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Review Article
Horseshoe crab and its medicinal values
Vikash Kumar*, Suvra Roy, A.K. Sahoo, B.K. Behera and A.P. Sharma
ABSTRACT
The medicinal value of horseshoe crab blood comes from its ability to clot in the
Keywords presence of bacteria, rendering the bacteria harmless. Even though bacteria are
usually destroyed by modern sterilization techniques, some bacteria, called
Horseshoe endotoxins, may survive the sterilization process. This blood-clotting ability of the
crab, horseshoe crab makes it very valuable in testing for injectable medicines, vaccines
Endotoxins, and sterile medical equipment. The horseshoe crab has also proved valuable to the
Blood- medical field in another way. Studies involving the nerve pathways in the eyes of
clotting, horseshoe crabs have led to many discoveries in human eye research. The outer
Chitin shell of a horseshoe crab is made primarily of chitin. Scientists discovered that
when used as a coating for suture material and burn dressings, chitin rapidly
increased wound healing, cutting the time by half.
Introduction
Horseshoe crabs are marine arthropods of it is not really a crab. Horseshoe crabs
the family Limulidae and order Xiphosura or belong to the phylum, Arthropoda, along
Xiphosurida, which live primarily in and with crabs, insects, and other invertebrates
around shallow ocean waters on soft sandy with jointed legs, but their closest living
or muddy bottoms. They occasionally come relatives are spiders and scorpions. True
onto shore to mate. They are commonly crabs have two pairs of antennae and a pair
used as bait and fertilizer. In recent years, a of mandibles, or jaws; horseshoe crabs lack
decline in the population has occurred as a these structures. Further, comparing the legs
consequence of coastal habitat destruction in of a true crab with the legs of a horseshoe
Japan and overharvesting along the east crab reveals the other significant difference.
coast of North America. Tetrodotoxin may True crabs classified as decapod
be present in the row of species inhabiting crustaceans, have five pairs of legs, which
the waters of Thailand [1]. Because of their include a pair of claws. Horseshoe crabs
origin 450 million years ago, horseshoe have seven pairs of legs under their helmet-
crabs are considered living fossils [2]. like shells; five of these seven pairs of legs
are equipped with claws. In adult males, the
Even though the horseshoe crab has a hard second pair of claws (pedipalps) has a
shell and numerous appendages with claws, “boxing-glove” appearance and is used to
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or dead fish, one of the claws picks it up and about 1000 ommatidia, plus a pair of median
pushes it toward the heavy, spiny eyes that are able to detect both visible light
projections that surround the mouth; the and ultraviolet light, a single endoparietal
horseshoe crab has no nose; but the tiny eye, and a pair of rudimentary lateral eyes
hairs on the spiny projections around its on the top. The latter becomes functional
mouth are chemoreceptors, allowing the just before the embryo hatches. Also, a pair
crab to smell prey. Since the horseshoe crab of ventral eyes is located near the mouth, as
has no jaws to chew its food, it must bring well as a cluster of photoreceptors on the
all of its legs together and use the spiny telson. Despite having relatively poor
projections around its month than the first eyesight, the animals have the largest rods
set of legs to crush the worm or clam. and cones of any known animal, about 100
Horseshoe crabs also have gizzards times the size of humans' [7, 8]. The mouth
containing sand and small bits of gravel to is located in the center of the legs, where
help grind their food. Horseshoe crabs their bases have the same function as jaws
continue to grow for nine to ten years until and help grinding up food. It has five pairs
they reach maturity. The young horseshoe of legs for walking, swimming, and moving
crabs molt, or shed, their outer skeleton food into the mouth, each with a claw at the
(exoskeleton) often, until they reach sexual tip except the last pair. The long, straight,
maturity, then molting slows, occurring only rigid tail can be used to flip it over if turned
about once annually. The animals increase upside down, so a horseshoe crab with a
in size from 25-30% with each molt by broken tail is more susceptible to desiccation
pumping in water to expand their new shells, or predation.
which will harden in approximately 24
hours. Males are sexually mature at their Behind their legs, they have book gills,
sixteenth molt or ninth year. Females need at which exchange respiratory gases and are
least 17 molts and mature in their tenth year. also occasionally used for swimming. As in
Unlike the blue crab, this breaks out of its other arthropods, a true endoskeleton is
old shell, the horseshoe crab crawls forward absent, but the body does have an
out of its shell through a split that develops endoskeletal structure made up of
along the junction of the dorsal (top) and cartilaginous plates that support the book
ventral (bottom) surfaces. gills [9].
Horseshoe crabs face dangers from a variety Horseshoe crab normally swims upside
of predators throughout their lifecycle. down, inclined at about 30° to the horizontal
These include mollusks, crustaceans, fish, and moving at about 10–15 cm/s (0.22–0.34
leopard sharks, eels, birds, sea turtles, and, mph) [10, 11, 12]. Despite this, they usually
maybe most importantly, the man. A partial are found on the ocean floor searching for
list of organisms known to prey on worms and molluscs, which are their main
horseshoe crabs during various stages of food. They may also feed on crustaceans and
their lives is given in Table 1 [4, 5, 6]. even small fish. Females are larger than
males; C. rotundicauda is the size of a
Anatomy and behavior human hand, while L. polyphemus can be up
to 60 cm (24 in) long (including tail). The
The entire body of the horseshoe crab is juveniles grow about 33% larger with every
protected by a hard carapace. It has two molt until reaching adult size [13].
compound lateral eyes, each composed of
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foreign cells, including bacterial cells, and commercialization, and use of LAL have
α2-macroglobulin, which inhibits the been an important improvement in the
proteases of invading pathogens. Blood is pharmaceutical industry. Prior to the use of
collected by direct cardiac puncture under LAL, compounds were tested for the
conditions that minimize contamination by presence of endotoxins in a variety of ways
lipopolysaccharide (endotoxin, LPS), a that involved living animals or living parts
product of the Gram-negative bacteria. A of animals [19]. Thus, LAL provides a
large animal can yield 200 - 400 ml of means to detect endotoxins without having
blood. Unlike vertebrates, horseshoe crabs to kill or disable animals [19].
do not have hemoglobin in their blood, but
instead use hemocyanin to carry oxygen. Limulus Amebocyte Lysate is extremely
Because of the copper present in useful in detecting those toxins that cause
hemocyanin, their blood is blue. Their blood fever – the bacterial “pyrogens” or
contains amebocytes, which play a role endotoxins. Endotoxins occur as a structural
similar to white blood cells of vertebrates in component of the cell wall of a large group
defending the organism against pathogens. of bacteria known as Gram negative [20].
Amebocytes from the blood of L. Most aquatic bacteria are of the Gram-
polyphemus is used to make Limulus negative variety, as studies at the Woods
amebocyte lysate (LAL), which is used for Hole Oceanographic Institution have shown
the detection of bacterial endotoxins in that seawater contains over 1 million Gram-
medical applications. The blood of negative bacteria per milliliter and that
horseshoe crabs is harvested for this purpose almost 1 billion bacteria can be found per
[15]. Harvesting horseshoe crab blood gram of sand near the shore [21]. Thus, the
involves collecting and bleeding the horseshoe crab habitat contains vast
animals, and then releasing them back into amounts of endotoxin, making it no
the sea. Most of the animals survive the coincidence that the horseshoe crab evolved
process; mortality is correlated with both the a vital system to protect itself against
amount of blood extracted from an endotoxins. The horseshoe crab blood
individual animal, and the stress experienced includes amebocytes that contain the
during handling and transportation [16]. clotting enzymes and other factors with the
Estimates of mortality rates following blood ability to immobilize and engulf an
harvesting vary from 3-15% [17] to 10-30% endotoxin [19]. When exposed to endotoxin,
[18]. the amebocytes change shape, adhere to the
sides of the vascular channels, and from the
The LAL test represents one of a number of resultant gel clot [4]. This phenomenon is at
pharmacological significant, chemical the heart of the LAL assay, as the formation
constituents found in marine flora and fauna of a clot shows the presence of endotoxin.
[19]. A wealth of significant compounds has
been isolated from marine animals. These The major use of LAL today is in the
include compounds derived from the sea detection of endotoxins in pharmaceutical
cucumber used in anti-cancer chemotherapy, products [20]. Since its original description,
hormones from gorgonians used for birth however, it has also been used in the
control, against peptic ulcers and asthma and diagnosis of endotoxemia in conjunction
lowering blood pressure, as well as with cirrhosis, cancer, meningitis, eye
compounds derived from red algae that can disease, dental problems, gonorrhea,
prevent atherosclerosis [19]. The discovery, boutonneuse fever, and water-quality
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analysis [21], as well as urinary tract mates during the spawning season. Each
infections [20]. In addition, new applications compound eye has about 1,000 receptors or
for LAL continue to be found, including the ommatidia [24]. The cones and rods of the
detection of bacterially contaminated meat, lateral eyes have a similar structure to those
fish, and dairy products, including frozen found in human eyes, but are around 100
items [20, 22]. times larger in size. The ommatidia are
adapted to change the way they function by
Study of blood chemistry day or night. At night, the lateral eyes are
chemically stimulated to greatly increase the
For a study of the plasma, blood cells are sensitivity of each receptor to light. This
immediately removed from the plasma by allows the horseshoe crab to identify other
centrifugation and the plasma can then be horseshoe crabs in the darkness. The
fractionated into its constituent proteins. The horseshoe crab has an additional five eyes
blood cells are conveniently studied on the top side of its prosoma. Directly
microscopically by collecting small volumes behind each lateral eye is a rudimentary
of blood into LPS-free isotonic saline (0.5 M lateral eye. Towards the front of the
NaCl) under conditions that permit direct prosoma is a small ridge with three dark
microscopic examination by placing one of spots. Two are the median eyes and there is
more LPS-free cover glasses on the culture one endoparietal eye.
dish surface, then mounting those cover
glasses in simple observation chambers Each of these eyes detects ultraviolet (UV)
following cell attachment. A second light from the sun and reflected light from
preparation for direct observation is to the moon. They help the crab follow the
collect 3 -5 ml of blood in an LPS-free lunar cycle. This is important to their
embryo dish and then explanting fragments spawning period that peaks on the new and
of aggregated amebocytes to a chamber that full moon. Two ventral eyes are located near
sandwiches the tissue between a slide and a the mouth but their function is unknown.
cover glass. In this preparation, the motile Multiple photoreceptors located on the
amebocytes migrate onto the cover glass telson constitute the last eye. These are
surface, where they can readily be observed. believed to help the brain synchronize to the
The blood clotting system involves cycle of light and darkness. The research
aggregation of amebocytes and the into their eyes has helped the study and
formation of an extracellular clot of a understanding of how the human eye works.
protein, coagulin, which is released from the
secretory granules of the blood cells. The horseshoe crab (also known as the king
Biochemical analysis of washed blood cells crab), is a hard-shelled invertebrate found on
requires that aggregation and degranulation the sea floor in warmer climates. Despite its
does not occur, which can be accomplished name, the horseshoe crab is not actually a
by collecting blood into 0.1 volumes of 2% crab (or crustacean) and is in fact more
Tween-20, 0.5 M LPS-free NaCl, followed closely related to arachnids such as spiders
by centrifugation of the cells and washing and scorpions. The horseshoe crab is a
with 0.5 M NaCl [23]. primitive-looking arthropod with a hard,
brownish-green exoskeleton and a spike-like
Horseshoe crabs have a total of 10 eyes (Fig tail. Horseshoe crabs have bright blue blood
1) used for finding mates and sensing light. and it gets clotted when it comes into
The most obvious eyes are the 2 lateral contact with foreign bacteria.
compound eyes. These are used for finding
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