Horseshoe Crab Tachypleus Gigas Research Articles

Genetic differentiation and heavy metal pollution influence on horseshoe crab Tachypleus gigas populations in Peninsular Malaysia: A comprehensive allozyme analysis

This study aimed study the genetic diversity and differentiation of Asian horseshoe crab Tachypleus gigas (Müller, 1785) populations across three distinct locations in Peninsular Malaysia: Kampung Pasir Puteh (KPPuteh), Kuala Sungai Ayam (KSAyam), and Kampung Pulau Sayak (KPSayak). A total of 28 individuals were collected, and their genetic structure was analyzed using starch gel electrophoresis. The study employed multiple buffer systems optimized for separating 20 different enzymes, each contributing to understanding the genetic variability within and between populations. The findings of this study, which revealed significant genetic differentiation, particularly between populations in polluted and less impacted environments, as reflected by high FST values and distinct clustering in the UPGMA dendrogram, are of paramount importance. The results indicate that environmental factors, especially metal-polluted sites at KPPuteh and KSAyam, have played a critical role in shaping the genetic structure of these populations. Populations from the two polluted sites, exhibited higher genetic diversity and greater deviations from Hardy–Weinberg equilibrium, suggesting localized selection pressures. Conversely, from a less polluted site, the unpolluted site at KPSayak population showed lower genetic variability and was genetically distinct from the others. These findings underscore the importance of considering environmental influences in conservation strategies for T. gigas, highlighting the need for site-specific management to preserve genetic diversity and ensure long-term survival of the species.

Morphometric variation of the horseshoe crab Tachypleus gigas (Xiphosura: Limulidae) from the Banyuasin estuarine of South Sumatra, Indonesia

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.

Comparative transcriptome profiling of horseshoe crab Tachypleus gigas hemocytes in response to lipopolysaccharides

Horseshoe crabs (HSCs) are living fossil species of marine arthropods with a long evolutionary history spanning approximately 500 million years. Their survival is helped by their innate immune system that comprises cellular and humoral immune components to protect them against invading pathogens. To help understand the genetic mechanisms involved, the present study utilised the Illumina HiSeq platform to perform transcriptomic analysis of hemocytes from the HSC, Tachypleus gigas, that were challenged with lipopolysaccharides (LPS). The high-throughput sequencing resulted in 352,077,208 and 386,749,136 raw reads corresponding to 282,490,910 and 305,709,830 high-quality mappable reads for the control and LPS-treated hemocyte samples, respectively. Based on the log-fold change of > 0.3 or < -0.3, 1338 genes were significantly upregulated and 215 genes were significantly downregulated following LPS stimulation. The differentially expressed genes (DEGs) were further identified to be associated with multiple pathways such as those related to immune defence, stress response, cytoskeleton function and signal transduction. This study provides insights into the underlying molecular and regulatory mechanisms in hemocytes exposed to LPS, which has relevance for the study of the immune response of HSCs to infection.

Mitochondrial DNA sequence of the horseshoe crab Tachypleus gigas

This paper reports on the complete mitochondrial (mt) genome of a horseshoe crab, Tachypleus gigas (T. gigas), in Kuala Kemaman, Terengganu, Malaysia. Whole-genome sequencing of hemocyte DNA was performed with Illumina HiSeq system and the generated reads were de novo assembled with ABySS 2.1.5 and reassembled using mitoZ against Carcinoscorpius rotundicauda and Limulus polyphemus, resulting in a contig of 15 Kb. Phylogenetic analysis of the assembled mt genome suggests that the Tachypleus gigas is closely related to Tachypleus tridentatus than to Carcinoscorpius rotundicauda.

Disruption of biofilm formation by fractions from book gills and carapace of horseshoe crab Tachypleus gigas

ABSTRACT Horseshoe crabs have survived for a long period due to their myriad defence mechanism. Thorough studies were conducted on their adaptive defence mechanisms but few highlighted their primary innate defence. This study aimed to determine the antifouling properties of the methanolic crude extracts and fractions obtained from the book gills and carapace of the Tachypleus gigas and to characterize their bioactive metabolites, using the detachment and preventive measurements. The active fractions recovered from the recycling HPLC were further analysed using GC-MS and major active constituents were identified. The results obtained indicated that the extract and fractions of T. gigas had significant detachment mode of actions for antifouling activities. Chemical interactions such as synergistic effects were observed in both the book gills and the carapace. Chemical profiles indicated the presence of organic acids such as amino acid, simple sugars and fatty acids. The observed activities might be caused by nutrient-induced biofilm dispersion mechanisms. These findings may provide new insight into the defence mechanism of the horseshoe crabs against biofilm formation. The isolated active fractions and the identified molecules from the horseshoe crab T. gigas may be applied as a dispersal antifouling agents. Further studies are needed to confirm these prospective potential applications.

Trailing the Spawning Horseshoe Crab Tachypleus Gigas (Müller, 1785) at Designated Natal Beaches on the East Coast of Peninsular Malaysia

Due to limited availability of literature on the spawning activity of Malaysian horseshoe crab, Tachypleus gigas (Muller, 1785), the reproduction behaviour and biology of this arthropod remains poorly understood. Hence, an investigation was carried out from April-July to trail spawning horseshoe crab amplexus at Balok and Cherating, the only known T. gigas spawning grounds on the east coast of Peninsular Malaysia. Through visual tracking during daytime full moon spring tides, the release of air bubbles indicate nest digging by female crabs. While air bubble formation aggravated, flagged aluminium poles were carefully driven into the sediment to mark the nest. Out of the 13 spawning T. gigas amplexus tracked, only one pair was able to dig up to 12 nests and release up to 2,575 eggs within the 2.5 hour spawning period. The female crabs dug nests at the intertidal regions, ranging 1.3-43.2 m from the highest tide marking on the beach. Interestingly after spawning, the horseshoe crabs returned to high seas during the residing tides as amplexus because the male crabs remained clinging onto the female crab’s opisthosoma. The incidence of recapturing a tagged female crab with different attached male crab at Cherating indicates this spawning ground as natal beach. In addition, the presence of 8 horseshoe crab amplexus at Balok compared to only 5 amplexus at Cherating highlight preferable beach settings for spawning and/or the presence of larger spawning crab assemblages at the former beach. Since only 13 spawning horseshoe crab pairs were witnessed at Balok and Cherating, identification of factors that alter beach sediment composition and water chemistry as well as gender-specific poaching and excessive boating activities should be regulated to restore wild T. gigas populations.

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.

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Epibiotic community of the horseshoe crab Tachypleus gigas

Horseshoe crabs act as moving substrata for simple to complex communities of small marine organisms. Amplexed adult pairs migrate for breeding once every 2 weeks from deep waters towards nearshore waters during highest high tide. Female horseshoe crabs bury themselves to the level of the lateral eyes to deposit eggs while the male crabs fertilize them. Subsequently eggs are buried by the female. Tachypleus gigas (Muller) is the most abundant horseshoe crab species above available along the Orissa coast (India). Adults reach terminal anecdysis once sexually mature and live with their carapace for 4 to 9 years. In spite of this, epibiosis is limited. In the current investigation, differences in the epibiotic community (diatoms and macro-epibionts) present on horseshoe crabs, according to gender, were evaluated, and the macro-epibiont population from different regions of the carapace was mapped. In general, female horseshoe crabs harbored fewer epibionts than the males. Among the diatoms, Navicula spp., Nitzschia spp. and Skeletonema sp. were dominant in both sexes. However, the abundance and diversity of diatoms was greater on the carapaces of male crabs. Among the macro-epibionts, the acorn barnacle (Balanus amphitrite Darwin) and encrusting bryozoan (Membranipora sp.) were the most dominant forms. Barnacles and bryozoans were greater in abundance in the “rough” zone (cardiopthalmic region and anterior region of the opisthosoma). Mapping of the macro-epibionts from different regions of the carapace revealed differential distribution in males and females. Such differentiated distribution of the macro-epibionts can be related to factors such as changing habitat by the horseshoe crabs during breeding, mechanical abrasion and surface availability during mating and nesting periods, requirements of epizootic larvae and surface properties of the carapace (wettability and roughness). In the case of females, mechanical abrasion and surface availability played an important role in the epibiotic community structure and distribution patterns. The surface wettability measurements indicated male carapace to be slightly more hydrophobic than the female carapace. Scanning electron microscopy revealed that the male carapace was comparatively rough compared to the smooth carapace of females. A comparison of surface properties of the carapace indicated that the male carapace is more conducive for epibiosis.

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.

A New Marine Triclad Ectoparasitic on Malaysian and Indonesian Horseshoe Crabs (Platyhelminthes, Turbellaria, Tricladida)

A new species of marine triclad, Ectoplana undata n. sp., ectoparasitic on the horseshoe crab Tachypleus gigas, is described. Cocoons of triclads were found on T. gigas as well as on the horseshoe crab Carcinoscorpius rotundicauda; since from the latter species no triclads were collected, it remains unknown whether these cocoons are those of E. undata or of another species. The genus Ectoplana, though poorly defined, is here retained, for stated reasons, pending a revision of the Procerodidae.

Coexistence of dyads and triads in an adult striated muscle.

AbstractElectron microscopic studies have revealed the presence of both dyads and triads in the accessory muscle of the walking leg of the horseshoe crab Tachypleus gigas. The dyads and triads are present in approximately equal numbers. The evolutionary significance of the coexistence of dyads and triads in such an ancient animal is discussed.

Epibiont Infestation on Horseshoe Crab Tachypleus gigas (Müller) at Pantai Balok in Peninsular Malaysia

A one year study was conducted at Pantai Balok in the east coast of Peninsular Malaysia to investigate the epibiont infestation on horseshoe crab Tachypleus gigas. Number of horseshoe crabs landed was enumerated and each prosoma size (prosoma length and width) was measured. Prosoma width of the horseshoe crabs was used as a morphometric proxy to estimate the approximate age and instar stage of the individual crabs. All horseshoe crabs examined were adult individuals. The carapaces of horseshoe crabs were carefully examined for epibiont infestation. The epibiont was identified to genus level. In this study, acorn barnacle Balanus, pedunculate barnacle Octolasmis, conical slipper shells Calyptraea and flat slipper shells Crepidula were found on the carapaces of horseshoe crab Tachypleus gigas collected from Pantai Balok. DOI: http://dx.doi.org/10.3126/on.v9i1.5725