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Three decades of horseshoe crab rearing: A review of conditions for captive growth and survival
Abstract
Threats to wild horseshoe crab populations and growing interest in their use for research, education and biomedical applications have prompted demand for improved techniques to rear and maintain crabs in captivity. Although numerous laboratory studies have been conducted to determine growth and survival of horseshoe crabs under various conditions, these data have not been compiled and summarized to inform culture practices. We surveyed the literature and analysed the range of available techniques to identify and define a consistent set of conditions for maximum growth and survival of horseshoe crabs in culture. We considered three age classes; embryo, juvenile and adult, and included all extant species (Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleus gigas and Tachypleus tridentatus). We discovered relatively few published studies that clearly related husbandry conditions to growth and survival. Comparison among studies was complicated by inconsistent data collection and reporting techniques. Most published sources reported data for younger age classes, and more studies considered L. polyphemus than Asian species. The most commonly reported variables (temperature, salinity, enclosure maintenance and diet composition) showed size dependent and in some cases species-specific effects on growth and survival that will be important in guiding culture efforts. We suggest that future studies give additional consideration to substrate type, water flow, dissolved oxygen concentrations, diet quality and the quantity and frequency of feeding. If laboratory-reared stocks are to be used for propagation and restoration activities, future studies will benefit from closing these data gaps and promoting international data sharing.
... Horseshoe crabs are "ecological generalists" with a wide tolerance of salinity, temperature and other environmental variables (Botton and Itow, 2009). They consume a diverse diet of clams, marine worms, small crustaceans, and other invertebrates (Botton, 2009;Carmichael and Brush, 2012). Age and growth studies in horseshoe crabs suggest that they require about 9-11 years to attain sexual maturity, and have an estimated life expectancy of at least 14-18 years (Shuster Jr. and Sekiguchi, 2003). ...
... Captive rearing has been most popular in Asia as part of restoration and associated public outreach campaigns to raise awareness of declining native horseshoe crab populations (Xu et al., 2021). These efforts have been dovetailed with ongoing research and educational activities in the United States and Asia (Carmichael and Brush, 2012). Since 2009, for example, the Ocean Park Conservation Foundation (OPCF), Hong Kong and City University of Hong Kong have maintained a Juvenile Horseshoe Crab Rearing Program in collaboration with local high schools, so that students can observe and monitor horseshoe crab growth and release them to the wild. ...
... Until recently (Xu et al., 2021), much of the information on horseshoe crab laboratory culture practices in Asia for the past four decades was unpublished or available only in the regional language (i.e., Chinese and Japanese), making their knowledge inaccessible in some cases. Despite these challenges, datasets are increasing and there has been an effort in recent years to increase the international exchange of data (Carmichael and Brush, 2012). ...
The four species of horseshoe crabs are true “living fossils” with an evolutionary history that pre-dates the dinosaurs. One species, Limulus polyphemus, is found along the Atlantic and Gulf of Mexico coasts of North America. Three other species, the tri-spine horseshoe crab, Tachypleus tridentatus; coastal horseshoe crab, Tachypleus gigas; and mangrove horseshoe crab, Carcinoscorpius rotundicauda, live in shallow-water coastal and estuarine environments in Asia from Japan to India. Horseshoe crabs are valuable for human health because their blood is used to test for the presence of bacterial contamination in vaccines and other biomedical products. Today, all four horseshoe crab species are imperiled by overharvesting, shoreline development, and the loss and degradation of their essential spawning and nursery habitats. Despite these challenges, we remain optimistic that efforts to enhance conservation, including increased fishery management practices, marine protected areas, scientific research, public awareness, and citizen science engagement may together curtail further damage to their populations and enable recovery of these irreplaceable animals.
... The global decline in T. tridentatus populations has attracted increasing interest in searching for appropriate management tools to revert the situation. Despite the introduction of marine protected areas and the establishment of new environmental legislation can be effective, stock enhancement initiative in some cases is considered as a more proactive approach to populate the exploited stocks (Carmichael and Brush, 2012;Wang et al., 2020;Zhu et al., 2020). Releasing programs using hatchery-bred T. tridentatus juveniles, mostly the newly hatched first-instars or second-instars, and occasionally adults have been attempted or are underway along the Chinese coast of Fujian, Guangdong, and Guangxi provinces in the Mainland and Taiwan (Hong, 2011;Zhu et al., 2020). ...
... There is considerable demand in the development of hatchery production technology for rearing horseshoe crab larvae and juveniles due to the decline in horseshoe crab populations (Carmichael and Brush, 2012). However, the capacity of hatcheries to producing a sufficient number of high-quality juveniles in a cost-effective manner is the prerequisite of any releasing activity. ...
... A broader scale of T. tridentatus culture efforts has later been attempted in China (e.g., Liang, 1987) and Japan (e.g., Tsuchiya, 2009) since the 1980s. While most previous studies concerned how diet compositions and water quality variables affect the survival and growth of horseshoe crab embryos and juveniles (Carmichael and Brush, 2012;Hu et al., 2013aHu et al., , 2014Hu et al., , 2018Liao et al., 2019b), the mass production of cost-effective, high-quality seedstocks has presented a bottleneck in the releasing programs for T. tridentatus (Kwan et al., 2014;Zhu et al., 2020). We compiled and discussed the available rearing techniques in maintaining T. tridentatus embryos and juveniles in hatcheries with the ultimate goal in defining effective culture conditions (Figure 2). ...
As a well-known example of “living fossil,” horseshoe crabs are ecologically significant macroinvertebrates in coastal and estuarine ecosystems. The tri-spine horseshoe crab, Tachypleus tridentatus, has been widely utilized for Tachypleus amebocyte lysate production and food consumption since the 1980s, which led to considerable population declines along the west coast of the Pacific Ocean. The declining horseshoe crab population is expected to have ecological and social impacts. Stock enhancement through captive rearing of juveniles is cited as an important alternative to repopulate the native T. tridentatus, which in turn supports sustainable resource utilization and research activities. The hatchery production techniques for this species have gradually developed following the mass culture efforts in Japan since the late 1980s. However, the previous studies have primarily concerned the feed types and husbandry conditions to maximize the growth and survival of the juveniles. Little is known about the practicability and effectiveness of releasing large numbers of hatchery-bred individuals through releasing programs. In this review, we (1) summarize the available captive breeding and rearing techniques, (2) discuss the release strategies that could potentially improve the survival of released juveniles, and (3) identify the future opportunities and challenges in establishing technical frameworks to support responsible stock enhancement programs for T. tridentatus. The information should benefit future horseshoe crab fisheries management efforts in the attempt to restore the severely depleted populations.
... Previous studies have determined the diet and food preferences of HSCs in their natural habitat, but there is relatively little information available regarding the diet of HSCs in aquaculture, with most captive diets composed of a natural feed sources, such as shrimp, krill, mussels, sprat, sand eels, squid, mackerel, and trash fish (Botton, 1984). Three decades of adult HSC aquaculture have yet to define optimal parameters required for long-term management in captivity (Carmichael and Brush, 2012). Sustained aquaculture (exceeding 6 months) has been associated with low survival rates, principally linked to poor water quality (due to high stocking density) and inadequate nutrition, both of which place the HSCs under stress and increase their susceptibility to pathogens and vulnerability to captivity-induced disease (Kautsky, 1982;Smith and Berkson, 2005;Defoirdt et al., 2007;Carmichael et al., 2009;Nolan and Smith, 2009;Schreibman and Zarnoch, 2009;Kwan et al., 2014). ...
... Initial observations revealed that HSCs fed at different rates per day (expressed as the percent of b/w of the HSC) depending on the diet. Feed rates were calculated as a percentage of HSC body weight eaten within 8 h (any feed not consumed was removed from the tanks), with a total daily feed rate of 3%, consistent with Carmichael and Brush (2012). We found that at this rate, feed was in slight excess and not limiting, and thus HSCs were more or less feeding at ad libitum throughout the day. ...
... Irrespective of feed type, adult HSCs consumed similar amounts of food energy per day across all groups (i.e., mean = 150 kJ per kg of HSC b/w per day), and the previously cited 3% feed rate was thus validated and employed (Carmichael and Brush, 2012; Table 1). Maintenance levels of CP for adult HSCs were found to be ∼5.25 mg per gram b/w per day when fed an 11-23% protein diet, which aligned with various fish, shrimp, and juvenile HSC aquaculture studies (Kuresh and Davis, 2000;Blanc and Margraf, 2002;Lupatsch et al., 2008;Berkson et al., 2009;Tzafrir-Prag et al., 2010). ...
The Limulus amebocyte lysate (LAL) isolated from cells in the horseshoe crab (HSC) hemolymph is a critical resource for global biomedical and pharmaceutical quality control and sterility testing. Given the necessity of and limitations associated with wild capture, a conservational approach to LAL harvesting would benefit the medical community that relies on the raw material while helping ensure species viability. We posited that aquaculture and year-round collection represented a sustainable alternative for the production of LAL from a finite HSC cohort, thereby averting the impact of current practices on wild populations. Given the specter of captivity diseases linked to diet, such as panhypoproteinemia, this work, at the outset, focused on optimizing a feed formulation to ensure animal vitality. In turn, each preparation required evaluation with respect to effects on LAL, as well as vital HSC health markers, so as to meet or exceed industry requirements and establish a new supply chain paradigm. In this controlled husbandry study, we conducted three 8-week feeding trials and demonstrated a ∼7-fold LAL reactivity range among the HSC feed groups. Relative protein abundance patterns of HSC amebocyte clotting factors (i.e., Factor C, Factor B, and proclotting enzyme) were influenced by diet in particular, and the up-regulation of specific LAL factors correlated with enhanced reactivity. These results also cite the discovery that coagulation Factor C, the LPS-sensitive serine protease proenzyme, may be a phosphoprotein.
... The temperature was 28-30 °C, seawater salinity was 30‰, water flow was maintained, more substrate was provided for digging, and the tank was much larger than that in Japan. The least time to hatch and greatest survival of the Asian species were observed in water temperatures of around 29°C (Carmichael & Brush 2012), and Chen et al. (2004) suggested 28-31 °C as the optimal seawater temperature for the year-round growth of the juvenile T. tridentatus. These observations were in line with the results of Yeh (1999, cited in Chen et al. 2010) and Lee & Morton (2005), who reported that ecdysis in T. tridentatus continues when the temperature remains at >28°C but stops at <22°C. ...
... In light of the fact that the growth increments per molt of T. tridentatus in the two nursery habitats were comparable, it is likely that the caging operations are responsible for the deviations observed in the laboratory studies. Significantly reduced growth increments at each molt and longer IMPs in the laboratory were observed in horseshoe crabs (Carmichael & Brush 2012) and various crustacean species (e.g., Hiatt 1948;Harms et al. 1994;Bonilla-Gómez et al. 2013). The effects of the holding time at the laboratory and the space available in the holding tank were reported (e.g., González-Gurriarán et al. 1998). ...
... The development of L. polyphemus in the laboratory was considerably slower than that in their natural habitatthe postembryonic growth time was nearly halved when the embryonic development took place in nature (Jegla & Costlow 1982). As shown for horseshoe crabs (Carmichael & Brush 2012;Hu et al. 2013) and several crustacean species (Hartnoll 2001;Chang et al. 2012), the IMP increased and the rate of increase in size decreased when the quantity or quality of food in culture was suboptimal. The diet of horseshoe crabs in nature is broad and highly mixed and they move up the food webs as they age and grow (Carmichael et al. 2004;Zhou & Morton 2004). ...
Populations of the Tri-spine Horseshoe Crab Tachypleus tridentatus have dramatically decreased over their distribution range and conservation efforts are now crucial. The implementation of appropriate management strategies and stock assessment rely on accurate growth-rate estimates. The postembryonic development of the species in the tropics, however, is not elucidated. To provide the information needed to assess the demographics of juvenile populations and to judge the status of T. tridentatus in the Philippines, we conducted a mark-recapture experiment in a nursery habitat on Palawan Island. The results obtained during the 10-month period provide the first consecutive data on the stepwise growth of the species in the Philippines and the first near comprehensive dataset collected within a single population of juveniles in the tropics. By analyzing size-frequency (prosomal width) distributions of 853 individuals and by using 94 juveniles that molted during the study, 13 molt stages were differentiated. Based on the intermolt periods of six instars, we estimated the growth curve of T. tridentatus following two models (non-linear and power function). The data support the assumption that growth continues year-round in the tropics and also indicate that the average age of mature male and female T. tridentatus in the Philippines ranges from three to four years. The agreement with a field study in Japan suggests that 14 postembryonic stages may be characteristic for the development of natural populations throughout the range of the species. Though more data are needed to validate these results, the study provides a sound baseline for future studies in the tropics.
... The success of this culture method depends on the survival and growth of the juveniles, which in turn relies on the provision of an optimal diet (Hu et al. 2014, John et al. 2017. The food and diet supplements used for juvenile horseshoe crab culture range from brine shrimp, bivalves, squid/fish, worms, crabs/shrimp, other invertebrates, and fish food to algae (Carmichael & Brush 2012). Tzafrir-Prag et al. (2009) estimated nutrient and protein requirements for the Atlantic horseshoe crab (Limulus polyphemus). ...
... Despite these abovementioned investigations, relevant research on Asian horseshoe crabs is still limited. Carmichael and Brush (2012) suggested that research on diet quality for laboratory-reared stocks should be performed if these stocks are to be used for propagation and restoration. The high cost of natural diet is a limitation; therefore, researchers need to focus on supply and quality of artificial food that will yield nutritionally balanced yet cost-effective feeds for successful mass culture. ...
... Survival and growth performance are the most important criteria for selecting the optimal feed formulation for certain animal species (Lovell 1989). The survival rates noted in the present study were generally in line with the reported survival data of greater than 50% on captive juvenile horseshoe crabs (Carmichael & Brush 2012). Based on the growth performance and feed utilization of the two species, the optimal FM replacement level for Tachypleus tridentatus was estimated at 50%, whereas that of Carcinoscorpius rotundicauda was at 75%. ...
A feeding experiment was undertaken for 84 days to investigate the growth performance and the feed utilization of two horseshoe crab species from partial replacement of fish meal (FM) with blood meal (BM), meat and bone meal (MBM), and poultry by-product meal (PBM) in their diet. Triplicate groups of Tachypleus tridentatus or Carcinoscorpius rotundicauda were fed nine isonitrogenous (digestible protein 40%) and isoenergetic (digestible energy 14 MJ kg⁻¹) diet treatments. For both species, the growth performance, that is, final individual body weight and thermal-unit growth coefficient (TGC), and feed utilization, that is, including feeding rate (FR) and feeding efficiency ratio, of the juveniles fed with PBM and MBM at 2:1 ratio were higher than those of juveniles fed with diets containing PBM and MBM at 1:1 ratio at the same FM replacement level. Final individual body weight, TGC, and FR of the two species fed with the 100% FM replacement diets were significantly lower than in other diet treatments. The optimal replacement levels ofFMby the combination of PBM, MBM, andBMwere 50% and 75% in the protein of T. tridentatus and C. rotundicauda, respectively. Based on literature data, FM replacement reduced feed costs by 15%-25% in aquaculture practices. © 2018 National Shellfisheries Association. All rights reserved.
... Intermediate culture of juvenile horseshoe crab (Tachypleus tridentatus) mixed with juvenile spotted babylon (Babylonia areolata) for restocking horseshoe crab populations 1,2 Changpo Chen, 3,4 Ruifang Chen, 2 Pohung Chen, 3,4 Haijuan Liu,5 Hweylian Hsieh crab juveniles in indoor culture facilities (Chen et al 2004;Hong et al 2009;Carmichael & Brush 2012) and due to a lack of restocking techniques (Chen et al 2014). ...
... First, what kinds of food should be used? Brine shrimp (Artemia spp.) have been fed to juvenile horseshoe crabs in most of the indoor culture experiments because it is a live zooplankton diet and easy to handle (Carmichael & Brush 2012). However, brine shrimp may not be an accessible diet for juvenile horseshoe crabs because the crabs are benthic and spend most of their time in sedimentary substrata. ...
... In coastal areas, the first instars hatch from nests on sandy beaches and disperse into mud flats . The first instars do not feed; the second instars commence feeding and forage on sand/mud flats (Sekiguchi 1988;Carmichael & Brush 2012). The third instars have a depleted yolk and must eat enough food to survive. ...
The populations of the ‘living fossil’ horseshoe crab Tachypleus tridentatus have decreased dramatically. Releasing juveniles in large quantities, preferably at up to the third instar stage and with better survival in the wild, would be an effective method for replenishing the populations. However, the mortality between second instar and third instar stages is high according to indoor, monospecies culture results. We conducted an outdoor culture of juvenile crabs mixed with juvenile spotted babylon (Babylonia areolata) and found that this technique could enhance the horseshoe crabs’ survival, accelerate their molting and produce large numbers of juveniles. At the final harvest time, after running the mixing culture for 150 days, horseshoe crab juveniles exhibited survival of 32.5% and production of 66,000 individuals with ages beyond the second instar. Spotted babylon had a survival rate of 87.3%. The resultant survival and abundance levels were greater than those previously reported. We propose that these profound results can be attributed to 1) abundant benthic algae grow in outdoor ponds under sunlight, 2) the commensal lifestyle of juvenile horseshoe crab and spotted babylon, which likely prefer different food particle sizes, 3) the behaviors of spotted babylon, such as burrowing and moving in the sediment substrata, probably create an environment that favors horseshoe crab growth, 4) large amounts of space in the outdoor ponds, which improves growth for unknown reasons, and 5) suitable weather conditions from November to April in Guangxi.
... It is generally agreed by scientists that horseshoe crabs are currently facing global threats for further population declines (Carmichael and Brush 2012). For example, a remarkably reduction in half of the spawning individuals of L. polyphemus was found in the surveys of New Jersey beaches, USA since 1990 (Swan et al. 1996). ...
... The experiment consisted of four diet treatments: the short-neck clam Ruditapes philippinarum, sandworm Marphysa sanguinea, mixed clam and sandworm (1:1 wet weight) and brine shrimp Artemia salina. The short-neck clam and sandworm are natural food sources of horseshoe crabs in the wild (Zhou and Morton 2004;Carmichael et al. 2009), whereas brine shrimp is the most common aquaculture food used for rearing juvenile horseshoe crabs (Hong et al. 2009;Hu et al. 2013;Carmichael and Brush 2012). ...
... (2012) that increasing carbohydrate levels of diets improved the FR of juveniles given that the protein levels were about or more than 40 %. These results implied that carbohydrate level in the diet may be an indicator for growth performance when juvenile horseshoe crabs are fed with high protein diets which are currently the most common practices in the culture of horseshoe crabs (Carmichael and Brush 2012). ...
Horseshoe crabs are regarded as an indicator species to reflect the health of coastal shore systems, because they play significant ecological roles as benthic predators and prey, bioturbators and hosts for epibionts. However, declining populations of Asian horseshoe crabs were evident as reported in East and Southeast Asia, owing to overexploitation for human consumption and biomedical use, along with habitat loss and marine pollution caused by construction of coastal defenses and anthropogenic activities. The present study attempted to provide insights into the perspectives of ecological and health status of juvenile Asian horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in Hong Kong, with the aim to supporting their conservation. Findings from this research will also be useful as reference for the establishment of marine protected areas (MPAs) locally and elsewhere, in which conservation of Asian horseshoe crabs is urgently needed. For ecological investigations, the present study updated the population abundance, growth and habitat condition for both juvenile horseshoe crab species, and estimated the home range and diet composition of these juveniles in the field. For health status studies, these included research on the use of hemolymph parameters as an indicator for wellness of cultured juveniles, addition of marine microalgae as diet supplement to improve hemolymph quality of juveniles in captivity, responses of juveniles to sublethal exposures of trace metals, and relationship of juvenile health and habitat pollution condition on the spawning/nursery shores.
To ascertain the present population status from past studies, the distribution and abundance data of juvenile Asian horseshoe crabs T. tridentatus and C. rotundicauda were re-examined and compared with historical records. The territory-wide surveys at 18 spawning/nursery beaches displayed a persistently low mean juvenile density from 0.16−2.19 ind/100 m2 in 2012 and 0.08−1.41 ind/100 m2 in 2014. These density data were within the same range of that reported in 2002 and 2007 (0.10−1.97 ind/100 m2), apart from a low record of 0.08−0.31 ind/100 m2 in 2004. The current total population of juvenile T. tridentatus and C. rotundicauda on these spawning/nursery shores is estimated in the range of 2,000−4,300 and 2,400−3,000 individuals, respectively. From the 2012−14 data, no new recruitment of early 1st−3rd instar juveniles was found.
The designation of MPAs for horseshoe crabs may resolve user conflicts from human activities and restore overexploited stocks and degraded areas. Such MPAs have been established in mainland China, Taiwan and Japan, though most of their designations have largely been an administrative process without scientific data back up or studies. To propose ecological-relevant MPAs for the protection of spawning/nursery shores, scientific information on juveniles’ diet composition and movement extent are necessary in determining the habitat and extent of the protected areas. The two-month field study at San Tau sandy mudflat found that the mean home range of the 150 individuals of 7th–10th instar juvenile T. tridentatus varied from 269 to 462 m2, based on the estimation derived from the adaptive kernel density approach. The diet composition and trophic position of 6th–11th instar juvenile T. tridentatus on the seagrass-covered intertidal mudflat at Ha Pak Nai were studied using dual stable isotopes δ13C and δ15N. Data of δ13C and δ15N in juvenile tissues and their potential food sources suggested that T. tridentatus consumed a mixed diet mainly comprised of a variety of polychaetes, crustaceans and bivalves that are abundantly available at the study site and largely supported by seagrass biomass in both summer and winter.
Apart from in-situ conservation measures, the study of the growth and survival of juvenile horseshoe crabs in the laboratory is important as an ex-situ effort in mass culture and subsequent release of the reared juveniles to the field, so as to increase the wild populations. Based on the juvenile diet composition results from the field, their natural food sources including the short-neck clam, sandworm as well as a mixture of clam and sandworm were provided to the cultured juveniles. While the hemolymph properties among these diets were similar at the end of the three-month experiment, significant declines in hemocyanin concentration, percentage of oxyhemocyanin, and ratio of granular-spherical to granular-flattened states of amebocytes were noted even under high-protein diets. Such a decrease in hemolymph quality indicated deleterious health effects due to prolonged culture, which might be attributed to captivity-related issues, such as deficiencies of essential diet compositions, movement constraints and absence of tidal rhythms. This was further supported from the finding that the hemolymph quality of the experimental juveniles was significantly poor compared to that of the juveniles collected from the field. Significant declines in the ratio of granular-spherical to granular-flattened states of amebocytes were also observed in all wild juveniles after 12-week culture period in the laboratory.
Since the survival and sustainable growth of cultured juveniles after the field release depend largely on their fitness and health status, the poor hemolymph quality and health condition of cultured juveniles may thus hinder the success of restocking the horseshoe crab populations in the field. As the early 2nd–3rd instar horseshoe crabs primarily feed on phytoplankton and/or microalgae in nature, four clam meat diets with either marine green flagellate Dunaliella tertiolecta or golden-brown flagellate Isochrysis galbana were provided to the 8th instar cultured juveniles to investigate the changes in hemolymph quality of juveniles under husbandry conditions. At the end of the three-month experiment, significantly higher percentages of amebocyte viability and granular-spherical amebocyte states were noted in the golden-brown microalgae I. galbana treatment, but not for those fed with green microalgae D. tertiolecta. Such differences were attributed to the presence of C18 and C20 polyunsaturated fatty acids (PUFAs) in I. galbana but absence in D. tertiolecta, suggesting the essential role of certain PUFAs in a protein-rich diet to improve the immune competence and health status of juvenile horseshoe crabs under culture prior to the release to the field.
Changes of juvenile hemolymph constituents are also demonstrated to be sensitive and useful in reflecting the health condition of habitats. Responses of growth endpoints and hemolymph constituents in juvenile T. tridentatus under treatments of 0.1 and 1 mg/l cadmium (Cd) and 0.01 and 0.1 mg/l tributyltin (TBT) were examined in a 12-week experiment. Various hemolymph quality indicators, including hemolymph plasma protein level, amebocyte viability and percentage of granular-spherical state of amebocytes of the juveniles exposed to TBT or Cd were significantly lower than the control. Such a decrease in hemolymph quality suggested deleterious effects of metal contaminant-induced stressors on the health status of the juveniles, even at low exposure levels. Given the high sensitivity of hemolymph indicators to contaminant levels, the hemolymph quality indicators of juvenile T. tridentatus and C. rotundicauda from five spawning and nursery shores in Hong Kong were further examined in relation to various environmental parameters. Results of correlation of juvenile hemolymph composition patterns with environmental parameters measured at the study sites showed that the lead concentration in sediments and nitrate level in interstitial waters appeared to best relate to the hemolymph constituents in juvenile T. tridentatus and C. rotundicauda, respectively.
In conclusion, the overall status of the juvenile horseshoe crabs in Hong Kong is fragile and susceptible to extinction, since apart from potential habitat loss and degradation, the existing populations are small and discrete, with relatively few new recruits. Their limited foraging range on the spawning/nursery shores is particularly vulnerable to localized habitat disturbances. The wide range of food sources for the juveniles is largely supported by seagrass biomass on the intertidal flats, which is also threatened by on-shore human activities. The changes in juvenile hemolymph quality, especially amebocyte viability and morphological states, are sensitive and useful as health status indicator in reflecting captivity stress due to prolonged laboratory culture, effects from environmental contaminants and general condition of the habitats. However, further research is needed to improve the hemolymph quality of the cultured juveniles to ensure their sustainable growth and survival if they are released to natural spawning/nursery shores for enhancing the local populations.
... Despite increasing interest in horseshoe crab culture in recent years, there are few consistently collected and detailed data available on best practices for captive rearing (Carmichael et al. 2009 ;Carmichael and Brush 2012 ). In particular, data are needed to better understand how horseshoe crab growth and survival are linked to environmental conditions and diet composition in culture (Chen et al. 2010 ;Carmichael and Brush 2012 ). ...
... Despite increasing interest in horseshoe crab culture in recent years, there are few consistently collected and detailed data available on best practices for captive rearing (Carmichael et al. 2009 ;Carmichael and Brush 2012 ). In particular, data are needed to better understand how horseshoe crab growth and survival are linked to environmental conditions and diet composition in culture (Chen et al. 2010 ;Carmichael and Brush 2012 ). Type of substrate and holding tank style are two variables that could affect the chemical and physical environment for horseshoe crabs in ways that affect growth and survival, but these effects are rarely directly studied. ...
... Growth rates of horseshoe crabs during this experiment were comparable to those reported in other culture studies (Carmichael and Brush 2012 ) and lower than reported for juvenile American horseshoe crabs in the wild (Carmichael et al. 2003 ). While there are few studies that similarly tested sediment type and tank shape, Hong et al. ( 2009 ) also found different rates of growth for Asian horseshoe crabs, T. tridentatus , reared on different substrates. ...
We measured the effects of two conditions, sediment type (natural or clean sand) and tank shape (rectangular or kreisel style), on growth, survival, and movement patterns of juvenile American horseshoe crabs (Limulus polyphemus). During the 9-week experiment, horseshoe crabs reared in natural sediment and/or in rectangular tanks had higher growth rates than crabs reared in kreisel style tanks with clean sand. Horseshoe crabs reared in natural sediment also had lower % mortality compared to crabs in clean sand. Hence, by the conclusion of the experiment, horseshoe crabs reared in kreisel style tanks with clean sand showed no growth and 100 % mortality. To determine whether sediment type affected growth and survival by affecting nutrition, we measured N and C stable isotope ratios in horseshoe crab tissues compared to organic matter in sediments and gel diet fed to crabs during the study. Sediment-derived organic matter made a significant contribution to the diet of crabs reared in natural sediments. Enriched N stable isotope ratios, along with the lack of growth and poor survival, suggested that crabs reared in clean sand experienced starvation. The movement patterns of juvenile horseshoe crabs within each tank also varied with sediment and tank type, suggesting links between these attributes and settlement or foraging behavior. Natural sediment may contribute to horseshoe crab success by providing an additional food source to juvenile crabs, and tank shape may have a secondary effect on growth, survival, and movements related to hydrodynamics that merits further study.
... • Tachypleus tridentatus, also the Chinese, Japanese or tri-spine horseshoe crab, is located in Southeast and East Asia, as shown in the Table 1 [5,6]. ...
... Collect samples with monitoring at 230 nm.5. Collect specific samples of corresponding peaks and dry the sample in a speed vac centrifugal evaporator.6. ...
Peptides are small biomolecule which acts a bioactive or host defense molecules. These peptides have a significant role in medicinal values because of their broad spectrum antimicrobial properties. Marine sources are the largest supply of peptides and natural small molecules, since they represent half of the total global biodiversity. Among the different species of crab, horseshoe crab is widely used sources for the extraction of the peptides. The horseshoe crab is a marine and brackish water arthropod; it plays a crucial role in the reel and whelk fishing industry and the production of important pharmaceutical bioactive components. This review highlights the details of extraction, purification of antimicrobial peptides from horseshoe crab. These peptides trends and prospects for the future, with special emphasis on pharmaceutical industries.
... For 450 million years, they maintained their original traits and lived in the benthic zone and were thus famous living fossils (Sekiguchi and Sugita, 1980;Rudkin and Young, 2009). Horseshoe crab types include only Limulus polyphemus (American horseshoe crab), Tachypleus tridentatus (trispine horseshoe crab), Tachypleus gigas (southern horseshoe crab), and Carcinoscorpinus rotundicauda (mangrove horseshoe crab; Carmichael and Brush, 2012;Mashar et al., 2017). L. polyphemus is mainly distributed in the American Atlantic coast and the Gulf of Mexico. ...
... In China and southeast Asia, T. tridentatus and T. gigas are regarded as food and traditional Chinese medicine and thus consumed by local people or travelers (Fu et al., 2019). With the progress in scientific research, specifically, biomedical research (Shuster, 1962), researchers explored and revealed the visual, endocrine, and physiological processes of horseshoe crabs (Berkson and Shuster, 1999;Zaldívar-Rae et al., 2009;Carmichael and Brush, 2012). Horseshoe crabs have medical importance given that they produce reagents for testing bacterial endotoxins (Levin and Bang, 1964;Cooper et al., 1971;Novitsky, 1984;Berkson and Shuster, 1999;Swan, 2002;Kreamer and Michels, 2009) and vaccines (Maloney et al., 2018). ...
This review provides the updated information and analysis of research development on horseshoe crabs over the past 30 years and raises some suggestions for future studies on horseshoe crabs. Horseshoe crab is a unique marine species and attracts many scholars in various research fields. However, to date, the development of horseshoe crab research has not been analyzed and reported from a bibliometric perspective. Bibliometric analysis is a unique tool for evaluating the development of a specific research area by analyzing relevant publications and researchers from all over the world. In this study, Web of Science, Google Scholar, and PubMed were used to collect relevant data on horseshoe crabs. VOSviewer software was used to visualize cooperation and major keywords in horseshoe crab research. From 1989 to 2019, scholars emphasized the physiological characteristics, medical value, and ecological conservation of horseshoe crabs. Some topics in this field have gradually developed because of some reasons explained in this review. Some important or recent studies have been discussed, and some potential research topics for future research have been suggested.
... Habitat degradation due to anthropogenic activities led to depletion of HSC populations around the world (Carmichael & Brush 2012;Nelson et al. 2016). In Malaysia, HSCs are facing unmonitored harvesting, exposure to pollution and habitat destruction (John et al.2012;Kamaruzzaman et al. 2011). ...
... Both of these two spawning sites has undergone destruction, for instances, roads and bridge construction at Tanjung Selongor and jetty construction at Pantai Balok has severely limited T. gigas spawning activities (Nelson et al. 2016). Captive rearing and releasing of HSC has been established to be a key approach in HSC conservation (Carmichael & Brush 2012;Hong et al. 2009). Captivity has been linked to a number of infectious and non-infectious diseases of HSCs, which cause mortality (Nolan & Smith 2009). ...
Horseshoe crab (HSC) populations around the world are declining in recent decades mainly due to destruction of breeding grounds and habitats. A short-term solution for this problem is captive breeding and artificial rearing. This experiment aimed at looking for a source of good sperm donors for in-vitro fertilization and captive breeding. Tachypleus gigas were collected from Balok Beach, Kuantan, Pahang, Malaysia. Crabs were divided to wild and captive groups. Wild T. Gigas sperm was collected on the same day, while sperm of captive T. Gigas was collected after 5 months of captivity to assess captivity effect on sperm traits. Sperm density and sperm viability were studied and correlated to morphometric measurements. The results indicated that T. Gigas from Balok is a good sperm donor. Captivity was found to affect sperm traits where sperm density dropped significantly and sperm viability increased slightly. Intraocular width to carapace width ratio (IO-Car) of the HSC body correlated to sperm density, hence can be used as an indicator for donor selection during sampling. In conclusion, T. Gigas from Balok can be used as a sperm donor for in-vitro fertilization for T. Gigas propagation. © 2019 Penerbit Universiti Kebangsaan Malaysia. All Rights Reserved.
... In DE, nesting adults become stranded under air and surface sand temperatures exceeding 31 and 37°C, respectively (Botton and Loveland 1989). In L. polyphemus from across the species' range, greatest development success of larvae occurs between 25 and 30°C (Jegla and Costlow 1982;Laughlin 1983), while temperatures higher than 35°C can be lethal (Ehlinger and Tankersley 2004;Carmichael and Brush 2012). Considering the effect of temperature alone (i.e., at the reference salinity and O 2 ), we found that embryos from both the DE and FGC populations showed reduced development success after exposure to temperatures above or below the reference temperature (30°C), but embryos from DE performed significantly better than embryos from FGC at the highest temperature (35°C). ...
... In L. polyphemus from across the species' range, greatest larval development success occurs between 20 and 30 ppt salinity (Jegla and Costlow 1982;Laughlin 1983;Ehlinger and Tankersley 2004;Botton et al. 2010;Carmichael and Brush 2012). Although we defined the reference salinity for our experiments as 34 ppt (approximately full-strength seawater), we found that development success was highest at moderate hyposalinity (15 ppt) for embryos from both the DE and FGC populations, whereas severe hyposalinity (5 ppt), whether alone or in combination with heat stress or hypoxia, greatly decreased development success in embryos from both populations. ...
The American horseshoe crab, Limulus polyphemus, is found along the Atlantic and Gulf of Mexico coasts in genetically-isolated populations. Eggs are laid in shoreline beaches that expose developing embryos to combinations of environmental stressors. Whether populations of L. polyphemus differ in multi-stressor tolerance had never been tested. We assessed the multi-stressor tolerance of L. polyphemus embryos from a population in Delaware Bay (DE) and determined whether these differed from the multi-stressor tolerance of embryos from a more southerly Florida Gulf Coast (FGC) population. We monitored the field sediment temperatures and determined multi-stressor tolerance of DE embryos, then compared these to published data for FGC embryos. For multi-stressor tolerance, we assessed development success of embryos in two-week exposures to 36 fully-factorial combinations of temperature (20, 25, 30, 35 °C), salinity (5, 15 and 34 ppt) and ambient O2 (5, 13 and 21% O2), followed by two-weeks in recovery conditions. Sediment temperatures in the DE site ranged from 9.5-46 °C, with extended periods exceeding 35 °C. Development success was similar between the DE and FGC populations in 14 of 26 multi-stressor combinations. The DE embryos were generally more successful in conditions that included high temperature or moderate hyposalinity, whereas the FGC embryos were generally more successful in conditions that included extreme hyposalinity. This suggests that although multi-stressor tolerances are generally similar between the two populations, specific differences exist that correlate more with differences in nest microenvironment than latitude.
... Although the development of horseshoe crab aquaculture is still in its infancy, various laboratory studies have reported culture conditions for juvenile horseshoe crab species in experimental investigations (Carmichael & Brush 2012;Shinn, M€ uhlh€ olzl, Coates, Metochis & Freeman 2015). Some of these studies have demonstrated that protein-rich diets of brine shrimp, clams, mussels and polychaetes can enhance the growth performance of juveniles (Carmichael, Gaines, Sheller, Tong, Clapp & Valiela 2009;Schreibman & Zarnoch 2009;Hu, Wang, Cheung & Shin 2012); however, Kwan, Chan, Cheung and Shin (2014) revealed that the haemolymph quality in juvenile Chinese horseshoe crabs under laboratory culture was significantly reduced over a 12-week experimental period even under high-protein diet treatments. ...
... Some of these studies have demonstrated that protein-rich diets of brine shrimp, clams, mussels and polychaetes can enhance the growth performance of juveniles (Carmichael, Gaines, Sheller, Tong, Clapp & Valiela 2009;Schreibman & Zarnoch 2009;Hu, Wang, Cheung & Shin 2012); however, Kwan, Chan, Cheung and Shin (2014) revealed that the haemolymph quality in juvenile Chinese horseshoe crabs under laboratory culture was significantly reduced over a 12-week experimental period even under high-protein diet treatments. High mortality in juvenile horseshoe crabs has often been noted after 6 months in culture (Brown & Clapper 1981;Carmichael & Brush 2012), and consistent with such observations, deficiencies in essential diet compositions, movement constraints, absence of tidal rhythms and high holding temperatures appear to affect haemolymph quality and health status of horseshoe crabs (Coates, Bradford, Krome & Nairn 2012;Kwan et al. 2014). The existence of such captivity-induced stress has been further supported by the finding that the haemolymph quality of cultured juveniles is significantly poorer than that of juveniles collected from the field (Kwan et al. 2014). ...
Five treatments (short-neck clam Ruditapes philippinarum and four formulated diets with the green
microalgae Dunaliella tertiolecta or the goldenbrown microalgae Isochrysis galbana in the ratio (dw/dw) of 5% or 10% to that of clam meat) were used to investigate the growth performance and haemolymph quality of juvenile Chinese horseshoe crabs Tachypleus tridentatus under laboratory culture. At the end of the 12-week study, various growth and moulting indicators as well as haemolymph biochemical parameters did not show any significant differences among the treatments. Significantly higher percentages of haemolymph amoebocyte viability and granular-spherical amoebocyte state were noted in both 5% and 10%
I. galbana-supplemented diets compared to that of clam meat. However, the percentages of these two
haemolymph parameters in juveniles fed with D. tertiolecta decreased significantly at the end of the experiment. Such differences were attributed to the presence of polyunsaturated fatty acid (PUFA), notably eicosapentaenoic acid (EPA), in I. galbana that was absent in D. tertiolecta and clam meat. Like many marine invertebrates, juvenile horseshoe crabs do not naturally obtain sufficient PUFAs from their diets to meet developmental and physiological needs; hence, supplementing microalgae with high levels of EPA in a protein-rich diet boosts immune competence and the health status of juvenile horseshoe crabs while under culture.
... 46 Mishra 2009; Schreibman and Zarnoch 2009; Tsuchiya 2009). Additionally, these 47 studies vary significantly in species studied, culture conditions, and reported 48 response variables, making it difficult to advance the field from these efforts 49 (Carmichael and Brush 2012). Thus, there is a need to more rigorously develop 50 culture methodologies and test culture variables to support future aquaculture 51 ...
... If you are 600 what you eat, then the nutritional content of brine shrimp, especially if eaten 601 preferentially, may displace more nutritious choices and unbalance the Limulus diet. 602Thus, the nutritional diversity of potential food supplements should be investigated 603(Carmichael and Brush 2012). If brine shrimp remain a preferred food, the 604 nutritional content can be varied by choice of strain and/or the food said brine 605 shrimp eat. ...
The New Jersey Aquaculture Innovation Center (AIC) at Rutgers University has instituted a program to enhance the population of the American horseshoe crab Limulus polyphemus in the Delaware Bay Estuary (DBE) by using aquaculture at the earliest stages in its life history, when losses are greatest and production costs are minimal. Located on the Cape May Canal, the AIC pumps provide both raw and treated (sand-filtered, UV-sterilized) DBE water throughout the facility. Eggs of L. polyphemus were collected from local, sub-optimal nesting beaches impacted by coastal development and rising sea level, and hatched in jars normally used for trout eggs. A hatching system was built that uses recirculating treated seawater and a header tank to provide constant, independently controllable flow through 12 jars. An experiment performed using the hatching system tested the effects of egg stocking density and seawater flow rates on hatching success. The results showed highest hatching success at high egg densities with low flow rates. Hatchlings were reared in downweller silos in a raceway tank with flow-through, raw seawater. Half of the hatchlings were allowed to feed on organic matter in the incoming raw seawater while the others were given a supplement of Artemia nauplii. Statistically, there was no difference in growth or development between the treatments. After 10 weeks, most hatchlings were at the 3rd instar stage. This suggests that aquaculture of the early stages of L. polyphemus, utilizing natural estuarine DBE water, maximizes the impact to the future population by protecting eggs and early instars, the most vulnerable life stages, at minimal cost.
... There has been an increasing interest in the captive rearing of horseshoe crab species to restore their depleted populations (Chen et al. 2010;Carmichael and Brush 2012;Xu et al. 2021). The horseshoe crab rearing and release program, particularly for L. polyphemus and T. tridentatus, has already been implemented in mainland China, Hong Kong, Taiwan and the USA (Kwan et al. 2017;Xu et al. 2021). ...
Context
Spawning beaches are important for coastal horseshoe crabs, Tachypleus gigas (Müller, 1785), to sustain their population. Over time, T. gigas natural spawning area has gradually diminished owing to human and natural causes. T. gigas is being artificially bred in captivity to enhance its population.
Aims
The study aims to determine the T. gigas spawning preference for three different sediment sizes.
Methods
Spawning experiments were carried out in an indoor concrete tank representing an artificial spawning ground, consisting of three combinations of sediment sizes: coarse, medium and fine. The spawning of 45 pairs of T. gigas samples was observed within 3 h, the nests were marked and excavated on the next day. The number of eggs per nest was counted individually after being cleaned.
Key results
The 45 pairs of T. gigas produced 27,266–30,859 eggs. Among these, on the basis of the number of nests produced, T. gigas prefers to spawn in medium to coarse sediment with a particle-size range of 0.25–1.00 mm.
Conclusions
Our findings indicated that medium to coarse sediments are optimal for T. gigas spawning in artificial spawning grounds, because they could produce a high number of nests and eggs, increasing the number of juveniles released.
Implications
These results could be used as a baseline for the conservation management of T. gigas in captivity.
... Genetic breeding of horseshoe crab is also feasible via the advancement in sequencing technologies. It is crucial to prevent inbreeding of HSCs by releasing juveniles from different cohorts of mating pairs when reintroducing them into the wild (Carmichael and Brush, 2012). ...
... The parameters for human care of horseshoe crabs as well as concerns for their welfare and survival have been described [5,[100][101][102][103][104][105][106][107]. However, in zoos and aquariums, horseshoe crabs are often included in touch pool exhibits, which allow for interaction between guests and their animal inhabitants. ...
Simple Summary
Invertebrates (animals without backbones) make up over 95% of the earth’s species yet compared with vertebrates (animals with backbones like fishes, amphibians, reptiles, birds, and mammals) our understanding of and efforts relating to the topic of welfare is relatively minimal. We have selected seven of the most economically important and widely recognized invertebrate taxa to focus the topic of animal welfare on. In these pages the reader will learn about coelenterates (jellyfishes, anemones, and corals), mollusks (snails, slugs, squid, and octopi), crustaceans (lobsters, crabs, and shrimp), echinoderms (sea stars, sea urchins, and sea cucumbers), chelicerates (spiders, scorpions, and horseshoe crabs), myriapods (centipedes and millipedes), and insects (butterflies, honeybees, and fruit flies). In addition to discussing the welfare of these species, other topics, including anatomy, physiology, husbandry, natural history, and environmental diseases, are reviewed.
Abstract
Invertebrates are a diverse group of animals that make up the majority of the animal kingdom and encompass a wide array of species with varying adaptations and characteristics. Invertebrates are found in nearly all of the world’s habitats, including aquatic, marine, and terrestrial environments. There are many misconceptions about invertebrate sentience, welfare requirements, the need for environmental enrichment, and overall care and husbandry for this amazing group of animals. This review addresses these topics and more for a select group of invertebrates with biomedical, economical, display, and human companionship importance.
... Also, their blood contains a chemical called Limulus amoebocyte lysate (LAL) that can be used to detect pathogens and their endotoxins. The growing interest in their use for research, education, biomedical applications, and habitat loss has threatened the wild horseshoe crab populations (Carmichael and Brush 2012). The horseshoe crabs are now included in the International Union for the Conservation of Nature's (IUCN) red list of threatened species (Laurie et al. 2019). ...
There were reports of the existence of horseshoe crabs in Palawan, Philippines, but there is a need to identify and confirm these species. A total of 128 adult horseshoe crabs were collected from Honda Bay, Palawan, Philippines. These were identified as Tachypleus tridentatus (68 males, 40 females) and Carcinoscorpius rotundicauda (2 males, 18 females). Morphology, morphometric measurements, and ratios of male and female of each species were described. Morphological characteristics in differentiating a male from a female crab involve examination of the appearance of the genitalia. Morphological characteristics in identifying the species include the presence or absence of notches in the frontal margin, arching in the frontal view, appearance of the chelae of the 2nd and 3rd prosomal appendages, number of lateral spines and the immovable spines in the opisthosoma, the presence of spinnerets on the surface of the telson, and shape of the telson and its cavity in cross-section. This study confirmed the identification of the two species found in Palawan, Philippines, and the morphological characteristics of the male and female of each species. In addition, a comparison was made of the size of the horseshoe crab species in Palawan with those found in other locations in the Asia-Pacific region.
... The global distribution of horseshoe crabs is thought to be constrained by continental geomorphology, temperature barriers, tidal types, and benthic currents [7,8]. Limulus polyphemus, Tachypleus tridentatus, T. gigas, and C. rotundicauda are types of horseshoe crab species [9,10]. ...
Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC-TOF-MS, multivariate analyses, Principal Component Analysis (PCA), and Partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP>1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p< 0.05. The metabolites were identified using several databases, and the literature review of the metabolites was reported in the manuscript. Thus, this study has provided further insights into the putative metabolites' presence in the hemocytes of horseshoe crabs that are stimulated and non-stimulated with LPS and their abundance in each species. Several putative metabolites showed they have medicinal values from previous studies.
... Therefore, domestic and foreign experts and relevant conservation departments are exploring ways to effectively restore the population of tri-spine horseshoe crabs. Previous study shows that artificially producing and releasing tri-spine horseshoe crabs are the most important and reliable protection methods for population restoration (Carmichael and Brush, 2012). T. tridentatus needs to molt about 15-16 times from the first instar to a sexually mature individual, and body size significantly increases after molting. ...
... T. tridentatus is mainly distributed in Guangdong, Guangxi and Fujian provinces * Corresponding author. E-mail: mhhu@shou.edu.cn and region in China (Carmichael and Brush, 2012). Unfortunately, habitat destruction, production of TAL, and overfishing have caused a sharp decline in the T. tridentatus population (Shin et al., 2007;Liao et al., 2019), and it is now classified as a Class II national protected species in China. ...
The population of tri-spine horseshoe crabs (HSCs) Tachypleus tridentatus has sharply decreased in recent decades. Domestication and manual releasing are now used to recover it. In order to accelerate the growth of T. tridentatus in indoor environment, in this study, gut microbiota of 2nd instar T. tridentatus was supposed to be improved by putting probiotics in water environment and thus promoted the health and growth of the host. Using 16S rRNA sequencing of gut samples, we examined the intestinal microbiome of juvenile HSCs and studied the effect of probiotics. Results showed that there was no significant difference between groups fed with high and low concentrations (1 × 107, 1 × 1012 CFU mL−1) of Bacillus subtilis and high concentration (1 × 1012 CFU mL−1) of Lactobacillus, and no significant difference compared with the control group, but the diversity of Lactobacillus group with low concentration (1 × 107 CFU mL−1) was significantly higher than those of other groups. Microbial-mediated functions predicted by PICRUSt showed that ‘Cellular Processes’, ‘Metabolism’ and ‘Organismal Systems’ pathways significantly increased in groups of Bacillus subtilis and Lactobacillus group with low concentration. The results showed that two types of probiotics with different concentrations have other effects on the structure, richness and function of intestinal microbes. By searching the literature and conducting pre-experiments, we forecasted the short time of breeding under the probiotic environment may not have much effect. The present study for the first time investigated the impact of probiotics on gut microbiota of juvenile HSCs and found them may benefit for the growth and health of HSCs.
... Erosion of the carapace is also an added indicator of parasitic infections. In prolonged captive conditions, such as employed in this study, non-infectious diseases such as deficiencies in certain dietary elements (Carmichael and Brush, 2012), limited movement in culture tanks, and absence of tidal rhythms also affects hemolymph quality (Kwan et al., 2014;Nolan and Smith, 2009). ...
Horseshoe crab (HSC) amebocyte cells degranulate to form a gel clot when in contact with endotoxins. This phenomenon is the basis of both the horseshoe crab immune system and the detection of endotoxin in biologicals. Horseshoe crab captive rearing has been suggested as a promising method for continuous amebocyte cell harvesting. Hence, we aimed to investigate the amebocyte cells quality in Tachypleus gigas pre and post bleeding under captivity. Wild and captive horseshoe crabs (5 months captivity) were fed with Meretrix meretrix (highly preferred diet by T. gigas) and bled in 6 anticoagulant formulations (A, B, C, D, E, and F). No profound difference in cell density was observed between captive and wild groups with the mean value of 0.883 × 107 a cells/mL and 0.917 × 107 cells/mL, respectively. Cell viability of the captive group was significantly lower than the wild group (F = 808.075, p < 0.001). Anticoagulant formulation greatly affected cell viability and cell morphology in captive and wild groups (p < 0.001). Amebocyte cells collected from the wild T. gigas using optimum anticoagulant (formula C) showed 0.6 × 107 cells/mL cell density and 86.9 % cell viability. Simultaneously, morphology analysis revealed the percentage of contracted, granular flattened, and degranulated flattened cells were 14.62 %, 71.39 %, and 14 %, respectively. The anticoagulant formulations showed varying capabilities in maintaining cell viability due to their buffering and chelating capacity. We postulate that selection of the best anticoagulant recipe is crucial to inaccurate blood profiling of horseshoe crabs. Prolonged captivity and single feed impact the quality of amebocyte cells in the crabs.
... A paucity of data for tracking post-bleeding fate beyond weeks or months has been an overriding reason for the lack of attention to long-term survival effects. The inability to maintain adult horseshoe crabs in captivity for long periods contributes to the absence of long-term studies of post-bleeding survival (Carmichael and Brush, 2012). The exception was a tagging data analysis conducted by Butler (2012), who found evidence of a positive effect of biomedical bleeding on annual survival, but the tag releases were not geographically constrained to control for regional differences in survival. ...
In the U.S., 525,000 horseshoe crabs (Limulus polyphemus) per year have been captured during 2013–2017, brought to biomedical facilities, and bled to produce Limulus amebocyte lysate (LAL), then mostly released to the area of capture. The Atlantic States Marine Fisheries Commission estimates short-term bleeding-induced mortality to be 15% (4% to 30%), resulting in mortality of approximately 78,750 horseshoe crabs annually in recent years comprising a minor portion (<13%) of the up to one million annual coastwide landings dominated by harvest for bait. However, the long-term effect of bleeding for LAL on annual survival and spawning behavior is unknown; thus, results from short-term studies alone might underestimate bleeding effects at the population level. To address this knowledge gap, we analyzed data from the U.S. Fish and Wildlife horseshoe crab tagging database to estimate the differences in survival and recapture rates of bled and not bled horseshoe crabs tagged in the same years and geographic area. Contrary to expectation, survival was not lower for bled crabs compared to unbled crabs. Differences varied, but survival estimates tended to be higher for bled crabs than for unbled crabs. However, biomedical culling and selection for younger or healthier animals could have resulted in biomedically tagged individuals representing a healthier subset of the overall population with subsequent higher survival. Furthermore, the tagging analysis revealed a post-bleeding reduction in capture probability, which could indicate decreased spawning activity, evident in males more than females. Continued tagging of bled and unbled crabs in the same geographic area while recording age class and sex will contribute to the further resolution of LAL production’s effect on horseshoe crab populations.
... A paucity of data for tracking post-bleeding fate beyond weeks or months has been an overriding reason for the lack of attention to long-term survival effects. The inability to maintain adult horseshoe crabs in captivity for long periods contributes to the absence of long-term studies of post-bleeding survival (Carmichael and Brush, 2012). The exception was a tagging data analysis conducted by Butler (2012), who found evidence of a positive effect of biomedical bleeding on annual survival, but the tag releases were not geographically constrained to control for regional differences in survival. ...
In the U.S., 525,000 horseshoe crabs per year have been captured during 2013-2017, brought to biomedical facilities, and bled to produce Limulus amebocyte lysate (LAL), then mostly released to the area of capture. The Atlantic States Marine Fisheries Commission estimates short-term bleeding-induced mortality to be 15% (4% to 30%), resulting in mortality of approximately 78,750 annually in recent years comprising a minor portion (<13%) of the up to one million annual coastwide landings dominated by harvest for bait. However, the long-term effect of bleeding for LAL on annual survival and spawning behavior is unknown; thus, results from short-term studies alone might underestimate bleeding effects at the population level. To address this knowledge gap, we analyzed data from the U.S. Fish and Wildlife horseshoe crab tagging database to estimate the differences in survival and recapture rates of bled and not bled horseshoe crabs tagged in the same years and geographic area. Contrary to expectation, survival was not lower for bled crabs compared to unbled crabs. Differences varied, but survival estimates tended to be higher for bled crabs than for unbled crabs. However, biomedical culling and selection for younger or healthier animals could have resulted in biomedically tagged individuals representing a healthier subset of the overall population with subsequent higher survival. Furthermore, the tagging analysis indicated a post-bleeding reduction in spawning behavior, evident in males more than females. Continued tagging of bled and unbled crabs in the same geographic area while recording age class and sex will contribute to the further resolution of LAL production's effect on horseshoe crab populations.
... In the case of HSCs, the most abundant food source in the wild may not be the most suitable feed source for aquaculture (Botton, 1984;Hu et al., 2013). Likewise, the nutritional requirements of HSCs in captivity based on controlled feeding trials have not yet been defined (Botton, 1984;Carmichael and Brush, 2012). Inadequate nutrition and poor water quality (due to high stocking density) can also contribute to HSC stress, increasing susceptibility to pathogens and vulnerability to captivity-induced disease (Kautsky et al., 2000;Carmichael et al., 2003;Smith and Berkson, 2005;Defoirdt et al., 2007;Nolan and Smith, 2009;Schreibman and Zarnoch, 2009;Kwan et al., 2014). ...
This study evaluated two approaches to the aquaculture of Limulus polyphemus with the ultimate goal of harvesting Limulus amebocyte lysate (LAL) at an industrial scale. To monitor Horseshoe crabs (HSCs), a combination of physical, biochemical and cellular components were examined for HSC cohorts in an indoor recirculating aquaculture system (RAS) and an outdoor on-bottom pen culture system (PCS) over a 6-month period. The metrics included body weight, hemocyanin (Hc) concentration, amebocyte density, and LAL reactivity. In addition, a simulated biomedical bleeding event (extracting 30% of the total hemolymph volume) was performed to assess the impact on physiochemical properties of the hemolymph and amebocytes. Overall, the HSCs fared better in the RAS compared to the PCS, with higher rebound kinetics with respect to Hc, amebocyte density, LAL reactivity, and with 100% survival in the RAS cohort. Further, hemolymph reinfusion (after amebocyte removal) was shown to improve HSC recovery time. In summary, outcomes of this research show that a RAS, coupled with adequate nutrition and monitoring can provide HSCs with a suitable environment for sustainable hemolymph extraction and year-round LAL production.
... To date, most conceptualizations of horseshoe crab aquaculture have been framed as stock enhancement through the release of juveniles into the wild (Schreibman and Zarnoch, 2009). Crabs are slow to reach maturity, and many studies report chronic mortality after 6 months in captivity (thought to be linked to diet) (Carmichael and Brush, 2012). Aside from the obvious implications on animal welfare, these challenges also pose significant economic barriers. ...
Endotoxin testing is a vital part of quality and safety control in pharmaceutical production. The primary method for this testing in North America and Europe is the limulus amebocyte lysate (LAL) test, a critical component of which is the blood of Atlantic horseshoe crabs (Limulus polyphemus). Procuring blood for LAL testing involves capturing and bleeding over 500,000 crabs from wild marine populations each year. Whilst efforts are made by manufacturers to return crabs to the sea following the collection of blood, there is a level of mortality and sub-lethal impact involved, prompting increasing discussions about welfare and ethics. The 3Rs – the ambition to where possible, replace, reduce, and refine the use of animals – are established and accepted worldwide as the best framework for governing animal-dependent science. However, the biomedical utilization of horseshoe crabs to produce the LAL test has rarely been viewed through a 3Rs framework. More recently, there has been a renewed attention on sustainable methods and alternatives to the LAL test. Drawing on in-depth qualitative interviews, this article examines stakeholder perspectives on opportunities for thinking with the 3Rs, considering current appetites to replace, refine, and reduce contemporary biomedical reliance on horseshoe crabs. The shape of conversations about the biomedical utilization of horseshoe crabs has shifted significantly in recent years, and the 3Rs are an important driver of change, offering the potential to advance the use of more sustainable methods, and realize the welfare considerations increasingly expected across science and society.
... Due to multifaceted threats, horseshoe crab populations have been in decline (Smith et al., 2017). There is a potential utility to multiple stakeholders in the maintenance of captive populations (Carmichael and Brush, 2012). However, very little is known of the microbial communities of either healthy or diseased horseshoe crabs. ...
The Atlantic horseshoe crab (Limulus polyphemus) is a common marine aquarium species and model organism for research. There is potential monetary and conservation value in developing a stable captive population of horseshoe crabs, however, one major impediment to achieving captivity is a lack of knowledge regarding captive diseases. We utilized 16S rRNA gene amplicon sequencing to track changes in the microbiomes of four body locations in three wild-caught (tracked over 14 months in captivity) and three tank-acclimated (>2 years in captivity) adult L. polyphemus in a touch tank at Shark Reef Aquarium at Mandalay Bay in Las Vegas, NV. The wild population hosted diverse and distinct microbiomes on the carapace (260 ± 96 amplicon sequence variants or ASVs), cloaca (345 ± 77 ASVs), gills (309 ± 36 ASVs), and oral cavity (359 ± 37 ASVs), which were dominated by classes Gammaproteobacteria, Bacteroidia, and Alphaproteobacteria. A rapid decline in richness across all body locations was observed within 1 month of captivity, with tank-acclimated (>2 years) animals having <5% of the initial microbiome richness and a nearly completely restructured microbial community. Tank-acclimated horseshoe crabs possessed distinct microbiomes that were highly uneven and low in species richness on the carapace (31 ± 7 ASVs), cloaca (53 ± 19 ASVs), gills (17 ± 2 ASVs), and oral cavity (31 ± 13 ASVs). The carapace, oral cavity, and gills of the tank-acclimated animals hosted abundant populations of Aeromonas (>60%) and Pseudomonas (>20%), both of which are known opportunistic pathogens of aquatic animals and can express chitinases, providing a plausible mechanism for the development of the carapace lesion pathology observed in this and other studies. The cloaca of the tank-acclimated animals was slightly more diverse than the other body locations with Aeromonas, Enterococcus, Shewanella, and Vagococcus dominating the community. These results provide an important baseline on the microbiomes of both wild and tank-acclimated horseshoe crabs and underscore the need to continue to investigate how native microbial populations may protect animals from pathogens.
... Despite representing the descendants of a long fossil lineage, they now face an extinction event. Extensive harvesting of specimens for their blood, and as a food source, as well as habitat modification have majorly impacted populations (Botton, 2001;Hsieh and Chen, 2009;Shin et al., 2009;Akbar John et al., 2011;Cartwright-Taylor et al., 2011;Carmichael and Brush, 2012;Nelson et al., 2015;Kwan et al., 2016;Fairuz-Fozi et al., 2018). Measures therefore need to be taken to prevent this group from an extinction event. ...
Horseshoe crabs are an iconic group of extant chelicerates, with a stunning fossil record that extends to at least the Lower Ordovician (~480 million years ago). As such, the group has retained significant biological and palaeontological interest. The sporadic nature of descriptive and systematic research into fossil horseshoe crabs over the last two centuries has spread information on the group across more than 200 texts dating from the early nineteenth century to the present day. We present the most comprehensive pictorial atlas of horseshoe crabs to date to pool these important data together. This review highlights taxa such as Bellinurus lacoei and Limulus priscus that have never been documented with photography. Furthermore, key morphological features of the true horseshoe crab (Xiphosurida) families—Austrolimulidae, Belinuridae, Limulidae, Paleolimulidae, and Rolfeiidae—are described. The evolutionary history of horseshoe crabs is reviewed and the current issues facing any possible biogeographic work are presented. Four major future directions that should be adopted by horseshoe crab researchers are outlined. We conclude that this review provides the basis for innovative geographic and geometric morphometric studies needed to uncover facets of horseshoe crab evolution.
... Despite representing the descendants of a long fossil lineage, they now face an extinction event. Extensive harvesting of specimens for their blood, and as a food source, as well as habitat modification have majorly impacted populations (Botton, 2001;Hsieh and Chen, 2009;Shin et al., 2009;Akbar John et al., 2011;Cartwright-Taylor et al., 2011;Carmichael and Brush, 2012;Nelson et al., 2015;Kwan et al., 2016;Fairuz-Fozi et al., 2018). Measures therefore need to be taken to prevent this group from an extinction event. ...
... Erosion of the carapace is also an added indicator of parasitic infections. In prolonged captive conditions, such as employed in this study, non-infectious diseases such as deficiencies in certain dietary elements (Carmichael and Brush, 2012), limited movement in culture tanks, and absence of tidal rhythms also affects hemolymph quality (Kwan et al., 2014;Nolan and Smith, 2009). ...
Horseshoe crabs amebocyte cells degranulate to form a gel clot when in contact with endotoxins. This phenomenon is the basis of both Horseshoe crab immune system and detection of endotoxin in biologicals. The present study investigates the amebocyte cells quality in Tachypleus gigas pre and post bleeding under captivity. Wild and captive horseshoe crabs (5 months captivity) were bled in 6 anticoagulant formulations (A, B, C, D, E and F). No profound difference in cell density between captive and wild groups with the mean value of 0.883×10 ⁷ a cells/mL and 0.917×10 ⁷ cells/mL, respectively. while, the cell viability of the captive group was significantly lower than the wild crabs ( F =808.075, p<0.001). Anticoagulant formulation significantly affected cell viability and cell morphology in both captive and wild groups ( p <0.001). Amebocyte cells collected from the wild T. gigas using optimum anticoagulant (formula C) showed 0.6 ×10 ⁷ cells/mL cell density and 86.9% cell viability, while morphology analysis revealed the percentage of contracted, granular flattened and degranulated flattened cells were 14.62%, 71.39% and 14%, respectively. The anticoagulant formulations showed varying capabilities in maintaining cell viability due to its buffering and chelating capacity. We conclude that captivity has a negative effect on the amebocyte cell quality.
HIGHLIGHTS
Captivity has a negative impact on amebocyte quality in horseshoe crab ( Tachypleus gigas ).
No significant difference in cell density between captive and wild crabs.
Anticoagulant formulations have significant impact on the cell density, viability and morphology
Captive crabs were immunocompromised due to single specific feed intake.
... As such, aquaculture has become a promising approach to restore dwindling populations. However, long-term efforts to husband adult HSCs have been largely unreported or unsuccessful for the last three decades (Carmichael and Brush, 2012). After 3-4 months of husbandry, research has indicated that hemolymph protein concentrations drop below reference ranges (3.4-11.8 ...
Horseshoe crab (HSC) hemolymph is the source of Limulus amebocyte lysate (LAL), a critical component in sterility testing that ensures drug and medical device safety for millions of patients every year. Wild HSC populations have been declining as a result of its use as whelk and eel bait, environmental changes, and its capture and bleeding for hemolymph by the biomedical industry, thus posing significant risks to species viability and the LAL raw material supply chain. We designed a controlled aquaculture habitat to husband HSCs and evaluated the effects of captivity on health markers (e.g., amebocyte density, hemocyanin levels, and LAL activity). We found HSC aquaculture to be practicable, with routine hemolymph harvesting resulting in high LAL quality, while safeguarding animal well-being with 100% HSC survival. Further, low-impact hemolymph harvesting via an indwelling catheter revealed rapid amebocyte rebound kinetics after consecutive 10% hemolymph extractions. Sustainable supplies of LAL could also be adapted to address daunting trends in septicemia and antimicrobial resistance. LAL is uniquely sensitive and specific for gram-negative bacteria, which represent 70–80% of pathogens that typically lead to sepsis. However, erratic results associated with interfering substances plagued efforts to adapt LAL for clinical use in the past. We report the development of a new LAL-based assay that can detect gram-negative bacteria and endotoxins in human blood without interference using aquaculture-derived LAL. Based on this research, sustainable LAL production from aquaculture could potentially satisfy industry needs with a fraction of one year’s current capture via year-round harvesting from a finite cohort of HSCs and expand raw materials supplies for potential future clinical applications.
... Limulus polyphemus from Florida could not tolerate colder temperatures; 17 conversely, those from Massachusetts suffered in warmer southern water temperatures. 18 Similar to L. polyphemus, temperature appears to have a considerable impact on the 19 embryonic development of T. tridentatus (Carmichael and Brush 2012). Among these 20 studies, Hong (2011) found that egg incubation at water temperature above 30 o C 21 could yield an impressively high survival rate (98%) and shorter hatching period (40 The present study investigates the survival rate, survival time and feeding of T. 8 tridentatus under short-term exposure to various temperatures. ...
Temperature appears to have pervasive effects on larval development, feeding and movement patterns of tri-spine horseshoe crabs, Tachypleus tridentatus. To investigate how temperature determines their geographic distribution range, we examined the survival rate (SR), survival time (ST) and food intake of subadult T. tridentatus under different temperature levels in a seven-day experiment, followed by a three-day recovery to room temperature at 25 oC. Significantly lower SR and ST of subadults were found at 40 oC after the end of the seven-day experiment. Meanwhile, SR and ST of individuals at 0 oC were negatively affected when the temperature was brought back to 25 oC. Mean food intake of the subadults was statistically lower at 0- 20 oC and 35-40 oC compared to that at 25 and 30 oC. After the subsequent three-day recovery to 25 oC, only the individuals previously exposed to 15 and 20 oC had recovered to their normal level of feeding activities, but not the other treatment groups. These findings may provide preliminary data on how thermal tolerance determines the present distribution of T. tridentatus, which are found in large numbers around Beibu Gulf (annual temperature variation: 22-30 oC), while considerably lower population densities are observed in the colder southern Sea of Japan and warmer seas of Southeast Asia.
... Four horseshoe crab species are known worldwide and are identified geographically. Generally, horseshoe crabs are classified as American (Limulus polyphemus) and Asian (Carcinoscorpius rotundicauda, Tachypleus gigas and Tachypleus tridentatus) species as per their availability in that region (Carmichael and Brush, 2012;Akbar-John et al., 2018). The global horseshoe crab distribution restrictions are thought limited by continental geomorphology, temperature barriers, tidal types and benthic currents (Sekiguchi and Shuster, 2009;Smith et al., 2017). ...
... Because horseshoe crabs analyzed for this study (>40 mm), are estimated to represent instars 9-10 at ages 4 years or older, crabs showed similar mean prosomal widths, apparent cohort distributions and male to female sex ratios. This finding is somewhat surprising given that mean and peak seasonal temperatures, which differed considerably among these sites, are thought to affect horseshoe crab growth and subsequent molting (Rudloe 1981, Sekiguchi 1988, and Carmichael and Brush 2012. Accordingly, previously reported spawning periods at Taunton Bay begin about one week later than spawning at apex habitats (Schaller et al. 2005, Schaller andDorsey 2011), and at Petit Bois Island spawning begins about two weeks earlier and extends about one week longer than at apex habitats (Smith et al 2002, Fulford andHaehn 2012). ...
The north central Gulf of Mexico (GOM) coastal region is a relatively unstudied habitat for the American horseshoe crab (Limulus polyphemus), which may become increasingly important to long-term survival of horseshoe crabs as natural and anthropogenic pressures alter habitat in other parts of the range. L. polyphemus is listed as a data deficient species by the International Union for Conservation of Nature. The study objectives were to 1) determine the presence of the American horseshoe crab at key locations in the northcentral GOM, 2) test the use of molts to evaluate population dynamics, 3) evaluate the spatial and temporal dynamics of habitat, and 4) develop a population sustainability model with biological and environmental components. Survey data were collected on molts during 2008, 2009 and 2011 and from live animals, molts, and carcasses during 2012 and 2013 at four sites: Horn Island, Petit Bois Island, Dauphin Island and Fort Morgan peninsula. Molts were found to be a useful indicator of juvenile population demographics. Petit Bois Island had the highest number of molts and live animals and using time to local extinction (Te) as a metric of population sustainability the most stable population. Fifty percent of the abundance index carrying capacity was found to be a threshold for resilience, and index increases above twenty percent could initiate exponential changes in Te. We found temporal autocorrelation among aquatic environmental variables for both years, with low persistence among meteorological variables in 2013. Shoreline land cover/land use was found to be significantly related to horseshoe crabs presence, with greater crab numbers in Bare Land and Estuarine Emergent Wetland classes in locations remote from urbanization and human presence. Salinity, water temperature and wind direction were also found to be significantly correlated with presence at some of the sites. A population sustainability index (PSI) was developed that may be applied to guide conservation of L. Polyphemus in the northcentral GOM and elsewhere. Climate change projections indicate a drying climate and rising sea level during the next 50 to 100 years; however, PSI results suggest salinity and temperature effects will not exceed limiting thresholds on habitat suitability.
... These horseshoe crabs distributed widely with one species confined to the Atlantic Ocean namely the American horseshoe crab Limulus polyphemus that distributed widely within the geographic ranges from 21° to 44° N and 68° to 90° W covers the area of Maine in the North of America (Sekiguchi and Shuster 2009;Shuster 2015), down to Yucatan in the Gulf of Mexico (Zaldivar-Rae et al 2009) with the highest population recorded in Delaware Bay (Sekiguchi and Shuster 2009;Walls et al 2002). The other three horseshoe crab species (known as the Asian horseshoe crabs) namely coastal horseshoe crab Tachypleus gigas, tri-spine horseshoe crab T. tridentatus and mangrove horseshoe crab Carcinoscorpius rotundicauda distributed in Indo-pacific Ocean that range from 6° S to 31° N and between 90° to 118° E (Sekiguchi and Shuster 2009) covering the Bay of Bengal in India to Philippines and from South-West Japan down to China, Thailand, Malaysia and Indonesia in the south (Carmichael and Brush 2012;Chatterji 1994). Recent recovery by Yang and Ko (2015) also reported T. tridentatus in the Korean waters. ...
Dwindling number of the tri-spine horseshoe crab, T. tridentatus has been reported globally. Similar case is also seen in Malaysia, specifically in Sabah where locals claim that the animal is less seen in the present compared to previous years. However, there is no scientific data on its population status. Capture-mark-recapture (CMR) method were carried out from October 2014 through September 2015 to estimate the abundance of adult T. tridentatus in Tawau, Sabah. Samples were also weighed and examined for their body parameters (i.e. prosomal width, carapace length, telson length, total length, intraocular distance). The estimated population sizes of T. tridentatus ranged from 182-1,095 with 95% confident limits of 56-42,942 individuals (Schnabel formula). Multivariate Discriminant Hotelling T2 test verifies the sexual size dimorphism among the adult T. tridentatus with 97.7% separation among sexes (Hotelling T2=778.49, F=152.85, p<0.001) with female being larger and heavier than the male individuals. The number estimated from the study is the first being reported for T. tridentatus in Malaysia, particularly in Sabah. Even though this number may slightly overestimate the actual population size in the area owing to the low number of individuals recaptured, for now it could serve as baseline data for horseshoe crab management purpose.
... Horseshoe crabs exist since the Ordovician period and have remarkable evolution history which stopped 250 million years ago [1]. These crabs are survived by Asian horseshoe crabs, Tachypleus tridentatus (Leach), Tachypleus gigas (Müller), Carcinoscorpius rotundicauda (Latreille) and the American relative, Limulus polyphemus (Linnaeus) [2,3]. In Malaysia, all three Asian horseshoe crabs are available, in which, T. gigas and C. rotundicauda are present in the coastal areas of Peninsular Malaysia whereas, T. tridentatus is restricted to East Malaysia at Sabah and Sarawak [4]. ...
Due to limited availability of literature on the spawning activity of Malaysian horseshoe crab, Tachypleus gigas (Müller, 1785), the reproduction behaviour and biology of this arthropod remains poorly understood. Hence, an investigation was carried out from April-July to trail spawning horseshoe crab amplexus at Balok and Cherating, the only known T. gigas spawning grounds on the east coast of Peninsular Malaysia. Through visual tracking during day-time full moon spring tides, the release of air bubbles indicate nest digging by female crabs. While air bubble formation aggravated, flagged aluminium poles were carefully driven into the sediment to mark the nest. Out of the 13 spawning T. gigas amplexus tracked, only one pair was able to dig up to 12 nests and release up to 2,575 eggs within the 2.5 hour spawning period. The female crabs dug nests at the intertidal regions, ranging 1.3-43.2 m from the highest tide marking on the beach. Interestingly after spawning, the horseshoe crabs returned to high seas during the residing tides as amplexus because the male crabs remained clinging onto the female crab’s opisthosoma. The incidence of recapturing a tagged female crab with different attached male crab at Cherating indicates this spawning ground as natal beach. In addition, the presence of 8 horseshoe crab amplexus at Balok compared to only 5 amplexus at Cherating highlight preferable beach settings for spawning and/or the presence of larger spawning crab assemblages at the former beach. Since only 13 spawning horseshoe crab pairs were witnessed at Balok and Cherating, identification of factors that alter beach sediment composition and water chemistry as well as gender-specific poaching and excessive boating activities should be regulated to restore wild T. gigas populations.
... 2. Since there is some evidence that horseshoe crabs are declining in Florida, reducing the number of animals that can be collected in the marine-life harvest would be appropriate. Horseshoe crabs are easy to rear, and it is particularly easy to rear them to the early juvenile stages (French 1979 ;Brown and Clapper 1981 ;Carmichael et al. 2009 ;Schreibman and Zarnoch 2009 ;Carmichael and Brush 2012 ). This means that the animals used by the aquaria trade could come from captive rearing. ...
American horseshoe crabs (Limulus polyphemus) breed in every coastal county of Florida, U.S.A., and yet the data available on the status of this species in Florida are limited. The east and west coasts of Florida have genetically distinct populations of horseshoe crabs, but it is not known if there is further genetic structuring. To fill these gaps, we compile data from a number of sources: genetic analysis of tissue samples from 13 sites, breeding activity from an on-line public database, population estimates from multi-year trawl and seine surveys, and spawning activity from a long-term study. Neighbor-joining and STRUCTURE analyses support at least five genetically distinct management units within both the Atlantic and Gulf coast populations of Florida horseshoe crabs. We find that in some parts of Florida, horseshoe crabs breed throughout the year, but in other parts there are distinct breeding seasons. Florida populations are generally smaller than those in more northern states, particularly along the east coast, but there are few indications of decline except in the Indian River area. Finally, we compile data on the harvest of horseshoe crabs in Florida and discuss the effect of the marine-life fishery, which has not been tracked in other states.
... Given that horseshoe crabs appear relatively easy to house ex situ (Smith and Berkson, 2005;Coates et al., 2012b), there is commercial aquaculture interest in maintaining and rearing sustainable captive held populations of L. polyphemus (Carmichael and Brush, 2012;Kwan et al., 2014). There remains, however, a lack of knowledge regarding the aetiologies/pathologies of infectious/ non-infectious diseases of captive and wild populations. ...
An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼ 96% (40,000) second/third instar larvae over a 61 day period. Peritich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10 ppm chlorine in freshwater for 1 h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343 bp (99.7% similarity to Z. duplicatum).
Copyright © 2014. Published by Elsevier Inc.
Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC–TOF–MS, multivariate analyses, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP > 1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p < 0.05. The metabolites were identified using several databases, and the literature review of the metabolites was reported in the manuscript. Thus, this study has provided further insights into the putative metabolites' presence in the hemocytes of horseshoe crabs that are stimulated and non-stimulated with LPS and their abundance in each species. Several putative metabolites showed they have medicinal values from previous studies.
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.
The American horseshoe crab, Limulus polyphemus, is a member of the phylum Arthropoda, subphylum Chelicerata, and class Merostomata. Although there are four horseshoe crab species worldwide, the American horseshoe crab is primarily found in the Atlantic Ocean along the East Coast of North America. The species ranges from Maine to Mexico, but is absent between Texas in the United States and Tabasco in Mexico. They mainly inhabit estuarine areas; however, juveniles and adults can venture into the shallow oceanic environments. Embryo development occurs in the intertidal zone of sandy beaches. Sandy or muddy bottom habitats are necessary for burrowing and benthic feeding. This publication will cover their natural history, aquaculture techniques, common diseases, and their uses and markets.
Tissues from bivalves are commonly fed to horseshoe crabs in captivity. In the laboratory, we fed two horseshoe crab species, Tachypleus gigas and Carcinoscorpius rotundicauda, with tissue from four bivalve species namely Scapharca cornea, Meretrix meretrix, Polymesoda erosa, and Magallana bilineata separately to determine the consumption (without choice) and were further fed with a mix of the same bivalve tissue to determine the consumption when a choice is available. Each bivalve was tested for proximate composition analysis. The highest number of bivalves consumed by male and female T. gigas were M. meretrix and M. bilineata with 1.93 and 1.62 individuals per day, respectively, when fed with no choice. Male C. rotundicauda consumed 2.51 individuals per day for M. meretrix and P. erosa, whereas female C. rotundicauda consumed 2.02 individuals of M. meretrix per day. There were no differences in feeding consumption for each type of bivalve tissue. Despite the difference in nutritional values, there are no significant differences in bivalve flesh selection by both sexes of T. gigas. Only male C. rotundicauda showed selection in bivalve consumption. These findings can be used to improve future hatchery management for horseshoe crabs in captivity in terms of feeding the correct quantity of feed to avoid feed wastage and fouling.
Transcriptome assemblies for the stomach, midgut, and fecal mucosa of Tachypleus tridentatus and Carcinoscorpius rotundicauda are reported for the first time in this study. Genome assembly resulted in 373069 unigenes with an N50 of 1314, while transcriptome assembly resulted in 736378 unigenes and an N50 of 2121. The annotated unigenes showed the highest similarity to fishes, amphibians, and reptiles, with most unigenes closely related to metabolism, translation, biogenesis, signal transduction, energy production, immune response, and secretion. Unigenes (4768) were mapped to KEGG pathways, and the most enriched pathways were involved in translation, environmental information signal processing, metabolism, endocrine system, immune system, nervous system, and varying metabolism. Totally 65889, 2001, and 7162 differentially expressed genes (DEGs) were identified in the stomach, midgut, and fecal mucosa between the two horseshoe crab species, respectively. Compared with C. rotundicauda, 99.95% of the DEGs in the T. tridentatus stomach were down-regulated, while 99.97% of the DEGs of T. tridentatus fecal mucosa and 55.42% of the DEGs of the T. tridentatus midgut were up-regulated. Most midgut DEGs were involved in hydrolase activity, protein metabolism, and cell cytoplasm, while most stomach and fecal mucosa DEGs were involved in catalytic activity and primary metabolic processes. Most stomach DEGs were assigned to the cellular component in cellular macromolecular complexes, while most fecal mucosa DEGs were assigned to the cellular component in cell and cell parts. These results will benefit the investigation of the molecular mechanisms of the digestive tract related to the feeding habits and environmental traces of horseshoe crabs.
Knowledge of dietary compositions is essential to the recovery of the dwindling populations of horseshoe crabs in China. The feeding habits of Tachypleus tridentatus and Carcinoscorpis rotundicauda of Pearl Bay in southwestern China were studied by stable isotope analysis. Hemolymph samples of the two species were obtained from two age groups living in different habitats (inner, central, and outer Bay). In addition, their potential food source samples were collected in May 2019. Results showed that the mean values of δ13C and δ15N in C. rotundicauda individuals ranged from −19.01‰ to −16.47‰ and from 10.49‰ to 13.5‰, respectively, and those of T. tridentatus ranged from −19.12‰ to −14.96‰ and from 8.78‰ to 13.48‰, respectively. These values indicated that the horseshoe crabs have a wide variety of food sources and therefore are highly omnivorous. No remarkably correlations were found between δ13C and δ15N values and individual widths in the two species. Thirteen potential food sources were selected, in which Sipunculus nudus, Batillaria zonalisi, and Ulva lactuca contributed largely to the dietary compositions of the two species of horseshoe crabs. No significant correlation was found between the feeding habits and habitat geochemical characteristics of the horseshoe crabs. All these results possibly indicate the high diversity of their food sources and the indiscriminate dietary compositions of the two Asian horseshoe crabs.
This chapter provides the information on natural history and taxonomy, taxonomic classification, anatomy and physiology, environmental diseases, infectious diseases, diagnostic techniques, health management, and anesthesia and surgery of horseshoe crabs. Horseshoe crabs have persevered essentially unchanged for more than 200 million years and are the closest living relatives of the trilobites. The horseshoe crab has been used as a laboratory animal model to study the structure, physiology, and function of its large eyes and simple nervous system, to study amoebocyte phagocytosis and innate immunology, and embryology of marine invertebrates. The body of the dorsoventrally flattened invertebrate is divided into three separate sections: a frontal prosoma, a posterior opisthosoma, and a telson. Infectious agents reported as causing health problems in horseshoe crabs include algae, fungi, colonial and filamentous cyanobacteria, Gram-negative bacteria, and variety of parasites. Evaluation of the health of a horseshoe crab can be very challenging because of the thick, nontransparent cuticle of the exoskeleton.
Endotoxins are bacterial components that can cause systemic toxicity if they enter the mammalian blood stream. Testing for the presence of endotoxins is vital for the safe use of vaccines, injectable medicines, and medical devices in human and veterinary medicine. In North America and Europe the primary method for endotoxin testing is the Limulus Amebocyte Lysate (LAL) test. This utilises the coagulative properties of Atlantic horseshoe crab (Limulus polyphemus) blood to detect endotoxins, linking this immunologically unique and ancient species to the global supply chains of modern health and medicine. The development of the LAL test served as a replacement to previous methods of endotoxin testing which involved injecting samples into rabbits (the Rabbit Pyrogens Test) – a significant reduction in harm to animals.
However, procuring blood for this (regulatory mandated) testing involves capturing & bleeding over 500,000 crabs from wild populations each year. The continued use – and potential rise in use given increasing global demand for pharmaceuticals – of horseshoe crabs is prompting growing questions around welfare and sustainability. There are growing debates around Limulus’ sentience and capacity to suffer, with animal protection groups posing welfare and ethical questions. Whilst the crabs are returned, alive, to the sea following the collection of their blood, and some view the bleeding process as harmless, there are increasing discussions about the impact that capture and bleeding can have on crab health and mortality.
This report explores what a social science perspective might add to understanding the debates surrounding the use of horseshoe crabs in endotoxin testing. It draws on qualitative research with stakeholders, alongside documentary and policy analysis to examine the various perspectives, positions, and sides of debates about horseshoe crabs. This report attempts to represent the wide diversity of stakeholder perspectives about the biomedical use of horseshoe crabs in a balanced manner.
The two major themes responsible for the current contestation – the impact that the biomedical industry has, or does not have, on horseshoe crabs, and the current replaceability of horseshoe crabs within contemporary pharmaceutical processes – are discussed. Building on this, the report then discusses the role that ideas about animal welfare have traditionally played in shaping conversations about horseshoe crabs, examining why the biomedical use of Limulus has remained outside of, and resistant to, an engagement with the welfare concerns that underpin the social acceptability of the biomedical usage of other animals. Bringing conversations about horseshoe crabs into connection with wider discussions about animal welfare more broadly offers many opportunities for restructuring debates in helpful ways. Particularly, the 3Rs framework – the ambition to, where possible, replace, reduce, and refine the biomedical use of animals – emerges as a useful and appealing concept to a wide variety of the stakeholders involved in discussions about horseshoe crabs. As conversations about the biomedical use of horseshoe crabs are starting to change there is also a need for more openness and transparency. This is increasingly a staple of best practice within the broader biomedical industry, and greater openness here would afford an important opportunity to reassure publics and other stakeholders about the level of care involved in contemporary biomedical use of horseshoe crabs. The pharmaceutical reliance on horseshoe crabs is growing as a topic of public interest, without assurances and openness about humane care, it may be that public opinion shifts from an understanding of the (currently perceived) need to utilise horseshoe crabs in a care-full manner, to one that rejects this as a tolerable practice. With welfare potentially rising on the agenda, there are opportunities – and demands – for animal welfare organisations to become involved in, and help shape, future discussions about horseshoe crabs.
This report is intended to be an overview of the topic. It is aimed at the multiple stakeholder and public audiences interested in the matter of horseshoe crabs and animal welfare. Additional scientific articles are in development in peer-reviewed academic journals and books, which will expand on the themes identified here.
【Article in Chinese with English abstract】Horseshoe crabs are an ancient group of marine arthropods. The Chinese horseshoe crab (Tachypleus tridentatus) is the largest among the four extant species. Tachypleus tridentatus is regarded as the indicator species of estuarine ecosystems. Meanwhile, its blood is used to manufacture biomedical testing kits, i.e., Tachypleus amebocyte lysate. The geographic distribution of T. tridentatus is constrained to the western Pacific Ocean, ranging from the Seto Inland Sea of Japan southwards to the North Jawa waters. A high historical population size of T. tridentatus was recorded in eastern China and southern Japan. Tachypleus tridentatus population decline has become apparent since the 1950s, and the species has been upgraded to “Endangered (EN)” in 2019 on the IUCN Red List, depicting its global declining trend. Habitat destruction and overfishing are identified as the primary threats. We reexamined current conservation measures targeting T. tridentatus, including marine protected area establishment, captive breeding and restocking, and awareness programs and regulations. We provide recommendations to enhance the conservation and management of global T. tridentatus populations through: (1) expediting population baseline data collection; (2) developing standards for population/habitat monitoring; and (3) establishing scientifically sound restocking programs.
Long-term and locally-based public education programs are essential in supporting conservation initiatives for horseshoe crab protection, particularly in Asia, where their populations have been largely exploited for food and biomedical applications. However, the implementation of such conservation initiatives can be difficult due to a community’s ingrained apathy towards the environment. In addition, the effectiveness of similar education programs to engage the community in conservation work has rarely been assessed. We described the implementation strategies of a conservation education program in Hong Kong, with the objective to improve the community’s awareness, engagement and environmental attitudes towards the conservation of Asian horseshoe crabs. This study evaluated the program’s outreach, outcomes and impacts between 2009 and 2016. By raising laboratory-cultured juvenile horseshoe crabs at local secondary schools, this long-term conservation education program succeeded in enhancing students’ knowledge on the biology and ecology of horseshoe crabs, as well as promoting positive attitudes and behaviors towards relevant conservation issues. Lessons and recommendations derived from this program can serve as guidance for similar campaigns in other places.
Horseshoe crabs have persisted for more than 200 million years, and fossil forms date to 450 million years ago. The American horseshoe crab (Limulus polyphemus), one of four extant horseshoe crab species, is found along the Atlantic coastline of North America ranging from Alabama to Maine, USA with another distinct population on the coasts of Campeche, Yucatán and Quintana Roo in the Yucatán Peninsula, México. Although the American horseshoe crab tolerates broad environmental conditions, exploitation and habitat loss threaten the species. We assessed the conservation status of the American horseshoe crab by comprehensively reviewing available scientific information on its range, life history, genetic structure, population trends and analyses, major threats, and conservation. We structured the status assessment by six genetically-informed regions and accounted for sub-regional differences in environmental conditions, threats, and management. The transnational regions are Gulf of Maine (USA), Mid-Atlantic (USA), Southeast (USA), Florida Atlantic (USA), Northeast Gulf of México (USA), and Yucatán Peninsula (México). Our conclusion is that the American horseshoe crab species is vulnerable to local extirpation and that the degree and extent of risk vary among and within the regions. The risk is elevated in the Gulf of Maine region due to limited and fragmented habitat. The populations of horseshoe crabs in the Mid-Atlantic region are stable in the Delaware Bay area, and regulatory controls are in place, but the risk is elevated in the New England area as evidenced by continuing declines understood to be caused by over-harvest. The populations of horseshoe crabs in the Southeast region are stable or increasing. The populations of horseshoe crabs in the Florida Atlantic region show mixed trends among areas, and continuing population reductions at the embayment level have poorly understood causes. Within the Northeast Gulf of Mexico, causes of population trends are poorly understood and currently there is no active management of horseshoe crabs. Horseshoe crabs within México have conservation protection based on limited and fragmented habitat and geographic isolation from other regions, but elevated risk applies to the horseshoe crabs in the Yucatán Peninsula region until sufficient data can confirm population stability. Future species status throughout its range will depend on the effectiveness of conservation to mitigate habitat loss and manage for sustainable harvest among and within regions.
In many regions of the world, horseshoe crab populations are sparse or difficult to study. We investigated the utility of molted shells, which may be more accessible than live animals, to provide essential data on population structure for juvenile horseshoe crabs. We collected and compared horseshoe crab (Limulus polyphemus) molts from two representative habitats near the edge of horseshoe crabs’ known range in the U.S., Taunton Bay, Maine (2005, 2006) and Petit Bois Island, Mississippi (2008, 2009, 2011). Molts were collected and measured every 2 weeks and characterized in terms of abundance, prosomal width, and sex. The greatest number of molts was found during full moon periods, corresponding to known spawning periods and following seasonal temperature increases at both sites. Molt prosomal width ranged from 3 to 169 mm at Petit Bois Island and 19–165 mm at Taunton Bay. Molt size frequency distributions and estimated growth increments were similar between sites and to size classes known for living juvenile L. polyphemus found elsewhere. Male to female sex ratios of molts ranged from 1.0 to 2.3, similar to sex ratios measured among live juveniles elsewhere and among live subadult and adult horseshoe crabs previously measured at the study sites. Molt numbers and size distributions on Petit Bois Island following the Deepwater Horizon oil spill confirmed survival of young crabs through the oil spill period. The overall similarity in molt frequency patterns and size distributions between distinct habitat types suggest some endogenous plasticity in horseshoe crab molting and subsequent growth patterns, despite regional differences in environmental attributes. Hence, data from molted shells have potential as an alternative to live animal studies by at least providing data on timing and rate of growth under different environmental conditions. These data will be particularly beneficial in habitats where live horseshoe crab populations are difficult to study or there are few background data to support live animal studies.
The three Southeast Asian horseshoe crab species are diminishing not only locally but also regionally and protection of them
is now an urgent matter. Two species of horseshoe crab, Tachypleus gigas and Carcinoscorpius rotundicauda, were artificially inseminated, and the eggs were incubated at 28±1°C and in the salinity of 33±2 ppt. The fertilized eggs
hatched after 42 days and 41 days with hatching rates of 98.1 and 98.9% for T. gigas and C. rotundicauda, respectively. This study reveals that in the identical laboratory condition, C. rotudicauda underwent more frequent molting than T. gigas. After 328 days of rearing, 63.8 and 22.9% of C. rotundicauda larvae had molted to the 6th and 7th instars, respectively, while 56.6 and 20.1% of T. gigas at the end of 355 days of rearing had molted to the 5th and 6th instars, respectively, but only 0.6% had molted to the 7th
instar. There is a wide variation in the molting rate among larvae obtained from synchronized inseminated eggs and reared
under uniform laboratory conditions.
This study aimed to determine the population structure of juvenile Tachypleus tridentatus, one of two species of horseshoe crabs found in Palawan, at Aventura Beach, Puerto Princesa City, Palawan. Surveys to assess the population in terms of size, density, size‐frequency, age structure, sex ratio and distribution were conducted from June to October 2002. The habitat was mapped and physical/chemical parameters taken to further describe it. The position of population members was plotted on the map. Individuals were measured, marked, sexed and released. Prosoma width was measured for living individuals and exuviae. Postembryonic stages were identified based on histograms with different interval size. The normal distribution of the animals per instar stage was tested with the Kolmogorov–Smirnov test. After the 5‐month survey a total of 125 juveniles was recorded and 40% of them were recaptured. Population size was estimated to be 150 individuals. Seven postembryonic stages were identified based on their prosoma width. Male: female ratio was 1:1.8 and the age of the first five postembryonic stages ranged from 5.4 months to 17.6 months. The population density was 1.47 individuals per 100 m. The majority of the animals of all postembryonic stages were found between 0 and 50 m from the mean high tide level. Further studies on the population structure of T. tridentatus with regards to the adult population are seen as a necessity.
The horseshoe crab (Limulus polyphemus) is the target of a commercial bait fishery that historically has lacked coordinated, coastwide monitoring and regulation. Because the catch has increased substantially in recent years, agencies have been increasingly pressured to manage the fishery more intensively. The diversity of interests involved (birders, environmentalists, commercial fishers, biomedical companies, coastal residents, and the Delaware Bay ecotourism industry) makes management especially challenging. In particular, the conflict between environmentalists and commercial fishers has been gaining public attention. With stronger management, the horseshoe crab population likely could meet the basic needs of all users, including fishers. Further delay or avoidance of coordinated, coastwide management increases the likelihood of a serious conservation problem that would injure the horseshoe crab population as well as various stakeholders and the species dependent on it (especially migratory shorebirds). The horseshoe crab, which has survived relatively unchanged for the past 350 million years, now is at the center of a resource tug of war. Regardless of our actions, the species is likely to persist beyond the current conflict. However, our desire and ability to manage the population will determine to what extent we will be able to meet society's many needs for horseshoe crabs.
Populations of horseshoe crabs Limulus polyphemus are widely distributed from Maine to the Gulf of Mexico, are commercially harvested, and are thought to have decreased in abundance in recent decades. To provide information needed to manage exploited populations of horseshoe crabs, we conducted comprehensive field sampling in a major shallow estuary containing preferred habitat for horseshoe crabs. The data we obtained are the first to comprehensively define abundance, determine sex ratios of horseshoe crabs across an entire estuary, and estimate cohort-specific growth and mortality of juvenile and adult horseshoe crabs. We found that juveniles were more abundant and suffered greater mortality than adults. Adults were largely found on the sediment surface, but 20% were buried shallowly in sediments. The male:female sex ratio in juveniles was 1.4: 1, but the adult sex ratio was 2.3:1. Juveniles grew faster than adults, and adult crabs may plausibly molt as frequently as once per year rather than have a terminal molt. Spawning appeared to span late March to mid-July, and juveniles hatched at semilunar intervals during Year 0 and grew to 16.6 +/- 0.9 mm prosomal width by the start of Year 1. The distinct semilunar cohorts of Year 0 coalesced into annual cohorts after Year 0. Females deposited large numbers of eggs, but only 0.001% survived to the end of Year 0, and approximately 78% of these juveniles reached adulthood.
Little is known about Mexican Limulus polyphemus, the southernmost population of the species. We present an overview of work on Mexican horseshoe crabs, their situation,
and perceived threats and opportunities regarding the conservation of the species. Horseshoe crabs occur along the western,
northern, and eastern coasts of the Yucatán peninsula, and are genetically distinct from populations in the United States.
Spawning aggregations and nests are found continuously throughout the year, commonly in protected lagoons where mangrove (Rhizophora mangle, Laguncularia racemosa, Avicennia germinans, and Conocarpus erectus) and sea grass (Thalassia testudinum) communities proliferate. Populations are thought to be dwindling since the 1960s and Limulus is listed as “in danger of extinction” in Mexican legislation since 1994. The most important localities are within protected
areas. Direct exploitation is not an important threat, but coastline modification (especially of mangrove areas and coastal
lagoons) for housing and tourism is a major concern. Additional potential threats are the oil industry and shrimp fishery
in the southern Gulf of Mexico, but their effects on horseshoe crab populations have not been assessed.
Horseshoe crabs are valued for economic, ecological, and educational purposes. These values have raised interest in managing
natural stocks and culturing crabs for conservation, research, and education. To inform these efforts, we used N and C stable
isotopes to define the natural diet of juvenile horseshoe crabs and then assessed effects of different diets on growth and
survival of juveniles in culture. In the natural environment, N and C isotope ratios in juvenile horseshoe crabs changed as
crabs grew, with larger crabs consuming larger prey. Linear mixing analyses suggested young crabs were supported by high quantities
of benthic and suspended particulate organic matter (POM), shifting between marine algae and salt marsh-based food webs, depending
on size. In culture, we tested the relative importance of algae, as a proxy for POM, in horseshoe crab diet by feeding juvenile
crabs different percentages of algae and prey animals. Initially, juvenile crabs showed a significant increase in size when
fed diets >70% protein, but showed a decrease in survival compared to algae-rich diets. Overall, growth rates and survival
declined through time during the 128-day study, regardless of diet composition. These data suggest horseshoe crabs require
foods from a combination of plant and animal sources. Successful culture or conservation of horseshoe crabs will depend on
understanding the relative importance of different food sources at different life stages as well as discerning the balance
between factors that increase growth, but reduce survival.
Atlantic horseshoe crabs,Limulus polyphemus, are currently harvested for biomedical, scientific, and bait purposes. In recent years, changes in population abundance
and magnitude of harvesting have raised concerns about the status of this resource. We found horseshoe crab harvest in Pleasant
Bay, Massachusetts, was selective by sex and size. Biomedical harvest preferred larger individuals and females, the scientific
harvest preferred smaller individuals and males, and the bait harvest preferred females. Total 2001 harvest for all purposes
accounted for the mortality of ∼1–2% the adult population. Biomedical harvest accounted for the greatest loss of horseshoe
crabs from Pleasant Bay, ∼1–1.6% of the total population. Although biomedical harvest had the lowest associated mortality
rate (∼10–15%), many more crabs were harvested from Pleasant Bay for biomedical purposes than for other uses. The scientific
harvest accounted for the mortality of ∼0.4% of the population, and bait harvest accounted for the smallest mortality at ∼0.06%
of the population. Harvest mortality rate was estimated to be lower in Pleasant Bay than in other Cape Cod areas and may be
lower than natural mortality in the population. This study is the first qualitative investigation of commercial harvest on
horseshoe crab populations and emphasizes that harvest pressures on different populations need to be individually evaluated.
Recent and dramatic declines have been documented in several shorebird populations that stage in Delaware Bay during spring
migration. As a result, considerable attention has been given to issues such as the reliance of sandpipers on Delaware Bay
American horseshoe crab (Limulus polyphemus) eggs for refueling and how to best manage the horseshoe crab fishery to insure adequate resources for migratory shorebirds.
In this chapter, we synthesize over 25 years of shorebird research and monitoring data in Delaware Bay to support the premise
that horseshoe crab eggs are an essential element for migrating sandpipers during northbound passage through the bay. We then
discuss long- and short-term trends in American horseshoe crab populations resulting from changes in demand and harvest regulations,
and how this has affected shorebird population viability. Regulatory actions have led to recent increases in some demographic
elements of the Delaware Bay crab population, but such changes have not yet translated into increased crab egg availability
or population recovery indices in shorebirds. Because reduced availability of horseshoe crab eggs has severe consequences
for migratory sandpipers at the individual and population levels, current conservation strategies that include harvest reductions
on American horseshoe crabs in the Delaware Bay region must persist into the foreseeable future to insure the recovery of
horseshoe crab populations and the long-term health of migratory sandpipers in Delaware Bay.
Invertebrate species have been used for many years in the laboratory and teaching environment. We discuss some of the most
commonly maintained invertebrates—the nematode (Caenorhabditis elegans), the California sea hare (Aplysia californica), the fruit fly (Drosophila melanogaster), terrestrial hermit crabs, the horseshoe crab (Limulus polyphemus), and cephalopods—and briefly describe general techniques for culturing them in captivity. The aim of this article is to
give potential users an idea of the materials, methods, and effort required to maintain each type of organism in a laboratory
or classroom setting.
Growth of the trilobite larvae of Tachypleus gigas was studied at salinity ranging from 10 to 40 ppt. In terms of body weight, the growth was higher (232 plus or minus 7.5 mg/week) (P < 0.05) at 40 ppt salinity, and the first moulting was also recorded on the 35th day. Though the maximum weight attained by the larvae was at salinity 10 ppt (260 plus or minus 5.9 mg/week) (P < 0.05), its moulting was not recorded until the 45th day. At 20 and 30 ppt, the weights attained by the larvae were 177 and 165 mg respectively, and moulting occurred on the 42nd day at both salinities.
The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.
The distribution of Limulus polyphemus in Delaware Bay (New Jersey) exposes it to salinities ranging from about 7 ppt to 30 ppt. Comparisons of osmotic concentrations of the blood to those of experimental solutions corresponding to such a salinity range indicate that Limulus blood changes markedly with changes in environmental salinity. Although some osmotic regulation may occur at lower salinities, the major adaptation to the estuarine habitat has been the development of an extremely wide tolerance to change in internal osmotic concentration.
Individuals of Limulus polyphemus and Tachypleus tridentatus reached, respectively, the fourteenth-instar and tenth-instar stages during our nine-year rearing experiment. Body sizes were measured using exuviae and body specimens. The results made their growth steps clear, and body sizes of the juveniles at further growth stages could be estimated. We conclude from these data that L. polyphemus males generally molt 16 times to reach maturity in the ninth year; females molt 17 times to reach maturity in the tenth year. Similarly, we conclude from the presumptive numbers of growth stages that T. tridentatus males generally molt 15 times to reach maturity in the thirteenth year; females molt 16 times to reach maturity in the fourteenth year. Although we have few data on growth stages of Tachypleus gigas and Carcinoscorpius rotundicauda, it is thought that T. gigas males molt 12 times to reach adulthood, females molt 13 times, and that C. rotundicauda reaches maturity after the thirteenth molt in both males and females.
Juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda with prosomal widths of between 17.1 and 91.1 mm were obtained from their nursery beaches in Hong Kong. They were kept in the laboratory and fed a mixture of squid, prawn and fish. Prosomal width and wet weight were measured weekly to obtain growth data. Over half of the individuals molted during a five and a half month captivity period. After every ecdysis, prosomal width and wet weight of T. tridentatus grew by averages of 24.2% and 71.5% over pre-molt measurements, respectively. Similar values were obtained for C. rotundicauda, i.e. 24.0% and 77.3%, respectively. Three T. tridentatus with prosomal widths of between 26.5 and 35.0 mm molted twice between 89 and 149 days, leading to an average growth rate of 0.1 mm·day−1 and 0.04 g·day−1 in terms of prosomal width and wet weight, respectively. A positive growth allometry (b coefficient=2.97) was identified, indicating that weight gain for T. tridentatus, and possibly C. rotundicauda, was faster than prosomal width growth after each ecdysis. The effect of temperature on growth was also determined by comparing the percentages of T. tridentatus which molted at ∼28 °C and ∼18 °C. Fifty percent of individuals molted at the former, but only 10% at the latter. This study indicates that Hong Kong horseshoe crabs take a shorter time to reach sexual maturity, as compared with conspecifics in Japan, because they can molt more frequently at higher sediment/water temperatures (∼28 °C) if food is available.
Horseshoe crab, Limulus polyphemus, eggs were reared through the first tailed stage in factorial combinations of temperature and salinity with respective values of 20, 25, 30, or 35°C and 10, 15, 25, or 35%o.Lowest survival occurred in low temperature, low salinity combinations (20°C,lO%o).Wet weight, and thus linear dimension, of the first tailed stage was only slightly different throughout the tem perature-salinity range tested. However, ash-free dry weights, indicating yolk utili zation, varied significantly with both temperature and salinity. The least yolk uti lization occurred at 30°C and increased in higher and especially lower temperatures. Yolk utilization also increased with a decrease in salinity. Temperature interacted with salinity to cause a greater salinity-dependent difference in yolk utilization at lower temperatures. Comparisons ofash-free dry weights (i.e., yolk utilization) with respiration rates indicate that L. polyphemus has little ability to compensate met abolically for the effects of temperature and, secondarily, of salinity. Nevertheless, the larvae are sufficiently provisioned with yolk to survive the prevailing ranges of these variables in the habitat where adults normally deposit eggs. This suggests that other unexamined physical variables, or more likely biological factors such as pre dation or competition among the feeding larval stages and adults, are important for larval survival of L. polyphemus.
Once common as a fishery resource, adult horseshoe crabs of two species occur in Hong Kong. A third, Tachypleus gigas, can no longer be found. Once common too were breeding and nursery beaches for horseshoe crabs in Hong Kong but overfishing of adults, pollution and coastal reclamation have reduced these to but three identified sandy mudflats and only one where juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda are sympatric. Nothing is known, however, of the conservation requirements of these two species locally and this study of the diets of these juvenile horseshoe crabs on two such nursery beaches aimed at providing information on this most elementary aspect of their biology. Gut contents of juvenile horseshoe crabs, i.e. nine Tachypleus tridentatus (8.5-67 mm prosoma width) and two Carcinoscorpius rotundicauda (50 and 52 mm prosoma width), were analysed and compared with the composition of the ambient assemblage of meiobenthos on a nursery beach at Pak Nai, Hong Kong. The obtained data suggest that juvenile horseshoe crabs of both species are selective benthic feeders and subsist mainly on insect larvae, polychaetes, oligochaetes, small crabs and thin-shelled bivalves. A strong, positive, preference for insect larvae (Chironomous sp.: Diptera: Chironomidae) was recorded, but with no preference for meiofauna over macrofauna.
Scientific studies of the Japanese horseshoe crab, Tachypleus tridentatus, from the Seto Inland Sea have been published for nearly a century, beginning with the pioneering work of Owhatari (1913).
Studies by Matsunari, Asano, Oka, Nishii, Sekiguchi, and many others established much of the basic reproductive biology of
the species in the vicinity of Kasaoka City. Oe-hama beach was designated as a “Horseshoe Crab Spawning Ground Natural Monument”
in 1928. In spite of this formal recognition, and in the face of opposition by various local conservation organizations, the
Kasaoka Bay Land Reclamation Project began in 1969. Horseshoe crab abundance since then has declined, which has stimulated
efforts to raise horseshoe crabs in captivity. The success of such projects, though small in scale, affords some hope that
horseshoe crab populations might experience recovery.
Horseshoe crab (Limulus polyphemus) harvest on Delaware Bay is not a new event. Early settlers to the New World reported use of horseshoe crabs by Native Americans
for food, tools, and to enrich soils for growing crops. Literature from the mid- to late 1800s documents the use of horseshoe
crabs for fertilizer and to supplement livestock feed. By the 1870s and for almost a century thereafter, well over a million
crabs were harvested annually from Delaware Bay, in support of a regionally significant “cancerine” (fertilizer) industry.
Subsequent to the cessation of the cancerine industry in the mid-twentieth century, relatively low-scale use of horseshoe
crabs as bait for American eel and other fisheries existed. This use exploded in the 1990s, as eel markets expanded and use
of horseshoe crabs for bait in a rapidly emerging whelk (Busycon spp.) pot fishery intensified along the East Coast of the United States. With horseshoe crabs spawning in mass along the
shores of Delaware Bay, and little or no regulations in place, harvest pressure once again approached levels of the fertilizer
use days. Simultaneously, an ongoing need for bleeding of horseshoe crabs to provide Limulus amebocyte lysate (LAL) for biomedical use, and growing recognition of the importance of horseshoe crab eggs on Delaware Bay
as a key stopover food source for migratory shorebirds prompted concerns about observed declines in the population, resulting
in implementation of significant management measures to ensure the sustainability of the species.
Horseshoe crabs Tachypleus gigas and Carcinoscorpius rotundicauda are distributed along the northeast coast of India extending from the extreme north of West Bengal through Orissa to the
northern coast of Andhra Pradesh. High congregations of these animals are found along the breeding beaches of Orissa and West
Bengal during full moon and new moon high tides. Both of these Indian species have significantly different breeding patterns
particularly in relation to their habitat and laying of eggs. Although at times both the species inhabit the same body of
water, C. rotundicauda selects mudflats of mangroves and T. gigas have the priority for sandy estuarine and creek zones for spawning.
Environmental conditions of horseshoe crabs along the Indian coast still remain free from any kind of pollution. Also there
is no known commercial exploitation for the production of amebocyte lysate or bait. But the degradation and destruction of
breeding beaches by excess human activities have been posing a serious threat. This is in turn affecting the breeding migration
of adult pairs (in amplexus) to come ashore for breeding. Over time, it has been observed that the number of nests (identified
by their concave shapes) following the high tide has been decreasing, indicating a decline in the number of breeding pairs
on the beaches. By taking the example of the status of T. tridentatus in Japan, a concerted effort should be made both at the national and international levels to protect the breeding beaches
of horseshoe crabs of the world and allow these precious guests to use the space as an incubator for their fertilized eggs
as ever before to grow up to trilobites and swim freely to their nourishing grounds.
The highly successful Green Eggs & Sand (GE&S) project is a joint effort of educators, scientists, resource users, and managers engaged in building a unique workshop/field
experience, a wealth of educational video, and a series of associated activity-based learning modules, centered on the current
horseshoe crab/shorebird phenomenon and management controversy on Delaware Bay. Over the past 8 years, more than 800 teachers,
non-formal educators, scientists, resource users, and managers from 20 states and 3 foreign countries have taken part in GE&S
workshops, which are now offered up and down the Atlantic coast each spring around lunar event peak times for horseshoe crab
spawning. These workshops are intensive, spanning 2–3 days, and feature a mix of hands-on field experiences, presentations
by experts, and demonstration of curricular components. This article provides a case-study overview of how GE&S was developed
and implemented, with an eye to how this approach might be adapted and modified for use elsewhere.
In China, horseshoe crabs have economic and biomedical value. However, the abundance of horseshoe crabs has declined markedly
because of uncontrolled exploitation and habitat loss. Artificial breeding and release of hatchery reared horseshoe crabs
might be a useful method for protecting and recovering the horseshoe crab population. To determine successful methods for
artificial propagation, juvenile horseshoe crabs (Tachypleus
tridentatus) were experimentally maintained in simulated sea environments using sand and mud. The experimental results show that juveniles
grow and survive better in the presence of sand compared to mud, and juveniles living in sea water with sediment (either sand
or mud) will grow and survive better than those living in sea water without sediment. These results provide some guidelines
for artificial propagation of juvenile horseshoe crabs.
An updated survey, using both random transect and walk-through search methods, at 17 shores in Hong Kong in summer and winter
showed that juvenile horseshoe crabs (Tachypleus tridentatus and Carcinoscorpius rotundicauda) were significantly reduced by over 90% in density since 2002. Of the shores where juveniles were found, the highest density
of T. tridenatus was 0.31 and lowest 0.08 ind 100 m–2. Juvenile C. rotundicauda was only found using the walk-through search method, with the highest record of 1.17 and lowest 0.17 ind hr–1 person–1. The mean prosomal width of juvenile T. tridentatus obtained from the walk-through survey varied from 2.6 to 5.5cm, which corresponded to an age of 4–8 years old. A larger
size range for C. rotundicauda was, however, noted, from 2.5 to 9.0cm.
The degree of human exploitation of adult horseshoe crabs (T. tridentatus) in Hong Kong was estimated through interviewing 34 seafood restaurants, 150 fish sellers and fish handlers, and fishermen
in two local fish wholesale markets over a 13-month study period. A total of 1,023 horseshoe crabs were caught in 2004–2005,
with 72% from mainland Chinese waters. Apart from releasing back to sea, an average sale of 17 horseshoe crabs per month was
estimated. While the sale of horseshoe crabs was low by comparison with other marine species of economic importance, human
exploitation still contributes a potential threat and puts further pressure on the mature population of horseshoe crabs in
Hong Kong due to their long maturity period and declining densities of the juveniles.
The fossil record of the basic xiphosurid horseshoe crab body plan has been extended back to the Late Ordovician Period, about
445 million years ago, demonstrating an origin that lies outside of the paraphyletic ‘synziphosurines.’ Horseshoe crab body
fossils are exceptionally rare and are found mostly in shallow coastal and marginal marine Konservat-Lagerstätten deposits.
Their sporadic occurrences document a post-Cambrian history of low overall diversity with a modest morphological and taxonomic
peak in the Late Paleozoic Era. Survival of a single xiphosurid lineage through the end-Permian mass extinction events was
followed by a minor secondary radiation during the Triassic Period. The Jurassic to Recent fossil record of horseshoe crabs
is relatively impoverished in both taxa and known occurrences. Overall, the rarity of fossil xiphosurids reflects both taphonomic
biases inherent in the unusual conditions required for preservation of their non-biomineralized exoskeletons and complex ecological
factors related to a long-term association with shallow marginal aquatic habitats. Focused paleontological investigations
should yield additional fossil horseshoe crab discoveries that will in turn inform research on their phylogeny, morphological
stasis, and ecological persistence.
Current knowledge of horseshoe crabs (HSC) has been derived, in large part, from field studies. Comprehending the biology
and conservation of HSC could be facilitated and augmented by understanding and improving their culture methods. Although
many researchers and even lay people are capable of getting animals to the early stages of development, very few are successful
in getting them to survive for longer periods of time. The Aquatic Research and Environmental Assessment Center (AREAC) has
been successful in rearing HSC in annual cohorts, some for more than 7 years. We have used indoor recirculating aquaculture
systems (RAS) containing medium prepared with artificial sea salts. Animals have been given various diets, including both
natural and specially formulated feeds. We have traced the earliest developmental stages through juvenile development in animals
that were derived from eggs fertilized in the field and laboratory. This chapter will discuss the problems and successes of
culturing adult and developing HSC in RAS, methods of fertilization, feed regimes, growth, and survivorship and observations
on HSC development from egg to juvenile.
Beyond their commercial importance for LAL and bait, and their status as a living fossil, it is often asserted that horseshoe
crabs play a vital role in the ecology of estuarine and coastal communities. How would the various ecological relationships
involving horseshoe crabs be affected if these animals were no longer abundant? Attempts to understand and generalize the
ecological importance of horseshoe crabs are hampered by several constraints. We know relatively little about the ecology
of juvenile horseshoe crabs. Most ecological studies involving adult Limulus polyphemus have been conducted at only a few locations, while much less is known about the three Indo-Pacific species. Furthermore,
we are attempting to infer the ecological importance of a group of animals whose numbers may have already declined significantly
(the so-called “shifting baseline syndrome”). Horseshoe crab shells serve as substrate for a large number of epibionts, such
as barnacles and slipper limpets, but the relationships between these epibionts and horseshoe crabs appear to be facultative,
rather than obligatory. Horseshoe crabs are dietary generalists, and adult crabs are ecologically important bivalve predators
in some locations. The most notable predator–prey relationship involving horseshoe crabs is the migratory shorebird–horseshoe
crab egg interaction in Delaware Bay. After hatching, the first and second instars are eaten by surf zone fishes, hermit crabs,
and other predators. Virtually nothing is known about predator–prey relationships involving older juveniles, but adult L. polyphemus are important as food for the endangered loggerhead turtle, especially in the mid-Atlantic region.
A study of the physical properties of horseshoe crab (Tachypleus tridentatus) nursery grounds indicated that juveniles preferred sediments consisting of fine sand with median grain size of 0.14–0.27mm
in diameter, 16.9–23.2% water content when tides receded, 0.23–0.41% TOC content, 0.04–0.07% TN content, 2.3–2.8 µg/cm2 chlorophyll a content, and poorly sorted substrates. Juvenile horseshoe crab density increased based on the amount of chlorophyll a content in the sediment and infaunal polychaete density, suggesting that the juveniles prefer nursery grounds containing
abundant prey and its supporting food web. An effort to restore horseshoe crab spawning grounds was conducted by covering
20cm deep mud substrate (0.23mm in diameter) with coarse sand (1.10mm in diameter). Although adults transferred to this
site succeeded in laying eggs, the hatching rate was only 33.9%. This low rate may be attributed to small tidal amplitude
at this restoration site.
Horseshoe crab populations along the northeast coast of India are under threat due to degradation of the breeding beaches. To augment the trend, attempts were made to culture the larvae of Tachypleus gigas and study its growth rate by enhancing the molting pattern in the laboratory condition. Trilobites of T. gigas were cultured on a controlled diet of brine shrimp (Artemia) and diatom (Chaetoceros gracilis) at 26–28°C and 32–34‰.
Trilobites could molt up to the fourth posthatched juvenile stage within a period of 180 days from the day of hatching of trilobite from the egg membrane as free swimming larval stage. The molting behavior was faster from the first to the third posthatched juvenile stage, i.e., within a period of 90 days. The average growth rate in terms of total body length from the first to second posthatched juvenile was about 63%, and from the second to third posthatched larva was about 38%. The growth rate was found to be about 25% from the third to fourth posthatched juvenile stage, and molting took place 180 days after the day of hatching of trilobite. All the posthatched juveniles had similar morphological features to the adults. The fourth posthatched juveniles exhibited more prominent morphological features with fully grown legs, spines, and segmentation, with a total body length of 45 mm.
Further studies on food-dependent molting patterns of juvenile instars may help to establish a standardized aquaculture method to grow horseshoe crabs in captivity. Sea ranching of these reared animals can be carried out regularly in the holding areas to increase horseshoe crab populations and conserve these precious organisms from the brink of extinction.
To discern the position of horseshoe crabs as a potentially important predator in estuarine food webs, we determined where they foraged and what they ate. We used N and C stable isotopes to link adult horseshoe crabs to their oraging locations and potential food sources in Pleasant Bay, Cape Cod. The δ15N in tissues of horseshoe crabs and their potential foods suggest crabs were loyal to local foraging sites and did not forage substantially in subestuaries receiving >110 kg N ha−1 year−1. Among locations where crabs foraged, δ13C values in potential foods showed that food webs in subestuaries subject to higher N loads were supported by algal producers, while food webs in subestuaries with lower N loads were also supported by Spartina. δ13C values in horseshoe crab tissue did not change with load, suggesting they ate a mixed diet, regardless of N load. N and C isotopes in horseshoe crab feces were similar to signatures of estimated diet, suggesting low assimilation efficiency, perhaps due to ingestion of low quality organic matter. Although horseshoe crabs were relatively opportunistic in foraging habits, conservation or culture of horseshoe crabs may require habitats with higher water quality, ample particulate organic matter, and supporting a variety of prey.
Often referred to as a living fossil, the American horseshoe crab, Limulus polyphemus, is one of the most-studied invertebrate animals in the world. It has served as a model in Nobel Prize-winning eye research, and researchers use a component of its blood to detect bacterial contamination in medical devices and drugs. The authors review the conditions necessary for housing these animals in the laboratory.
1. Functional responses -- the relationship between resource intake rate and resource abundance -- are widely used in explaining predator-prey interactions yet many studies indicate that resource availability is crucial in dictating intake rates. 2. For time-stressed migrant birds refuelling at passage sites, correct decisions concerning patch use are crucial as they determine fattening rates and an individual's future survival and reproduction. Measuring availability alongside abundance is essential if spatial and temporal patterns of foraging are to be explained. 3. A suite of shorebird species stage in Delaware Bay where they consume horseshoe crab Limulus polyphemus eggs. Several factors including spawning activity and weather give rise to marked spatial and temporal variation in the abundance and availability of eggs. We undertook field experiments to determine and contrast the intake rates of shorebird species pecking for surface and probing for buried eggs. 4. Whether eggs were presented on the sand surface or buried, we demonstrate strong aggregative responses and rapid depletion (up to 80%). Depletion was greater at deeper depths when more eggs were present. No consistent give-up densities were found. Type II functional responses were found for surface eggs and buried eggs, with peck success twice as high in the former. Maximum intake rates of surface eggs were up to 83% higher than those of buried eggs. 5. Caution is needed when applying functional responses predicted on the basis of morphology. Our expectation of a positive relationship between body size and intake rate was not fully supported. The smallest species, semipalmated sandpiper, had the lowest intake rate but the largest species, red knot, achieved only the same intake rate as the mid-sized dunlin. 6. These functional responses indicate that probing is rarely more profitable than pecking. Currently, few beaches provide egg densities sufficient for efficient probing. Areas where eggs are deposited on the sand surface are critical for successful foraging and ongoing migration. This may be especially true for red knot, which have higher energetic demands owing to their larger body size yet appear to have depressed intake rates because they consume smaller prey than their body size should permit.
A multi-year retrospective of methods and approaches to horseshoe crab (Limulus polyphemusandTachypleus tridentatus) culture and husbandry in Japan (research results: raising horseshoe crabs from the egg to maturity)
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New progress on horseshoe crab rearing
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Coping with environmental changes: physiological challenges
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The conservation of horseshoe crabs in Hong Kong (Masters thesis) Department of Biology and Chemistry
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