Female Horseshoe Crabs Research Articles

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.

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.

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.

New records of cheilostome Bryozoa from the eastern coast of India encrusting on the exoskeleton of live horseshoe crabs of Indian Sundarbans

Bryozoans are common commensal on hard surfaces and cover slow-moving animals like molluscans, sea turtles, brachyuran crabs, and horseshoe crabs. A total of six species of bryozoans belonging to four genus under three families of order Cheilostomatida were recorded encrusting on the carapaces of horseshoe crabs collected from Indian Sundarbans along the east coast of India and two among them, viz., Biflustra savartii (Audouin, 1826) and Sinoflustra arabianensis (Menon & Nair, 1975) are reported for the first time. Additionally, Jellyella tuberculata (Bosc, 1802) is reported for the first time from West Bengal coastal waters, previously known only from the Odisha coast of India. Both male and female horseshoe crabs were found to have been encrusted with bryozoan mats, although adequately not known about the life stages of their encrustation.

Characteristics of chemical compounds of horseshoe crabs Tachypleus gigas in different body proportions

Horseshoe crabs are a marine animal that has been widely used empirically, but in Indonesia the exact bioactive compounds are unknown. People poisoning caused by consuming horseshoe crabs has also been reported. The aims of this study was to observe the bioactive compounds in every part of horseshoe crabs which suspected to be related to toxicity. The work was conducted to measure morphometrics, to count body proportions, and to determine phytochemical compositions, toxicity, and antioxidants in different body proportions. Female horseshoe crabs with average weight more than 500 g were obtained from the waters of Lamongan Regency in July 2018. The highest proportion of the different body parts was the shell and the lowest was viscera. Edible portions of horseshoe crabs were 45.39%. Meat contains low fat 1.97% and high fat in gonads 4.87%. The solvent that produces the best extracts from the gonads was ethanol and used to extract meat, viscera, and gills. Viscera extracts were toxic with values of LC50 109.44 ppm, gonad and meat extracts have not shown to be toxic with values of LC50 1251.30 and 1101.79 ppm. Antioxidant activity of meat is weak (164.50±23.16 ppm), and gonads are very weak (330.47±02.60 ppm).

Feeding ecology and food preferences of Cherok Paloh, Pahang horseshoe crab, Tachypleus gigas

Intensive previous studies on horseshoe crabs feeding ecology were mostly focus on Limulus polyphemus. Their food preference might be different depend on the availability and abundance of feeds in the particular environment. This paper aims to investigate the feeding ecology and food preferences of Cherok Paloh, Pahang Tachypleus gigas. Ten samples of male and female horseshoe crabs, Tachypleus gigas were trapped in fishing net during the incoming high tide and 20 samples of male and female were hand-harvested at the spawning beach; 10 samples during pre-mating and 10 samples during post-mating. Their gut content was analysed by the Electivity Index. Results showed that Tachypleus gigas coming to spawn with full gut content. Echinoderm served as a main food composition in the gut of males (50%) and females (51.94%) during the open sea migration phase. The main composition was substituted by macrophyte (males: 59.51% to 65.15%; females: 36.36% to 58.10%) as they arrived to shore. Based on Electivity Index, male crabs showed positive preference toward polychaete (EI: 0.04) and macrophyte (EI: 0.19) at the spawning site while, the females showed positive preference toward bivalve (EI: 0.46). Further study on feeding ecology is needed in order to improve the population of Tachypleus gigas in Malaysia.

Pyritized in situ trilobite eggs from the Ordovician of New York (Lorraine Group): Implications for trilobite reproductive biology

Abstract Despite a plethora of exceptionally preserved trilobites, trilobite reproduction has remained a mystery. No previously described trilobite has unambiguous eggs or genitalia preserved. This study reports the first occurrence of in situ preserved eggs belonging to Triarthrus eatoni (Hall, 1838) trilobites from the Lorraine Group in upstate New York, USA. Like other exceptionally preserved trilobites from the Lorraine Group, the complete exoskeletons are replaced with pyrite. The eggs are spherical to elliptical in shape, nearly 200 µm in size, and are clustered in the genal area of the cephalon. The fact that the eggs are smaller than the earliest-known trilobite ontogenetic (protaspis) stage suggests that trilobites may have had an unmineralized preliminary stage in their ontogeny, and that the protaspis shield formed only after hatching. The eggs are only visible ventrally with no dorsal brood pouch or recognized sexual dimorphism. The location of the eggs is consistent with where modern female horseshoe crabs release their unfertilized eggs from the ovarian network within their head. Trilobites likely released their gametes (eggs and sperm) through a genital pore of as-yet unknown location (likely near the posterior boundary of the head). If the T. eatoni reproductive biology is representative of other trilobites, they spawned with external fertilization, possibly the ancestral mode of reproduction for early arthropods. Because pyritization preferentially preserves the external rather than internal features of fossils, it is suggested that there is likely a bias in the fossil record toward the preservation of arthropods that brood eggs externally: arthropods that brood their eggs internally are unlikely to preserve any evidence of their mode of reproduction.

Sublethal Behavioral and Physiological Effects of the Biomedical Bleeding Process on the American Horseshoe Crab, Limulus polyphemus

The hemolymph of the American horseshoe crab, Limulus polyphemus, is harvested from over 500,000 animals annually to produce Limulus amebocyte lysate (LAL), a medically important product used to detect pathogenic bacteria. Declining abundance of spawning Limulus females in heavily harvested regions suggests deleterious effects of this activity, and while mortality rates of the harvest process are known to be 10%-30%, sublethal behavioral and physiological effects are not known. In this study, we determined the impact of the harvest process on locomotion and hemocyanin levels of 28 female horseshoe crabs. While mortality rates after bleeding (18%) were similar to previous studies, we found significant decreases in the linear and angular velocity of freely moving animals, as well as changes in their activity levels and expression of circatidal behavioral rhythms. Further, we found reductions in hemocyanin levels, which may alter immune function and cuticle integrity. These previously unrecognized behavioral and physiological deficits suggest that the harvest of LAL may decrease female fitness, and thus may contribute to the current population decline.

Horseshoe crab eggs: A rare resource for predators in Long Island Sound

In Delaware Bay, the spawning of several million horseshoe crabs (Limulus polyphemus) coincides with the arrival of migratory shorebirds that feed on their eggs. High horseshoe crab spawning densities and resulting high egg densities drive egg availability and predation rates. At high spawning densities, female horseshoe crabs perturb previously deposited clutches causing eggs to rise to the sediment surface (surface egg densities average 100,000eggs m−2). At the surface (0–5cm), the eggs are quickly depleted by shorebirds and other predators. This interaction between egg density and egg predation has not been explicitly explored on beaches with low spawning densities such as on the Connecticut (CT) shore of Long Island Sound (LIS). Spawning indices in LIS (range: 0.002 to 0.02females m−2) are two to three orders of magnitude less than in Delaware Bay (0.7–1.0females m−2). Given the low spawning density and correspondingly low subsurface egg density (x=1.5eggs cm−2), we predicted that the frequency of egg predation would be rare. A series of exclosures was constructed on two known horseshoe crab spawning beaches to test the frequency and rate of predation on horseshoe crab eggs. There was no significant difference between egg masses initially placed in artificial nests and recovered at the conclusion of each experiment. While shorebirds were observed probing sediments within and outside of experimental plots, eggs were never observed at the surface during this study. Foraging by shorebirds for buried eggs may be energetically prohibitive given the lack of eggs on the surface and the low subsurface egg densities on CT beaches in LIS (range=0.6–2.4egg cm−2). Limited egg predation by fish was observed but tidal scouring of eggs buried below mean tide primarily drove egg availability for these predators. We conclude that horseshoe crab egg predation is a rare occurrence in Connecticut due to low egg density as a direct result of low spawning densities.

Alternative reproductive tactics in female horseshoe crabs

Consistent differences among females in mating with one (monandrous) or multiple males (polyandrous) may be a product of male behavior or may reveal the existence of female alternative reproductive tactics (ARTs). The distinction is an important one for understanding the evolution of sexually selected behavior. We evaluated whether ARTs exist in female horseshoe crabs (Limulus polyphemus), a species in which male alternative reproductive tactics are well known. In this species, attached pairs migrate to shore and spawn on high tides; the male fertilizes the female’s eggs externally with free-swimming sperm as the eggs are being laid. Unattached males are attracted to pairs by visual and chemical cues and become satellites of some females while ignoring others. We used multiple lines of evidence, including mark/resighting; measurements of size, physical condition, and eggs laid; and field manipulations of female response to satellite males. We show that even at high nesting densities with intense male–male competition, some females mated only with their one attached male, and females were consistently monandrous or polyandrous across multiple nestings. Monandrous females did not attract satellites but when males were experimentally manipulated to join monandrous pairs, some females stopped nesting and left rather than nest with a satellite male. These females tended to be smaller than polyandrous females. Our results suggest condition-dependent differences between monandrous and polyandrous females that result in different context-dependent mating decisions by monandrous and polyandrous females to

Isolation by the 5′ anchored PCR technique and characterization of eighteen microsatellite loci in horseshoe crab (Tachypleus gigas)

The horseshoe crab, a well-known ‘living fossil’, is represented by two species (Tachypleus gigas and Carcinoscorpius rotundicauda) in peninsular Malaysia. One of these, T. gigas inhabits shallow marine waters and migrates to intertidal sandy beaches for breeding during high tides at every full and new moon throughout the year (Hajeb et al. 2009). This species is being used as bait by local fishermen to catch crabs or shrimps by netting or cage fishing methods. Due to its low commercial value in comparison to other marine economic species such as fishes, crabs and shrimps, most of the animals caught in fishing nets or cages are released immediately back to sea by fishermen or left to die in the sun. In certain states of Malaysia, such as Kelantan, Penang and Johore, adult female horseshoe crabs are sold to local seafood restaurants and in fish markets due to consumption of their eggs by the local people. Horseshoe crab populations are rapidly declining around the world due to various reasons such as water pollution, loss of their living and spawning habitats and human exploitation of adult horseshoe crabs (Li et al. 2009; Shin et al. 2009). Assessing the genetic variability of horseshoe crab is important as it has direct benefit for the conservation and management of wild populations of the species. However, there is no information available on the population genetic structure and diversity of T. gigas in Malaysia. To address this research need, we have isolated and developed the first set of polymorphic microsatellite markers for T. gigas by using the 5′ anchored PCR technique. A total of 130 individuals of horseshoe crabs were collected from five locations along the coastal areas of peninsular Malaysia, namely Port Dickson (Negeri Sembilan), Pantai Bersih (Penang), Sungai Muar (Johor), Pantai Balok

Estimation of Short‐Term Tag‐Induced Mortality in Horseshoe Crabs Limulus polyphemus

AbstractHorseshoe crabs Limulus polyphemus range along the East Coast of the United States and over 150,000 of them have been marked with U.S. Fish and Wildlife Service disk tags. It has been assumed that the tags do not harm the animals and are similar to common epibionts often found on the shells of horseshoe crabs. We investigated whether newly tagged adult female horseshoe crabs would exhibit higher short‐term mortality rates than untagged adult females. All crabs were collected from a beach in Connecticut and then were transported to a laboratory for the experiment. Tagging involved drilling a small hole through the carapace in the lower back corner of the prosoma and then inserting the tag into the hole. Overall mortality of tagged and untagged females held in flow‐through raceways for 44 d was minimal (0% mortality among 53 tagged crabs; 4% mortality among 52 untagged crabs). None of the horseshoe crabs lost or shed their disk tags over the course of the experiment. In field mark–recapture studies of horseshoe crabs, typically between 11% and 20% of the initial numbers of tagged crabs are recaptured. Crabs that are recaptured but reported dead are unlikely to have died from the initial tagging process. Our results indicate that newly tagged adult horseshoe crabs have the potential to survive as well as untagged crabs through the Connecticut spawning season (∼30–45 d). Recapture data also suggest that these crabs can survive for as long as 8–10 years with the tags in place.

High resolution tracking of adult horseshoe crabs Limulus polyphemus in a New Hampshire estuary using fixed array ultrasonic telemetry

Abstract While several studies have documented the large-scale, seasonal movements of horseshoe crabs, little is known about their fine-scale, daily movement patterns. In this study we used a fixed array ultrasonic telemetry system to track the movements of 12 male and 16 female horseshoe crabs in the Great Bay estuary, New Hampshire. Data were obtained during the mating season, as well as during the remainder of the summer and fall, in the years 2005-2008. During the mating season animals were often, but not always, active during the high tides when they were approaching and leaving the spawning beaches. On average, both males and females approached mating beaches during 33% of the high tides they experienced and they most often made the transition from being inactive to active during the last two hours of an incoming tide. From April-October horseshoe crabs were significantly more active during high tide periods vs low tide periods, with no clear preference for diurnal vs nocturnal activity. After the mating season ended horseshoe crabs continued to move into shallower water at high tide and then return to deeper water at low tide. Observations by SCUBA divers suggest that during these excursions into the mudflats horseshoe crabs were digging pits in the sediment while foraging for food. Thus, the tidal rhythm of activity that has been so well documented during the mating season probably persists into the fall, and primarily involves foraging activities.

Costs of multiple mates: an experimental study in horseshoe crabs

Why do females mate with multiple males? Since females often acquire all the sperm they need in one mating and since mating is usually costly, the evolution of polyandry is puzzling. Females may gain from mating multiply by ensuring fertilization, increasing offspring diversity, improving mate quality (‘good genes’) or increasing genetic compatibility. Alternatively, multiple mating may result in a net cost to females as a by-product of male–male competition. We report on field experiments in which we evaluated whether spawning with multiple males is costly or beneficial for female horseshoe crabs, Limulus polyphemus . Unpaired male horseshoe crabs crowd around some spawning pairs (polyandrous) as satellites while ignoring others (monandrous). We conducted three field experiments in which we compared the success of (1) unmanipulated polyandrous and monandrous females, (2) polyandrous females with and without satellites (satellites removed) and (3) monandrous females without and with satellites (forced to be polyandrous by adding satellites). When polyandrous females lost their satellite males, they laid more eggs than did polyandrous females that retained their satellites. When a satellite male arrives at a monandrous pair, some females are tolerant and will nest as a polyandrous female, but many females are intolerant of satellite males and leave immediately, thus laying fewer eggs. These results indicate that spawning with multiple males is costly in terms of nesting success for female horseshoe crabs. Yet, natural monandrous and polyandrous females did equally well, suggesting that there are compensating benefits from multiple males for polyandrous females. We suggest that monandry and polyandry may be alternative reproductive tactics for female horseshoe crabs.

Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: implications for fisheries management

Horseshoe crabs (Limulus polyphemus) are bled for biomedical purposes to produce Limulus Amebocyte Lysate (LAL), a valuable material used to detect endotoxins in medical devices and implants. Previous studies generally found mortality from the bleeding process of 5–15% with one study reporting 29% for females; the 15% value is now used for management purposes. However, most of these studies looked only at males, did not mimic handling practices typical of the biomedical facility in Massachusetts, or harvested females after the spawning season when much of the actual biomedical harvest occurs. Female mortality is of particular concern because they have greater importance than males for population dynamics, are preferentially harvested, and are more likely to be physiologically stressed than males because of energy outlay for egg production. We examined the mortality of unbled female horseshoe crabs versus that of crabs bled by the local biomedical company and either returned directly to water, or held overnight as is the current practice. Mortality of Control (unbled) crabs was low (3%) and differed significantly (p < 0.001) from that of either bled group (22.5% and 29.8% mortality, respectively); the two bled groups did not differ significantly from each other (p = 0.31). Mortality rates in bled treatment groups were double those used in current management of the biomedical fishery. The bait and biomedical fisheries are managed differently because biomedical is considered a low-mortality “catch and release” fishery. States and interstate management agencies may want to re-examine policies surrounding biomedical horseshoe crab harvest based on these results.

Acoustic-Tracking and Radio-Tracking of Horseshoe Crabs to Assess Spawning Behavior and Subtidal Habitat Use in Delaware Bay

Abstract This study used telemetry to determine spawning behavior and subtidal habitat use of horseshoe crabs Limulus polyphemus. We attached combined acoustic and radio transmitters to 12 gravid female horseshoe crabs at Ted Harvey Beach and 12 at North Bowers Beach (both on the western shore of Delaware Bay) over a 5-d period before peak spawning on the new moon. Horseshoe crabs were acoustically tracked and radio-tracked daily for 8 d during both high tides and during the incoming dominant (higher) high tide. All horseshoe crabs were relocated at least once, and 83% of females spawned from two to six times (x = 3.35, SE = 0.18). Of these females, 85% spawned on two to five consecutive nights (x = 3.31, SE = 0.59). Most (95%) females spawned on the beaches where they were initially tagged. Typically, the shoreline used by an individual for spawning ranged from 70 to 1,160 m (x = 351 m, SE = 38 m). Between spawning events, horseshoe crabs remained 50–715 m offshore (x = 299 m, SE = 57 m) from their estab...

Larval hatching in the horseshoe crab, Limulus polyphemus: facilitation by environmental cues

Female horseshoe crabs, Limulus polyphemus (Linnaeus), lay their eggs in nests on sandy beaches near the high water line. Embryos develop within the sand, hatch into trilobite larvae, and enter the water column when the nest is inundated. Given the diversity of tidal and shoreline inundation patterns that populations of L. polyphemus experience throughout their range (semidiurnal and diurnal tides, microtidal, and nontidal), hatching may also be facilitated by environmental triggers that serve to synchronize hatching and larval emergence with periods of high water. The objective of this study was to determine if larval hatching in L. polyphemus is triggered or facilitated by environmental cues. Stage 21 embryos were subjected to one of seven different treatments that simulated conditions experienced during inundation: (1) hydration, (2) agitation, (3) hydration and agitation, (4) hydration and agitation with sand, (5) osmotic shock, (6) terrestrial hypoxia, and (7) aquatic hypoxia. Hatching rates increased significantly under all simulated tidal conditions compared to controls and were highest (96%) for eggs simultaneously exposed to both hydration and agitation with sand. Measurements of the osmolarity of the perivitelline fluid of developing eggs collected from the field indicated that it is hyperosmotic to the ambient seawater and porewater. Thus, when inundated, eggs also experience a hypoosmotic shock, which would likely facilitate hatching by causing the eggs to swell, rupturing the egg membrane and thereby increasing the likelihood that larvae would hatch and enter the water column during periods of high water.

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.

The Impact of Biomedical Bleeding on Horseshoe Crab, Limulus polyphemus, Movement Patterns on Cape Cod, Massachusetts

The purpose of this study was to determine if bleeding, for biomedical purposes, influenced the behavior of horseshoe crabs, Limulus polyphemus. In the summer of 2001, ten bled and ten control (unbled) female horseshoe crabs were tracked for 26 days using acoustic telemetry in a small estuary on Cape Cod, Massachusetts. All but three crabs, two bled and one control, were located during the study period. No mortality was observed in the control group, while 20% mortality was observed within the bled group. There was no significant difference in the average rate of movement or in the spatial distribution within the estuary between the two groups. However, a difference was detected in the movement patterns. Horseshoe crabs from the bled group had a random direction of movement compared to the directional movement pattern of the control group, suggesting that the bled crabs experienced more disorientation.