Limulus Polyphemus Research Articles

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.

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.

The American horseshoe crab (Limulus polyphemus) spawns regularly in salt marshes

While considered ecological generalists in most aspects of their life history, horseshoe crabs (Limulus polyphemus) have traditionally been regarded as reproductive habitat specialists, given that spawning is thought to occur primarily on beaches where conditions for embryonic development are considered optimal. Observations of horseshoe crabs spawning in other habitats were deemed isolated and the behavior non‐adaptive. Here, we used spawning and egg surveys to compare the use of beach and salt marsh habitats for spawning by the horseshoe crab in three US states along the Atlantic coast. We found similar spawning and egg densities in both habitats but were more likely to find eggs in marsh habitats, indicating that spawning in marsh habitats is common and geographically widespread. These results suggest that the conservation of salt marshes may be critical for the protection of this species and that management strategies should be revised to incorporate this generalist behavior.

A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals.

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals.

Traumatic telson avulsion and attempted external stabilization in Atlantic horseshoe crabs (Limulus polyphemus).

Atlantic horseshoe crabs (Limulus polyphemus) are prevalent in public aquarium touch pools. Despite their popularity, the literature concerning medical management under managed care is sparse. Noninfectious conditions include trauma to the exoskeleton and compound eyes; however, injury to the soft tissue connecting the telson and opisthosoma has not been previously reported. This report describes telson avulsion in three Atlantic horseshoe crabs at a public aquarium and attempted external stabilization with a vascular silicone tie in two of three affected animals. The horseshoe crab that received no veterinary intervention suffered a complete telson amputation 1 month after the initial injury. Although the two other horseshoe crabs did well postoperatively, and external stabilization prevented further avulsion or amputation, they still could not right themselves if flipped into dorsal recumbency. All three horseshoe crabs were ultimately euthanized due to animal welfare concerns. To avoid potentially serious telson ligament injury, horseshoe crabs should never be picked up by the telson. The authors recommend that telson ligament injuries be addressed promptly, any inciting cause be mitigated, and external stabilization be considered earlier.

Bioaccumulation of Some Metals and Metalloids in Laughing Gulls (Leucophaeus atricilla): Increases in Mercury and Decreases in Selenium from 2019 to 2022/2023.

The elements in blood normally reflect the levels in prey, indicating a recent exposure. Laughing gulls (Leucophaes atricilla) eat mainly horseshoe crab eggs (Limulus polyphemus) in the spring in Delaware Bay, New Jersey. The levels of arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), and selenium (Se) in the blood of laughing gulls foraging on crab eggs were examined in Delaware Bay to provide information on a species that is normally a generalist, and to determine if the levels of these elements were similar in 2019 and 2022/2023, were intercorrelated, and were related to those in crab eggs. Hg increased from 2019 (136 ± 31 ng/g) to 2022/2023 (473 ± 75 ng/g), while Cd and Se decreased. There were some significant correlations among elements and a close relationship between the element levels in blood and those in crab eggs collected in the same month (except for As). The levels differed between laughing gulls and three species of shorebirds for As and Cd. The elements in the blood of gulls and shorebirds should be similar because they eat mainly the same eggs in the same places. A significant proportion of laughing gull blood samples had levels of Hg and Se that were above the levels associated with adverse effects, which requires further examination.

Laboratory investigations into alternative baits for the channeled whelk (Busycotypus canaliculatus) fishery

Channeled whelks (Busycotypus canaliculatus) are fished along the eastern US coast using traps typically baited with pieces of female horseshoe crabs (HSC; Limulus polyphemus). However, the use of HSC as bait, particularly females, is controversial because they are already heavily fished because their blood contains a substance that the biomedical industry uses to manufacture a test for pathogenic bacteria. Therefore, the goals of this study were to: 1) develop a cost-effective alternative bait that does not rely on HSCs and 2) determine whether whelk are more attracted to female HSC pieces than male HSC pieces. Seventeen different baits were tested in a total of 209 laboratory bait trials over a total of 5496 h. Whelk attraction to bait was measured by using time-lapse video to quantify the amount of time a whelk interacted with a bait bag containing one of the baits. While whelks were attracted to many of the alternative baits tested, those containing surf clam and green crab parts, were the most attractive. One such alternative bait composed of green crabs and clam bellies (60 GC/40 CBel Mix) resulted in greater whelk interaction than female HSC Prosoma did over a 24 hr period (mean ± SEM whelk interaction time: 60 GC/40 CBel Mix, 9.31 ± 0.52 hrs; female HSC Prosoma, 8.4 ± 4.6 hrs). We also found that male HSC pieces were as attractive to whelks as female HSC pieces, indicating that it may be possible to reduce fishing pressure on female HSCs to aid in conservation of the species. Field testing of these alternative baits is currently underway to determine whether these laboratory findings successfully translate to commercial fishing operations.

Long-term circatidal rhythms of heart rate, gill ventilation, and locomotion in the American horseshoe crab, Limulus polyphemus (Arthropoda: Chelicerata: Merostomata: Limulidae).

While several marine species exhibit biological rhythms of heart rate, gill ventilation, or locomotion, the relationship between these three measures in any species remains unexplored. The American horseshoe crab, Limulus polyphemus, Linnaeus, 1758, expresses circalunidian locomotor rhythms and circadian eye sensitivity rhythms but it is not clear if either heart and ventilation rates are controlled on a circadian or circatidal basis or the nature of the relationship between these three measures. The goal of this study was to determine the extent to which the heart and ventilation rates of Limulus polyphemus are coordinated with its endogenous rhythms of locomotion. To address this goal, rhythmic beating of the heart and movements of the gill plates were recorded in freely moving horseshoe crabs. Most animals exhibited robust circatidal rhythms of locomotion, heart rate, and ventilation that were highly correlated with each other over three weeks of continuous recording. These results are the first showing long term rhythms of all three measures in any marine species and suggest that heart rate and ventilation rhythms are coordinated in Limulus polyphemus both with each other, and with locomotion, and thus are all modulated on a tidal basis.

Reconstitution of (1→3)-β-D-glucans measurement system using recombinant Limulus polyphemus Factor G.

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-β-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and β from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for β. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and β subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and β subunits for BDG-dependent activation.

A walk on the beach: exploring iNaturalist observations of the Atlantic horseshoe crab, Limulus polyphemus, as a tool for conservation

The Atlantic horseshoe crab, Limulus polyphemus, is an ecologically and commercially important species occurring across the south and northeastern coast of the United States. However, populations of this species are experiencing declines due to overcollection, use as bait, and other anthropogenic factors. Citizen science holds great potential as a useful complementary tool to traditional monitoring efforts, so we evaluated horseshoe crab observations uploaded to the citizen science platform iNaturalist. Across 10,094 observations, the majority of which occurred primarily during summer months, the number of observations increased annually from 2008 to 2022, presumably as this platform is gaining popularity in overall use by citizen scientists. Analyzing a subset of images from this dataset, we found evidence of scavenging, ranges in the number of individuals, and additional organisms (molluscs or barnacles) attached to specimens, with 52.7% live versus 38.9% dead, and most images showing a complete specimen (61.6%) and adult life stages (80.3%). Most observations were taken on sand (75.7%), with some in shallow water (9.4%) or in vegetation (7%). We recommend researchers incorporate this freely available database as a complementary conservation tool to document presence and natural history and to monitor population trends both in this species and other marine species.

Water temperature and season length interact to explain a rare non‐linear ecogeographic cline in body size

AbstractAimAdult body size often exhibits patterns across large‐scale environmental gradients, creating ecogeographic clines. However, the form of body size clines varies across taxonomic groups, with linear and non‐linear patterns in body size observed in nature. Non‐linear body size clines have received less study, and questions remain about how environmental gradients interact to produce non‐linear clines. We examined the body size of the American horseshoe crab (Limulus polyphemus), a widely distributed marine arthropod, and evaluated the hypothesis that temperature and active season length can interact multiplicatively to result in a dome‐shaped distribution.LocationFourteen states in the United States of America and three Mexican states, representing the entire geographic range of the species.MethodsWe compiled environmental data and body size measurements from more than 49,000 individual horseshoe crabs. For each location, we extracted from the literature or calculated from raw data the mean male prosoma width and the mean female prosoma width. We applied a general additive modelling (GAM) approach to characterize the body size cline, test a hypothesis regarding temperature and season length, and explore evidence for the influence of additional environmental factors.ResultsModel results indicate temperature and season length could act multiplicatively to produce dome‐shaped clines, and these findings align with and quantify previous anecdotal reports of a strong dome‐shaped body size cline across latitude for horseshoe crabs.Main ConclusionsActive season length appears to become relatively more influential on horseshoe crab body size in the northern part of their range, while temperature effects per se appear to dominate in southern latitudes. For horseshoe crabs, the pattern of size variation is consistent with the predictions of Optimal Resource Allocation models, but more study is needed to elucidate mechanistic underpinnings. Considering climate change projections, results from our study suggest future shifts in horseshoe crab body sizes.

Application of a catch multiple survey analysis for Atlantic horseshoe crab Limulus polyphemus in the Delaware Bay

AbstractObjectiveThis paper applies a catch multiple survey analysis (CMSA) to Atlantic horseshoe crabs Limulus polyphemus in the Delaware Bay to generate robust population estimates for harvest management. Currently, horseshoe crabs along the U.S. Atlantic coast are harvested as bait for other fisheries and collected for their blood, which is used in a biomedical industry. The Delaware Bay is home to the largest population of horseshoe crabs and is a significant stopover for shorebirds to rebuild energy by consuming horseshoe crab eggs prior to completing their northward migration. To address this interrelationship, the Adaptive Resource Management (ARM) Framework has been used since 2013 to ensure that horseshoe crab harvest within the region takes into account the forage needs of migratory birds. Since its inception, the ARM Framework has used a single trawl survey's swept area‐based population estimates of horseshoe crab relative abundance and a theoretical population model developed primarily from literature‐derived values. With more data collected in the region in recent years and other sources of mortality that can now be quantified, a catch survey model can provide horseshoe crab population estimates going forward.MethodsA CMSA was used to estimate male and female horseshoe crab population size for 2003–2021 using all quantifiable sources of mortality and three fishery‐independent indices of abundance.ResultThe CMSA results indicated that adult abundance of male and female horseshoe crabs was stable from 2003 to 2013 and then began to increase through 2017, a result that is consistent with stock rebuilding following a period of harvest restrictions as recommended by the ARM Framework. Population estimates were lower in recent years but remained above the levels estimated before implementation of the ARM Framework. In 2021, the CMSA estimated that there were over 6 million mature females and nearly 16 million mature male horseshoe crabs in the region.ConclusionThe CMSA provides the best and most comprehensive population estimates of horseshoe crabs in Delaware Bay and will improve modeling efforts within the ARM Framework going forward.

Microsatellite and mitochondrial DNA based evidence reveals a single horseshoe crab (Limulus polyphemus) population on the northern Yucatan Peninsula, Mexico

In Mexico, the American horseshoe crab, Limulus polyphemus, is regarded as in danger of extinction, with a distribution range limited to the Yucatan Peninsula. Population genetics knowledge of this species in Mexico is incipient. We conducted a base-line genetic survey of eight localities on the northern coast of the Yucatan Peninsula to assess patterns of genetic structure, genetic variation, and gene flow among these localities. We analyzed DNA samples from 190 L. polyphemus specimens using 14 nuclear-encoded microsatellites and a region of the mitochondrial Cytochrome Oxidase I (COI) gene. Total allelic richness was 5.432, similar to previous figures for populations at the northern and southern extremes of the species’ global distribution. Observed heterozygosities (Ho) were 0.540–0.590, while expected heterozygosities (He) were 0.479–0.532, and there was no evidence of population bottlenecks. We found 129 mtDNA haplotypes (121 of which were found in single individuals) and 275 polymorphic sites, and the mean haplotype diversity among sites was high (0.852 ± 0.027 SD). AMOVAs based on both microsatellite and mtDNA data revealed that L. polyphemus from the northern Yucatan Peninsula show low genetic structure (Fst < 0.05, p > 0.05) and high gene flow (Nm = 10.967) favored by the absence of geographic barriers between coastal lagoons, suggesting that they constitute a single population. This was further confirmed by discriminant analyses of principal components using microsatellite data. This information is useful in establishing coordinated conservation efforts among federal and state protected areas.

Assessment of recovery potential for the American horseshoe crab (Limulus polyphemus): An application of the IUCN green status process

Abstract According to an International Union for Conservation of Nature (IUCN) Red List assessment (RLA), the American horseshoe crab (Limulus polyphemus), an iconic coastal species, is at risk of extirpation in some regions within its range where small and vulnerable populations occur. However, the RLA does not consider future status beyond viability and does not attempt to identify the conservation necessary to effectively mitigate threats and recover the species to full ecological functionality. To aid in conservation planning for vulnerable species, the IUCN developed the Green Status of Species assessment (GSA) process to complement the RLA. This paper describes the application of the GSA process to assess the recovery potential of the American horseshoe crab. First, specific Limulus populations within spatial units for conservation were delineated, and their statuses were defined based on viability and ecological functionality. Then conservation actions were identified that would promote recovery and affect their near‐ and long‐term population status under different conservation scenarios. Horseshoe crab conservation has relied on, and will continue to depend on, effective harvest regulation. However, as currently conceived, conservation is not expected to mitigate habitat loss at the scale required to restore range‐wide ecological functionality, primarily because habitat loss is widespread and affected by climate change. Thus, the GSA results, while indicating that there is potential for near‐term recovery gains, reveal that long‐term recovery is in doubt owing to expected loss of habitat. To conserve critical habitats for spawning and early life stages and achieve ecological functionality, it is imperative to identify and develop conservation plans at appropriate spatial scales. Unfortunately, such plans do not currently exist and need to be established. The GSA Green Score can then serve as a metric for monitoring recovery and gauging the effectiveness of conservation implementation.

Relations of coral reef benthos composition and horseshoe crabs’ gut content

Intensive earlier studies on horseshoe crab feeding ecology are focused on the Atlantic species, Limulus polyphemus (Linnaeus, 1758). However, the distribution of species, food abundance, and availability of benthos in a particular environment might influence the horseshoe crab's preference for diet in a particular region. The aims of this research are to analyse the benthos composition in Tachypleus gigas spawning site and coral reefs located near the spawning ground in Cherok Paloh, Pahang, and compare it with the food composition inside the T. gigas digestive track. A total of 30 samples of male and female horseshoe crabs, T. gigas respectively, were harvested by using a fishing net during the incoming high tide. Their guts were dissected, and the contents were analysed according to Squires and Dawe criteria. Results showed that echinoderm was the main food composition in the gut of males (54.27%) and females (51.44%) during the open sea migration phase. Amuk-amuk coral reef (3°41'51"N 103°24'13"E), Raja Muda coral reef (3°38'01"N 103°28'23"E) and Air Leleh coral reef (3°35'38"N 103°29'00"E) are the nearest sea urchin habitats to the sampling area. Benthos analysis showed higher echinoderm composition in those coral reefs (31.58 – 42.11%). There is no echinoderm composition found in the spawning site sediment. Further study is needed to provide reliable data on the significance of coral reefs in the horseshoe crab's foraging ecology.

Metal Levels in Delaware Bay Horseshoe Crab Eggs from the Surface Reflect Metals in Egg Clutches Laid beneath the Sand.

Understanding variations in metal levels in biota geographically and under different environmental conditions is essential to determining risk to organisms themselves and to their predators. It is often difficult to determine food chain relationships because predators may eat several different prey types. Horseshoe crab (Limulus polyphemus) eggs form the basis for a complex food web in Delaware Bay, New Jersey, USA. Female horseshoe crabs lay thumb-sized clutches of eggs, several cm below the surface, and often dislodge previously laid eggs that are brought to the surface by wave action, where they are accessible and critical food for migrant shorebirds. This paper compares metal and metalloid (chromium [Cr], cadmium [Cd], lead [Pb], mercury [Hg], arsenic [As] and selenium [Se]) concentrations in horseshoe crab eggs collected on the surface with concentrations in eggs from clutches excavated from below the sand surface, as well as examining metals in eggs from different parts of the Bay. The eggs were all collected in May 2019, corresponding to the presence of the four main species of shorebirds migrating through Delaware Bay. These migrating birds eat almost entirely horseshoe crab eggs during their stopover in Delaware Bay, and there are differences in the levels of metals in blood of different shorebirds. These differences could be due to whether they have access to egg clutches below sand (ruddy turnstones, Arenaria interpres) or only to eggs on the surface (the threatened red knot [Calidris canutus rufa] and other species of shorebirds). Correlations between metals in clutches were also examined. Except for As and Cd, there were no significant differences between the metals in crab egg clutches and eggs on the surface that shorebirds, gulls, and other predators eat. There were significant locational differences in metal levels in horseshoe crab eggs (except for Pb), with most metals being highest in the sites on the lower portion of Delaware Bay. Most metals in crab eggs have declined since studies were conducted in the mid-1990s but were similar to levels in horseshoe crab eggs in 2012. The data continue to provide important monitoring and assessment information for a keystone species in an ecosystem that supports many species, including threatened and declining shorebird species during spring migration.

Infection prevalence, intensity, and gill coverage by the parasitic flatworm, Bdelloura candida, in the American horseshoe crab (Limulus polyphemus)

Infection prevalence, intensity, and gill coverage by the parasitic flatworm, Bdelloura candida, in the American horseshoe crab (Limulus polyphemus)

Comparative physicochemical analysis of chitin quality of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus

Recently, chitin biopolymer has received much attention as a wide variety of biomedical application for this and its derivatives have been reported, in fact, the study of non-conventional species as alternative sources of them compounds has taken particular interest. Here, we present a comparative physicochemical survey of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus: the prosoma and the opisthosoma, collected in Yucatán, Mexico. The characterization included CHNSO analysis, FTIR, TGA, DSC, XRD, and SEM. The CHNSO analysis revealed that C is present in the highest proportion (∼45 %) and that chemical composition did not show significant differences (P < 0.05) between the two tagmata. FTIR spectra of two tagmata presented a wide characteristic band of the chitin between 3600 and 3000 cm−1, confirming the presence of this biopolymer in the exoskeleton studied. TGA and DTGA profiles resulted very similar for both tagmata being the residual mass at 650 °C of around 30 % for both samples; these values were associated to the presence of minerals. SEM micrographs showed a porous matrix with infinite large number of irregularly shaped particles. Results show that both tagmata are made up of chitin, and they seem to have a high mineral content.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Morphometry of Limulidae (Oscar E. Brown) in Leidong, Sei Berombang, and Tanjung Tiram, North Sumatera

Limulidae (Horseshoe crab), or Mimi in the local name, are known; there are four species, namely Limulus polyphemus in North America and Asia, namely Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. In Indonesia, these species are protected by the Decree of the Minister of Forestry Number 12/KPTS-II/1987; meanwhile, illegal fishing still occurs. This study aims to identify Limulidae species and analyze Mimi's morphometric characteristics and distribution in North Sumatra to conserve the species. This research was conducted using a quantitative descriptive method in Leidong, Sei Berombang, Tanjung Tiram - North Sumatera, from September to December 2021. Biometric measurements were performed on 98 Mimi, and the data were statistically analyzed using the Kruskal Wallis test, Mann-Whitney test, and Multinomial logistics. The results showed that Mimi in the study area was identified as Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. The most commonly caught was T. tridentatus at the Tanjung Tiram. Based on the maximum width of the prosoma and body length, the largest to most petite sizes are T. tridentatus at Leidong (10.31 cm), T. gigas at Leidong, and Sei Berombang (10 cm) and C. rotundicauda at Sei Berombang (7.56 cm) respectively. The cluster analysis based on 27 morphometric characters showed that T. tridentatus had almost the same characteristics as T. gigas, while C. rotundicauda had its features. It can be concluded that the largest to most petite sizes of Mimi in the study area were T. tridentatus, T. gigas, and C. rotundicauda, respectively.