Histologic Findings in Captive American Horseshoe Crabs ( Limulus polyphemus )

Inflammatory Response and Infectious Agents

The inflammatory cell of HSCs is the hemocyte. ^4,9 Some texts refer to these cells as granulocytes or amebocytes. ¹⁰ The authors prefer the term hemocyte to denote a circulating immune cell (“-cyte”) in the hemolymph (“hemo-”). Hemocytes had abundant, brightly eosinophilic, cytoplasmic granules when they were in the vasculature (Fig. 1) but varied in the amount of cytoplasmic granules during an inflammatory response. In large inflammatory foci, the margins of the lesions were infiltrated by hemocytes with abundant cytoplasmic granules and round nuclei, while hemocytes closer to the centers of inflammatory foci frequently had few to no evident cytoplasmic granules and occasionally had elongate/oval nuclei. ⁹ For descriptive purposes, hemocytes with granules were called “granular hemocytes,” and those without granules and/or nuclear changes were called “reactive hemocytes.” Centers of large inflammatory foci were filled with amorphous, pale eosinophilic material (which represents coagulin gel) ⁹ admixed with a small amount of karyorrhectic debris; for descriptive purposes, this mix of hemocytes, coagulin, and necrotic cellular debris was called a “hemocyte coagulum” (Fig. 2). Rarely in the gills, large inflammatory foci had loose aggregates of plump spindle cells (Fig. 3). Infectious agents and cutaneous wounds were often embedded in and occluded by hemocyte coagula, respectively. Fungi were frequently seen invading from the external body wall or external surface of the gills and associated with a robust inflammatory response. Bacteria were commonly associated with small to moderately sized inflammatory foci randomly scattered throughout internal organs. Metazoan parasites frequently had minimal to no associated inflammation and are described further in each system below.

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Figures 1–10.

American horseshoe crabs (HSCs; Limulus polyphemus). Figure 1. Normal hemocytes, vascular sinus, case 14. Hemocytes are within vasculature and have abundant, eosinophilic cytoplasmic granules. Hematoxylin and eosin (HE). Figure 2. Hemocyte coagulum, hepatopancreas, case 27. A large hemocyte coagulum infiltrates the hepatopancreas and has a central region of eosinophilic acellular material that encases bacteria (arrow). Hemocytes surround the coagulum. HE. Figure 3. Branchitis, gill, case 44. Vascular chamber of a branchial lamella is markedly expanded by infiltrating hemocytes and clear space (edema) at the top of photo. Large numbers of spindle cells form loosely aggregated streams in the center of the area of inflammation (bottom of photo). The significance and function of these spindle cells are unknown, but they presumably function in the reparative response. Inset: Higher magnification of spindle cells. HE. Figure 4. Normal hepatopancreas, case 11. The hepatopancreas is composed of collecting tubules and interstitial cells. Gonads (ovaries represented by round oocytes) interdigitate with this organ. HE. Inset: Higher magnification of interstitial cells. Figure 5. Interstitial cell globule depletion, hepatopancreas, case 23. Hepatopancreatic interstitial cells have few eosinophilic globules in the cytoplasm. Aggregates of interstitial cells are highlighted with an asterisk. Hemocyte sinuses containing hemocytes (left of photo) and collecting tubules are also visible in this image. HE. Figure 6. Metacercarial cyst, skeletal muscle, case 25. Parasitic cyst with a double cyst wall and a folded, acoelomate body with sucker (arrow) is within autolyzed skeletal muscle. HE. Figure 7. Bacterial coagula, hepatopancreas, case 40. Several small coagula centered on bacterial rods are within the hepatopancreatic interstitium. HE. Inset: Bacterial rods are acid-fast positive. Zhiel Neelson. Figure 8. (a) Crystalline material, hepatopancreas, case 50. Hepatopancreatic interstitium has multifocal aggregates of brown crystalline material that sometimes appears intracellular as it is rarely associated with nuclei and forms polygonal shapes. HE. (b) Crystalline material is birefringent when viewed with polarized light. Figure 9. Normal collecting tubule, hepatopancreas, case 32. The collecting tubule is lined by 2 cell types: dark cells have cytoplasmic zymogen granules, and light cells have spherical, sometimes multilayered, deeply basophilic granules. The collecting tubule is surrounded by a thin muscular layer. HE. Inset: Higher magnification of dark and light cells. Figure 10. Bacterial tubulitis, hepatopancreas, case 56. A mix of bacteria (arrow), eosinophilic material, and light cell granules occlude the tubular lumen; dark and light cells are effaced. The tubule is surrounded by granular and reactive hemocytes that rarely invade the muscular layer. HE.

Hepatopancreas

The hepatopancreas occupies the majority of the prosoma (ie, largest and most anterior body segment) and was examined histologically in 57 cases. It interdigitated with skeletal muscle of the legs and the reproductive tissue (ie, testes or ovaries) and surrounded multiple hemocoel sinuses containing hemocytes (Fig. 4). The hepatopancreas comprises interstitial cells and collecting tubules. Interstitial cells (also called storage cells or reserve cells) ⁵ occupied the majority of the hepatopancreas and were interrupted regularly by collecting tubules.

Interstitial cells were polygonal and large with abundant, vacuolated cytoplasm; variable numbers of eosinophilic, spherical globules; and fewer, smaller basophilic globules that varied in size and shape. The amount of eosinophilic globules varied by case and was not uniform across different regions of the hepatopancreas. In some cases, eosinophilic globules were remarkably sparse (12/57 cases; 21%), and this was interpreted as eosinophilic globular depletion (Fig. 5). Hemocytes formed aggregates and coagula in the hepatopancreatic interstitium of 16 of 57 cases (28%). Inflammatory foci in the hepatopancreas were usually associated with identifiable infectious agents (see below). Encysted, variably degraded parasites resembling trematodes were identified in the hepatopancreatic interstitium of 22 of 57 cases (39%), as well as in many other tissues (see “Other Organs”). These cysts were 80 to 150 microns in diameter with a double wall and infolding of an acoelomate body that sometimes had recognizable suckers (Fig. 6). The immune response to these parasites ranged from small aggregates of granular hemocytes, to a combination of granular and reactive hemocytes, to a small amount of surrounding, compressed mesenchymal cells (possible fibrosis), to no identifiable host response. Bacterial infections in the hepatopancreatic interstitium were identified in 10 of 57 cases (18%); bacteria were associated with multifocal, randomly dispersed, small to moderately sized hemocyte aggregates and/or coagula. In 1 case, an acid-fast stain revealed intralesional acid-fast bacilli (Fig. 7). Fungal infections in the hepatopancreas were rare (2/57 cases; 4%) and always occurred at the margin of the organ due to extension of cutaneous infection through the body wall (see “Body Wall” section). Rarely (4/57 cases; 7%), the hepatopancreatic interstitium had granular, birefringent, brown crystalline material that was not associated with any identifiable host reaction and appeared both intra- and extracellular (Fig. 8).

Collecting tubules varied from 50 to 500 microns in diameter and were lined by simple, tall, columnar epithelial cells surrounded by a thin muscular network composed of circular and longitudinal striated muscle. ⁶ The 2 primary cell types in the tubule epithelium were light cells (also called acidophils) and dark cells (also called basophils) (Fig. 9). ⁵ Dark cells had secretory granules that were variable in number depending on the animal. Dark cells are analogous to the exocrine pancreas of mammals and secrete enzymes such as lipase, amylase, and trypsin; as such, their granules may be referred to as “zymogen granules.” ⁵ Light cells were much more numerous (approximately 4 times) than dark cells. Collecting tubules were sometimes distended with amorphous granular, basophilic material (presumed digestive contents) admixed with zymogen granules. Inflammation with peripheral granular and central reactive hemocytes surrounded and rarely infiltrated the muscular layer of tubules in 6 of 57 cases (11%), all of which had intratubular bacteria with effacement of tubular epithelium (Fig. 10).

Body Wall

Body wall was histologically identified in 60 animals. The cuticle was organized into 3 acellular layers: an external, thin, poorly staining epicuticle; a thick, chitinous exocuticle; and an inner, deeply eosinophilic endocuticle (Fig. 11). The epidermis was composed of melanized columnar cells. The dermis had large secretory glands that emptied through the outer cuticle.

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Figures 11–21.

American horseshoe crabs (HSCs; Limulus polyphemus). Figure 11. Normal cuticle, case 32. The outermost epicuticle is thin, acellular; the middle layer (exocuticle) is thick, chitinous, acellular; the inner endocuticle is similar in thickness to the exocuticle. Inset: The epidermis is composed of packets of columnar cells with apical melanization. The dermis contains large secretory dermal glands that empty into dermal gland ducts, which extend through the outer carapace and comprise vacuolated, basophilic trichogen cells (arrowhead). Hematoxylin and eosin (HE). Figure 12. Ulcerative fungal dermatitis, cuticle, case 14. The epidermis is diffusely ulcerated with inflammation of the dermis and skeletal muscle. The ulcerated epidermis is overlain by a crust of numerous granular and reactive hemocytes and extracellular, eosinophilic, homogeneous protein (coagulen; arrow). HE. Figure 13. Fungal dermatitis, cuticle, case 14. Higher magnification of fungi from Fig. 12 shows numerous, nonpigmented, approximately 8- to 10-micron-diameter, fungal hyphae with occasional septation and variable angle branching (frequently right angle). HE. Figure 14. Green algal dermatitis, cuticle, case 30. Algae are pleomorphic with frequent septation, argyrophilic internal structure, nonparallel walls, and bulbous distentions. Gomori methenamine silver. Figure 15. Dysecdysis, cuticle, case 4. The previous molt (brown) is retained on the caudal aspect of the opisthosoma and over the entire telson. An ectoparasite is on the retained molt (arrow). The newly molted portion of the cuticle is gray. Image credit: Dr. Tabitha Viner. Figure 16. Dysecdysis, cuticle, case 4. Previous molt (top) overlies the new cuticle with some debris between the two. HE. Figure 17. Rhabdomyolysis, skeletal muscle, case 56. All skeletal myocytes have fragmented sarcoplasm with loss of cross-striations and internalization of nuclei with rare karyorrhexis; edema is represented by pale eosinophilic fluid between sarcoplasmic fragments. HE. Figure 18. Amoebic myositis, skeletal muscle, case 61. Moderate numbers of hemocytes infiltrate skeletal muscle with scattered necrotic cellular debris. Amoeba (arrowheads) are within and around inflamed regions. HE. Inset: Higher magnification shows amoebas (arrowheads) among hemocytes and necrotic debris. HE. Figure 19. Normal book gills, case 53. Parallel chitinous lamellae are held apart by pillar cells (arrow) and contain vascular spaces (asterisks). HE. Figure 20. Fungal branchitis, gills, case 43. Gill lamellae are markedly expanded by hemocytes, eosinophilic fluid (edema), and numerous branching fungal hyphae (arrows) that invade through chitinous walls (inset). HE. Figure 21. Biofilm, gill, case 19. Thick mats of biofilm include bacteria and algae consistent with blue-green algae (ie, cyanobacteria). Blue green algae have algal cells that are 15 to 30 microns in diameter and spherical to indented with internal granules. HE. Inset: Higher magnification of algal cells (arrowheads). Periodic acid–Schiff.

The most common lesions in the body wall were loss of a portion of the epicuticle (termed erosion), parasitic cysts, and ulceration of the cuticle and epidermis with inflammation in the dermis and surrounding cuticle (Fig. 12). Erosion without ulceration was identified in 10 of 60 cases (17%) and was invariably associated with superficial (ie, noninvasive) bacterial colonization without any appreciable inflammation. Ulceration was present in 34 of 60 cases (57%) and was consistently associated with infectious organisms; associated fungi were usually invasive and bacteria were usually superficial, suggesting that fungal agents were more likely causes of ulcerative lesions. Fungi had variable morphology. All were filamentous with septate hyphae. Branching was generally acute angle, and walls were either parallel or nonparallel. Size varied widely from approximately 6 to 30 microns in diameter (Fig. 13). Parasitic cysts, as described in the hepatopancreas, were identified in soft tissues of the body wall in 19 of 60 cases (32%).

An invasive algal infection was identified in the body wall in 1 of 60 cases (2%). Histomorphology of this infection was consistent with previous reports of green algal infection. ^3,8 Algae had highly pleomorphic morphology with occasional septation, budding, and internal structure. Gomori methenamine silver stain revealed argyrophilic internal contents of the algae, but algal walls had minimal or no argyrophilia (Fig. 14). Algae were not associated with appreciable inflammation but appeared to cause defects in the cuticle as they were associated with ulceration and invaded into subjacent dermis and skeletal muscle. Superficial bacteria were also present in algal lesions.

Retention of molt was seen in 3 of 60 cases (5%) (Fig. 15). Histologically, an extra layer of cuticle (ie, previous molt) was overlying the body wall, which had a thin cuticle lacking normal layering and some chitinous debris between the old and new cuticle (Fig. 16). Retention of molt was also observed in the chitinous sections of the gut in those cases. Four cases had swollen myofibers with loss of cross-striations and occasional karyorrhectic nuclei (ie, rhabdomyolysis) in large sections of skeletal muscle without substantial inflammation (Fig. 17). Abundant hemocytic inflammation in the skeletal muscle with some myocellular necrosis (ie, myositis) was observed in 4 cases, 3 of which were associated with bacteria, and 1 was centered on presumptive amoeba, the latter of which were identified by their shape (amoeboid), spherical nucleus, size (20–35 microns), and cytoplasm with phagocytosed contents (Fig. 18). Superficially, the body wall was occasionally overlaid by arthropods, algae, protozoa, and other metazoan parasites, none of which were associated with inflammation or substantial cuticular changes.

Book Gills

Book gills were identified histologically in 48 of 61 cases (79%) and composed of lamellae that arose from opercula. Gill lamellae were arranged in 2 simple, flattened epithelial layers that enclosed a vascular lumen containing hemolymph. These epithelial layers were held apart by pillar cells (Fig. 19). ¹ The most common branchial lesion was inflammation (29 of 48 cases; 60%). Hemocyte coagula were frequently multifocal and led to widening of the lamellae and sometimes ulceration of the lamellar tips (Fig. 20). Inflammation was variably associated with infectious agents, including bacteria in 21 of 29 cases (72%) and fungi in 19 of 29 cases (66%); no infectious agents were seen on HE-stained sections in 5 of 29 cases (17%) of branchitis, but special stains for infectious agents were not performed on branchial sections in those cases. Fungal and bacterial morphology were similar to those described above (see “Hepatopancreas” and “Body Wall”). Parasitic cysts embedded in connective tissue as described above were seen in the gills of 1 case (2%). Mats of bacteria, algae, fungi, and parasites (including arthropods, nematode larvae, and trematode larvae) occasionally formed on the surface of lamellae, were not associated with inflammation, and may have been incidental. In 4 of 48 cases (8%), these mats included algae consistent with blue-green algae (ie, cyanobacteria) characterized by 15- to 30-micron, spherical to indented algal cells with internal granules (Fig. 21). ⁸ Algae were consistently superficial and were not identified invading into gills.

Other Organs

Parasitic cysts as previously described were identified in a wide range of tissues in addition to those previously mentioned, including brain, chitinous gut, compound eye, heart, and nonchitinous gut. Lesions in the compound eye reflected the range of lesions seen in the body wall to include fungal and/or bacterial ophthalmitis with variable ulceration. Enteritis was identified in the chitinous gut in 4 of 40 cases (10%) and nonchitinous gut in 3 of 42 cases (7%) for which these organs were identified histologically. Bacteria were associated with enteritis in 5 of 7 cases (71%), 1 of which had acid-fast bacilli, and fungi were associated with enteritis in 1 of 7 cases, which was a mixed bacterial and fungal enteritis; infectious agents were not visibly associated with enteritis in the remaining 2 of 7 cases (29%). Lesions were not identified in the gonads or coxal gland in any case.

Culture Results

Bacterial or fungal culture results were reported in the necropsy records of 6 cases. Pseudomonas putrefaciens and Aeromonas hydrophila were isolated from hemolymph from cases 2 and 4, respectively. Micrococcus sp. was isolated from hepatopancreas from case 18. Fusarium spp. were isolated from gills from cases 14 and 15 and from an unspecified location from case 8.