Download IJMS 45(10) 1234-1244

Indian Journal of Geo-Marine Sciences
Vol. 45(10), October 2016, pp. 1234-1244
Review Article
Horseshoe crabs: biomedical importance and its potential use in developing
health-care products
Vikash Kumar1*, Suvra Roy1, A.K. Sahoo1 & Vikas Kumar2
1
2
Central Inland Fisheries Research Institute (CIFRI), Barrackpore, 700120, India
Division of Aquaculture, Kentucky State University, Frankfort, KY, 40601, USA
*[E-mail: [email protected]]
Received 10 February 2015; revised 23 March 2016
Horseshoe crabs have been a model for many biomedical science studies. Medicinal value of horseshoe crabs
comes from its blue blood, eye and exoskeleton (chitin). Ability of blood to clot in the presence of bacteria,
rendering the bacteria harmless has created its biomedical importance. Blood cells (amebocytes) carry Factor C,
which binds lipopolysaccharide (LPS), undergoes a structural reorganization, then auto-proteolytically activates
itself to initiate the clotting pathway that eventually results in a proteolytic modification of the zymogen,
coagulogen, which then self-polymerizes into the insoluble fibrils of the extracellular blood clot. Blood-clotting
ability of the horseshoe crab makes it very valuable in testing for injectable medicines, vaccines and sterile medical
equipment. Secondly, the nerve pathways in the eyes of horseshoe crabs have led to many discoveries in human eye
research. Furthermore, the outer shell of a horseshoe crab is made primarily of chitin and being used as a coating for
suture material and burn dressings, rapidly increases the wound healing, cutting the time by half.
[Keywords: horseshoe crab, amebocytes, Factor C, lipopolysaccharide, chitin]
Introduction
Limulus polyphemus (Linnaeus, 1758), commonly
known as Atlantic horseshoe crabs along with
Limulus albus (Bosc, 1802), Limulus americanus
(Leach, 1819), Limulus cyclops (Fabricius, 1793),
Limulus occidentalis (Lamarck, 1801), Limulus
sowerbii (Leach, 1815), Tachypleus gigas (Müller,
1785), Limulus rotundicauda (Latreille, 1802),
Tachypleus tridentatus (Leach, 1819), and Monoculus
polyphemus (Linnaeus, 1758), were originally
classified as horseshoe crabs. They have wide
geographical distribution e.g mangrove horseshoe
crab (Carcinoscorpius rotundicauda) reported from
Bay of Bengal, Thailand, Malaysia, Philiphines,
Borneo and Torres Straits; Atlantic horseshoe crab
(Limulus polyphemus) from Atlantic Coast of North
America from Maine to Yucatan; Southeast Asian
horseshoe crab (Tachypleus gigas) from Bay of
Bengal (North-East Coast), Thailand, Malaysia,
Philiphines, Borneo & Torres Straits and Tri-spine
horseshoe crab (Tachypleus tridentatus) from Western
& Southern Japan, Taiwan, Philiphines & North
Borneo, Malaysia1. They are a distant relative of
crustaceans external link, and are more closely related
to arachnids such as spiders, scorpions and ticks.
Although they look prehistoric, and ancient relatives
of Limulus polyphemus were present 520 million
years ago as evidenced by fossils, this species has
only been around for about 20 million years, which is
not enough time to consider this animal a "living
fossil" as they are sometimes called. Despite
inhabiting the planet for so long, horseshoe crab body
forms have changed very little over all of those years.
The strange anatomy of the horseshoe crab is one of
this animal's most notable aspects. Unfortunately, the
long, thin, spike-like tail of horseshoe crabs has given
this species an unfavorable reputation. Many people
view horseshoe crabs as dangerous animals because
they have sharp tails but in reality, horseshoe crabs
are harmless. Their tails are used primarily to flip
themselves upright if they are accidentally
overturned2.
The horseshoe crab plays a vital (little-known) role
in the human medication. An extract of the horseshoe
1235
KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE
crab's blood is used by the pharmaceutical and
medical device industries to ensure that their
products, e.g., intravenous drugs, vaccines, and
medical devices, are free of bacterial contamination.
Horseshoe crabs have 10 eyes located all over their
bodies and most of the eyes are located on the back or
sides of the animal. Some eyes contain photoreceptors
such are located on their tails. The eyes found on the
back are having about 1,000 photoreceptor clusters or
ommatidia, each with a lens, cornea and
photoreceptor cells. Horseshoe crabs have the largest
rods and cones of any known animal that are about
100 times the size of humans and research work is
going on to reveal the understanding of the horseshoe
crab eyes working mechanism. Similarly, the outer
membranes know as chitin is mostly used for coating
for suture material and burn dressings, rapidly
increase the wound healing, cutting. This review
highlights the fundamental mechanism, that involve
blood clotting and clotting factors, mechanism of eye
for biomedical research and potential use in health
care products.
Horseshoe crab model for innate immune system
The innate immune system is considered as first
line of inducible host defense against bacterial,
fungal, and viral pathogens3. This defense system is
essential for the survival and perpetuation of all
multicellular organisms4,5. Invertebrates, which do not
possess immunoglobulins, have developed unique
modalities to detect and respond to microbial surface
antigens like lipopolysaccharides (LPS), lipoteichoic
acids, lipoproteins, peptidoglycan (PGN) and (1 → 3)
β-D-glucans6. Because both invertebrates and
vertebrates respond to these substances, it is likely
that a system recognizing these epitopes emerged at
an early stage in the evolution of animals7,8.
Moreover, it is well known that various microbial cell
wall components elicit a variety of responses that
depend on the species and cell type4,9. The major
biological host defense systems of invertebrates
includes hemolymph coagulation system, Prophenoloxidase (pro-PO) activating system, lectincomplement system, agglutinin-lectin system,
antibacterial, antifungal, and antiviral systems
mediated by toll-like receptors and peptidoglycan
binding protein (PGBP), reactive oxygen-producing
system and phagocytic system8,10.
After antigen recognition, several mechanism i.e.,
toll-like receptor-mediated antimicrobial peptide
production11, hemolymph coagulation12, melanin
formation13, and lectin-mediated complement
activation14 involve in the process of immune defence
in invertebrates. In addition to these enzyme cascades,
a variety of agglutinin-lectins and reactive oxygen
producing and phagocytic systems cooperate with
immune reactions to kill invading pathogens15. Figure
1 shows the principal defense systems associated with
phagocytosis. Invaders detected by these systems are
ultimately engulfed by phagocytes, such as
macrophage-like, neutrophils like and dendritic cells,
and are then internalized as phagosomes and finally
killed10,16.
Antimicrobial peptides
Phagocyte
Toll-like receptor system
Protease cascade
Phagocyte
Lectin complement
network
Biosensor
Invaders
Biosensor
Biosensor
Protease cascade
Phenol oxidase system
Coagulation cascade
clot system
Melanin formation
Antimicrobial substances
Phagocyte
Fig. 1- The principal host defense systems associated with
phagocytosis in invertebrates.
The major innate immune systems include;
hemolymph coagulation, melanization mediated by
phenoloxidase, the expression of antimicrobial
peptides mediated by Toll-like receptors and the
immuno deficiency (IMD) pathway, and the
lectin/complement pathway mediated by bacterial cell
wall components. Invaders detected by these systems
are ultimately engulfed by phagocytic cells, such as
macrophage-like, neutrophil-like, or dendritic cells,
and then internalized, processed, and killed10.
Hemolymph and circulating hemocytes
The innate immune system of horseshoe crab is
mainly involved in defence response by employing
unique and highly efficient host defense systems17.
Hemolymph and hemocytes plays fundamental
defence mechanism during infection. Hemolymph
plasma of this animal contains many soluble defense
molecules, such as hemocyanins, various lectins, and
C-reactive proteins, and thioester bond containing
proteins (α2-macroglobulins), in addition to a large
numbers of granular hemocytes (amebocytes), which
undergo a rapid degranulation on contact with
pathogens18. Hemocytes, which are composed of more
than 99 % of circulating cells, contain a variety of
1236
INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016
defense molecules, which are located in two types of
secretary granules viz large (L)-and small (S) (Fig.
2)19,20. L-granules selectively store more than 25
defense components with molecular masses between
8 and 120 kDa. These include clotting factors, a
clottable protein coagulogen, proteinase inhibitors,
lectins, and antimicrobial proteins. In contrast, the Sgranules contain at least six antimicrobial peptides
and several proteins of molecular mass <30 kDa.
These peptides include large amounts of hairpin-like
tachyplesin (17-18 amino acid residues, >10mg per
individual), tachystatins (41-44 amino acid residues),
tachycitins (73 amino acid residues) and big defensins
(79 amino acid residues), which are highly active
against Gram-negative and -positive bacteria and
fungi21,22.
Fig. 2- Electron micrograph of horseshoe crab (T. tridentatus)
hemocytes, and major defense molecules that have been identified
in large and small cell granules (Source: Iwanaga and Lee 2005).
Various proteins and peptides that are identified in
T. tridentatus hemocytes and hemolymph plasma are
summarized in Table 1. Hemolymph plasma of
Tachypleus tridentatus contains three predominant
protein types, namely, hemocyanin (O2 transporter),
C-reactive
proteins
(CRP)23,
and
α2macroglobulins24,25,26.
Moreover,
circulating
hemocytes are extremely sensitive to bacterial LPS,
and respond by degranulating a number of granular
components after LPS-mediated stimulation, which
results in the formation of hemolymph clot. This rapid
clotting response is believed to be important for the
animal’s host defense, which involves engulfing of
invading microbes, and in addition prevents
hemolymph leakage27.
Table 1 Defense molecules found in hemocytes and hemolymph
plasma of the horseshoe crab
and
Proteins
peptides
Coagulation
factors
Factor C
Factor B
Factor G
Proclotting
enzyme
Coagulogen
Protease inhibitors
LICI-1
LICI-2
Mass
(kDa)
Function/
specificity
Localization
123
64
110
54
Serine protease
Serine protease
Serine protease
Serine protease
L-granule
L-granule
L-granule
L-granule
20
Gelation
L-granule
48
42
L-granule
L-granule
LICI-3
Trypsin inhibitor
LTI
LEBP-PI
Limulus cystatin
53
6.8
16
12
12.6
α2-Macroglobulin
180
Serpin/factor C
Serpin/clotting
enzyme
Serpin/factor G
Kunitz-type
New type
New type
Cystatin family
2
Complement
Chymotrypsin
inhibitor
Antimicrobial
substances
Anti-LPS factor
Tachyplesins
Polyphemusins
Big defensin
10
ND
12
2.3
2.3
8.6
GNB
GNB, GPB, FN
GNB, GPB, FN
GNB, GPB, FN
Tachycitin
Tachystatins
Factor D
Lectins
Tachylectin-1
8.3
6.5
42
GNB, GPB, FN
GNB, GPB, FN
GNB
27
Tachylectin-2
Tachylectin-3
27
15
Tachylectin-4
470
Tachylectin-5
Limunectin
18K-LAF
380-440
54
18
Limulin
300
LCRP
TCRP-1
TCRP-2
TCRP-3
Polyphemin
TTA
300
300
330
340
ND
ND
Liphemin
Carcinoscorpin
GBP
400-500
420
40
(KDO),
LPS
LTA
GlcNAc, LTA
(OLPS
antigen)
LPS
(Oantigen), LTA
N-acetyl group
PC
Hemocyte
aggregation
HLA/PC, PE,
SA, KDO
PC, PE
PE
HLA/PE, SA
HLA/SA, KDO
LTA, GlcNAc
SA, GlcNAc,
GalNAc
SA
SA, KDO
Gal
L-granule
ND
ND
L-granule
L-granule
Plasma & Lgranule
Plasma
L-granule
S-granule
S-granule
L
&
Sgranule
S-granule
S-granule
L-granule
L-granule
L-granule
L-granule
ND
Plasma
L-granule
L-granule
Plasma
Plasma
Plasma
Plasma
Plasma
Plasma
Plasma
Hemolymph
Hemolymph
Hemolymph
KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE
PAP
(1→ 3) β-D-glucan
binding protein
Others
Transglutaminase
(TGase)
8.6 kDa protein
Pro-rich proteins
(Proxins)
Limulus kexin
40
168
Protein A
Pachyman,
cardlan
Hemolymph
Hemocyte
86
Cross-linking
Cytosol
8.6
80
TGase substrate
TGase substrate
L-granule
L-granule
70
ND
Hemocyanin
3600
Toll-like receptor
(tToll)
L1
L4
110
Precursor
processing
O2 transporter
(PO activity)
ND
11
11
Unknown
Unknown
L-granule
L-granule
Plasma
Hemocyte
Mechanism of clot formation
Vertebrate and invertebrate animals have evolved
efficient molecular mechanisms to form clots through
a sequential process using blood components. This is
vital in preventing loss of blood in case of injury and
as defence mechanism against certain microbes. All
vertebrates have similar coagulation systems based on
the proteolytically induced aggregation of fibrinogen
into insoluble fibrin28,29. The fibrin aggregates, which
initially are noncovalently associated, are further
stabilized by intermolecular covalent crosslinks
formed by a proteolytically activated transglutaminase
(TGase), factor XIIIa. TGases (EC 2.3.2.13) are Ca2+dependent enzymes capable of forming covalent
bonds between the side chains of specific lysine and
glutamine residues on certain proteins30,31. Among the
invertebrates, which is a more diverse group, different
coagulation mechanisms seem to have evolved, and
detailed information on the coagulatory mechanisms
at the molecular level is lacking in most groups. One
exception is the hemocyte (blood cell)-derived
clotting cascade in horseshoe crabs, which has been
characterized in detail32. The clotting system in
horseshoe crab is activated by microbial
lipopolysaccharides or β-1,3-glucans, and it has some
resemblance to the vertebrate coagulation system, as it
is based on a proteolytic cascade leading to the
conversion of a soluble protein (coagulogen) into an
insoluble aggregate (coagulin). However, the proteins
participating in the Limulus clotting system are all
from the hemocytes and are not homologous to the
vertebrate plasma coagulation proteins. A TGase has
been characterized and cloned from Limulus
hemocytes, but it does not appear to recognize the
coagulogen as a substrate33,34, and its role during
1237
clotting is unclear35.
In crustaceans the clotting reaction are
characterized and observed that TGase-mediated
cross-linking of a specific plasma clotting protein
(CP)36,37. The crayfish CP, has been biochemically and
functionally characterized36,38. It is a very high density
lipoprotein (VHDL)38 consisting of two identical 210kDa subunits held together by disulfide bonds36. Each
one of the 210-kDa subunits has both lysine and
glutamine side chains, which are recognized and
become covalently linked to each other by TGases36.
Clotting is induced when a TGase is released from
hemocytes or tissue becomes activated by the Ca2+content in plasma, and starts cross-linking the plasma
CP molecules into large aggregates. The hemocytes
also contain components of the so-called
prophenoloxidase activating system (proPO system),
that constitutes an important part of the immediate
immune response in crustaceans39,40. Components of
the proPO system cause degranulation and lysis of
hemocytes, and as a result more proPO components
and TGase are released39,40. In this way, the proPO
system could affect the clotting reaction by causing
the release of TGase activity. However, the proPO
system and the clotting reaction do not appear to share
a common activation pathway, as the proPO system is
activated by a proteolytic cascade [triggered by
microbial polysaccharides], and the initiation of the
clotting reaction requires no proteolytic processing
(only Ca2+, which activates the TGase)36.
In lobster the N terminus of the fibrinogen (the CP
homologue in lobster) was reported to have sequence
similarity to vitellogenins (VTGs)41, which are
proteins expressed only in females of egg-laying
animals (vertebrates as well as invertebrates)42,43.
Besides having similar functions, the CP does not
appear to share any characteristics with fibrinogen or
coagulogen, the proteins forming clots in vertebrate
animals and horseshoe crabs, respectively. This
indicates that the crayfish CP36,38 and its homologues
in other crustaceans37,41 constitute a separate group of
blood CPs35. Recently Sahoo et al.44 reported
hemolymph clot in shimp, p.monodon against the
white spot syndrome virus infection. Further, author
hypothesized that hemolymph clot may progress to
melanin formation which is having antimicrobial
activity.
Hemolymph clotting system in horseshoe crab
The hemolymph-clotting phenomenon was first
1238
INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016
identified as a prominent defense system in the
horseshoe crab (Limulus polyphemus) by Bang45.
When Gram-negative bacteria invade the hemolymph,
hemocytes detect LPS molecules on their surfaces46,
and then release, via rapid exocytosis, the contents of
L- and S-granules47. These released granular
components include two biosensors, named factors C
and G. These two factors are serine protease
zymogens and are autocatalytically activated by LPS
or (1→ 3)-β-D-glucan, which are major components
of the cell walls of Gram-negative bacteria and fungi,
respectively. In 1996, Tamura et al.48 reported that
hemocytes contain a (1→ 3)-β-D-Glucan binding
protein, which differs from factor G as it does not
participate in the hemolymph clotting cascade. One of
the authors of this review has previously described in
detail LPS and (1→ 3)-β-D-glucan-mediated clotting
cascades and their molecular structures, and the
functions of the five clotting factors, factor C, factor
G, factor B, proclotting enzyme, and clottable
coagulogen, which all participate in clotting
cascades17,19,27,47. Figure 3 illustrates the LPS and (1→
3)-β-D-glucan-mediated clotting cascades of the
hemolymph of T. tridentatus21, and includes limulus
intracellular coagulation inhibitors (LICI), which act
as regulators of the cascade reaction49. These clotting
cascades both involve four serine protease zymogens,
factors C (123 kDa), B (64 kDa), G (110 kDa),
proclotting enzyme (54 kDa), and coagulogen (20
kDa)50,51. In the presence of LPS or synthetic lipid A
analogs, factor C is autocatalytically activated to an
active form, factor C17,52. Factor B zymogen is then
activated by factor C to its active form (factor B),
which activates proclotting enzyme to clotting
enzyme53. Clotting enzyme then converts coagulogen
to an insoluble coagulin gel, which is composed of
non-covalent homopolymers, through head to tail
interaction54. On the other hand, factor G zymogen
consisting
of
two
heterosubunits and
is
autocatalytically activated in the presence of (1→ 3)β-Dglucan, in the absence of any other protein55. The
resulting active factor G activates proclotting enzyme
directly, resulting in coagulin gel formation56.
Recently, Osaki et al.57 found that non-covalent
coagulin homopolymers are cross-linked by bridging
hemocyte cell surface proteins, named proxins, in the
presence of hemocyte-derived transglutaminase58,59.
This indicates that cross-linking is important at the
final stage of hemolymph clotting to facilitate
hemostasis and wound healing, as has been reported
in the mammalian blood clotting system47.
Interestingly, the NH2-terminal portions of zymogen
factor B and of proclotting enzyme contain a small
compact domain containing three disulfide bonds,
called the clip domain10,19. A similar clip domain has
also been reported in the NH2-terminal proenzyme
regions of Drosophila-derived serine proteases.
Moreover, the folding pattern of the three disulfide
bridges located in the clip domain is identical to that
of big defensin, which was recently identified as an
antimicrobial peptide in T. tridentatus hemocytes. As
the COOH-terminal end of the clip domain in
proclotting enzyme constitutes a hinge region
susceptible to proteolysis, the clip domain, in the same
manner as defensin, might be released during the
activations of serine protease zymogens, in order to
act as an antimicrobial substance. In fact, the clip
domain derived from the prophenoloxidase activated
serine protease of freshwater crayfish has an
antimicrobial activity similar to that of human βdefensin60. Thus, the clotting cascade could also
produce antimicrobial agents, and thus provide a dual
action clotting and killing system against invaders61,62.
Lipopolysaccharide
(LPS)
β-1,3-Glucan
Factor C
Factor C
LICI 1
Factor B
Factor G
Factor G
LICI 3
Factor B
Clotting enzyme
Proclotting
enzyme
LICI 2
Gelation
Cell agglutination
etc.
Coagulin
Coagulogen
Fig. 3- LPS- and (1→ 3)-β-D-glucan mediated clotting cascades
found in horseshoe crab (T. tridentatus) hemocytes. LICI,
(Limulus intracellular coagulation inhibitor).
The biochemical principle of the so called limulus
test, which is used for detecting bacterial endotoxins
are shown in figure 3. The method was developed by
Levin and Bang63 based on a finding that a trace
amount of endotoxin coagulates the hemocyte
(amebocyte) lysate of the American horseshoe crab,
Limulus polyphemus. This gelation reaction has been
widely employed as a simple and highly sensitive
KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE
1239
assay for endotoxins (LPS). The limulus test is 3-15% to 10-30 %67. The LAL test represents one of a
dependent on the protease cascade reaction shown in number of pharmacological significant, chemical
the figure, and is being used extensively in constituents found in marine flora and fauna68. A
combination with new technology10,12,64,65.
wealth of significant compounds has been isolated
from marine animals. These include compounds
derived from the sea cucumber used in anti-cancer
Potential application of horseshoe crab in human
chemotherapy, hormones from gorgonians used for
medicine
The horseshoe crab has the best-characterized
birth control, against peptic ulcers and asthma and
immune system of any long-lived invertebrates. When
lowering blood pressure, as well as compounds
a foreign object (bacteria) enters through a wound in
derived from red algae that can prevent
their body, it almost immediately clots into a clear, gel
atherosclerosis68. The discovery, commercialization,
like material, thus effectively trapping the bacteria. If
and use of LAL have been an important improvement
the bacterium is harmful, the blood will form a clot.
to the pharmaceutical industry. Prior to the use of
Horseshoe crabs are proving to be very helpful in
LAL, compounds were tested for the presence of
finding remedies for diseases that have built
endotoxins in a variety of ways that involved living
immunities against penicillin and other drugs64.
animals or living parts of animals68. Thus, LAL
provides a means to detect endotoxins without having
The study of immunity in horseshoe crabs has
been facilitated by the ease in collecting large
to kill or disable animals64.
volumes of blood and from the simplicity of the
blood. Horseshoe crabs show only a single cell type in
the general circulation, the granular amebocyte. The
plasma has the salt content of sea water and only three
abundant proteins, hemocyanin, the respiratory
protein, the C-reactive proteins, which function in the
cytolytic destruction of foreign cells, including
bacterial cells, and α2-macroglobulin, which inhibits
the proteases of invading pathogens. Blood is
collected by direct cardiac puncture under conditions
that minimize contamination by lipopolysaccharide
(endotoxin, LPS), a product of the Gram-negative
bacteria. A large animal can yield 200 - 400 ml of
blood (Fig. 4). Unlike vertebrates, horseshoe crabs do
not have hemoglobin in their blood, but instead use
hemocyanin to carry oxygen. Because of the copper
present in hemocyanin, their blood is blue. Their
blood contains amebocytes, which play a role similar
to white blood cells of vertebrates in defending the
organism against pathogens. Amebocytes from the
blood of L. polyphemus are used to make Limulus
amebocyte lysate (LAL), which is used for the
detection of bacterial endotoxins in medical
applications. The blood of horseshoe crabs is
harvested for this purpose66. Harvesting horseshoe
crab blood involves collecting and bleeding the
http://www.wired.com
animals, and then releasing them back into the sea.
Fig. 4- Extraction of blood from horseshoe crabs
Most of the animals survive the process; mortality is
correlated with both the amount of blood extracted
Limulus Amebocyte Lysate is extremely useful in
from an individual animal, and the stress experienced
detecting
those toxins that cause fever – the bacterial
during handling and transportation. Estimates of
pyrogens
or endotoxins. Endotoxins occur as a
mortality rates following blood harvesting vary from
1240
INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016
structural component of the cell wall of a large group
of bacteria known as gram negative69. Most aquatic
bacteria are of the gram-negative variety, as studies at
the Woods Hole Oceanographic Institution have
shown that seawater contains over 1 million Gramnegative bacteria per milliliter and that almost 1
billion bacteria can be found per gram of sand near the
shore70. Thus, the horseshoe crab habitat contains vast
amounts of endotoxin, making it no coincidence that
the horseshoe crab evolved a vital system to protect
itself against endotoxins. The horseshoe crab blood
includes amebocytes that contain the clotting enzymes
and other factors with the ability to immobilize and
engulf an endotoxin68. When exposed to endotoxin,
the amebocytes change shape, adhere to the sides of
the vascular channels, and form the resultant gel
clot71. This phenomenon is at the heart of the LAL
assay, as the formation of a clot shows presence of
endotoxin. The major use of LAL today is in the
detection of endotoxins in pharmaceutical products69.
Since its original description, however, it has also
been used in the diagnosis of endotoxemia in
conjunction with cirrhosis, cancer, meningitis, eye
disease, dental problems, gonorrhea, and water-quality
analysis70, as well as urinary tract infections69. In
addition, new applications for LAL continue to be
found, including the detection of bacterially
contaminated meat, fish, and dairy products, including
frozen items72.
Blood chemistry
For study of the plasma, blood cells are
immediately removed from the plasma by
centrifugation and the plasma can then be fractionated
into its constituent proteins. The blood cells are
conveniently studied microscopically by collecting
small volumes of blood into LPS-free isotonic saline
(0.5 M NaCl) under conditions that permit direct
microscopic examination by placing one of more
LPS-free cover glasses on the culture dish surface,
then mounting those cover glasses in simple
observation chambers following cell attachment. A
second preparation for direct observation is to collect
3 -5 ml of blood in a LPS-free embryo dish and then
explanting fragments of aggregated amebocytes to a
chamber that sandwiches the tissue between a slide
and a cover glass (Fig. 5). In this preparation, the
motile amebocytes migrate onto the cover glass
surface, where they can readily be observed.
Separation of amoebocytes
Horseshoe crab
Pharmaceuticals products
Extraction of blood
Diagnosis/ treatment
Food products
Clinical diagnosis
Eye disease
Spinal meningitis
Body and mental exhaustion
Drowsiness after sea bathing
Gastroentric symptoms
Pain in the body
Urinary infection
Rheum atism
Fig. 5- Use of Limulus amebocyte lysate (LAL)
The blood clotting system involves aggregation of
amebocytes and the formation of an extracellular clot
of a protein, coagulin, which is released from the
secretory granules of the blood cells. Biochemical
analysis of washed blood cells requires that
aggregation and degranulation does not occur, which
can be accomplished by collecting blood into 0.1
volumes of 2% Tween-20, 0.5 M LPS-free NaCl,
followed by centrifugation of the cells and washing
with 0.5 M NaCl73.
Principle of Limulus test
Limulus test, a test for detecting nano gram of
bacterial endotoxins, was invented by Levin and Bang
based on their finding that a trace amount of endotoxin
coagulates hemocyte lysate of the horseshoe crab,
Limulus polyphemus63. This gelation reaction has been
widely employed as a simple and very sensitive assay
method for bacterial endotoxins. The original method is
qualitative or semi-quantitative; the presence of
endotoxin is determined by reading the formation of gel
clot after incubation of a sample with the hemocyte
lysate at 37 °C for 1 hour (Limulus gelation test).
During the past decade, the studies on molecular
mechanism of hemolymph coagulation in horseshoe
crab, established a protease cascade described above.
Because the Limulus lysate contains all the enzymes
described above, the Limulus test reacts with (1,3)-β-D-
KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE
glucan as well as endotoxin. The latter activates factor
C, whereas the former activates factor G; both pathways
converge on proclotting enzyme, ensuing its activation
and hydrolysis of a chromogenic peptide substrate12.
The chromogenic substrate used for specific assay of
bacterial endotoxins is Boc-Leu-Gly-Arg-p-nitroanilide
(pNA). The sequence of this substrate originates from
the sequences located close to the site cleaved during
the gelation of coagulogen by Limulus clotting enzyme.
The chromogenic substrate is hydrolyzed by clotting
enzyme to release pNA. By measuring the absorbance
of released pNA at 405 nm, endotoxin concentration in
the samples can be determined. Endotoxin
concentration can also be determined by measuring the
absorbance at 545 nm after the diazo coupling of pNA,
when a yellowish color in samples interferes with the
measurement at 405 nm64. The methods described
above are a 100 times more sensitive than the limulus
gelation test and are very reproducible. If this technique
is to be applied to blood samples, however, the
activities of limulus test-interfering factors in the
samples, such as thrombin, blood coagulation factor Xa,
and α1-antitrypsin, need to be abolished. To remove
such interferences, various methods have been studied
and applied to blood samples, such as pretreatment with
chloroform, ether, acid, or alkali and heating65.
Horseshoe crabs eye anatomy and importance
Horseshoe crabs have a total of 10 eyes used for
finding mates and sensing light. The most obvious
eyes are the 2 lateral compound eyes. These are used
for finding mates during the spawning season. Each
compound eye has about 1,000 receptors or
ommatidia74. The cones and rods of the lateral eyes
have a similar structure to those found in human eyes,
but are around 100 times larger in size. The
ommatidia are adapted to change the way they
function by day or night. At night, the lateral eyes are
chemically stimulated to greatly increase the
sensitivity of each receptor to light. This allows the
horseshoe crab to identify other horseshoe crabs in the
darkness. The horseshoe crab has an additional five
eyes on the top side of its prosoma. Directly behind
each lateral eye is a rudimentary lateral eye. Towards
the front of the prosoma is a small ridge with three
dark spots. Two are the median eyes and there is one
endoparietal eye. Each of these eyes detects ultraviolet
(UV) light from the sun and reflected light from the
moon. They help the crab follow the lunar cycle. This
is important to their spawning period that peaks on the
1241
new and full moon. Two ventral eyes are located near
the mouth but their function is unknown. Multiple
photoreceptors located on the telson constitute the last
eye. These are believed to help the brain synchronize
to the cycle of light and darkness. The research into
their eyes has helped the study and understanding of
how the human eye works.
Chitin
Chitin, a cellulose-like component from the shell
of the horseshoe crab, is non-toxic, biodegradable and
used in contact lenses, skin creams and hair sprays. It
is also used to make chitin-coated sutures and wound
dressings for burn victims. The chitin-coated sutures
reduce healing time by 35% to 50%. When chitin is
processed, another substance, called chitosan, is
produced and can be used as a raw material to
manufacture a variety of important products.
Conclusion
Horseshoe crabs are chelicerates, distant relatives
of spiders. They are often referred to as living fossils,
as they have changed little morphologically in the last
445 million years. There are four extant species of
horseshoe crabs. The species Limulus polyphemus
occurs only along the eastern coast of the USA. The
other three species, Tachypleus tridentatus,
Tachypleus gigas and Carcinoscorpius rotundicauda
live along the coast of the Indo-West Pacific. In Asian
waters, habitat degradation especially the loss of
spawning and nursery grounds, marine pollution and
human exploitation have resulted in a decline in
horseshoe crab populations.
Horseshoe crab having bright blue blood contains
blood cells amebocytes carry protein called coagulogen
which plays an important role in blood clotting and
trapping of bacteria when it comes into contact with
foreign bacteria. The mechanism of blood clotting and
entrapment of foreign bacteria drawing attention to
many researchers and many healthcare products are
been getting developed like pharmaceutical drugs etc. in
human medicine. Horseshoe crabs have 2 large
compound eyes located on the top of the shell. These
eyes are made up of a thousand light sensors that see in
shades of gray. The crab combines all these separate
sensors together as an image that they see. These eyes
are probably used for finding a mate. The compound
eyes are larger and have an optical nerve that is easy to
identify making the crabs ideal for studying how an eye
works. The eyes have the ability to detect UV light and
1242
INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016
are sensitive enough that the horseshoe crab sees as
well at night as it does during the day. Scientist and
researchers are putting effort in horseshoe crab eyes to
know the mechanism how their eyes work so that it can
give some light in human eyes working mechanism.
Acknowledgements
Authors are thankful to all the Central Inland Fisheries
research Institute (CIFRI) for ample help and support.
16
17
18
19
20
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Sadava D., Heller H.C., Hillis D.M. and Berenbaum M., Life:
the Science of Biology (9th ed.). W. H. Freeman, (2009) p.
683. ISBN 978-1-4292-1962-4.
Kumar, V., Roy, S., Sahoo, A.K., Behera, B.K. and Sharma,
A.P., Horseshoe crab and its medicinal values, Int. J. Curr.
Microbiol. App. Sci., 4(2)(2015) 956-964.
Hoebe, K., Jansen, E. and Beutler, B., The interface between
innate and adaptive immunity. Nat. Immunol., 5(2004) 971974.
Hoffmann, J.A., Kafatos, F.C., Janeway, C.A.Jr. and
Ezekowitz, R.A.B., Phylogenetic perspectives in innate
immunity. Science, 284(1999) 1313-1318.
Salzet, M., Vertebrate innate immunity resembles a mosaic of
invertebrate immune responses. Trends Immunol., 22(2001)
285-288.
Begum, N., Matsumoto, M., Tsuji, S., Toyoshima, K. and
Seya, T., The primary host defense system across humans,
flies and plants, Current Trends in Immunology, 3(2000) 5974.
Medzhitov, R. and Janeway, C. Jr., Innate immune
recognition: mechanisms and pathways. Immunol. Rev.,
173(2000) 89-97.
Aderem, A. and Ulevitch, R., Toll-like receptors in the
induction of the innate immune response, Nature, 406(2000)
782-787.
Cooper, E.L., Kauschke, E. and Cossarizza, A., Digging for
innate immunity since Darwin and Metchnikoff, BioEssays,
24(2002) 319-333.
Iwanaga S. and Lee B.L., Recent Advances in the Innate
Immunity of Invertebrate Animals. Journal of Biochemistry
and Molecular Biology, 38(2)(2005) 128-150.
Krutzik, S. R., Sieling, P.A. and Modlin, R.L., The role of
Toll-like receptors in host defense against microbial infection,
Curr. Opin. Immunol., 13(2001) 104-108.
Iwanaga, S., Morita, T., Harada, T., Niwa, M., Takada, K.,
Kimura, T. and Sakakibara, S., Chromogenic substrates for
horseshoe crab clotting enzyme. Its application for the assay
of bacterial endotoxins, Haemostasis, 7(1978) 183-188.
Sugumaran,
M.,
Comparative
biochemistry
of
eumelanogenesis and the protective roles of phenoloxidase
and melanin in insects, Pigment Cell Res., 15(2002) 2-9.
Fujita, T., Evolution of the lectin-complement pathway and its
role in innate immunity, Nat. Rev. Immunol., 2(2002) 346353.
Bogdan, C., Röllinghoff, M. and Diefenbach, A., Reactive
oxygen and reactive nitrogen intermediates in innate and
specific immunity, Curr. Opin. Immunol., 12(2000) 64-76.
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Greenberg, S. and Grinstein, S., Phagocytosis and innate
immunity, Curr. Opin. Immunol., 14(2002) 136-145.
Iwanaga, S., Miyata, T., Tokunaga, F. and Muta, T.,
Molecular mechanism of hemolymph clotting system in
Limulus, Thrombosis Res., 68(1992) 1-32.
Iwanaga, S., Kawabata, S. and Muta, T., New types of clotting
factors and defense molecules found in horseshoe crab
hemolymph: their structures and functions, J. Biochem.,
123(1998) 1-15.
Muta, T. and Iwanaga, S., The role of hemolymph coagulation
in innate immunity, Curr. Opin. Immunol., 8(1996b) 41-47.
Iwanaga, S. and Kawabata, S., Evolution and phylogeny of
defense molecules associated with innate immunity in
horseshoe crab, Front. Biosci., 3(1998) 973-984.
Iwanaga, S., Muta, T., Shigenaga, T., Miura, Y., Seki, N.,
Saito, T and Kawabata, S., Role of hemocyte-derived granular
components in invertebrate defense, Ann. NY Acad. Sci.,
712(1994a) 102-116.
Iwanaga, S., The molecular basis of innate immunity in the
horseshoe crab, Curr. Opin. Immunol., 14(2002) 87-95.
Iwaki, D., Osaki, T., Mizunoe, Y., Wai, S. N., Iwanaga, S. and
Kawabata, S., Functional and structural diversities of
Creactive proteins present in horseshoe crab hemolymph
plasma, Eur. J. Biochem., 264(1999) 314-326.
Iwaki, D., Kawabata, S., Miura, Y., Kato, A., Armstrong, P.
B., Quigley, J. P., Nielsen, K. L., Dolmer, K., Sottrup-Jensen,
L. and Iwanaga, S., Molecular cloning of limulus alpha 2
macroglobulin, Eur. J. Biochem., 242(1996) 822-831.
Armstrong, P.B.,, The contribution of proteinase inhibitors to
immune defense, Trends Immunol., 22(2001) 47-52.
Husted, L. B., Sorensen, E. S., Armstrong, P. B., Quigley, J.
P., Kristensen, L. and Sottrup-Jensen, L., Localization of
carbohydrate attachment sites and disulfide bridges in Limulus
α2-macroglobulin: Evidence for two forms differing primary
in their bait region sequences, J. Biol. Chem., 277(2002)
43698-43706.
Muta, T. and Iwanaga, S., Clotting and immune defense in
Limulidae, Prog. Mol. Subcell. Biol., 15(1996a) 154-189.
Furie, B. and Furie, B.C., The molecular basis of blood
coagulation, Cell, 53(4)(1988) 505-18.
Davie, E.W., Fujikawa, K. and Kisiel, W., The coagulation
cascade: initiation, maintenance, and regulation, Biochemistry,
30(43) (1991): 10363-70.
Lorand, L. and Conrad, S.M., Transglutaminases, Mol Cell
Biochem., 58(1-2)(1984) 9-35.
Greenberg, C.S., Birckbichler, P.J. and Rice, R.H.,
Transglutaminases: multifunctional cross-linking enzymes
that stabilize tissues, FASEB J., 5(15)(1991) 3071-7.
Kawabata, S-i, Muta, T. and Iwanaga, S., In: New Directions
in Invertebrate Immunology. Söderhäll K, Iwanaga S, Vasta G
R, editors. Fair Haven, NJ: SOS Publications; (1996). pp.
255–283.
Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.L., HiranagaKawabata, M., Muta, T., Iwanaga, S., Ichinose, A. and Davie,
E.W., Limulus hemocyte transglutaminase. Its purification and
characterization, and identification of the intracellular
substrates, J Biol Chem., 268(1)(1993a) 252-61.
Tokunaga, F., Muta, T., Iwanaga, S., Ichinose, A., Davie,
E.W., Kuma, K. and Miyata, T., Limulus hemocyte
transglutaminase. cDNA cloning, amino acid sequence, and
tissue localization, J Biol Chem., 268(1)(1993b) 262-8.
KUMAR et al.: HORSESHOE CRABS: BIOMEDICAL IMPORTANCE AND ITS POTENTIAL USE
35 Hall, M., Wang, R., Van Antwerpen, R., Sottrup-Jensen, L.
and Soderhall, K., The crayfish plasma clotting protein: a
vitellogenin-related protein responsible for clot formation in
crustacean blood, Proc. Natl. Acad. Sci. USA, 96(1999) 1965–
1970.
36 Kopácek, P., Hall, M. and Söderhäll, K., Characterization of a
clotting protein, isolated from plasma of the freshwater
crayfish Pacifastacus leniusculus, Eur J Biochem. 213(1)
(1993) 591-7.
37 Komatsu, M. and Ando, S., A very-high-density lipoprotein
with clotting ability from hemolymph of sand crayfish, Ibacus
ciliates, Biosci. Biotechnol. Biochem., 62(3)(1998) 459-63.
38 Hall, M., van Heusden, M.C. and Söderhäll, K., Identification
of the major lipoproteins in crayfish hemolymph as proteins
involved in immune recognition and clotting, Biochem
Biophys Res Commun., 216(3)(1995) 939-46.
39 Söderhäll, K., Cerenius, L. and Johansson, M.W., In: New
Directions in Invertebrate Immunology. Söderhäll K, Iwanaga
S, Vasta G R, editors. Fair Haven, NJ: SOS Publications;
(1996). pp. 229–253.
40 Söderhäll, K. and Cerenius, L., Role of the prophenoloxidaseactivating system in invertebrate immunity, Curr Opin
Immunol., 10(1)(1998) 23-8.
41 Doolittle, R.F. and Riley, M., The amino-terminal sequence of
lobster fibrinogen reveals common ancestry with
vitellogenins, Biochem Biophys Res Commun., 167(1)(1990)
16-9.
42 Chen, J.S., Sappington, T.W. and Raikhel, A.S., Extensive
sequence conservation among insect, nematode, and
vertebrate vitellogenins reveals ancient common ancestry, J
Mol Evol., 44(4)(1997) 440-51.
43 Sappington, T.W. and Raikhel, A.S., Molecular characteristics
of insect vitellogenins and vitellogenin receptors, Insect
Biochem Mol Biol. 28(5-6)(1998) 277-300.
44 Sahoo, A.K., Thakur, P.C., Shankar, K.M., Mohan, C.V.,
Sharma, S.R. and Corsin, F., Histopathological findings on
innate responses of white spot disease positive Penaeus
monodon (Fabricius) under semi‐intensive culture. Journal of
fish diseases, 38(1)(2015) 91-95.
45 Bang, F.B., A bacterial disease of Limulus polyphemus, Bull.
Johns Hopkins Hosp., 98(1956) 325-351.
46 Ariki, S., Koori, K., Osaki, T., Motoyama, K., Inamori, K. and
Kawabata, S., A serine protease zymogen functions as a
pattern-recognition receptor for lipopolysaccharides, Proc.
Natl. Acad. Sci. USA, 101(2004) 953-958.
47 Iwanaga, S., The Limulus clotting reaction, Curr. Opin.
Immunol., 5(1993) 74-82.
48 Tamura, H., Tanaka, S., Oda, T., Uemura, Y., Aketagawa, J.
and Hashimoto, Y., Purification and characterization of a
(13)-β-D-glucan binding protein from horseshoe crab
(Tachypleus tridentatus) amoebocytes, Carbohyd. Res.,
295(1996) 103-116.
49 Agarwara, K.L., Kawabata, S., Miura, Y., Kuroki, Y. and
Iwanaga, S., Limulus intracellular coagulation inhibitor type 3.
Purification, characterization, cDNA cloning, and tissue
localization, J. Biol. Chem., 271(1996) 23768-23774.
50 Bergner, A., Muta, T., Iwanaga, S., Beisel, H. G., Delotto, R.
and Bode, W., Horseshoe crab coagulogen is an invertebrate
protein with a nerve growth factor-like domain, Biol. Chem.,
378(1997) 283-287.
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
1243
Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke,
D., Huber, R. and Bode, W., Crystral structure of a
coagulogen, the clotting protein from horseshoe crab: a
structural homologue of nerve growth factor, EMBO J.,
15(1996) 6789-6797.
Tan, N.S., Ho, B. and Ding, J.L., High-affinity LPS binding
domain(s) in recombinant factor C of a horseshoe crab
neutralizes LPS-induced lethality, FASEB J., 14(2000) 859870.
Koshiba, T., Hashii, T. and Kawabata, S., A structural
perspective on the interaction between lipopolysaccharide and
factor C, a receptor involved in recognition of Gram-negative
bacteria, J. Biol. Chem., 282(2007) 3962–3967.
Kawasaki, H., Nose, T., Muta, T., Iwanaga, S., Shimohigashi,
Y. and Kawabata, S., Head-to-tail polymerization of coagulin,
a clottable protein of the horseshoe crab, J. Biol. Chem.,
275(2000) 35297-35301.
Muta, T., Seki, N., Takaki, Y., Hashimoto, R., Oda, T.,
Iwanaga, A., Tokunaga, F. and Iwanaga, S., Purified
horseshoe crab factor G. Reconstitution and characterization
of the (1→3)-beta-D-glucan-sensitive serine protease cascade,
J. Biol. Chem., 270(1995) 892-897.
Takaki, Y., Seki, N., Kawabata, S., Iwanaga, S. and Muta, T.,
Duplicated binding sites for (1→ 3)-beta-D-glucan in the
horseshoe crab coagulation factor G: Implications for a
molecular basis of the pattern recognition in innate immunity,
J. Biol. Chem., 277(2002) 14281-14287.
Osaki, T., Okino, N., Tokunaga, F., Iwanaga, S. and
Kawabata, S., Proline-rich cell surface antigens of horseshoe
crab hemocytes are substrates for protein cross-linking with a
clotting protein coagulin, J. Biol. Chem., 277(2002) 4008440090.
Tokunaga, F. and Iwanaga, S., Horseshoe crab
transglutaminase, Methods Enzymol., 223(1993) 378-388.
Osaki, T. and Kawabata, S., Structure and function of
coagulogen, a clottable protein in horseshoe crabs, Cell Mol.
Life Sci., 61(2004) 1257-1265.
Wang, R., Lee, S. Y., Cerenius, L. and Söderhäll, K.,
Properties of the prophenoloxidase activating enzyme of the
freshwater crayfish, Pacifastacus leniusculus, Eur. J.
Biochem., 268(2001) 895-902.
Krem, M.M. and Cera, E.D., Evolution of enzyme cascades
from embryonic development to blood coagulation, Trends
Biochem. Sci., 27(2002) 67-74.
Theopold, U., Schmidt, O., Söderhäll, K. and Dushay, M.S.,
Coagulation in arthropods: defense, wound closure and
healing, Trends Immunol., 25(2004) 289-294.
Levin J. and Bang F.B., The role of endotoxin in the
extracellular coagulation of Limulus blood, Bull. Johns
Hopkins Hosp., 115(1964) 265–274.
Tanaka, S. and Iwanaga, S., Limulus test for detecting
bacterial endotoxins, Methods Enzymol., 223(1993) 358-364.
Obayashi, T., Tamura, H., Tanaka, S., Ohki, M., Takahashi,
M., Arai, M., Matsuda, M. and Kawai, T., A new
chromogenic endotoxin-specific assay using recombined
Limulus coagulation enzyme and its clinical application, Clin.
Chim. Acta., 149(1985) 55-65.
Funkhouser, D., Crab love nest, Scientific American, 304
(4)(2011) 0411-29.
1244
INDIAN J. MAR. SCI., VOL. 45, NO. 10 OCTOBER 2016
67 Hurton, L., Reducing post-bleeding mortality of horseshoe
crabs (Limulus polyphemus) used in the biomedical industry
(M.Sc. thesis). Virginia Polytechnic Institute and State
University, 2003.
68 Mikkelsen, T., The Secret in the Blue Blood, Science Press,
1988.
69 Novitsky, T.J., Discovery to commercialization: the blood of
the horseshoe crab, Oceanus, 27(1984) 13–18.
70 Rudloe, A., The effect of heavy bleeding on mortality of the
horseshoe crab, Limulus polyphemus, in the natural
environment, Journal of Invertebrate Pathology, 42(1983)
167–176.
71 Shuster, C.N., Jr., A pictorial review of the natural history and
ecology of the horseshoe crab, Limulus polyphemus, with
72
73
74
reference to other limulidae. pp. 1–52. In: Physiology and
Biology of Horseshoe Crabs: Studies on Normal and
Environmentally Stressed Animals. (Bonaventura, J., C.
Bonaventura, and S. Tesh, Eds.). New York: Alan R. Liss,
Inc, 1982.
Walls, Elizabeth A. Berkson, J. and Smith Stephen A., The
Horseshoe Crab, Limulus polyphemus: 200 Million Years of
Existence, 100 Years of Study, Reviews in Fisheries Science,
10(1)(2002) 39–73.
Armstrong, P. and Conrad, M., Blood Collection from the
American Horseshoe Crab, Limulus Polyphemus, Journal of
Visualized Experiments, 20(2008) e958-e958.
Anatomy of the Horseshoe Crab, Maryland Department of
Natural Resources. Retrieved 3 December 2015.