Mark L Botton

Fordhant University, New York, USA

Horseshoe crabs are one of evolution's success stories. These living fossils have contributed
immensely to biomedical research, and their eggs are a critical link in the tnigration cycle of
New World waders (shorebirds). Can they endure threats posed by a growing commercial
fishery and the loss of essential spawning habitats?

Zoologists have long been intrigued by animals such as croc-
odiles, turtles and sharks because they represent living
fossils - animals whose form and function have seemingly
stood still over long marches of evolutionary time.
Horseshoe crabs are paradigms of living fossils, with exam-
ples of distant relatives dating back to the Cambrian.
Remarkably, 150 million-year-old fossils oi MesolimuluH
walchi, collected from the famous Solnhofen beds in
Bavaria, are so markedly similar in form to the extant
horseshoe crabs that they could easily be confused with one
another (Figure 1). Though appearing somewhat flatter
than our modern horseshoe crab, the basic tripartite body
plan is clearly evident in Mesnlimulus. A dome-like prosoma
shields the mouthparts and walking legs, and also contains
the majority of the digestive and reproductive organs, along
with the hrain and eyes. The somewhat more flattened
opisthosoma shields the book-gills, and bears spines and an
array of sensory receptors along its margin. The terminal
portion of the hody is the telson, or tail, used hy horseshoe
crabs to right themselves if they are overturned.
Horseshoe crahs are placed into the suhphylum
Chelicerata, along with spiders, ticks, and scorpions (the
true crahs are classified within the subphylum Crustacea).
There are four living species of horseshoe crabs
(Xiphosurans) in the world today. Three species,
Tachypleus tridentatus, T. gigas and Carcinoscorpius
rotundicauda, are distributed throughout southeast Asia,
ranging from India to southern Japan. The fourth species,
known as the American, or Atlantic horseshoe crab
{Limulufi polyphemus), occurs along the east coast of North
America, from Maine to the Gulf of Mexico, where its
southern limit is Mexico's Yucatan Peninsula. Limulus is
most ahundant along the middle Atlantic coast of the US,
especially in the Delaware Bay area. Sporadic reports of
Limulus from the Atlantic coast of Europe or the
Mediterranean have been attributed to specimens
discarded by fishermen returning from trips off North
America (Shuster, 1979).
Why have the horseshoe crabs persevered, while their
arthropod cousins, the trilobites and the eurypterids {giant
sea scorpions), fell to extinction? One factor that has
clearly favoured the persistence of horseshoe crahs is that
they are ecological generalists. Modern horseshoe crahs,
and presumably their ancestors as well, can tolerate wide
fluctuations in temperature, salinity, and other physical
variables; likewise, they are not tied to highly specialised
habitats or food resources (Shuster, 1982). Consequently,
environmental changes since the Jurassic, which have led
to the extinction of many animals, have been mere speed
bumps for the horseshoe crahs.
There is another curious feature to the evolution of
horseshoe crabs: as palaeontologists such as Daniel Fisher
and Niles Eldredge have pointed out, if the Limulidae have
been so successful in persisting over geological time, why

Biologist (2002) 49 (5) 193

H o r s e s h o e crab

scarcely fmd even small remnants of a

horseshoe crab carapace from the previ-
ous spring's spawning cycle.
Furthermore, any horseshoe crab cara-
paces that might escape decomposition
would be unlikely to fossilise on the
unconsolidated sandy beaches and
(iunes wbere they tend to accumulate. I
should tbink the best hope of finding a
fossil oi' Liinulus polyphemus might be
if a chance specimen (perhaps a larva
or juvenile) were to be preserved in
anaerobic salt-maT'sh sediments, where
decay processes might be less rapid.

Reproduction and life history

Adult horseshoe crabs migrate from
deeper waters to estuarine sborelines
in tbe spring, most likely stimulated
by increasing day length. Within the
estuary, they await favourable
temperatures and tides before coming
ashore to spawn, Peaks of horseshoe
Figure I. A specimen. o/'Mesolimulus, photographed by the author from the coUeetion of ihe crab mating activity generally coincide
horseshoe crab museum in Kasaoka, Japan. vvith tbe highest tides, which occur on
the days surrounding the full and new
have they failed to diversify? In fact, we have only four moon phases. Limulus males attach to females using a pair
contemporary species of horseshoe crab, and there is no of specially modified legs called claspers, which grip the
fossil evidence of especially greater biodiversity at any rear of the female's opisthosoma so tightly that they leave
time in the past (Fisher, 1984). It had once been hypothe- a visible imprint called a 'mating scar' (curiously, among
sised that Limulus had reached an evolutionary dead end the Asiatic horseshoe crabs, males each have two pairs of
because it lacked sufficient genetic diversity with which to claspers!. Mated pairs are often intruded upon by crowds of
evolve new forms. However, in one of the first population unpaired satellite males that vigorously attempt to wedge
genetic studies employing protein elec-
trophoresis, Selander et al. (1970)
showed that levels of hetcrozygosity in
LimuluH were similar to other
animals. Furtberniore, there appears
to be considerable genetic variation
among Limulus populations inhabit-
ing different estuaries along the coast
(Pierce et al., 2000). Perhaps these
studies only deepen the mystery, for if
horseshoe crabs have levels of genetic
variability comparable to mammals or
fish, why aren't there as many species?
The dearth of any fossils of Li?7iulus
polyphemus itself, or of any of the
three Asiatic species, also seems
anomalous for such an ancient animal.
My colleagues, Bob Loveland and Carl
Shuster. and I have often speculated
about the lack of fossil Liinulus as we
walk along the shores of Delaware Bay
in May and June, which are cluttered
with thousands of horseshoe crabs
that have been overturned by the
waves and left stranded on the beach.
We think that their absence from the
fossil record is related to two factors.
Unlike the true crabs, or for that
matter, the trilobites, exoskeletons of
horseshoe crabs are not mineralised.
They consist solely of chitin, and are
Figuri- 2. Mating hor.'^csiiuc '.rubs mi u ^andy bench in Ih'laiviirc Bay. To the right is a mated
more effortlessly decomposed than pair, with lhe smaller male attaehed to the opiMhosama of the female. On the left is c clus-
shells that incorporate calcium ter con.'ii sting of a mated pair ivith four unpaired satellite males. The female, at the centre of
carbonate. By late winter, we can the cluster, is almost completely buried an she lays her eggs.

194 Biologist (2002) 49 (5)


Figure 3. Delaware Bay is a inlal stopaver area for migrant waders each spring as they
migrate north tu the Arctic. Two of the most common species are the Ruddy Turnstones {fore-
ground, with orange legs and harlequin faces) and Red Knots.
themselves near the female as she is depositing her eggs
(Figure 2). Despite the intense male-male competition,
actual displacements ofthe mated male by a satellite
almost never take place. But, by using genetic markers,
Jane Brockmann and colleagues were able to demonstrate
that male competition takes a mo7-e subtle route. In horse-
shoe crabs, fertilisation is external and sperm contributed
by satellite males competes with sperm from the mated
male. Thus, a given batch of eggs frequently has mixed
paternity. So, the satellite male strategy can be profitable
even if these males don't ever become part of a mated pair
(Brockmann et al,, 1994).
Females use their spade-like pusher legs to dig a 'nest'
about 10-20 cm deep on a sandy heach. The female
releases about 4000 eggs that mix with sperm, and are
moulded into a mass along with adherent sand grains. She
may lay several egg clutches on a given high tide, before
retreating to the bay with her mated male in tow. The
process can then be repeated over the next few days,
perhaps longer, until she has laid her entire yearly comple-
ment of about 80 000 eggs. The greenish-blue eggs are
about 1.5 mm in diameter and develop within the sand,
reaching tbe 'tj'ilobite' larval stage after about three to four
weeks, depending on the temperature. On high tides,
particularly when the sand is disturbed by wave action,
trilobites will emerge from the beach and enter the plank-
ton for a short while before settling to tbe bottom. We actu-
ally know rather little about the ecology of juvenile horse-
shoe crabs, but evidence suggests that they grow quite
slowly and do not attain sexual maturity until they are at
least nine years of age (Shuster, 1979).
Biologist (2Q02) 49 (5)
H o rs e s h c r a h s
Perhaps the most spectacular aspect
of the horseshoe crab's ecology is tied
to the exploitation of its eggs by migra-
tory waders (shorebirds) in the
Delaware Bay. In the New World,
many waders migrate from wintering
grounds as far south as the coasts of
Chile and Argentina, to Arctic breed-
ing grounds in northern Canada.
Along the migration route, these birds
may undertake nonstop flights of 1000
km or more. To complete their journey,
wadtirs need to have dependable, high-
quality food resources at these inter-
mediate stopover points in order to
regain the fats and proteins that they
have used up en route. Delaware Bay
is the most important wader 'staging
area' on the Atlantic coast during their
spring (northbound) migration, host-
ing some 425 000 to 1 000 000
migrants from May to early June
(Figure 3). Waders feed almost exclu-
sively on Limulus eggs during their
brief stopover in Delaware Bay, and on
this diet, waders can increase their
body mass by as much as 70-80% in a
three week period. Therefore, a deple-
tion in the supply of horseshoe crab
eggs could be catastrophic to wader
populations. It is this concern that has
enlisted many conservationists to the
cause of horseshoe crab preservation.
Horseshoe crabs in
biomedical research
Lhnulus eyes are valuable experimental models in vision
research. The horseshoe crab has two prominent lateral
eyes on its prosoma. Each lateral eye has about 1000
photoreceptive units called ommatidia - these are large
enough to be seen with the naked eye. Pioneering
researchers, such as H K Hartline at the Marine Biological
Laboratory in Woods Hole, took advantage of this to
develop instrumentation to record electrical signals from
the horseshoe crab's eyes, leading to fundamental discover-
ies about tbe physiological hasis of pbotoreceptors.
Horseshoe crabs also have two median eyes (ocelli) near
tbe midline ofthe prosoma. Eyes on tbe ventral surface of
tbe prosoma are especially prominent in larval horseshoe
crabs, and tbey probably help the larvae to orient while
swimming. Horseshoe crabs have additional photorecep-
tors near tbeir mouth and in the telson.
Robert Barlow, a student of Hartline, determined tbat
Limulus lateral eyes had circadian rhythms that increased
their visual sensitivities up to a million-fold in darkness. In
effect, tbe horsesboe crab can see just about as well at
night as it can during the day, despite the much reduced
amount of ambient ligbt. This remarkable discovery
suggested a possible behavioural role for vision in mating,
since it was long known that the densest aggregations of
spawning horsesboe crabs occur after dark. In an inge-
nious field experiment. Barlow and colleagues sbowed that
male horseshoe crahs could detect and orient toward
cement castings of females at an underwater distance of
about one meter, under both light and dark conditions
(Barlow et al., 1988). Now, with the invention of the 'crab
cam' (Figure 4), Barlow's team can directly link borsesboe
195
Ho rs es h o e era
Figure 4. A 'crab cam.' mounted above the right lateral eye of a horseshoe crab, is tinked to An endotoxin assay based on recom-
researchers by a 40' tether. A suction etectrode is implanted anterior to the eye that enables binant Factor C, rather than whole
scientists to gather data from a single optic nerve fiber while the animal behaves underwater. blood, has the potential to help
Photograph courtesy ofDr Robert Barlow.
crab behaviour under field conditions to what the animals
are actually seeing underwater (Passaglia et al., 1997).
Biomedical researchers have also found that the blood of
horseshoe crabs has extraordinary properties. Its blood has
a pale blue colour because of the copper-based respiratory
protein, hemocyanin. Hemocyanin is dispersed in the blood
plasma rather than being contained within erythrocytes,
as is the case in mammals. In fact, horseshoe crabs have
only one type of circulating hlood cell, an ovoid amoebocyte
about 15 to 20 jam long. These amoehocytes are the basis of
the horseshoe crab's defense mechanism. When a crab is
injured and bacteria enter its blood, the secretory granules
within the amoebocytes undergo exocytosis and release
their contents into the external environment, causing the
cells to became adhesive. This leads to the formation of a
gel-like blood clot. This clotting reaction is triggered hy the
presence of surface marker molecules on the pathogen,
notably lipopolysaccharides (endotoxins) from Gram-nega-
tive bacteria. The clotting process helps to seal off the
wound and prevent excessive blood loss, and it also traps
bacteria, preventing them from circulating beyond the site
of the injury. Amoebocytes also release a suite of antimi-
crobial proteins that constitute an efficient system of
innate immunity (Iwanaga et al., 1998).
Gram-negative bacterial contamination of pharmaceuti-
cals, intravenously-delivered medications, and implanted
prosthetic devices such as beart valves may cause endotox-
emia, a potentially life threatening systemic toxicity.
Careful study of the reaction of Limulus blood to endotox-
ins by Jack Levin and Frederik Bang in the early 1960s
eventually led to the development of a commercially impor-
tant product. At that time, the standard test for endotoxins
involved the injection of material into rabbits: if bacteria
(pyrogens) were present, they would induce a fever. By the
early 1970s, there was a proliferation of studies on the
potential utility of the so-called Limu/i/.s amoebocyi;e lysate
(LAL) test for endotoxins. These led to refinements in the
LAL protocol by 1977 that met the approval of the US Food
and Drug Administration (USFDA). ilie LAL test had two
obvious advantages over the rabbit pyrogen test: it was
less expensive and more precise. LAL testing is now the
state of the art method used throughout North America
196
and Europe to detect Gram-negative
bacterial endotoxins. The LAL test is
based on the simple principle that a
gel clot will only form when endo-
toxin is present in the sample.
A reliable supply of whole horse-
shoe crab blood is required for the
commercial production of LAL. It is
estimated that about 250 000 horse-
shoe crabs are bled annually for the
production of LAL in the US, most of
which are returned to the water
shortly after bleeding. Fortunately,
studies have shown that horseshoe
crabs are extremely hardy, and about
90% of them survive after 'donating'
their blood, Ding and Ho (2001) have
recently succeeded in producing
genetically engineered Factor C, an
endotoxin-sensitive protein involved
in the Limulus coagulation sequence.
conserve natural populations of
horseshoe crabs.
The commercial fishery for horseshoe crabs
There is surprisingly little edible muscle on a horseshoe
ci'ab; most of the soft mass within the prosoma consists of
the digestive and reproductive tissue. In parts of Southeast
Asia, horseshoe crab eggs are considered to be a delicacy,
though it carries the risk of food poisoning caused by
tetrodotoxin (the same algal toxin that is sometimes found
in fugu, or puffer fish). Though never an important source
of food for people in the US, Limulus was the basis of an
extensive commercial fishery, centered in the Delaware
Bay area, from the mid-19th century to the first three
decades of the 20th century, Massive catches were facili-
tated by the use of pound-nets or weirs that were set out on
the tidal flats to intei-cept horseshoe crabs as they came
ashore to spawn. At low tides, the fishermen retrieved the
crabs, and stacked them in large holding pens on shore
for drying (Figure 5). Then, they were processed into
fertiliser (known locally as 'cancerine'), or used to feed
chicken and livestock. Catches of more than 1.2 million
crabs per year were the norms from the 1870s through the
1920s. By the 1960s, commercial manufacturing of cancer-
ine had ceased, a victim of competition from cheaper chem-
ical fertilisers and decades of declining horseshoe crab
populations in the bay.
Beginning in the mid-1990s, there was a large and unex-
pected increase in the quantity of horseshoe crabs collected
by watermen and used for bait in eel pots and whelk traps.
This bait fishery had always been regarded as a small-time
industjy, and the numbers of crabs collected for bait was
considered to be trivial compared to the vast population
within Delaware Bay. But seemingly out of nowhere,
teams of workers were converging on small bayshore
towns, and loading up large refrigerated trucks with thou-
sands of horseshoe crabs that were pulled off the heach as
fast as they came ashore to spawn. In a matter of a few
years, the horseshoe crah bait fishery went from 'too small
to be recorded' to close to one million animals per year.
And. since egg-bearing females made the best eel bait, the
fishery was preferentially removing tbe component of the
population that was most valuable in terms of reproduc-
Biologist (2002) 49 (5)

Figure 5. A large homeshoe crab factory on the New Jer.^ey shore of Delaware Bay, proba-
bly from ihe 1920s or 1930s. Original glass slide photograph by Dr Thurlow Nehon, cour- the elimination of the Japanese horse-
tesy ofDr Harold Haskin.
tion. Seagoing fishing vessels also began to target horseshoe
crahs as they trawled the middle Atlantic continental shelf.
With horseshoe crabs fetching US $0.40 to $1.25 apiece, a
'gold iTJsh' mentality prevailed in the mid-1990s. Outraged
conservationists, convinced that watermen were endanger-
ing the food supply for migratory waders, were furious that
there were no regulations in the states bordering Delaware
Bay to control the harvesting of horseshoe crahs.
The conservation of horseshoe crabs
The authority to regulate marine fisheries in the United
States is delegated to individual states for animals caught
within their jurisdiction, and to the National Marine
Fisheries Service (NMFS), which is empowered to establish
quotas and other statutes for animals caught outside the
three nautical mile boundary that divides State and Federal
waters. For several years, fishery managers gr'appled with
the dilemma of how to regulate the rapidly growing horse-
shoe crab fishery, in the absence of hard data on both the
numbers of animals caught and the size ofthe population
(Berkson and Shuster, 1999}. A turning point came in
February 1996, when more than 80 biologists, fishery
managers, conservation advocates, Limulus lysate produc-
ers, and watermen met at a two-day conference at the
University of Delaware to review the status ofthe horseshoe
crab fishery and resomxe (Farrell and Martin, 1997).
It was axiomatic that regulators had the responsibility of
managing the horseshoe crab population to sustain the
commercial needs for LAL and bait. But beyond that goal,
it was seen as critical to conserve enough horseshoe crab
eggs to sustain tbe migratory waders in Delaware Bay. The
Delaware Forum also reinforced the point that horseshoe
crabs crossed jurisdictional boundaries as they migrated
from the estuaries out to the continental shelf, necessitat-
ing a coordinated management strategy to eliminate loop-
holes in the mosaic of state-by-state regulations that had
been enacted over tbe prior two years. In 1998. tbe Atlantic
States Marine Fisheries Commission (ASMFC). a consor-
tium of State resource managers, voted to enact the first
coast-wide fishery management plan for the horseshoe
crab. At the present time, regulations on the fishery
Biologist (2002) 49 (5)
H o r s e s h o e c r a b s
include quotas on total catch, limita-
tions on tae numbers of permits issued,
and restrictions on the number of days
and/or areas in which watermen are
permitted to harvest horseshoe crabs.
ASMFC also mandated detailed report-
ing of all horseshoe crabs caught, and
made recommendations for coordinated,
statistically rigorous monitoring of the
horseshoe crah population. Finally, in
October 2000, NMFS enacted a rule
that prevented fishermen from collect-
ing horseshoe crabs in a Federally
designated 'sanctuary,' a 1500 square
nautical mile area off the mouth of
Delaware Bay.
No discussion of horseshoe crab
conservation should omit the critical role
of habitat preservation, particularly
during a time of global sea level rise. The
extensive loss of spawning hahitats,
lesulting from land reclamation projects
and th(! construction of dikes, has caused
shoe crab (TachypleuK tridentatus) from
much of its former range in Japan
(Botton, 2001). In general, horseshoe crabs are extremely
hardy creatures, but when it comes time for spawning,
females are actually ratber choosy, preferring to lay their
eggs on beaches with a deep layer of well-aerated sand.
Beach erosion is surpri.'iingly rapid on estuarine beaches,
and we have observed significant erosion on a number of
beaches along the Delaware Bay shoreline. During a time of
rising sea level in an uninhabited area, this would pose no
special problem, as the beach would simply tend to migrate
landward. However, beach front houses and roads set a
practical upper limit to beach movement; some beaches are
now little more than slivers of sand terminating at a wooden
or steel hulkhead. Fortunately, large areas of good quality
sandy beaches in Delaware Bay have been preserved though
the efforts of the National Park Service, The Nature
Conservancy, and the States of New Jersey and Delaware.
Outlook for the future
Horseshoe crabs have been resilient through geological
time, yet they face a dual threat from overfishing and habi-
tat loss. Fishery managers have enacted a series of increas-
ingly more stringent I'egulations on the commercial fishery
for Limulu.s, but it remains to be seen what the long-term
impacts of this industry will be. There may have been fewer
horseshoe crabs laying eggs on Delaware Bay beaches
beginning in the m\d-1990s compared to the prior two
decades, but the year-classes associated with these eggs
won't he sexually mature until 2005. at the earliest.
Monitoring ofthe population will continue, driven in large
part by the concern that the fragile linkage between waders
and horseshoe crab eggs cannot withstand further diminu-
tions in the crab population.
The robust 'jack of all trades' strategy has served the
horseshoe crah quUe well over the ages. As long as we
preserve good spawning sites and compel prudent limits to
the fishery, the horseshoe crab will likely continue its inex-
orable march through time.
References
Barlow R B. Powers M K and Kass L (1988). Vision and mating
197
H o r s e s h o e c r a b s
behavior in Limulus. In: Sensory Biology of Aquatic Animals.
Atema J, Fay R R, Popper A N and Tavolgna N (eds). New York:
Springer-Verlag.
Berkson J and Shuster C N (1999) The horseshoe crab: The battle
for a true multiple-use resource. Fisheries, 24. 6 - 11.
Botton M L (2001) The conservation of horseshoe crabs: What can
we learn from the Japanese experience? In: Limulus in the
Limelight. Tanacredi J (ed). New York: Kluwer Academic/
Plenum.
Brockmann H J, Colson T and Potts W (1994) Sperm competition
in horseshoe crabs {Limulus polyphemus). Behavioral Ecology
and Sociobiology, 35, 153 - 160.
Ding, J L and Ho B (2001) A new era in pyrogen testing. Trends in
Biotechnology, 19, 277 - 281.
Farrell J and Martin C (eds) (1997) Proceedings ofthe Horseshoe
Crab Forum: Status of the Resource. University of Delaware Sea
Grant College Puhlication DEL-SG-05-97.
Fisher D C (1984) The Xiphosurida: archetypes of bradytely? In:
Living Fossils. Eldredge N and Stanley S M (eds). New York:
Springer-Verlag.
Iwanaga S, Kawabata S and Muta T (1998) New types of clotting
factors and defense molecules found in horseshoe crab
hemolymph: their structures and functions. Journal of
Biochemistry. 123, I - 15.
Pas.saglia C, Dodge F, Herzog E, Jackson S and Barlow R (1997)
Deciphering a neural code for vision. Proceedings of the
National Academy of Sciences USA, 94, 12 649 - 12 654.
Pierce J C, Tan G and Gaffney P M (2000) Delaware Bay and
Chesapeake Bay populations of the horseshoe crab Limulus
polyphemus are genetically distinct. Estuaries, 23, 690 - 698.
Selander R K, Yang S Y, Lewontin R C and Johnson W E (1970)
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a phylogenetic 'relic'. Evolution, 24, 402 - 414.
Shuster C N Jr (1979) Distribution ofthe American horseshoe
•crab', Limulu.t polyphemus (L.). In: Biomedical Applications of
the Horseshoe Crab (Limulidae). Cohen E (ed). New York: Alan
R Liss.
Sh uster C N Jr (1982) A pictorial review ofthe natural history and
ecology of the hor.seshoe crab Limulus polyphemus, with refer-
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(eds). New York: Alan R. Liss.
Websites
www. mfai.edu/animats/index.html
Ttie Marine Biological Laboratory In Woods Hole has been home to
many of the most significant breakthroughs in biomedical research
with horseshoe crabs. This well-illustrated site chronicles the history
of these discoveries.
www.horseshoecrab.orQ/
An extensive websfte emphasising horseshoe crab ecology and
conservation Issues, sponsored by Ecological Research and
Development Group, a nonprofit organisation.
Dr Mark L Botton is Professor of Biology in the Department
of Natural Sciences, Fordham College at Lincoln Centre,
113 West 60th Street
New York, NY 10023
USA
botton@fordham.edu
Biologist (2002) 49 (5)