger of extinction in China. To better estimate how genetic structure can be used to obtain a conservation perspective of the species, genetic variation was examined in nine locations covering its distributing range in the coast of Chinese mainland using ten nuclear microsatellite DNA loci and mitochondrial DNA control region (CR) sequences. Moderate levels of genetic diversity were detected (expected heterozygosity from microsatellites was 0.635, haplotype diversity from mitochondrial DNA was 0.800) as a whole. Significant genetic differentiation was detected only by mitochondrial DNA ( F ST = 0.0693, P < 0.01), while microsatellite markers indicated nuclear genetic homogeneity of these locations. Probably, nuclear genetic homogeneity was caused by outbreeding among different groups due to artificial transporting. Very weak genetic differentiation indicates that reintroduction programs of the movement and mixing of horseshoe crab from different locations will result in minimal negative genetic effects. Upon four management units were inferred from the results of CR analysis, accordingly four or more nature reserves should be established to conserve this endangered animal along the Chinese coast. Haplotype network pattern indicated that T. tridentatus population in Chinese coast has undergone historic population expansion and very recent historic population recession. Mismatch distributions analysis also revealed existence of historic demographic expansion. Keywords : Tachypleus tridentatus , microsatellites, mitochondrial DNA, population structure, genetic diversity African Journal of Biotechnology Vol. 12(16), pp. 2088-2099

Regarded as a protected species on the IUCN list of endangered species, and classified as data deficient species, the stone crayfish (Austropotamobius torrentium) is one of the five European species from the Astacidae family that lives in freshwater ecosystems across Bosnia and Herzegovina. The aim of this study was to investigate the variability of selected morphometric characteristics of male and female crayfish of the A. torrentium species from the upper reaches of the Mlinska River. The crayfish were sampled in June 2021 when 62 individuals (22 males and 40 females) were caught. They were analyzed fresh in the field and in the laboratory. The treatment included the measurement of 12 morphometric features: total body length (TBL), weight (W), claw length (CLL), claw width (CLW), carapace length (CPL), carapace width (CPW), rostrum length (ROL), rostrum width (ROW), abdominal length and width (ABL, ABW) and telson length and width (TEL, TEW). The mean body length ± SD was 79.05 ± 7.35 in males and 60.54 ± 10.03 mm in females. The mean recorded body weight ± SD was 17.51 ± 8.3 g in males and 7.02 ± 4.41 g in females. Using the linear regression method, a positive correlation was found between body length and weight (♂ R2=0.7644; ♀ R2=0.8222), body weight and claws length (♂ R2=0.876; ♀ R2=0.9661), as well as carapace width and body length (♂ R2=0.7216; ♀ R2=0.8411). Using the T-test, a statistically significant difference of the analyzed morphometric parameters was found between the sexes, which is explained by the pronounced sexual dimorphism of stone crayfish. The calculated values for Condition factor and the Crayfish constant indicate that the males are of better fitness, which is in line with the results of previous research in Europe. Data presented in this paper can serve as a basis for further research of A. torrentium in this area.

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.

The heterogeneity of arthropod hemocyanins was studied by polyacrylamide gel electrophoresis and immunochemical techniques. The spider (Argiope bruennichii), the scorpion (Heterometrus sp.), and the horseshoe crab (Tachypleus tridentatus) were found to have 4, 5, and 5 minimum functional units of hemocyanin, respectively, the apparent molecular weights of which were 79,000, 81000, and 80,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The marine horseshoe crab (Tachypleus tridentatus) has been considered as food and traditional medicine for many years. Kynurenic acid (KA) was isolated from horseshoe crab in this study for the first time in the world. A previous study in 2018 reported that intraperitoneal administration of KA prevented high-fat diet- (HFD-) induced body weight gain. Now, we investigated the effects of intragastric gavage of KA on HFD mice and found that KA (5 mg/kg/day) inhibited both the body weight gain and the increase of average daily energy intake. KA reduced serum triglyceride and increased serum high-density lipoprotein cholesterol. KA inhibited HFD-induced the increases of serum low-density lipoprotein cholesterol, coronary artery risk index, and atherosclerosis index. KA also suppressed HFD-induced the increase of the ratio of Firmicutes to Bacteroidetes (two dominant gut microbial phyla). KA partially reversed HFD-induced the changes in the composition of gut microbial genera. These overall effects of KA on HFD mice were similar to that of simvastatin (positive control). But the effects of 1.25 mg/kg/day KA on HFD-caused hyperlipidemia were similar to the effects of 5 mg/kg/day simvastatin. The pattern of relative abundance in 40 key genera of gut microbiota from KA group was closer to that from the normal group than that from the simvastatin group. In addition, our in vitro results showed the potential antioxidant activity of KA, which suggests that the improvement effects of KA on HFD mice may be partially associated with antioxidant activity of KA. Our findings demonstrate the potential role of KA as a functional food ingredient for the treatment of obesity and hyperlipidemia as well as the modulation of gut microbiota.

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.

The interaction between lipopolysaccharide (LPS) and an LPS-sensitive serine protease zymogen, factor C, purified from horseshoe crab (Tachypleus tridentatus) hemocytes, was investigated to elucidate the LPS-mediated activation of factor C. The rate of activation of the zymogen factor C was highly dependent on the concentration of LPS and on temperature, and the curve of amount of LPS versus activation showed saturation at 37 degrees C. Moreover, a high-molecular-mass complex formed between factor C and LPS was found in a gel-filtration experiment on a Sepharose 4B column. This complex formation was also confirmed by double diffusion analysis on agarose plates. Triton X-100, which destroys LPS micelles, strongly inhibited the LPS-mediated activation of factor C but not activated factor C. These results indicate that the binding of factor C with LPS is required for its activation and that only LPS-associated factor C generates the active factor C. On the other hand, the LPS-mediated activation of factor C was strongly inhibited by the S-alkylated heavy chain derived from factor C. In contrast, the S-alkylated factor C-light chain did not show any inhibitory effect on the activation of factor C, suggesting that the heavy chain located in the NH2-terminal portion of factor C contains an LPS-binding region.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.

A clotting enzyme associated with the hemolymph coagulation system of Japanese horseshoe crab (Tachypleus tridentatus) was highly purified from the amebocyte lysate. The method for purification consisted of gel-filtration of the lysate on a pyrogen-free Sepharose CL-6B column and affinity chromatography of the endotoxin-treated clotting enzyme on a benzamidine-Sepharose 4B column. Through these procedures, about 3 mg of the purified enzyme was obtained from 70 ml of the lysate and about 390-fold purification was achieved. The purified preparation was found to give a single major band, respectively, on polyacrylamide-gel disc electrophoresis at pH 3.2 in the presence of 6 M urea and on sodium dodecyl sulfate (SDS)-gel electrophoresis in the presence and absence of 2-mercaptoethanol. It also gave a single symmetrical peak on QAE-Sephadex A-25 column chromatography. The molecular weight of the clotting enzyme was estimated to be approximately 42,000 for the unreduced sample by SDS-gel electrophoresis. For the reduced sample, it was 30,000, suggesting that the protein consists of plural polypeptide chains bridged by disulfide(s). The Tachypleus clotting enzyme was a glycoprotein, as shown by the positive periodic acid-Schiff reaction for the protein band on SDS-gel and the amino acid analysis. The purified clotting enzyme transformed Tachypleus coagulogen to coagulin gel and hydrolyzed a chromogenic peptide substrate, Tos-Ile-Glu-Gly-Arg-p-nitroanilide for Factor Xa, liberating p-nitroaniline. The enzyme was sensitive to DFP and benzamidine. It was also inhibited partially by PCMB. Antithrombin III and alpha 2-plasmin inhibitor (alpha 2-antiplasmin) were very effective inhibitors of this enzyme among ten kinds of naturally occurring proteinase inhibitors tested. The clotting enzyme had a restricted specificity towards protein substrates and activated only prothrombin among plasma zymogens including Factor IX, Factor X, fibrinogen, plasminogen and prekallikrein. The cleavage sites of bovine prothrombin for this enzyme were the same Arg-Thr and Arg-Ile linkages as those for Factor Xa, resulting in the formation of alpha-thrombin. These results indicate that the horseshoe crab clotting enzyme is a Factor Xa-like serine proteinase rather than alpha-thrombin. It seems likely that the Tachypleus clotting enzyme is a prototype of mammalian serine proteinases participating in blood coagulation.

Abstract. Fauziyah, Mustopa AZ, Fatimah, Purwiyanto AIS, Rozirwan, Agustriani F, Putri WAE. 2021. Morphometric variation of the horseshoe crab Tachypleus gigas (Xiphosura: Limulidae) from the Banyuasin estuarine of South Sumatra, Indonesia. Biodiversitas 22: 5061-5070. Morphological studies are essential for fish resource management, ecology, conservation, and stock assessment. This research was conducted to characterize the morphological variation of the coastal horseshoe crab (Tachypleus gigas) from the Banyuasin estuarine, South Sumatra, Indonesia. The body weight (BW), prosoma width (PW), carapace length (CL), telson length (TEL), and total length (TL) were measured for describing the morphometric variability of T. gigas. A total of 70 T. gigas (32 females and 38 males) were found using trammel net fishing and bottom gillnet during the survey. Multivariate and allometric methods analyzed their morphometric characters. Based on Kruskal- Wallis tests, the result showed a significant difference in somebody measurements (TL and CL) between males and females. The principal component analysis (PCA) results showed that all morphometric characters had a strong correlation in both sexes, so the first principal component (PC1) values were 92% and 72%. In comparison, the second principal component (PC2) values were 5% and 20% in females and males, respectively. The results of discriminant function analysis (DFA) suggested that only one (BW) of the five morphometric characters was significant in separating both sexes. Both sexes were also revealed variations in growth patterns based on the allometric analysis results. These results were expected to be used as a basis for managing the horseshoe crab population-based conservation.