Horseshoe crab

Horseshoe crabs are marine and brackish water arthropods of the family Limulidae and are the only surviving xiphosurans. Despite their name, they are not true crabs or crustaceans. Rather, they are chelicerates. This makes them more closely related to arachnids like spiders, ticks, and scorpions.

The body of a horseshoe crab is divided into three main parts: the cephalothorax, abdomen, and telson. The largest of these, the cephalothorax, houses the majority of the animal's eyes, limbs, and internal organs. It's also where the animal gets its name, as its shape somewhat resembles that of a horseshoe.

Horseshoe crabs primarily live on the bottom of shallow coastal waters, but can swim if needed. These animals are occasionally used as fishing bait, but they are also eaten in some parts of Asia. More commonly, horseshoe crabs are caught for their blood, something valuable to the medical industry. This use comes from the Limulus amebocyte lysate, a chemical in their blood used to detect bacterial endotoxins.

In recent years, these animals have experienced a population decline. This is mainly due to coastal habitat destruction and overharvesting.

Phylogeny and Evolution
The fossil record of xiphosurans extends all back to the Ordovician, or around 445 million years ago. For modern horseshoe crabs, their earliest appearance was approximately 250 million years ago during the Early Triassic. As their bodies have barely changed between then and the present, extant forms have been described as "living fossils".

Horseshoe crabs resemble crustaceans but belong to a separate subphylum of the arthropods, Chelicerata. Horseshoe crabs are closely related to the extinct eurypterids (sea scorpions), which include some of the largest arthropods to have ever existed, and the two may be sister groups. The enigmatic chasmataspidids are also thought to be closely related to the horseshoe crabs.

The radiation of horseshoe crabs occurred rapidly and resulted in 88 known lineages, of which only 4 remain. The Atlantic species is sister to the three Asian species, the latter of which are likely the result of two divergences relatively close in time. The last common ancestor of the four extant species is estimated to have lived about 135 million years ago in the Cretaceous.

The Limulidae are the only extant family of the order Xiphosura and contains all four living species of horseshoe crabs:


 * Carcinoscorpius rotundicauda, the mangrove horseshoe crab, found in South and Southeast Asia
 * Limulus polyphemus, the Atlantic or American horseshoe crab, found along the Atlantic coast of the United States and the Southeast Gulf of Mexico
 * Tachypleus gigas, the Indo-Pacific, Indonesian, Indian or southern horseshoe crab, found in South and Southeast Asia
 * Tachypleus tridentatus, the Chinese, Japanese or tri-spine horseshoe crab, found in Southeast and East Asia

Genera
After Bicknell et al. 2021 and Lamsdell et al. 2020


 * Incertae sedis
 * †Albalimulus? Bicknell & Pates, 2019 Ballagan Formation, Scotland, Early Carboniferous (Tournaisian) (Considered Xiphosura incertae sedis by Lamsdell, 2020 )
 * †Casterolimulus Holland, Erickson & O'Brien, 1975 Late Cretaceous (Maastrichtian) Fox Hills Formation, North Dakota, USA (Inconsistently placed in this family)
 * †Heterolimulus gadeai Vıa & Villalta, 1966 Alcover Limestone Formation, Spain, Middle Triassic (Ladinian)
 * †Limulitella Størmer, 1952 Middle-Upper Triassic, France, Germany, Tunisia, Russia
 * †Sloveniolimulus Bicknell et al., 2019 Strelovec Formation, Slovenia Middle Triassic (Anisian)
 * †Tarracolimulus Romero & Vıa Boada, 1977 Alcover Limestone Formation, Spain, Middle Triassic (Ladinian)
 * †Victalimulus Riek & Gill, 1971 Lower Cretaceous (Aptian) Korumburra Group, NSW, Australia
 * †Yunnanolimulus Zhang et al., 2009 Middle Triassic (Anisian), Guanling Formation, Yunnan, China
 * †Mesolimulus Middle Triassic-Late Cretaceous England, Spain, Siberia, Germany, Morocco
 * †Ostenolimulus Lamsdell et al. 2021 Early Jurassic (Sinemurian) Moltrasio Limestone, Italy
 * †Volanalimulus Lamsdell, 2020 Early Triassic, Madagascar.
 * Subfamily Limulinae Leach, 1819
 * †Crenatolimulus Feldmann et al., 2011 Upper Jurassic (upper Tithonian) Kcynia Formation, Poland. Lower Cretaceous (Albian) Glen Rose Formation, Texas, USA
 * Limulus O. F. Müller, 1785 Pierre Shale, United States, Late Cretaceous (Maastrichtian), Atlantic North America, Recent
 * Subfamily Tachypleinae Pocock, 1902
 * Carcinoscorpius Pocock, 1902, Asia, Recent
 * Tachypleus Leach, 1819 Upper Cretaceous (Cenomanian) Haqel and Hjoula Konservat-Lagerstatten, Lebanon, Upper Eocene Domsen Sands, Germany, Asia, Recent

Phylogeny
The horseshoe crab's position within Chelicerata is complicated, though most morphological analyses have placed them outside Arachnida. This assumption was challenged when a genetics-based phylogeny found horseshoe crabs to be the sister group to the ricinuleids, thereby making them an arachnid. Nonetheless, a more recent paper has again placed horseshoe crabs as separate from the arachnids. This new study utilized both new and more complete sequencing data while also sampling a larger number of taxa.

Below is a cladogram showing the internal relationships of Limulidae (modern horseshoe crabs) based on morphology. It contains both extant and extinct members.

Adaptation to Freshwater
According a phylogeny from 2015, now-extinct xiphosurans traveled to freshwater at least five times throughout history. This same transition happened twice in the horseshoe crabs Victalimulus and Limulitella, with both inhabiting environments such as swamps and rivers. In contrast, all extant species are predominantly marine (though mangrove horseshoe crabs can survive in brackish water).

Morphological Stasis
As generalists, horseshoe crabs can have a broad diet and live in diverse habitats, meaning they are more likely to survive and produce viable offspring in more places. Horseshoe crabs also have an incredibly efficient immune system, and can also be successful in areas with high concentrations of bacteria. Amoebocytes in the bloodstream attack bacterial cells, and in doing so act as coagulants around the foreign body, preventing them from multiplying. This trait is an adaptation to their often bacteria-rich environment. Their ability to succeed in many environments limits selective forces, as there are few, if any, mutations that would result in more beneficial alleles that would make horseshoe crabs more suited for survival.

In the Atlantic horseshoe crab, microRNA’s exist on 7 loci, comparatively high to the 2 loci in spiders and scorpions, meaning horseshoe crabs have comparatively high rates of gene regulation, which could contribute to their morphological status. Additionally, several other gene clusters are present in at least 6, and often 7 loci.

Whole Genome Duplication
The common ancestor to Arachnids underwent a whole genome duplication event, which was likely followed by another duplication event in the common ancestor to the 4 extant species of horseshoe crabs about 135 million years ago, and evidence points to two additional events having occurred since then. However, many of these ancient genes have likely undergone either neo-functionalization or sub-functionalization as a result of functional divergence, meaning their expression is not the same as it was following the WGD event, as seen in other chelicerate lineages.

Evidence from Genomic Sequencing

 * The genome assembly of two species of horseshoe crabs, C. rotundicauda and T. tridentatus, suggests a genome size of 1.72 Gb for both species. The relatively large size of these genomes is believed to be another result of multiple rounds of whole genome duplication. In C. rotundicauda, the number of Hox genes is 43, which mainly exist on five Hox clusters. There are 36 hox genes on 3 clusters in T. tridentatus. Both species contain two ParaHox clusters. The L. polyphemus similarly contains 4 hox clusters. The evidence of at least 3 hox clusters in these three species indicates at least two rounds of whole genome duplication.


 * Homeobox genes are present in more than four copies. Furthermore, most chromosomes in C. rotinducauda exhibit synteny with between 4 and 8 chromosomes, also indicating that at least two, and likely 3 rounds of whole genome duplication occurred in the common ancestor of the 4 extant species between 135 and 500 million years ago.


 * Many of the ancient genes present due to past whole genome duplication events have experienced ongoing mutations, and as a result, have become pseudogenes, and lost their ability to code for proteins. Evidence for this phenomenon was found in a population of C. Rotundica in Hong Kong, in which 9/10 individuals sequenced exhibited deletion of the paralog of the Unpg-A1 gene, indicating an ongoing mutative process of pseudogenization in C. rotundicauda.

Sexual Size Dimorphism
Several hypotheses have been proposed as possible mechanisms for the size difference between male and female horseshoe crabs. The sexual size dimorphism of horseshoe crabs that results in a larger average size in females than males is likely a result of the amalgamation of many different aspects of these hypotheses and more:


 * 1) Horseshoe crabs exhibit self-similar size preferences when choosing a mate. Inverse relationships in which the male is larger than the female are rare. The maintenance of size difference in mating partners is likely in part a result of the stability of this self-similar preference and dimorphism.
 * 2) Mature females undergo an additional year of maturation and an additional molt to males, a contributing factor in their larger average size to mature males.
 * 3) Larger female horseshoe crabs can house more eggs within their bodies, and therefore pass on more genetic material than smaller females during each mating cycle.
 * 4) Satellite males found occupying the ideal mating position were found to be on average in better condition than those in other mating positions, indicating a significant relationship between reproductive success and the male’s condition, as satellite males account for nearly 40% of fertilization.
 * 5) There is no evidence of assortative mating, indicating sexual selection is likely not the main factor in this sexual size dimorphism. Rather than favoring size, selection favors the ability to switch to satellite behavior, as individuals like these can contribute more genetic material to the next population, which will increase the number of individuals with the ability to engage in both types of mating behaviors.

General Body Plan
Like all arthropods, horseshoe crabs have segmented bodies with jointed limbs, which are covered by a protective cuticle made of chitin. They have heads composed of several segments, which eventually fuse as an embryo.

Horseshoe crabs are chelicerates, meaning their bodies are composed of two main parts (tagma): the cephalothorax and the opisthosoma. The first tagma, the cephalothorax or prosoma, is a fusion of the head and thorax. This tagma is also covered by a large, semicircular, carapace that acts like a shield around the animal's body. It's shaped like the hoof of a horse, giving this animal its common name. In addition to the two main tagmata, the horseshoe crab also possesses a long tail-like section known as the telson.

In total, horseshoe crabs have 6 pairs of appendages on their cephalothorax. The first of these are the chelicerae, which give chelicerates their name. In horseshoe crabs, these look like tiny pincers in front of the mouth. Behind the chelicerae are the pedipalps, which are primarily used as legs. In the final molt of males, the ends of the pedipalps are modified into specialized, grasping claws used in mating. Following the pedipalps are three pairs of walking legs and a set of pusher legs for moving through soft sediment. Each of these pusher legs is biramous or divided into two separate branches. The branch closest to the front bears a flat end that looks like a leaf. This end is called the flabellum. The branch towards the back is far longer and looks similar to a walking leg. However, rather than ending in just a claw, the back branch has four leaf-like ends that are arranged like a petal. The final segment of the cephalothorax was originally part of the abdomen but fused in the embryo. On it are two flap-like appendages known as chilaria. The opisthosoma or abdomen of a horseshoe crab is composed of several fused segments. Similar to a trilobite, the abdomen is made up of three lobes: a medial lobe in the middle, and a pleural lobe on either side. Attached to the perimeter of each pleural lobe is a flat, serrated structure known as the flange. The flange on either side is connected by the telson embayment, which itself is attached to the medial lobe. Along the line where these lobes meet are six sets of indentations known as apodeme. Each of these serve as a muscle attachment point for the animal's twelve movable spines.

On the underside of the abdomen are several biramous limbs. The branches closest to the outside are flat and broad, while the ones on the inside are more narrow. Closest to the front is a plate-like structure made of two fused appendages. This is the genital operculum and is where horseshoe crabs keep their reproductive organs. Following the operculum are five pairs of book gills. While mainly used for breathing, horseshoe crabs can also use their book gills to swim. At the end of a horseshoe crab's abdomen is a long, tail-like spine known as a telson. It's highly mobile and serves a variety of functions. If severed from the body, lost legs or the telson may slowly regenerate, and cracks in the body shell can heal.

Central Nervous System
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Eyes
Horseshoe crabs have two compound lateral eyes, each composed of about 1,000 ommatidia, plus a pair of median eyes that are able to detect both visible light and ultraviolet light, a single parietal eye, and a pair of rudimentary lateral eyes on the top. The latter becomes functional just before the embryo hatches. Also, a pair of ventral eyes is located near the mouth, as well as a cluster of photoreceptors on the telson. Having relatively poor eyesight, the animals have the largest rods and cones of any known animal, about 100 times the size of humans', and their eyes are a million times more sensitive to light at night than during the day.

Other Senses
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Circulation and Respiration
Behind its legs, the horseshoe crab has book gills, which exchange respiratory gases. As in other arthropods, a true endoskeleton is absent, but the body does have an endoskeletal structure made up of cartilaginous plates that support the book gills.

Feeding, Digestion, and Excretion
Horseshoe crabs use their chelicerae—a pair of small appendages—for moving food into the mouth. The mouth is located in the center of the legs, whose bases are referred to as gnathobases, and have the same function as jaws and help grind up food.

Locomotion
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Distribution and Habitat
In the modern day, horseshoe crabs have a relatively limited distribution. The three Asian species mainly occur in South and Southeast Asia along the Bay of Bengal and the coasts of Indonesia. A notable exception is the tri-spine horseshoe crab, whose range extends northward to the coasts of China, Taiwan, and Southern Japan. The American species lives from the coast of Nova Scotia to the northern Gulf of Mexico, with another population residing around the Yucatán Peninsula.

Extant horseshoe crabs generally live in salt water, though one species, the mangrove horseshoe crab (Carcinoscorpius) is often found in more brackish environments.

Diet
Horseshoe crabs are more often found on the ocean floor searching for worms and molluscs, which are their main food. They may also feed on crustaceans and even small fish. Foraging usually takes place at night. Gravel and sand particles are ingested to further grind up food in a gizzard before passing it into the stomach.

Growth and Development
Females are about 20–30% larger than males. The smallest species is C. rotundicauda and the largest is T. tridentatus. On average, males of C. rotundicauda are about 30 cm long, including a tail (telson) that is about 15 cm, and their carapace (prosoma) is about 15 cm wide. Some southern populations (in the Yucatán Peninsula) of L. polyphemus are somewhat smaller, but otherwise this species is larger.

In the largest species, T. tridentatus, females can reach as much as 79.5 cm long, including their tail, and up to 4 kg in weight. This is only about 10-20 cm longer than the largest females of L. polyphemus and T. gigas, but roughly twice the weight.

The juveniles grow about 33% larger with every molt until reaching adult size. Atlantic horseshoe crabs molt in late July.

Reproduction
During the breeding season (spring and summer in the Northeast U.S.; year-round in warmer locations or when the full moon rises), horseshoe crabs migrate to shallow coastal waters. There they spawn on beaches and salt marshes. The smaller male horseshoe crab clings to the back or opisthosoma of the larger female using specialized front claws and fertilizes the eggs as they are laid in the sand. Additional males called "satellite males" which are not attached to the female may surround the pair and have some success in fertilizing eggs. Young female horseshoe crabs can be identified by the lack of mating scars.

The female can lay between 60,000 and 120,000 eggs in batches of a few thousand at a time. The eggs may be inseminated within 20 to 30 minutes. In L. polyphemus, the eggs take about two weeks to hatch; shore birds eat many of them before they hatch. The larvae molt six times during the first year and annually after the first 3 or 4 years.

Natural breeding of horseshoe crabs in captivity has proven to be difficult. Some evidence indicates that mating takes place only in the presence of the sand or mud in which the horseshoe crab's eggs were hatched; it is not known with certainty what is in the sand that the crabs can sense or how they sense it. Artificial insemination and induced spawning have been done on a relatively large scale in captivity, and eggs and juveniles collected from the wild are often raised to adulthood in captivity.

In order to preserve and ensure the continuous supply of the horseshoe crab, a breeding center was built in Johor, Malaysia where the crabs are bred and released back into the ocean in the thousands once every two years. It is estimated to take around 12 years before they are suitable for consumption.

Consumption
While not having much meat, horseshoe crabs are valued as a delicacy in many parts of East and Southeast Asia. The meat is white, has a rubbery texture similar to lobster, and possesses a slightly salty aftertaste. Horseshoe crab can be eaten both raw and cooked, but must be properly prepared to prevent food poisoning. Furthermore, only certain species can be eaten. There have been numerous reports of poisonings after consuming mangrove horseshoe crabs (Carcinoscorpius rotundicauda) as its meat contains tetrodotoxin.

Horseshoe crab is commonly prepared by grilling or stewing, but the meat can also be pickled in vinegar or stir-fried with veggies. Many recipes involve the use of various spices, herbs, and chilies give the dish more flavor.

In addition to the meat, horseshoe crabs are also valued for their eggs. Much like the meat, only the eggs of specific species can be eaten. Much like its meat, the eggs of mangrove horseshoe crabs also contain tetrodotoxin.

Use in Fisheries
Horseshoe crabs are used as bait to fish for eels (mostly in the United States), whelk, or conch. Nearly 1 million (1,000,000) crabs a year are harvested for bait in the United States, dwarfing the biomedical mortality. However, fishing with horseshoe crab was banned indefinitely in New Jersey in 2008 with a moratorium on harvesting to protect the red knot, a shorebird which eats the crab's eggs. A moratorium was restricted to male crabs in Delaware, and a permanent moratorium is in effect in South Carolina.

A low horseshoe crab population in the Delaware Bay is hypothesized to endanger the future of the red knot. Red knots, long-distance migratory shorebirds, feed on the protein-rich eggs during their stopovers on the beaches of New Jersey and Delaware. An effort is ongoing to develop adaptive-management plans to regulate horseshoe crab harvests in the bay in a way that protects migrating shorebirds.

Use in Medicine
Horseshoe crabs use hemocyanin to carry oxygen through their blood. Their blood contains amebocytes, which play a similar role to the white blood cells of vertebrates in defending the organism against pathogens. Amebocytes from the blood of Limulus polyphemus are used to make Limulus amebocyte lysate (LAL), which is used for the detection of bacterial endotoxins in medical applications. There is a high demand for the blood, the harvest of which involves collecting and bleeding the animals, and then releasing them back into the sea. Most of the animals survive the process; mortality is correlated with both the amount of blood extracted from an individual animal, and the stress experienced during handling and transportation. Estimates of mortality rates following blood harvesting vary from 3–15% to 10–30%. Approximately 500,000 Limulus are harvested annually for this purpose. Declining horseshoe crab populations on the East Coast of the United States endanger certain bird species which feed upon their eggs.

Bleeding may also prevent female horseshoe crabs from being able to spawn or decrease the number of eggs they are able to lay. Up to 30% of an individual's blood is removed, according to the biomedical industry, although NPR reported that it "can deplete them of more than half their volume of blue blood." The horseshoe crabs spend between one and three days away from the ocean before being returned. As long as the gills stay moist, they can survive on land for four days. Some scientists are skeptical that certain companies return their horseshoe crabs to the ocean at all, instead suspecting them of selling the horseshoe crabs as fishing bait.

The harvesting of horseshoe crab blood in the pharmaceutical industry is in decline. In 1986, Kyushu University researchers discovered that the same test could be achieved by using isolated Limulus clotting factor C (rFC), an enzyme found in LAL, as by using LAL itself. Jeak Ling Ding, a National University of Singapore researcher, patented a process for manufacturing rFC; on 8 May 2003, synthetic isolated rFC made via her patented process became available for the first time. Industry at first took little interest in the new product, however, as it was patent-encumbered, not yet approved by regulators, and sold by a single manufacturer, Lonza Group. In 2013, however, Hyglos GmbH also began manufacturing its own rFC product. This, combined with the acceptance of rFC by European regulators, the comparable cost between LAL and rFC, and support from Eli Lilly and Company, which has committed to use rFC in lieu of LAL, is projected to all but end the practice of blood harvesting from horseshoe crabs.

In December 2019, a report of the US Senate which encouraged the Food and Drug Administration to "establish processes for evaluating alternative pyrogenicity tests and report back [to the Senate] on steps taken to increase their use" was released; PETA backed the report.

In June 2020, it was reported that U.S. Pharmacopeia had declined to give rFC equal standing with horseshoe crab blood. Without the approval for the classification as an industry standard testing material, U.S. companies will have to overcome the scrutiny of showing that rFC is safe and effective for their desired uses, which may serve as a deterrent for usage of the horseshoe crab blood substitute.

Vaccine research and development during the COVID-19 pandemic has added additional "strain on the American horseshoe crab."

In 2023, the U.S. Fish and Wildlife Service halted the harvesting of horseshoe crabs in the Cape Romain National Wildlife Refuge, South Carolina, from March 15 to July 15 to aid their reproduction. This decision was influenced by the importance of horseshoe crab eggs as a food source for migratory birds and the ongoing use of horseshoe crabs for bait and their blood in medical products. The ban supports the conservation goals of the refuge, spanning 66,000 acres (26,700 hectares) of marshes, beaches, and islands near Charleston.

Conservation Status
Development along shorelines is dangerous to horseshoe crab spawning, limiting available space and degrading habitat. Bulkheads can block access to intertidal spawning regions as well.

The population of Indo-Pacific horseshoe crabs (Tachypleus. gigas) in both Malaysia and Indonesia has decreased dramatically since 2010. This is primarily due to overharvesting, as horseshoe crabs are considered a delicacy in countries like Thailand. The individuals most likely to be targeted are gravid females, as they can be sold for both their meat and eggs. This method of harvesting has led to an unbalanced sex ratio in the wild, something that also contributes to the area's declining population.

Because of the destruction of habitat for shoreline development, use in fishing, plastic pollution, status as a culinary delicacy, and use in research and medicine, the horseshoe crab is facing both endangered and extinct statuses. One species, the tri-spine horseshoe crab (Tachypleus tridentatus), has already been declared extirpated from Taiwan. Facing a greater than 90% population decrease in T. tridentatus juveniles, it is suspected that Hong Kong will be the next to declare tri-spine horseshoe crabs as extirpated from the area. This species is listed as endangered on the IUCN Red List, specifically because of the overexploitation and loss of critical habitat that's lead to its steep decline.