Elise E.B. LaDouceur1, Michael M. Garner2, Katie J. Roorda3, and Alisa L. Newton4,5 1 Joint Pathology Center, Silver Spring, MD, USA 2 Northwest ZooPath, Monroe, WA, USA 3 Johns Hopkins University, Baltimore, MD, USA 4 Wildlife Conservation Society, Bronx, NY, USA 5 Disney’s Animals, Science and Environment, Orlando, FL, USA This class of chelicerate arthropods has only four extant species: one from North America (Limulus polyphemus,American horseshoe crab) and three from Asia (Tachypleus tridentatus, tri‐spine horseshoe crab, Tachypleus gigas, Indo‐Pacific horseshoe crab, and Carcinoscorpius rotundicauda, mangrove horseshoe crab) (Smith et al. 2017). Although termed “crabs,” these animals are more closely related to spiders and scorpions, which are also chelicerate arthropods, than true crabs, which are crustaceans. Horseshoe crabs (HSC) are often referred to as “living fossils” as current forms resemble fossils dating back to the Jurassic Period approximately 200 million years ago (Walls et al. 2002). HSC are marine invertebrates that feed on algae, marine worms, clams, other molluscs, and fish. They migrate to deep ocean waters in the winter, and move to shallow waters to spawn in the summer. L. polyphemus molts approximately 16–17 times in 9–10 years to reach maturity. Tachypleus spp. and C. rotundicauda both require relatively fewer molts (approximately 13) to reach maturity (Fahrenbach 1999). Juvenile HSC are morphologically similar to mature adults but have shorter telsons and are smaller. During spawning, HSC eggs serve as a major source of food for migratory bird populations. Sources of mortality in wild populations of L. polyphemus include complications of hemolymph collection for biopharmaceutical production, natural predation, commercial harvest for bait, spawning habitat loss, pollution, bycatch, climate change, and collection of specimens for research, education, and aquaria (Smith et al. 2017). HSC are used heavily by the biopharmaceutical industry for an enzyme in their hemolymph called limulase amebocyte lysate (LAL), which detects small quantities of endotoxin. Due to this property, the Food and Drug Administration requires that nearly all pharmaceutical products are tested with LAL prior to market to screen for contamination (Anderson et al. 2013; Walls and Berkson 2003). The body of HSC may be divided into three parts: prosoma (cephalothorax), opisthosoma (abdomen), and telson (tail). Dorsally, on the prosoma, there are two medially located ocelli and two laterally located compound eyes (Figure 9.1). A hinge joint connects the prosoma and opisthosoma. The margin of the opisthosoma is lined bilaterally by six spines that are movable by a hinge joint, but are not under nervous control. A dorsal midline ridge extends most of the length of the prosoma and opisthosoma and is called the keel. An inhalant channel separates the opisthosoma and prosoma, and aids in water flow to the book gills. Ventrally HSC have 14 pairs of specialized appendages (Figure 9.2). The first six reside in the prosoma while the rest are opisthosomal appendages. The first pair, the chelicerae, consist of a small pair of pinchers used for collecting food. The second, the pedipalps, help define the sex of HSC. Males have a larger hook‐shaped bulbous claw used to attach themselves onto females during mating whereas females’ claws appear the same as the other walking legs. The walking legs comprise appendages three through five. The sixth appendage, also known as the pusher leg or swimmer leg, is used to propel the animal through water. Located centrally in the prosoma is the gnathobase or mouth, which is made up of the coxa leg segments. Figure 9.1 External photograph of the dorsum of a horseshoe crab, Limulus polyphemus. The body is divided into prosoma, opisthosoma, and telson. Figure 9.2 External photograph of the ventrum of L. polyphemus. Walking and swimmer legs are attached to the prosoma, and the gills are attached to the opisthosoma. The opisthosomal appendages include the genital operculum, gill opercula, and telson. The genital operculum covers and protects the book gills (Figure 9.3). The eighth through thirteenth appendages are gill opercula which individually protect and contain the ventrolateral paired book gills. Finally, the last appendage is known as the telson, which functions to right the animal if it has been flipped upside down. Figure 9.3 External photograph of the ventrum of L. polyphemus. A gill operculum is reflected to reveal the subjacent book gills. An excellent tutorial on the anatomy and dissection of HSC is provided by Bergdale with images, an abridged version of which follows (Figure 9.4) (Bergdale 2017). The authors prefer a ventral approach as organs remain in situ during examination, and the internal viscera are more easily accessed ventrally. For a ventral approach, place the animal in dorsal recumbency. Use pruning shears to cut around the entire edge of the prosoma, approximately ½ cm from the margin of the prosoma. Gently peel back the ventral, inner carapace to reveal the interdigitating hepatopancreas and gonads, which may be distinguishable in gravid females. Cut the muscular attachments that extend through the hepatopancreas and attach to the ventral carapace, as well as those that extend through the hepatopancreas and attach to the dorsal carapace; cut as close to the carapace as possible both dorsally and ventrally to avoid damaging the viscera. The hepatopancreas and gonads should be completely separable from the ventral carapace (Figure 9.5). Push the gnathobase laterally to expose the mouth. Make a circular cut around the mouth in the soft tissue. Observe the excretory system (i.e., coxal glands) if still intact; these are bright orange, paired structures with four lobes each on either side of the arterial ring. Observe the arterial ring, located directly posterior, which completely houses the brain. Next cut through all the gill opercula to completely separate the internal anatomy from the carapace. Starting at the mouth, cut up through the esophagus to expose the gizzard. Flip the specimen to the dorsal side and continue opening the intestinal tract. Observe the heart and pericardial sac located anteriorly to the midgut. Figure 9.4 Illustrated sagittal section of L. polyphemus. am, arthrodial membrane; an, anus; bl, bladder; br, brain; ca, coelomic artery; cg, coxal gland; cr, crop; cs, cartilage shelf (endosternite); es, esophagus; gh, glandular hepatic ceca; gl, gill operculum; gn, gnathobase; go, genital operculum; gz, gizzard; he, heart; hg, hepatopancreas and/or gonadal tissue; hi, hindgut; mg, midgut; mo, mouth; pr, protocerebrum; ps, pericardial sac; pv, pyloric valve; re, rectum; tm, telson muscles; tr, tritocerebrum, vn, ventral nerve cord. Source: Illustration by Katie J. Roorda. Bergdale (2017). Table 9.1 outlines the organs for histologic examination horseshoe crabs. The body of a horseshoe crab is organized into three segments: prosoma, opisthosoma, and telson. The prosoma is the largest and most anterior body segment. The opisthosoma is smaller than the prosoma; it is the midbody, and houses the book gills. The telson is the tail‐like structure attached to the caudal end of the opisthosoma. The entire body, except for joints, is overlain by the carapace, which provides a physical protective barrier for the internal body. Skeletal muscle attaches to the inner carapace and is under nervous control, permitting movement of body parts (e.g., legs, telson, etc.). The cuticle ranges from thin and flexible to thick and stratified, such as over the dorsal prosoma. It is composed of protein and chitin. In thick regions, the entire cuticle is up to 1500 μm thick and is subdivided into three layers (Figure 9.6). The outermost epicuticle is thin (approximately 5–15 μm thick), acellular, and frequently does not stain well on routine HE, paraffin‐embedded sections. The middle layer (exocuticle) is thick, chitinous, acellular, birefringent, and pale eosinophilic. This layer is laminated and approximately 450 μm thick. The inner endocuticle is deeply eosinophilic, and approximately 550 μm thick. The basilar aspect of the inner endocuticle has a variably present, 2–10 μm thick, granular, basophilic region at the junction with the epidermis. A thin (3–15 μm diameter), hard, cuticle is over the gills and lacks discrete layering. A thin, bilayered, relatively flexible cuticle covers parts of the ventral prosoma, arthrodial membranes, and the mucosa of the fore‐ and hindgut. It is composed of endocuticle and epicuticle, which are demarcated by a feathered borderline (Figure 9.7) (Fahrenbach 1999). There are several adornments and structures that extend through and from the cuticle. Skeletal muscle attaches directly to the underside of the cuticle (i.e., myocuticular junction), resulting in a focal deviation at the site of attachment. Long, striated myofibers extend directly from the basilar aspect of the epidermis deep into the body wall (Figure 9.6). Tendinous attachments, which are fewer than muscular attachments, appear similarly. Long, infolded pillars of hard cuticle extend into the body wall and serve as a muscular attachment for the gills and telson. Thin pores extend from the base of the cuticle through the epicuticle. These pores appear to originate at the epidermis. They can be straight or wavy and are generally thicker in the endocuticle with narrowing at the epicuticle. A variety of setae, bristles, and spines emanate from the surface of the cuticle multifocally, particularly on the ventral aspect of HSC. The cuticle overlying the eyes is altered into a lens or cuticular cones to facilitate vision (see section 9.3.9). Figure 9.5 Illustrated ventral dissection approach of L. polyphemus. The ventral carapace has been removed by cutting the muscular attachments between the hepatopancreas and the carapace, and cutting around and removing the gnathobase (not pictured). Deep to the region of the gnathobase is the mouth surrounded by an arterial ring. The brain resides entirely within the arterial ring, and most nerves reside inside the arterial system. Lateral to the brain are four pairs of coxal glands, which are bright orange. After the nervous structures and coxal glands are removed, the genital opercula can be removed to expose the remainder of the digestive tract (not pictured). Source: Illustration by Katie J. Roorda. Bergdale (2017). a Alternative names for organs are provided parenthetically, in italics. Figure 9.6 Body wall of L. polyphemus. The cuticle is organized into a poorly staining epicuticle, densely staining exocuticle, and inner endocuticle. Immediately deep to the cuticle are the pigmented epidermis, glands, and skeletal muscle, the latter of which attaches directly to the cuticular wall. 40×. HE. Figure 9.7 Arthrodial membrane of L. polyphemus. The cuticle is focally thin and flexible representing the arthrodial membrane, which is demarcated by a feathered margin from the flanking hard cuticle. 100×. HE. The epidermis is composed of packets of columnar cells arranged in a single layer on a basal lamina. Nuclei are oval and basilar. Epidermal cells associated with the hard cuticle generally have apical melanization, while those associated with thin, flexible cuticle are generally nonpigmented. The dermis contains two types of glands. Mucous glands are oval, horizontally oriented, and directly underneath the epidermis. They are up to 250 μm in diameter and contain droplets that vary in size and color (Figure 9.8). Serous glands are smaller, pear‐shaped, and surround a 10 μm diameter duct. Serous glands are most common under the flexible cuticle (Fahrenbach 1999). Both glands are associated with acellular ducts that traverse the overlying cuticle. Ducts coalesce to form larger canals on the outer surface of the carapace. Approximately 10–12 ducts may empty into one large canal (Stagner and Redmond 1974). The remaining tissue of the dermis is composed of interwoven, small vascular channels, loose connective tissue, and reserve cells (Fahrenbach 1999). Figure 9.8 Epidermis of L. polyphemus. The epidermis is a simple layer of cells with basilar, round nuclei, and abundant pigment granules. A mucous gland and small vascular spaces containing hemocytes are also pictured. 400×. HE. A cartilage shelf (also called endosternite) is present dorsal and lateral to the ventral nerve cord, and lateral and ventral to the intestines. The caudal portion of the shelf is adjacent to the anterior‐most book gills. The cartilage is composed of pale basophilic chondroid matrix studded regularly with lacunae that contain a large clear vacuole and peripheralized chondrocytes. Larger lacunae may be separated by partitions (Figure 9.9). The cartilagenous matrix is immediately surrounded by fibrous connective tissue that is further surrounded by hepatopancreas, dermis, or skeletal muscle. All muscle in HSC, both visceral and structural, is striated, similar to other arthropods. Muscle fibers vary in size (ranging from 10 to 130 μm in diameter) and are surrounded by bundles of collagen, fibroblasts, and axons. Myocytes can have both peripheral and central nuclei.
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Arthropoda: Merostomata
9.1 Introduction
9.2 Gross Anatomy
9.2.1 Dissection
9.3 Histology
9.3.1 Body Wall/Musculoskeletal
9.3.1.1 Cuticle
Organ system
Organs
Body wall/musculoskeletal
Cuticle, epidermis/dermis, cartilage, arthrodial membrane, skeletal muscle
Digestive
Alimentary canal
Mouth, esophagus, proventriculus (crop and ventriculus [gizzard]), intestines (midgut), rectum (hindgut)
Hepatopancreas
Interstitial cells, collecting tubules (midgut diverticula)
Excretory
Coxal glands (kidneys, coxae)
Circulatory
Heart, hemocoel, arteries
Immune
Hemocytes
Respiratory
Book gills
Nervous
Brain, circumesophageal ring, ventral nerve cord, peripheral nerves
Reproductive
Male
Testis
Female
Ovary, oviduct
Special senses/organs
Compound eyes, ocelli (median eyes), rudimentary eyes, ventral (endoparietal) eye
9.3.1.2 Epidermis and Dermis
9.3.1.3 Connective Tissues and Muscle
9.3.2 Digestive System
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