Horseshoe Crab Research Articles

Seven Years of Monitoring the Development of an Oyster Reef Living Shoreline

The implementation of nature-based solutions (NbS), including living shorelines, to mitigate estuarine habitat loss is increasing at a pace exceeding the evaluation of their long-term success. Constructed oyster reefs (CORs) made of shell, concrete, stone, and other materials are one living shoreline tactic that is widely utilized, yet few studies have been conducted to understand the development of CORs within the context of both physical and ecological parameters over longer time scales (4 + years). A COR-based living shoreline project at the Gandy’s Beach Preserve (GBP) in Delaware Bay, NJ, USA, had dual goals of coastal protection and habitat provisioning, which prompted the development of a goal-driven monitoring framework to track project objectives. Methods were developed to quantify the following multi-disciplinary metrics over 7 years: elevations of CORs, waves (height, period, and direction), shoreline elevations, change in extent of vegetation patches, oyster density and size, nekton richness and community composition, and horseshoe crab impingement. The CORs met most of their habitat provisioning objectives as they were colonized by a multi-generational population of shellfish and created habitat for nekton, while posing negligible hazards to horseshoe crabs. However, none of the coastal protection objectives was fully achieved including material stability, wave attenuation, and sediment elevation increase. Results highlight the value of longer-term monitoring to understand performance and the need to match the scale and type of NbS tactic(s) with both the scale of the landscape and the site-specific hydrodynamic conditions to meet project goals.

Kinetic Bacterial Endotoxin Testing Methods: Specifics of Analytical Conditions Exemplified by Medicinal Products from Different Classes

INTRODUCTION. Kinetic bacterial endotoxin (BE) testing methods based on amoebocyte lysate from the haemolymph of the horseshoe crab can quantify BEs in parenteral medicinal products across a broad range of concentrations. To develop a specific analytical procedure that is based on these methods and can be implemented into pharmaceutical quality control and standardisation, it is required to ascertain the conditions for its use, in particular, to determine the lowest product dilution that would neutralise interfering factors.AIM. This study aimed to develop an analytical procedure for the quantitative determination of BEs by the kinetic chromogenic method.MATERIALS AND METHODS. The study examined samples of medicinal products containing active substances from different classes, including a monoclonal antibody (mepolizumab) and a direct-acting anticoagulant (nadroparin). The BE limits for mepolizumab and nadroparin were ≤20 EU/mL and ≤95 EU/mL, respectively. The samples were tested at different dilutions, which did not exceed the maximum valid dilution. The measurements were performed using a BioТek EL×800 photocolourimeter and EndoScan-V software.RESULTS. The results for mepolizumab samples show that the kinetic chromogenic method can quantify BEs in mepolizumab medicinal products in the dilution range of 1:10 to 1:40. The study detected BEs in quantities of 0.0078–0.224 EU/mL within this dilution range. However, at a dilution ratio of 1:50 or higher, the method is suitable only for qualitative BE tests. For nadroparin medicinal products, the quantitative determination of BEs should start at a dilution of 1:1000 (the study demonstrated no influence of interfering factors at this dilution).CONCLUSIONS. The authors developed analytical procedures for the determination of BEs in mepolizumab and nadroparin medicinal products by the kinetic chromogenic method (method D). When developing analytical procedures for the quantification of BEs in individual medicinal products, the developer should determine the lowest sample dilution that would neutralise interfering factors. Subsequently, the dilution should be specified in the documentation for the analytical procedure. This approach can enhance assay accuracy and save time and resources in routine testing.

Population structure and feeding habits of Carcinoscorpius rotundicauda in the bay environment, Southern Gulf of Thailand

This study presents the first comprehensive assessment of the population structure and feeding habits of Carcinoscorpius rotundicauda in Thailand. Sample collection was conducted using crab gill nets at seven sampling sites characterized by distinct ecosystem attributes in Pattani Bay between February 2019 and March 2020. Our findings indicate the presence of a single Asian horseshoe crab (HSC) species within the bay, with a sex ratio of approximately 1:1 (48.1 % males, 51.9 % females) that did not deviate significantly from parity. Habitat variation within the bay did not influence HSC abundance (P > 0.05) whereas monthly abundance fluctuations were highly significant (P < 0.001), peaking in March and reaching the lowest in September. Positive correlations were observed between HSC abundance and environmental factors including dissolved oxygen, temperature, water pH, water depth, and chlorophyll c concentrations. Linear regression analysis revealed a positive correlation between morphometric characteristics and body weight. However, multiple linear regression analysis indicated a negative relationship between body weight and telson length (TEL) and opisthosoma width (OW) of males, as well as TEL of females. The size at which 50 % of the HSC population reached maturity (L50) was estimated to be 12.2 cm for males and 13.5 cm for females based on prosomal width (PW). The average PW measurements of HSC at different egg stages (1–4) from developing to mature females was 14.4 cm, with corresponding body weights ranging from 224 to 243 g. Females at egg of stages 3 and 4 were found throughout the year, indicating multiple spawning events, particularly prevalent in January-April, and predominantly abundant at stations 1, 2, 5 and 6. HSC primarily consumed bivalves (22.1±21.2 %), followed by fishes (5.9±13.0 %) and crustaceans (2.9±9.0 %) by volumetric contribution (%V). This comprehensive study provides novel insights into the population dynamics and feeding ecology of HSC within Thailand. The findings establish a foundational framework for future research endeavors and offer critical data to inform the management and conservation of their habitats.

A novel antimicrobial peptide CpAMP identified from Chinese horseshoe crab, Tachypleus tridentatus.

Antibacterial peptide (AMP) is a crucial component of the innate immune system in most organism, play an important role in host defense processes. Many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a novel AMP named CpAMP was identified using transcriptome analysis in Chinese horseshoe crab. The preprotein of CpAMP consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. And the putative mature peptide CpAMP was 4.95 kDa with a theoretical isoelectric point (pI) of 8.73. The mature CpAMP showed α-helix and irregularly curled structure in the cys-stabilized region. CpAMP exhibited a broad spectrum of antimicrobial activity against Gram-negative, -positive bacteria and antibiotic-resistant strains. In addition, CpAMP can significantly increase the survival rate of zebrafish infected with bacteria. Due to its broad-spectrum antibacterial activity and the sensitivity of drug-resistant strains, CpAMP could be used as a new type of drug for inhibition of pathogenic microorganisms or as an immune enhancer in animals.

On the Recovery of Malformed Horseshoe Crabs Across Multiple Molting Stages

On the Recovery of Malformed Horseshoe Crabs Across Multiple Molting Stages

A-274 The Yield of the Limulus Amebocyte Lysate (LAL), Which is Used for Endotoxin Detection and Fungal Diagnosis is Increased by Caffeine Collection Buffer

Abstract Background Limulus Amebocyte Lysate (LAL), which is a major component of Fungal infection diagnosis kit, is used for the adjunct diagnosis of invasive fungal infection. This study used different blood collection buffers to find the optimal blood collection buffer to maximize the yield of LAL. The PBS collection buffer was found to prevent degranulation during blood collection procedure and the PBS-derived Limulus Amebocyte Lysate (LAL) was observed to work in both the chromogenic test and the turbidimetric method assays. A similar phenomenon was observed with the caffeine collection buffer. Methods To further confirm this observation, we used a caffeine collection buffer to collect the blood of horseshoe crabs in the 2023 bleeding season (from July to October). Once a week, 3 crabs were bled for a total of 30 crabs. The blood of each crab was collected using the caffeine collection buffer and the 3% NaCl collection buffer, respectively. The cell pellets were resuspended into LAL reagent water (LRW) or 5 mM CaCl2. The final LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The results showed that a caffeine collection buffer could prevent degranulation for over 1 hr and the activity yield of caffeine-derived LAL is much higher than that of the 3% NaCl solution. There was no discernible impact upon activity yield of crab size, period of the bleed season, or the vendors. Interestingly, caffeine-derived LAL was observed to work in both the chromogenic and turbidimetric assays. The enzyme characteristic of the LAL was also described. Conclusions Caffeine collection buffer prevents amebocyte degranulation during blood collection and processing. The activity of caffeine LAL is much higher than that of 3% NaCl. Caffeine LAL works in both chromogenic test and turbidimetric assay.

Concomitant melanoma and keratoma affecting the equine digit: clinical, pathological, and long-term follow-up findings

BackgroundThis case report details a long-term follow-up of a hoof melanoma with dermo-epidermal activity (resembling Spreading Superficial Melanoma (SSM)) in a bay horse with a history of a right front hoof keratoma. Melanomas involving the horse's foot are seldom reported and usually diagnosed as anaplastic melanomas based on signalment and post-mortem examination. The clinical-pathological characteristics of the foot melanoma in this bay horse are consistent with SSM-like described in humans, which is considered an intermediate malignant tumour attending their biological behaviour. However, a definitive diagnosis is limited by the single case and the lack of references in horses.Case presentationA 12-year-old bay Andalusian gelding underwent keratoma removal on the lateral aspect of the hoof wall. A partial resection of the hoof wall was performed for this purpose. Additionally, a plaque-like, hyperkeratotic pigmented lesion, 2 × 2X0,4 cm in size, was observed at the lateral aspect of the coronary band and was also resected for histopathological examination. Microscopically, a melanocytic tumour, characterised by small nests of large polygonal or epithelioid cells infiltrating the basal and suprabasal epidermis, the dermo – epidermal junction, and the superficial dermis, was observed. The neoplastic cells exhibited large euchromatic nuclei, prominent nucleoli, moderate pleomorphism and 4 mitotic figures per 2,37mm2; variable amounts of dark granules (melanin) were present in the cytoplasm, as well as in numerous peritumoral macrophages. The immunophenotype of the tumour cells was PNL2 + + + , S100 + + , AE1/AE3-. A diagnosis of melanoma with dermo-epidermal junction and marked intraepidermal activity (consistent with superficial spreading melanoma) was made.A magnetic resonance imaging (MRI) performed, revealed no further invasion into surrounding structures. Treatment was based on surgical resection and multiple local chemotherapy sessions with cisplatin were applied. The biopsies obtained after treatment showed partial regression of the tumour and different stages of healing. After 26 months of follow-up, there was no signs of malignant spreading into surrounding structures including the pedal bone and distal metastasis but a dark – coloured area persists over the lateral aspect of the coronary band.ConclusionsThis case presents a concomitant keratoma and melanoma with dermo – epidermal activity, resembling a spreading superficial melanoma. After a follow – up of 26 months the horse remains healthy and sound providing new information for clinicians and pathologists. Despite the poor prognosis associated with foot malignant melanocytic tumours, it is important that an early and accurate diagnosis is reached through different diagnostic modalities such as advanced imaging techniques and histopathology. Additionally, these findings demonstrate that the current classification and prognosis for equine foot melanomas are insufficient.

Tracking animal movements via collaborative acoustic telemetry networks: Multiscale habitat use, phenology, and management insights.

Estuaries support diverse fish and invertebrate communities, including resident species that rely on estuarine habitats year-round and transient migratory species. The unique movement patterns of these animals connect habitats within and far beyond the estuary and are integrally linked to fisheries management objectives. With a focus on Chesapeake Bay, this study leveraged data from collaborative acoustic telemetry networks in the northwest Atlantic to assess habitat use and phenology of movements for seven species of fish (cownose rays, dusky sharks, smooth dogfish, alewife, striped bass, common carp, and blue catfish) and one invertebrate (horseshoe crabs). A total of 288 acoustically tagged individuals were detected >3.2 million times (6,743 to 2,095,717 detections per species) on receivers across ~20.5 degrees of latitude spanning the North American Atlantic seaboard from Florida, USA, to New Brunswick, Canada. Common metrics of movement and phenology grouped these species as resident (common carp, blue catfish, horseshoe crabs), primarily resident in estuaries (juvenile striped bass), and coastal migrant (cownose rays, dusky sharks, smooth dogfish, alewife); maximum distance traveled varied by three orders of magnitude among these species. Further analysis of phenology for coastal migrants elucidated the timing and duration of these species' use of Chesapeake Bay. Collectively, movements linked habitats within Chesapeake Bay and connected the estuary to coastal ecosystems both to the north (e.g., alewife) and south (e.g., cownose rays), creating networks of fisheries management jurisdictions that varied in complexity and identified opportunities for enhancement to current management or co-management of some species. Our results elucidate the importance of estuaries to species with diverse movement behaviors, identify scales and pathways of habitat connectivity via animal movements, and highlight the utility of collaborative acoustic telemetry networks for quantifying movements relevant to both ecological research and fisheries management.

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.

An effective way to monitor the population of juvenile horseshoe crabs in the Beibu Gulf of China

Population conservation of horseshoe crab is urgently required in China as their population is declining dramatically. However, conservation efforts are severely constrained by the lack of information on the population dynamics of the species. Here, a citizen science-based investigation was conducted to continuously monitor the population dynamics of juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda in the Beibu Gulf of China over five years. A low abundance of horseshoe crabs was observed at all stations. T. tridentatus was primarily observed in the eastern of Beibu Gulf, with an abundance (0.179 ind/100 m2) larger than that of C. rotundicauda (0.007 ind/100 m2). The prosomal width range of T. tridentatus was between 9.0 and 138.0 mm while that of C. rotundicauda was between 20.0 and 111.0 mm, representing their different age distribution patterns. A continuous distribution pattern from 3rd to 14th instar was observed in the T. tridentatus population, whereas a discontinuous distribution pattern from 6th to 16th instars was observed in the C. rotundicauda population. This instar distribution pattern implies that these areas are crucial nursery habitats for the two species. No horseshoe crab within the 1st to 2nd instars (for T. tridentatus) or before 6th instar (for C. rotundicauda) were observed within five years, indicating a declining age structure with few natural births. It was concluded that the declining population would exacerbate the risk of endangered horseshoe crab. There is an urgent need to strengthen the protection of horseshoe crab in the Beibu Gulf, particularly to protect their nursery habitats. The value of long-term citizen science based-monitoring was highlighted, which greatly increased public awareness of horseshoe crab protection.

An evolutionarily conserved function of C-reactive protein is to prevent the formation of amyloid fibrils.

C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-β (Aβ) and prevents the formation of Aβ fibrils. It is unknown whether the Aβ-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aβ at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aβ. Both Li-CRP-I and Li-CRP-II bound to Aβ in the fluid phase also and prevented the fibrillation of Aβ. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aβ is an ancient function of CRP. In invertebrates, the Aβ-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aβ-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aβ-like structures after deposition at places where they are not supposed to be.

The Evolutionary Biology of Chelicerata.

Chelicerata constitutes an ancient, biodiverse, and ecologically significant group of Arthropoda. The study of chelicerate evolution has undergone a renaissance in the past decade, resulting in major changes to our understanding of the higher-level phylogeny and internal relationships of living orders. Included among these conceptual advances are the discoveries of multiple whole-genome duplication events in a subset of chelicerate orders, such as horseshoe crabs, spiders, and scorpions. As a result, longstanding hypotheses and textbook scenarios of chelicerate evolution, such as the monophyly of Arachnida and a single colonization of land by the common ancestor of arachnids, have come into contention. The retention of ancient, duplicated genes across this lineage also offers fertile ground for investigating the role of gene duplication in chelicerate macroevolution. This new frontier of investigation is paralleled by the timely establishment of the first gene editing protocols for arachnid models, facilitating a new generation of experimental approaches.

Mapping the habitat distribution of horseshoe crabs (Tachypleus gigas) in Pamekasan, Madura Island, Indonesia

Abstract Horseshoe crabs, often known as Belangkas or Mimi, is one of the ancient marine faunas with protected conservation status. Horseshoe crabs are distributed in coastal waters of Indonesia of the islands of Sumatra, Java, Kalimantan, and Sulawesi, which are often caught as bycatch. Horseshoe crabs’s bloods were uniquewhich contain amebocytes as a test material for the presence of endotoxin bacteria. The purpose of this study was to identify the species of horseshoe crabs and to analyze environmental conditions, as well as the distribution of horseshoe crabs in the coastal waters of Pamekasan, Madura Island. This study was conducted in June 2023 by collecting data from crab fishers. ArcGIS was applied to delineate the distribution of horseshoe crab. The identification results showed that there was one species horseshoe crab of the Tachypleus gigas in the waters of Polagan Village, Galis District, Pamekasan Regency. The environmental condition of the waters in Polagan Village, Galis District, and Pamekasan Regency was relatively good. Habitat mapping is one way to protect these animals from the threat of extinction and support conservation efforts.

Haplotype diversity of coastal horseshoe crab (Tachypleus gigas, Muller 1785) based on 16S rRNA gene markers as a basis for conservation

Abstract Tachypleus gigas is one of horseshoe crab species found in Indonesia, but does not have sufficient molecular biology information available. This study aims to predict the populations health of T. gigas from the waters of Langkat, Semarang, and Bintan; through analysis of haplotype diversity based on 16S rRNA gene markers. The observation was conducted from November 2022 until April 2023. Sample’s total DNA was isolated, extracted, amplified and then sequenced to obtain the nucleotide base sequence. The BLASTn results showed that the samples were validated as Tachypleus gigas and the genetic distance between locations showed a close kinship relationship between the three. The population health of the horseshoe crabs from Langkat and Semarang were indicated good with high haplotype diversity values, meanwhile the Bintan indicated poor health with low haplotype diversity values. This study of haplotype diversity provided the starting point to the conservation of horseshoe crab T. gigas

Morphometric Character of Horseshoe Crab (Tachypleus) at Mempawah Mangrove Park Coastal Area, West Kalimantan, Indonesia

Abstract In Mempawah Mangrove Park (MMP) coastal area, fishers are still often catch horseshoe crabs as a bycatch. One of its genera is Tachypleus. This research aimed to assess the morphometric character of Tachypleus from MMP Coastal area, West Kalimantan. A total of 14 parameters were measured from samples that were collected using a trammel net. The sampling frequency was three times from July to September 2022. Physical and chemical parameters of the waters are also studied to support the ecological information. Two species of Tachypleus were found in these waters, i.e. Tachypleus gigas (8 males and 4 females) and Tachypleus tridentatus (6 males and 11 females). In terms of size, females were larger than males in both species. Based on morphometric comparison between females and males of T. gigas, there was a significant difference in CL/PW ratio (p&lt;0.05). On the other hand, for T. tridentatus, the ratio of PL/OL differed significantly (p&lt;0.05).

Cloning and functionally characterization of TtVtg2-like in horseshoe crab Tachypleus tridentatus: A special focus on ovarian development

Horseshoe crabs are living fossils. In recent decades, the population of horseshoe crabs, especially the tri-spine horseshoe crab Tachypleus tridentatus, has decreased significantly and was listed as an ‘endangered species’ under the IUCN Red List in 2019. In order to improve the reproduction of T. tridentatus to facilitate stock enhancement, it is important to understand their ovarian development. In this study, a novel TtVtg2-like gene from T. tridentatus was cloned and functionally characterized. The total legth of TtVtg2-like was 5469 bp, encoding a protein consisting of 1822 amino acid with a pI value of 6.51 and a molecular weight of 208.68 KDa. The TtVtg2-like was highly expressed in the ovary and yellow connective tissues, mainly localized in cytoplasm and endoplasmic reticulum vesicles of oocytes and yellow connective tissues, respectively. RNA interference of TtVtg2-like caused the accumulation of ROS, DNA damage, and apoptosis of ovarian primary cells. The results of this study provide useful baseline information for future studies on ovarian development in horseshoe crabs.

Early warning of trends in commercial wildlife trade through novel machine-learning analysis of patent filing

Unsustainable wildlife trade imperils thousands of species, but efforts to identify and reduce these threats are hampered by rapidly evolving commercial markets. Businesses trading wildlife-derived products innovate to remain competitive, and the patents they file to protect their innovations also provide an early-warning of market shifts. Here, we develop a novel machine-learning approach to analyse patent-filing trends and apply it to patents filed from 1970-2020 related to six traded taxa that vary in trade legality, threat level, and use type: rhinoceroses, pangolins, bears, sturgeon, horseshoe crabs, and caterpillar fungus. We found 27,308 patents, showing 130% per-year increases, compared to a background rate of 104%. Innovation led to diversification, including new fertilizer products using illegal-to-trade rhinoceros horn, and novel farming methods for pangolins. Stricter regulation did not generally correlate with reduced patenting. Patents reveal how wildlife-related businesses predict, adapt to, and create market shifts, providing data to underpin proactive wildlife-trade management approaches.

In the Eye of the Beholder-Visual Search Behavior in Equestrian Dressage Judges.

This study investigated the visual search behavior of equestrian dressage judges at different expertise levels during the assessment of Grand Prix horse-rider combinations. Twenty judges (11 foundational level, 9 advanced level) participated in the study, with their eye movements recorded using Tobii Fusion Eyetracker as they evaluated video recordings of dressage tests. Fixation metrics, namely Total Duration of Fixation (TDF), Average Duration of Fixation (ADF), and Total Number of Fixations (TNF), were analyzed across four Areas of Interest (AOIs): front, back, rider, and horse's feet. Statistical analysis utilized linear mixed-effects models. Results demonstrated that judges consistently focused more on the front of the horse, with additional differences in fixation duration and frequency based on judge experience and specific movements. Advanced judges focused more on the horses' feet, suggesting they draw meaning from specific areas indicative of performance quality. Conversely, foundational level judges focused more on the rider, reflecting different evaluative priorities at lower levels of the sport. These findings suggest that judges focus on a limited number of highly relevant areas, differing across movements and expertise levels. The study underscores the necessity of understanding both gaze behavior and subsequent interpretations of visual information to increase judging transparency, fairness, and equine welfare.

Morphometric and DNA-based identification of horseshoe crab Carcinoscorpius rotundicauda (Latreille, 1802) and Tachypleus gigas (Müller, 1785) from Bangladesh coast

Horseshoe crabs are the living fossils of xiphosurans that have survived for over 200 million years without morphological change and records fossil dating back 500 million years. A study was conducted on morphometric and molecular characterization of horseshoe crab Carcinoscorpius rotundicauda (Latreille, 1802) and Tachypleus gigas (Müller, 1785) from Bangladesh waters. A total of ten samples of C. rotundicauda and one sample of T. gigas were collected from Dublar Char, Sundarbans and Cox’s bazar from June 2016 to May 2017 and February 2024, respectively. Among them, four specimens were male and seven female. Morphometric measurements were taken from all the collected samples and differentiated male and female based on the second pair of legs, the pedipalps. The species were genetically identified using mitochondrial cytochrome c oxidase subunit I (COI) gene. An average length of about 670.5 bp nucleotide sequences were obtained. Average percentage of nucleotide frequencies were T (30.3), C (16.3), A (34.4) and G (19.0) for C. rotundicauda and T (34.45), C (22.01), A (27.91) and C (15.63) for T. gigas. GC content analysis showed that average GC 36.20% and AT 63.80%. The average GC content at the 1st, 2nd and 3rd codon position were 38.82%, 37.87% and 31.92% respectively. This is the first report of DNA barcoding of a living fossil C. rotundicauda and T. gigas from Bangladesh and will serve as baseline information for future research, conservation and management of this invaluable primitive creature. Bioresearch Commu. 10(2): 1489-1494, 2024 (July)

Environmental variables driving horseshoe crab spawning behavior in a microtidal lagoon in Florida.

The timing of American horseshoe crab (Limulus polyphemus) spawning behavior along the coast of Florida (United States) is generally associated with the highest tides during the spring and fall lunar cycles. All Florida estuaries support horseshoe crab populations, but tidal characteristics vary markedly among locations, which may influence the timing of horseshoe crab spawning behavior. The Indian River Lagoon is a large microtidal estuary on Florida's east coast. Given the microtidal nature of the lagoon, it is unclear which environmental factors affect horseshoe spawning. In 2019, volunteers of Florida Horseshoe Crab Watch conducted daily surveys at two sites in the northern Indian River Lagoon during peak spawning months (February-April). During each survey, volunteers counted all spawning horseshoe crabs and recorded environmental variables, including water temperature, air temperature, wind speed, wind direction, salinity, and tide height. We developed a suite of negative-binomial regression models to quantify relationships between the number of spawning horseshoe crabs and environmental factors. Modeling results indicated a positive relationship between onshore wind speed and number of spawning horseshoe crabs. Our study suggests that in the absence of tidal cues, onshore wind speed may be an important driver of horseshoe crab spawning activity in microtidal estuarine systems.