Classroom ApplicationsUsing Lim ulus polyphem us -The American Horseshoe Crab Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021

FrederickC. Pearson III MarlysWeary

The Horseshoe Crab T HROJUGHOUT its 300 million-yearhistory, the Amer- ican horseshoe crab (Limulus polyphemus) has been This 'living fossil," so-called because it has existed regarded at best as a scientific curiosity, and at worst as virtually unchanged since the Ordovician period, is indi- a nuisance. However, recent scientific publications have genous to the eastern coast of North America. Related changed that image. In 1970, Jack Levin of Johns Hop- species-Tachypleus tridentatus, Tachypleus gigas, and kins Universitypublished findings of a test for gram-nega- Carcinoscorpius rotundicaudo-are found in certain tive bacterialsepsis and endotoxemia based on an extract, areas of the Far East. Because the animals exist on a Limulus amebocyte lysate (LAL), made from the blood diet of shellfish and other marine invertebrates, they are cells of this ancient animal (Levin, Tomasulo, and Oser poorly regarded by fishermen, who hold them respon- 1970). The excitement generated in the scientific com - sible for everything from decimating valuable shellfish munity by this discovery has elevated the status of the beds to fouling nets. Consequently, in certain areas, lowly horseshoe crab to that of a valuable commodity, wholesale destruction of the crabs is encouraged. In Mass- and thus a unique naturalresource. achusetts, for example, clam diggers are instructed to throw any horseshoe crabs they find over the high water FrederickPearson is Manager- line, where the animals ultimately dehydrate and die. Pyrogen Technology and Microbiological Support, Cor- In addition, eel and conch fishermen use them as bait. porate Microbiology at Trav- As a result, some 60,000 crabs are destroyed annually. enol Laboratories, Morton The horseshoe crab is not a true crab at all, but is more Grove, Illinois 60053. He received his B.S. degree in OP"7 closely related to other members of its phylum, Arthro- biology from Nasson College poda, particularlyscorpions. The four extant species have and his Ph.D. degree in micro- a subclass, Xiphosura, to themselves. biology from University of New Hampshire. He has been Although their physical structure is relatively simple, at Travenol Laboratoriessince 1978 and heads a group which is respon - horseshoe crabs function very efficiently. Their bodies, sible for Limulus amebocyte lysate test development, among other which are covered by an exoskeleton called the carapace, things. He holds many professional memberships, both here and in the United Kingdom. He has published extensively. Marlys Weary is Senior are divided into two major portions-the prosoma and Supervisor-Pyrogen Technology and MicrobiologicalSupport at Trav- the opisthosoma (fig. 1). Although they can swim, adult enol Laboratories. She received her B.A. degree in biology from Val- crabs are bottom dwellers, and their usual mode of loco- paraiso University, her M.S. degree in anatomy from the University of Illinois, and her M.B.A. degree from Lake Forest School of Manage- motion is walking. ment. She has been at Travenol Laboratories since 1962 and has The horseshoe crab's tubular heart, located beneath worked in the field of pyrogen testing for 15 years. She is a member of the carapace and above the intestine, is large in relation to the New York Academy of Sciences and is chairman of the Biological Testing Division of the Pharmaceutical Manufacturers' Association. its body (measuring from five inches in length in an adult She also publishes and lectures on the subject of pyrogen testing. male to six inches in a female, and from 1/2 to 3/4 inch

440 THE AMERICAN BIOLOGYTEACHER, VOLUME43, NO. 8, NOVEMBER1981 Limulus polyphemus

ventral view p dorsal view pedipalp simple eye \\ chelicera compound eye prosomaI mot -,-~gnathobase

FIGURE1. Dorsal and ventral views of the | genital operculum American horseshoe crab, Limulus pOly- gillgil lamelle phemus. _a_Ia I \ [anus opisthosoma _ s ^ - - ~~caudalspinel Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021

detail of terminal segments of pedipalps

in diameter). Gaseous exchange takes place in the lam- The AnnualCycle ellae of the gills, which are also called "book lungs" be- The crabs hibernate during the winter months, digging cause of their resemblance to the leaves in a book. into the ocean bottom, and are found at water depths of The crab's blood is brightblue in color due to the pres- up to fifty to sixty feet. They have been shown to be ence in the plasma of a copper compound, hemocyanin, highly sensitive to heat and cold, and will dig into the sand that carries oxygen. It contains only one type of circulat- if the temperature drops even five degrees in twenty-four ing cell, the amebocyte. Amebocytes agglutinate in the hours. Further evidence of this sensitivity is provided by presence of lipopolysaccharides (endotoxin) found in the fact that the largest specimens and populations are the cell walls of gram negative bacteria. This agglutina- found within the most temperate areas of the animal's tion response is the basis of Levin's LAL test. habitat. Salinity also appears to affect size and distribution The horseshoe crab has two functional pairs of eyes. (Shuster 1967, 1970). There have been few studies of A third pair is degenerated in the adult, although it may either the extent or habits of typical populations. serve a function in the larval stage. The multilensed com- The average lifespan of a horseshoe crab has been es- pound eye, which is sensitive to polarized light, has been timated to be between fourteen and nineteen years, and of great interest to scientists because its structure allows the animals probably reach sexual maturity at between the relationship between light stimulus and axon firing nine and eleven years of age. Crabs breed during the to be studied easily. It seems probable that the eye can spring, males and females coming up to the beaches in detect movement, but is not capable of image formation tandem (fig. 2) during the spring tide (the highest of the (Kaestner 1968). There is no evidence that the eye aids high tides on the full of the moon). The female deposits the crab in navigation (Shuster 1979). some 200-300 eggs in each of a series of ten to fifteen There are chemoreceptors on the base of the crab's nests hollowed out in the sand. The eggs are fertilized legs (pedipalps) that are used for taste. Food is partially externally by the male and then covered. The next tide crushed by gnathobases at the base of the legs and on the full of the moon, roughly a month later, triggers pushed into the animal's mouth with the fifth pair of legs. the hatching of trilobite larvae. These larvae, which re- Further grinding takes place in the gizzard, aided by bits semble trilobites and the adult horseshoe crab, are im- of shell and sand. Actual digestion begins in the midgut mature specimens with incomplete digestive, nervous, and is completed in the digestive gland. Horseshoe crabs and circulatorysystems. The larvae receive nourishment do most of their feeding during the breeding season. from a yolk sac.

HORSESHOE CRAB 441 Maturation,which involves several moltings (ecdysis), segment of the scientific community. The LAL test uses although not completely understood, appears to be con- material lysed from the amebocytes following the method trolled by hormonal secretions. The animals first molt developed by Levin and Bang (1972) to detect the pres- as early as three weeks after hatching, at which time the ence of endotoxin (pyrogens), a lipopolysaccharide rudiments of a telson first appear. The molting cycle per- present in the cell walls of gram-negative bacteria. Endo- sists until the crabs reach maturity, a period covering ten toxin can cause a variety of symptoms in man ranging to eleven years. Young crabs undergo frequent molts, from fever to shock and death if it is introduced directly each time increasing their size by roughly 25%. The cycle into the circulatory system. The LAL test therefore has is divided into four phases: premolt, molt, postmolt, and proved invaluable in the pharmaceutical industry where intermolt. The premolt phase is triggered by a hormonal a large variety of devices and preparations must be secretion of the Y-glands that lie in the head. This secre- certified as pyrogen-free. In addition, the test may be tion is probably related to the steroid ecdysone, which used as a diagnostic tool. One of the most promising initiates molting in insects (Russell-Hunter 1969). Action applications, in fact, is its use in the detection of gram- of the Y-glands is, in turn, inhibitedby hormones from the negative bacterial meningitis. The speed of the LAL test X-organ, a structure located within each eye stalk. It is makes it particularly valuable. As a test for meningitis, only when the latter hormones are absent that molting it can be completed in one hour, which compares favor- may begin. ably with eight to twenty-four hours for more traditional Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021 methods (Siegel and Nachum 1977). It may also be used Limulus Amebocyte Lysate in the diagnosis of urinaryinfections, bacteremia, mastitis, certain corneal infections, and possibly endotoxemia Much of the data on horseshoe crabs has been (Levin,Tomasulo, and Oser 1970). gathered by a relatively small group of marine biologists Studies have shown the LAL test to be remarkably and naturalists.It was only with the development of the sensitive to small quantities of endotoxin. As little as LAL test that the animal aroused the interest of a broader 10-15 pg/ml can be detected (Sullivan, Valois, and Wat-

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FIGURE 2. Male and female adult horseshoe crabs in the tandem mating position. The female is the larger of the pair.

442 THE AMERICAN BIOLOGYTEACHER, VOLUME43, NO. 8, NOVEMBER1981 son 1976). In addition, it is more sensitive than the U.S. Pharmacopeia rabbittest, which is the pyrogen assay still widely used in the pharmaceutical industry. In one study, the LAL test was found to be five to ten times more sen- sitive than the rabbitassay, while in another it was shown to be capable of detecting marginally pyrogenic solutions that may be passed by the other assay (Cooper, Levin, and Wagner 1971; Weary and Baker 1977). The LAL test has been officially sanctioned by the Food and Drug Administration'sBureau of Biologics and Bureau of Med- ical Devices. The Bureau of Drugs has recently published guidelines for the use of LAL as a final release for qual- ified human and veterinary drugs. To obtain the lysate, crabs are bled in specially de- signed racks. Blood is removed with an 18-gauge needle

inserted into the cardiac chamber. A study is currently Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021 underway to determine the precise effects on these "blood donors." However, it appears that as much as 200 milliliterscan be removed from an adult specimen without apparent harm to the animal, and blood volume is quickly restored once the crab is returned to sea water. Given the wide application of the LAL test, it is hardly surprising that interest in maintaining or increasing cur- rent populations has been generated.

Breeding There have been attempts to breed crabs in captivity, FIGURE 3. Fertilized Limulus polyphemus eggs. The developing embryos are seen through the transparentegg case. an eventuality that will prove particularlyimportant be- cause large areas of the world do not have indigenous populations. Dr. Kathleen A. French (1979) reported cul- tivating fertilized eggs gathered during the spawning Classroom Applications season, as well as artificiallyinseminating eggs collected in the laboratory, and raising them in a variety of media Much of our work with Limulus can be duplicated in a (fig.3). She managed to maintain several specimens classroom situation. The experiments that follow will through the firstor second juvenile molt stages. help students develop an appreciation for the growing At Travenol Laboratories we were able to induce the importance of this "livingfossil." first successful breeding of horseshoe crabs out of their Raising fertilized eggs-Fertilized eggs are available native habitat. Four adult specimens (two male and two June through August from Marine Biological Laboratory, female) were stored at a temperature of 4?C for two Woods Hole, MA 02543. The eggs should be placed in months. The animals were then placed in a tank filled an aquarium that is filled with aerated artificialseawater with Instant Ocean artificialseawater to a depth of two maintained at room temperature (approximately 68- feet and maintained at a temperature of 69-70?F. 70?F), and lined with estuary mud or fine sand. In about Not long after they were placed in the tank, the crabs thirty days, the eggs should hatch and larvae will be ob- were observed in the tandem mating posture, and served swimming on their backs, flapping their book lungs roughly a month later both trilobite larvae and fertilized to propel themselves. At firstthe larvae subsist on the yolk eggs were discovered in the tank. sac, but after they have molted into the first juvenile Because the animals are known to be highly sensitive tailed stage they should be fed either brine shrimp or to temperature, it is hypothesized that the breeding cycle tubifex worms. On the eastern seaboard, eggs can be col- was triggered by the extremes of heat and cold to which lected during the spawning season (May-June). Nests the crabs were exposed, and by the fact that they went are craterlike areas of recently disturbed mud or sand from a very cold to a very warm environment. In com- about the size of a dinner plate and are usually located parison, in earlier attempts at inducing breeding, horse- near the high-tide line. The eggs, which are olive green shoe crabs were exposed to a range of temperatures spheroids about one millimeter in diameter, can be re- similar to those they would encounter in their natural moved by carefully sifting through the sand, or by gently habitat,yet these breeding efforts were not successful. agitatingthe loose nest material in a bucket of seawater.

HORSESHOE CRAB 443 Fertilizing eggs in vitro-Eggs can also be fertilized procedure for controls. The last dilution at which a solid in vitro. Sperm or eggs are removed by applying gentle clot forms gives the end point. This, multiplied by the sen - pressure in the area behind the genital flap (fig. 4). These sitivityof the lysate Oistedon the vial) gives the endotoxin should be released into artificialseawater and allowed concentration, e.g., if the 1:20 dilution is the last to clot to stand for about half an hour, after which the eggs can and the sensitivity of the lysate is 0.05 ng/ml, the solution be placed in an aquarium following the procedure out- contains 1.0 ng/ml endotoxin (0.05 x 20 = 1.0). Any lined above. solution (drinking water, seawater, distilled water, etc.) Testing water samples-Another interesting project can be tested using the method described, provided the is testing water samples for the presence of endotoxin test is not inhibited or potentiated. If it is potentiated, the (fig. 5). Limulus amebocyte lysate (LAL) is available for negative control will form a clot, and if inhibited,the posi- simple clot testing from Mallinckrodt, St. Louis, MO tive control will fail to gel. A common cause of potentia- 63134; Difco, Detroit,MI 48232; Associates of Cape Cod, tion is glassware contaminated by endotoxin. Woods Hole, MA 02543; and M.A. Bioproducts, Walkers- ville, MD 21793. Reconstituted according to the manu- References facturer's instructions, it can be used as a semiquanti- COOPER, J.F., LEVIN, J., and WAGNER, H.V. 1971. Quanti- tative assay for the presence of endotoxin. A positive tative comparison of in vitro and in vivo methods for the Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021 control, consisting of 0.1 ml of LAL and 0.1 ml of endo- detection of endotoxin. J. Lab. Clin. Med. 78:138. toxin standard (included in the Mallinckrodt test kit) FRENCH, K.A. 1979 Laboratory cultivation of embryonic and should be used. This is gently mixed in a pyrogen-free juvenile Limulus. In Cohen E. (ed.) Biomedical applications test tube and incubated undisturbedin a 37TCwater bath of the horseshoe crab New York: Alan R. Liss. for one hour. (To be sure glassware is pyrogen-free, it KAESTNER, A. 1968. Invertebrate zoology. New York: Inter- science. should be wrapped in aluminum foil and heated for one LEVIN, J., and BANG, F.B. 1968. Clottable protein in Limulus: hour at 250?F. The foil should not be removed until the Its localization and kinetics of its coagulation by endotoxin. glassware is used.) If a 37?C water bath is not available, Thrombosis et Diathesis Haemmorrhagica 19:1790. the tube can be incubated at room temperature for 1-1/2 (Continued on p. 472) hours. A firm clot that does not separate when the tube is inverted 180 degrees should be formed. A negative control, 0.1 ml of LAL and 0.1 ml of sterile water for in- jection, treated in the same way, may also be used. In this instance, a clot should not form. To perform the assay, twofold serial dilutions of the sample and sterile water for injection should be made in nonpyrogenic glassware. Samples known to contain a high level of endotoxin should be diluted 1:10 with sterile water for injection. One-tenth ml of each dilution should be mixed with 0.1 ml LAL and incubated following the

FIGURE 4. In preparation for fertilizingeggs in vitro, apply pressure to FIGURE 5. Results of an endotoxin Limulus amebocyte lysate clot the genital flap to release sperm or eggs. test. The tube on the right contains the gel clot, a positive reaction for endotoxin. The tube on the left is the negative control.

444 THE AMERICAN BIOLOGY TEACHER, VOLUME43, NO. 8, NOVEMBER1981 Platyfish ... continued from p. 429 CLARK, E., ARONSON, L., and GORDON, M. 1954. Mating behavior patterns in two sympatric species of Xiphophorin fishes: Their inheritance and significance in sexual isolation. Conclusions Bulletin of the American Museum of Natural History 103: I have described only a few of the ways platyfish could 135-226. be of value to biology teachers. My students have used GORDON, H., and GORDON, M. 1957. Maintenance of poly- them in conditioning experiments, studies of dominance morphism by potentially injurious genes in 8 natural popu- lations of the platyfish, Xiphophorus maculatus. Journal of orders, and studies of nutrition.They have also observed, Genetics 55:1-44. during a lab exercise on circulation, blood passing HAMILTON,T.R. 1979. Swordtail fish as subjects for animal through capillaries in the caudal fin of living specimens. behavior studies. American Biology Teacher 41(7):426-430. Even if the fish are not used in experiments, their KALLMAN, K.D. 1973. The sex determining mechanisms of presence in the classroom cannot help but be an asset. the platyfish, Xiphophorus maculatus. In Schroder, J.H. (ed.), Genetics and mutagenesis of fish. Berlin: Springer- Certainly the interest generated by a platyfish giving Verlag. birth during class would be hard to stimulate in some . 1975. The platyfish Xphophorus maculatus. In King, other way, and the abstractions of genetics can seem a R.C. (ed.), Handbook of genetics. Vol. 4, Vertebrates of little less difficultif living things can be used as examples, genetic interest. London: Plenum Press. LEGLER, K.F., BARDACH, J.E., and MILLER, R.R. 1962. especially when the topic is being introduced. Downloaded from http://online.ucpress.edu/abt/article-pdf/43/8/440/39357/4447345.pdf by guest on 25 September 2021 Ichthyology. New York: John Wiley & Sons, Inc. SOHN, J.J., and CREWS, D. 1977. Size-mediated onset of References genetically determined maturation in the platyfish, Xipho- ANDERS, F. 1967. Tumor formation in platyfish-swordtail phorus maculatus. Proceedings National Academy of hybrids as a problem of gene regulation. Experentia 23:1-10. Science 74:4547-4548.

Membership Horseshoe Crabs Application ... continued from p. 444 The National Association of Biology Teachers is the only LEVIN, J., professional association devoted exclusively to the con- TOMASULO, A., and OSER, R.A. 1970. Detection of endotoxin in cerns of people in biology education. Active Member- human blood and demonstration of an inhibitor.J. Lab. Clin. Med. ship is open to any person sympathetic to our purposes, 75:103. without regard to sex, race, color, creed, or nationality. RUSSELL-HUNTER,W.D. 1969. A biology of higher inverte- brates. New York: Dues are $25 per year, of which $15 is allocated for a MacmillanPublishing Co. C. subscription to The American Biology Teacher. Benefits SHUSTER, 1967. Xiphosura (with special reference to include two bimonthly newsletters, an annual convention, Limulus polyphemus). Geological Society of America an insurance program, and much more. Memoir 1:1171. . 1979. Distribution of the American "crab,"Limulus O Please enroll me as an Active Member of NABT polyphemus. In Cohen, E. (ed.) Biomedical applications of beginning January 1982. My check for $25 (payable the horseshoe crab. New York:Alan R. Liss. to NABT) is enclosed. SIEGEL,S.E., and NACHUM, R. 1976. Use of Limulus amebo- cyte lysate assay (LAL)for the detection and quantitation of L SPECIAL OFFER-Please enroll me beginning endotoxin. In Bernheimer, A.W. (ed.) Bacterial toxicology. December 1981 through December 1982-a full New York: John Wiley & Sons. 13 months. My check for $30 (payable to NABT) SULLIVAN, J.D., VALOIS, F.W., and WATSON, S.W. 1976. is enclosed. (Offerexpires 12/1/81.) Endotoxin: The Limulus amebocyte lysate system. In Bern- heimer, A.W. (ed.) Bacterial toxicology. New York: John Name Wiley & Sons. WEARY, M., and BAKER, B. 1977. Utilizationof the Limulus Address amebocyte lysate test for pyrogen testing large volume parenterals, administration sets, and medical devices. Bul- letin Parenteral Drug Association 3:127.

City State Zip

Send to: NABT 11250 Roger Bacon Drive No. 19 Reston, VA 22090

472 THE AMERICAN BIOLOGYTEACHER, VOLUME43, NO. 8, NOVEMBER1981