Invertebrate Anatomy OnLine
Limulus polyphemus ©
Copyright 2001 by
This is one of many exercises available from Invertebrate Anatomy OnLine , an Internet laboratory manual for courses in Invertebrate Zoology. Additional exercises can be accessed by clicking on the link to the left. A glossary and chapters on supplies and laboratory techniques are also available through this link. Terminology and phylogeny used in these exercises correspond to usage in the textbook, Invertebrate Zoology, by Ruppert, Fox, and Barnes (2004). Hyphenated figure callouts refer to figures in the textbook. Callouts that are not hyphenated refer to figures embedded in the exercise. The glossary includes terms from this textbook as well as the laboratory exercises.
Arthropoda P, Chelicerata sP, Euchelicerata, Xiphosura C, Limulidae F (Fig 16-15, 18-47)
Arthropoda, by far the largest and most diverse animal taxon, includes chelicerates, insects, myriapods, and crustaceans as well as many extinct taxa such as Trilobitomorpha. The segmented body primitively bears a pair of jointed appendages on each segment. The epidermis secretes a complex cuticular exoskeleton which must be molted to permit increase in size. Extant arthropods exhibit regional specialization in the structure and function of segments and appendages but the ancestor probably had similar appendages on all segments. The body is typically divided into a head and trunk, of which the trunk is often further divided into thorax and abdomen.
The gut consists of foregut, midgut, and hindgut and extends the length of the body from anterior mouth to posterior anus. Foregut and hindgut are epidermal invaginations, being derived from the embryonic stomodeum and proctodeum respectively, and are lined by cuticle, as are all epidermal surfaces of arthropods. The midgut is endodermal and is responsible for most enzyme secretion, hydrolysis, and absorption.
The coelom is reduced to small spaces associated with the gonads and kidney. The functional body cavity is a spacious hemocoel divided by a horizontal diaphragm into a dorsal pericardial sinus and a much larger perivisceral sinus. Sometimes there is a small ventral perineural sinus surrounding the ventral nerve cord.
The hemal system includes a dorsal, contractile, tubular, ostiate heart that pumps blood to the hemocoel. Excretory organs vary with taxon and include Malpighian tubules, saccate nephridia, and nephrocytes. Respiratory organs also vary with taxon and include many types of gills, book lungs, and tracheae.
The nervous system consists of a dorsal, anterior brain of two or three pairs of ganglia, circumenteric connectives, and a paired ventral nerve cord with segmental ganglia and segmental peripheral nerves. Various degrees of condensation and cephalization are found in different taxa.
Development is derived with centrolecithal eggs and superficial cleavage. There is frequently a larva although development is direct in many. Juveniles pass through a series of instars separated by molts until reaching the adult size and reproductive condition. At this time molting and growth may cease or continue, depending on taxon.
Chelicerata is a large taxon that includes spiders, scorpions, pseudoscorpions, ticks, mites, horseshoe crabs, sea spiders, and many others. The group originated in marine habitats but almost all modern chelicerates are terrestrial.
The body is divided into an anterior cephalothorax with six pairs of appendages and a posterior abdomen which, in most groups, does not bear appendages or has highly modified appendages. The first appendages of the cephalothorax are the chelicerae. Antennae are not present and the brain has two regions rather than the three found in mandibulates. Appendages are primitively biramous but are uniramous in almost all Recent taxa.
The segments of the cephalothorax are fused and covered by a dorsal shield, or carapace. Two median eyes are present.
Xiphosurans are primitive marine chelicerates represented today by the horseshoe crabs. They exhibit many of the characteristics expected of the ancestral chelicerates including a body divided into a cephalothorax and abdomen, both of which bear appendages, and they are aquatic. The abdominal segments are fused and their appendages are biramous book gills. The body terminates posteriorly with a spike-like telson. Four saccate nephridia on each side share a common nephridiopore.
Horseshoe crabs are common in shallow water along the east coast of the United States with the largest population in Delaware Bay. They feed on small invertebrates, polychaetes, crustaceans, and molluscs gathered from the sediment by their chelate cephalothoracic appendages. Mating occurs in the spring during a spring high tide with males riding in tandem behind the female waiting spawning to occur. Females dig large hemispheric depressions in the high intertidai on silty sand beaches. She buries about 4000 eggs in each depression and may lay as many as 80,000 eggs in a season. Spring high tides a month later wash the trilobite larvae out of the depression. Birds and fishes feed on the eggs and recently hatched larvae. It has been estimated that migrating shorebirds crossing Delaware Bay on the way to their Arctic breeding grounds consume over 500 metric tons of horseshoe crab eggs. The lamellae of the book gills may support large populations of commensal (not parasitic) flatworms, Bdelloura candida and, less often, Syncoelidium pellucidum.
Horseshoe crabs are harvested by whelk fishers who use them for bait. Limulus are also harvested and their blood sold to the biomedical industry. Limulus amoebocyte lysate is extracted from horseshoe crab blood and used to test drugs and medical devices used internally. Horseshoe crabs are killed by clam fishers to prevent predation on juvenile clams.
Limulus polyphemus , the common horseshoe crab of the American east coast, is one of only four living members of Xiphosura. The other three species inhabit the western margin of the Pacific Basin. Today's horseshoe crabs closely resemble those known from the Silurian. They inhabit soft bottoms in shallow marine waters and feed on small invertebrates gathered from the sediment.
External anatomy can be studied using a living, dried, or preserved Limulus. Living specimens are, as usual, preferable to preserved but are usually not available at a cost within the budget of most invertebrate zoology courses. Dried specimens are suitable for external anatomy and, if handled carefully, can be reused year after year. Exuviae can also be used. If the study is to include internal anatomy then dried specimens or exuviae cannot be used and living or preserved specimens are needed. If you are studying a small individual, you may sometimes feel the need for magnification. Small specimens can be placed directly on the stage of the dissecting microscope without benefit of a dissecting pan or dish. If the specimen is wet, drape a towel over the stage first and be careful to get NO seawater on the microscope.
The body is divided into two regions, or tagmata. The anterior tagmata is the large, shieldlike cephalothorax (= prosoma) (Fig 1, 18-1). Posterior to it is the shorter abdomen (= opisthosoma). The long spikelike tail spine, or telson, extends posteriorly from the abdomen. Flexible articulations between the two tagmata and between the abdomen and tail spike permit flexion of the body. Both tagmata are composed of several fused segments, most of which bear paired appendages.
Figure 1. Dorsal view of a juvenile horseshoe crab. The abdominal segments are numbered. Merostome9La.gif
The body is completely covered by an extracellular cuticle, or exoskeleton. That of horseshoe crabs is composed of chitin and protein and is not calcified. In adults it is tough and hard but juveniles have flexible exoskeletons.
The dorsal exoskeleton of the cephalothorax is the carapace, which is strongly convex (Fig 1). The cephalothorax is shaped like a horse's hoof with a smoothly curved anterior margin and a posterior recess into which the abdomen fits. The anterior edge of the carapace resembles a bulldozer blade and functions like one, enabling the animal to push through soft sediments. There are three longitudinal ridges on the dorsal surface of the carapace. The one on the midline is the median ridge and it has a lateral ridge on either side. All three ridges bear smallteeth.
Figure 2. Ventral view of a juvenile horseshoe crab. The appendages of the left side have been omitted for clarity. The cephalothoracic appendages on the right have been numbered 1-6. The articles of the appendages have also been numbered, thus 1 = coxa, 2 = trochanter, 3 = femur, 4 = patella, 5 = tibia, 6 = tarsus. Merostome10L.gif
The carapace bears a pair of lateral compound eyes and two anterior median ocelli , or simple eyes (Fig 1). Each compound eye is located on the lateral side of a tooth on a lateral ridge. The ocelli are located on either side of a tooth at the anterior end of the median ridge.
The ventral surface of the cephalothorax is strongly concave and bears six pairs of appendages (Fig 2, 18-1). The anteriormost appendages are the characteristic chelicerae of chelicerate arthropods. The chelicerae are relatively small and end with a pincer, or chela. Each chelicera is composed of three articles, the distal two forming the chela. The distalmost article is a movable finger and the penultimate article is an immovable finger. Note how they oppose each other to form a prehensile, or grasping, organ. The chelicerae attach to the body anterior to the mouth.
Figure 3. A chelicera. The articles are numbered. Merostome11L.gif
The next four appendages are similar to each other and are chelate walking legs although the first of them is sometimes referred to as a pedipalp (Fig 2, 18-1). In females the pedipalp (Fig 4) is almost identical to the other walking legs but in mature males it is modified and is not chelate. Instead, it has a terminal bulbous swelling and a hook used to hold the female prior to and during mating. What sex is your specimen? _________
The pedipalps are followed by three pairs of unspecialized walking legs (Fig 2, 4). The pedipalps and walking legs are similar in morphology and each is composed of six articles which are, in order from proximal to distal, the coxa, trochanter, femur, patella, tibia, and tarsus (Fig 4). Each coxa bears a spiny gnathobase on its medial surface and attaches to the body proximally. The walking legs are prehensile with chelae formed of the tibia and tarsus. The tarsus is a movable finger that closes against the immovable finger extending from the tibia.
The sixth cephalothoracic appendages are pushers that differ from the more anterior limbs (Fig 5). They are used to push the animal over the soft surface of the substratum. The fifth article of the pusher bears four petal-like spines which flare apart when pushed against the sand. The gnathobase of this leg bears a heavily cuticularized set of teeth resembling a crustacean mandible and presumably it functions as a mandible to crush and cut large food items into smaller particles. Limulus thus has a "mandible" at the posterior end of the food groove, rather than at the more customary anterior end.
Laterally the coxa of the pusher leg bears a narrow, spatulate flabellum, supposedly used to clean the gills but it seems to be too short to be effective in this role. Perhaps, since it is located in the inhalant water channel, it is sensory. The flabellum articulates with the coxa and is sometimes considered to be an exopod thus making the limb biramous. The sixth article bears a pair of short spines. In juveniles one of these spines is long and biarticulate.
Figure 4. The unspecialized pedipalp of an immature horseshoe crab. The walking legs are similar to this leg. Merostome12L.gif
The right and left rows of five gnathobases each lie beside the ventral midline and form the lateral borders of the food groove (Fig 2). The mouth is a large funnel-like opening between the coxae of the second pair of legs. It is directed posteriorly facing the anterior end of the food groove (Fig 8, 18-2). It is overhung anteriorly by a large upper lip, or labrum.
When feeding the crab uses its chelae to capture small invertebrates and place them in the food groove. Feeding and walking motions of the legs move the coxae, thereby causing the gnathobases to crush and grind any food in the groove. The spines move the food inexorably toward the mouth.
Note the rows of spines of the gnathobases and that they point to the mouth. If you have an anesthetized living or fresh specimen, insert a probe into the mouth and demonstrate that it opens into an esophagus that runs anteriorly, rather than dorsally or posteriorly. The gut is thus J-shaped, with the mouth facing posteriorly toward the anterior end of the food groove. Trilobites and the ancestral crustaceans also had posteriorly facing mouths, J-shaped guts, food grooves, and gnathobases and presumably fed in a similar fashion.
Excretion is accomplished by four pairs of saccate nephridia, known as coxal glands, in the cephalothorax. The four on each side drain by a single duct to an inconspicuous nephridiopore located at the base of the fifth appendage.
Figure 5. The sixth cephalothoracic appendage, or pusher, of a horseshoe crab. Merostome13L.gif
The abdomen is composed of nine segments and bears seven pairs of appendages. Study the ventral surface first. Like the cephalothorax, the abdomen is strongly concave ventrally (Fig 2). The concavity is the branchial chamber, which contains the book gills and is ventilated by movements of the appendages which bear the gills.
The first abdominal appendages are the chilariae on the posterior cephalothorax (Fig 2). They are the appendages of the vestigial first abdominal segment. They are small, oval, and composed of only one article. They have the important function of closing the posterior end of the food groove and perhaps also help manipulate or hold food in position for the mandible-like surfaces of the pusher gnathobases.
The second abdominal appendages are two broad lamellae fused together to form the genital operculum, which covers and protects the remaining abdominal appendages (Figs 2, 6, 18-3). Lift the operculum, if possible, to demonstrate that it is a single unit composed of two appendages fused together on the midline. In dried specimens the operculum usually cannot be lifted without breaking it. The two gonopores are located on the hidden posterior face of the genital operculum near its articulation with the body. (The posterior surface faces posteriorly when the operculum is extended straight down from the body. It faces dorsally when the operculum is folded back against the more posterior appendages.) The gonopores cannot be seen in dried specimens without breaking the operculum. Do not do so. Living specimens must be anesthetized in isotonic magnesium chloride order to lift the genital operculum to see the gonopores and the book gills.
Figure 6. The posterior surface of the genital operculum. Merostome14L.gif
Each limb of the genital operculum (and the remaining abdominal appendages) is biramous and the two limbs of each pair are fused along the midline (Figs. 6, 7). Each consists of a basal protopod from which arise two distal rami, an endopod and an exopod. The large protopod attaches to the body. The wide, short, uniarticulate exopod articulates with the protopod along most of its distal lateral margin. A more slender and biarticulate endopod attaches on the medial corner of the distal margin.
The next five pairs of delicate, flaplike appendages bear book gills (Figs 2, 7). These appendages are broad and each bears numerous flat, cuticular gill lamellae, on the posterior surface of the protopod. The lamellae resemble the pages of a book and are ventilated by movements of their respective appendages. These appendages may be difficult to study in dried or preserved specimens because of the rigidity of their articulations. Blood circulates through the interior of the lamellae and gases are exchanged over the thin permeable cuticle.
Dorsally the abdomen bears a median longitudinal row of three teeth (Fig 1). Lateral to this row, on either side, is a row of six small muscle insertion depressions, which mark the attachment points of muscles (Fig 1).
A large immovable spinous process is situated laterally on each side of the anterior end of the abdomen and a row of six movable spines can be seen along each lateral margin of the abdomen. These, and other, features are external manifestations of the segmentation of the abdomen, which consists of nine fused segments. Segment 1 is vestigial and is the segment of the chilariae, segment 2 bears the spinous process and the genital operculum, segments 3-8 bear book gills (absent on 8), dorsal muscle depressions, and lateral spines, and segment 9 has no appendages or lateral spines.
The tail spine extends posteriorly from the abdomen. The anus is a ventral opening in the soft flexible articular membrane connecting the abdomen and the telson (Fig 2).
Figure 7. The posterior surface of the 1 st pair of book gills. Merostome15L.gif
The ventilating current enters the ventral, abdominal respiratory chamber via two narrow inhalant channels located laterally and dorsally between the cephalothorax and abdomen (Fig 1, 18-1). It exits the chamber posteriorly via exhalant channels on either side of the tail spine.
>1a. If your specimen is living and active, place some carmine/seawater suspension in the intake to the inhalant channel and observe its fate. Watch for its exit via the exhalant channels.<
In most undergraduate invertebrate zoology labs the internal anatomy of horseshoe crabs is not studied. Juvenile specimens, with their flexible exoskeleton, are easily dissected however and a brief account of internal anatomy follows (Fig 8, 9, 18-2A, 18-4). Juveniles can be anesthetized in isotonic magnesium chloride.
" Use a preserved or anesthetized specimen and cut a semicircle (more or less) out of the middle of the dorsal surface of the cephalothorax. The straight hinge between cephalothorax and abdomen should form the straight edge of the semicircle. From one end of this hinge make a shallow circular cut through the exoskeleton, passing lateral to the compound eyes and simple eyes and roughly paralleling the margins of the cephalothorax and ending at the other end of the hinge. The cut should be no deeper than the exoskeleton.
Lift the anterior edge of the semicircle and reach beneath it with a scalpel or needle to free it from the underlying body wall and the many muscles that attach to it. Once freed of these connections it can be removed and set aside.
Now make two longitudinal cuts through the cuticle, parallel to the sides of the dorsal abdomen. Each should extend from the base of the spinous process to the base of the telson. Free this piece from the underlying tissue and remove it. Organs and structures will be described as they are encountered.
Looking into the cephalothorax you should see a large horseshoe of olive brown tissue located peripherally around the anterior and lateral margins of the cephalothorax (Fig 9, 18-4). This is the digestive cecum composed of myriad tiny branching tubes connected ultimately with the midgut. In some areas the tubules of the digestive cecum may be accompanied by bright white deposits of calcium phosphate which may obscure the brown color of the cecum. Calcium phosphate is removed from the blood and released into the lumen of these tubules to eventually be discarded in the feces. In mature specimens tubules of the gonad, very much like those of the digestive cecum, are intermingled with those of the cecum and the two occupy roughly the same areas of the cephalothorax.
Figure 8. Median sagittal section of the horseshoe crab, Limulus. Redrawn and modified from Owen (1873). Merostome23L.gif
A large oval, pale area on the midline in the anterior cephalothorax is the proventriculus (crop/gizzard) of the gut (Fig 8, 9). It is surrounded on three sides by digestive ceca.
Two oblique rows of circular white masses extend posterolaterally, one on each side, from the proventriculus. These are some of the muscles that operate the cephalothoracic appendages. In life they originate on the inner surface of the carapace and insert on the appendages.
The coelom is reduced to small spaces associated with the coxal glands and gonads and the chief body cavity is a hemocoel. The long tubular heart (Fig 8), enclosed in a thin-walled pericardial sinus, extends from the posterior end of the proventriculus almost to the tail spike. It lies on the dorsal midline. Anteriorly it narrows to form an anterior aorta that immediately bifurcates to form two major arteries to the brain and anterior cephalothorax. Numerous segmental arteries exit the heart. Blood enters it from the pericardial sinus via nine pairs of ostia.
The gut lies immediately ventral to the heart but is longer, extending past the heart both anteriorly and posteriorly. You have already seen the proventriculus at the anterior end. Carefully remove the heart to reveal the entire length of the gut (Fig 9). The gut is distinctly J-shaped but that will not be immediately apparent to you. Like the gut of other arthropods, it is composed of an anterior ectodermal foregut, an endodermal midgut, and a posterior ectodermal hindgut. The foregut consists of the mouth, esophagus, and proventriculus and its lumen is lined with cuticle. The midgut is sometimes called the intestine or stomach-intestine but will be referred to here simply as the midgut. It is long and narrow and extends from the proventriculus posteriorly through the cephalothorax and anterior abdomen to the short hindgut, or rectum. In living specimens the midgut is brownish whereas the foregut and hindgut are whitish, at least in juveniles.
" Use your fine scissors to make a longitudinal incision along the dorsal midline of the proventriculus. Look inside and note the thin transparent cuticle lining its lumen and the conspicuous, bumpy, longitudinal ridges on the walls of its posterior end. This part is sometimes called the gizzard.
The mouth opens into a short esophagus which extends anteriorly and dorsally to join the much larger proventriculus. The walls of the esophagus also bear longitudinal ridges. The esophagus enters the ventral portion of the proventriculus, sometimes called the crop, whose walls are not ridged, rather are wrinkled irregularly. The proventriculus forms the bend in the "J" of the gut. The esophagus is the short arm of the "J" and the midgut begins the long arm.
The posterior dorsal end of the proventriculus (sometimes known as the gizzard) narrows and extends into the lumen of the anterior midgut. This arrangement looks like a nozzle and forms a one-way valve from proventriculus to midgut.
" Extend the longitudinal incision posteriorly from the proventriculus for the length of the midgut. This will expose the valve.
The walls of the midgut are much thinner than those of the proventriculus. Two pairs of hepatic ducts from the digestive ceca open into the anterior midgut. The first pair, the anterior hepatic ducts, open ventrolaterally just posterior to the anterior end of the midgut. The posterior hepatic ducts open laterally and are a little farther posterior. Both pairs of ducts join the anterior fourth of the midgut. These four ducts lead to the abundant fine tubules of the digestive ceca which you saw earlier. The lumina of the digestive ceca tubules are continuous with that of the midgut. The digestive ceca arise in the embryo as diverticula of the midgut.
The midgut is the secretory and absorptive region of the gut. If your specimen is alive you may notice a distinct iridescence associated with the midgut epithelium. This is a manifestation of the brush border of microvilli on the midgut epithelium. These microvilli act as a diffraction grating to produce the colors. At about the level of the third movable abdominal spine the midgut joins the rectum.
Figure 9. Dorsal dissection of Limulus featuring the digestive system. The heart and hemal system have been removed the reveal the gut. Adapted from Owen (1873). Merostome24L.gif
" Extend the longitudinal, mid-dorsal incision of the gut tube posteriorly to open the rectum.
The character of the gut wall changes dramatically as the transversely folded, brown midgut wall gives way to the cuticularized, white, unfolded wall of the rectum. The rectum is short and empties ventrally via the anus. The anus opens at the base of the tail spine.
The nervous system is well-developed and consists of a cephalized central nervous system and abundant peripheral nerves.
" Expose the nervous system by carefully removing the midgut and proventriculus.
The double ventral nerve cord of the abdomen will be visible immediately upon removal of the midgut. The nerve cord of the cephalothorax however is hidden beneath a tough white sheet of connective tissue that covers the midline.
You must cut through this sheet with your fine scissors to expose the central nervous system ventral to it. Make a longitudinal incision along the midline, reflect and pin aside the cut edges of the connective tissue sheet. Most of the central nervous system will now be exposed. The esophagus is also exposed. Trace the esophagus posteriorly from the cut proventriculus to the mouth.
Many fused ganglia, connectives, and commissures form a large oval circumesophageal nerve ring around the esophagus at its junction with the mouth. The nerve ring is on the floor of the cephalothorax (Fig 8, 18-2A). Free the esophagus from the connective tissue strands holding it in place. Move it aside so you can see the nerve ring all the way around the mouth. Many nerves exit the ring to the appendages, sense organs, muscles, and organs of the cephalothorax. The nerve ring consists of the dorsal brain and the paired ganglia of the cephalothoracic and anterior abdominal appendages. The six pairs of cephalothoracic appendages, the chilaria, and the genital operculum are innervated from ganglia of the nerve ring. If you have a good dissection with easily visible nerves, you may want to trace some of them to their target organs. The nerve ring, nerve cord, and many peripheral nerves are enclosed in arteries.
Two longitudinal ventral nerve cords extend posteriorly from the nerve ring to the abdomen. They lie side by side and touch each other for part of their length. Five pairs of segmental ganglia can be seen as swellings along the nerve cord in the abdomen. From these arise nerves to the five pairs of book gills. The last of these five pairs of ganglia is also the end of the nerve cord and from it arise nerves not only to the fifth book gill but to many other posterior structures including the tail spine. This terminus is located at the level of the fourth and fifth book gills.
The excretory organs are four pairs of saccate nephridia, also known as coxal glands. Coxal glands are modified metanephridia resembling those of crustaceans, other chelicerates, and a few insects.
The four glands on each side share a common duct to a common nephridiopore on the fifth cephalothoracic appendage. The nephridiopores have already been found. The coxal glands lie at the bases of the cephalothoracic appendages 2-5 but are difficult to demonstrate in juvenile specimens. They are brick red in adults.
The reproductive system of juveniles is not developed. In adults the paired gonads, either testes or ovaries in these gonochoric animals, are composed of highly branched, arborescent tubules extending throughout the hemocoel, mostly in association with the similar tubules of the digestive ceca. Spermatozoa are flagellated as you can observe if you make a wetmount of testis from a mature living specimen. Each gonad empties via a gonoduct to a genital aperture on the genital operculum.
Fertilization is external and development produces a trilobite larva.
Look at a wholemount slide of a trilobite larva (or living or preserved specimens) using the dissecting microscope or low power of the compound microscope (Fig 10, 18-5).
The larva closely resembles the adult in most features. The body is divided into cephalothorax and abdomen but the tail spine is very small (Fig 10). The cephalothorax bears a median simple eye and a pair of lateral compound eyes on the dorsal surface. The margins of the abdomen bear six lateral spines and a spinous process.
Figure 10. Dorsal view of a trilobite larva. Merostome16L.gif
Focus on the ventral surface where you will find the same appendages you saw on the adult. Anteriorly on the cephalothorax is a pair of small chelicerae followed by four pairs ofwalking legs and a pair of pushers. The first pair of walking legs is homologous to the pedipalps of other chelicerates. A pair of tiny chilaria is located between the bases of the pushers. The outlines of the genital operculum are apparent on the venter of the abdomen. The posterior edges of some of the book gills can be seen extending beyond the posterior edge of the operculum.
Bonaventura J, Bonaventura C, Tesh S (eds.). 1982. Physiology and biology of horseshoe crabs: Studies on normal and environmentally stressed animals. Alan R. Liss, New York. 334p.
Bullough WS . 1958. Practical invertebrate anatomy (2 nd ed). MacMillan, London. 483p.
Lochhead JH. 1950. Xiphosura polyphemus, pp 360-381, in Brown FA. (ed) 1950. Selected invertebrate types. Wiley, New York. 597p.
Odell J, Mather ME, Muth RM. 2005. A biological approach for analyzing environmental conflicts: A case study of horseshoe allocation. BioScience. 55(9):735-748.
Owen R. 1873. On the anatomy of the American king-crab (Limulus polyphemus, Latr.). Trans. Linnaean Soc. London 28:459-506, pl 36-39.
Ruppert EE, Fox RS, Barnes RB. 2004. Invertebrate zoology, A functional evolutionary approach, 7 th ed. Brooks Cole Thomson, Belmont CA. 963 pp.
Walls EA, Berkson J, Smith SA . 2002. The horseshoe crab, Limulus polyphemus: 200 million years of existence, 100 years of study. Reviews Fisheries Science 10:39-73.
Dissecting microscope (if using small specimens)
Living, preserved, or dried Limulus, exuviae can also be usedTrilobite larvae, wholemount slides or living or preserved specimens