NOTES Gill Trematodes (Flukes) in Wild-Caught Killifish ( heteroclitus)

DAVID R. GOULDING, BS, RLAT,1* TERRY L. BLANKENSHIP-PARIS, DVM, MS, DIPLOMATE, ACLAM,1 GREGORY A. LEWBART, MS, VMD, DIPLOMATE, ACZM,2 PAGE H. MYERS, BS, LATG,1 TRACY K. DEMIANENKO, BS, RLAT,1 JAMES A. CLARK, LATG,1 AND DIANE B. FORSYTHE, DVM, DIPLOMATE, ACLAM1

Three wild caught killifish (Fundulus heteroclitus) on an Institutional Animal Care and Use Committee-approved protocol were found dead within 2 days after being received. The fish were housed in two separate aquaria. Aquarium water was evaluated, and pH, salinity, ammonia, nitrate, and nitrite levels were within acceptable parameters. Several remaining fish appeared to be slow-moving and were presented for necropsy. Multiple, scattered, ulcerated skin lesions (diameter, 1 to 5 mm) were noted at necropsy and were cultured. No pathogenic bacteria were isolated. Wet-mount samples of the gills revealed multiple cysts at the gill margins, each containing a motile organism. No other gill parasites were detected. A diagnosis of trematodiasis was made. The cysts were identified as encysted metacercariae of a digenetic trematode. We surmise that the large numbers of gill flukes combined with the stress of recent shipment likely caused the observed morbidity and mortality.

Sixty-two wild-caught killifish (Fundulus heteroclitus) obtained from Duke Marine Center (Beaufort, N.C.) were group-housed upon receipt in two 38-gallon aquaria containing conditioned brack- ish water. Thirty-six fish were placed in tank A, and 26 fish were placed in tank B. One day after arrival, two fish were found dead in tank B, and 2 days after arrival, one fish was found dead in tank A. In addition, on the second day, one fish from each tank presented with inactivity. The killifish had an average weight of 1 g and an average length of 6 cm. Water-quality monitoring occurred twice weekly and included the evaluation of pH, salinity, nitrate, nitrite, and ammonia levels. All indices were within acceptable levels in both tanks; the pH ranged from 8.0 to 8.3, salinity was 28 to 30 ppt, ammonia was 0 ppm, nitrate was 20 to 40 ppm, and nitrite was 0 ppm. Dissolved oxygen parameters were not monitored. The two fish that presented with inactivity were euthanized with an anes- thetic overdose (3 g/liter) of NaOH-buffered MS222 (Sigma Chemical Co., St. Louis, Mo.) for diagnostic evaluation and necropsy. Gross external examination revealed multiple, scattered, ulcerated dorsolateral skin lesions (diameter, 1 to 5 mm). Skin lesions were cultured on blood, MacConkey, PEA, Pseudomonas, and potato dex- trose media. Gross lesions were not present on the gills, although the gills did appear red and inflamed. No other gross lesions were noted. The gills were excised using a pair of fine scissors. Saline wet mounts were prepared of the gills and skin lesions. Several samples were stained with methylene blue. Both stained and unstained wet mounts were examined under light microscopy (magnification, ×40, ×100, and ×200). Microscopic examination of the gill wet mounts re- vealed multiple, encysted, motile, organisms located in the primary lamellae of the gills. No abnormalities were observed in skin wet mounts. Figure 1. A digenetic trematode metacercaria excised from a Fundulus heteroclitus A diagnosis of trematodiasis was made. In light of their location × and morphologic characteristics, the encapsulated parasites were di- lamella. Wet mount prep stained with methylene blue (magnification, 400). agnosed as metacercariae of a digenetic trematode (Fig. 1). No pathogenic bacteria were cultured from the skin lesions. evaluated by a pathologist who confirmed the presence of approxi- Sections of the gills were fixed in 10% neutral buffered formalin, mately 20 metacercariae scattered across the entire gill section (Fig. 2 processed, and stained with hematoxylin and eosin. The gills were and 3). Trematodes (flukes) are organisms classified into two subclasses, Comparative Medicine Branch,1 National Institute of Environmental Health Sciences, MD monogenetic and digenetic. Monogenetic trematodes complete their C1-06, Research Triangle Park, North Carolina 27709 and the Department of Clinical entire development in a single host (1). Digenetic trematodes re- Sciences, 2 North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606 quire at least one intermediate host in order to mature (1). *Corresponding author Digenetic trematodes (digeneans) are monecious; they have re-

32 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science Volume 43, No. 2 / March 2004 Figure 2. Histopathology of digenetic trematode metacercariae located in the Figure 3. Histopathology of digenetic trematode metacercariae located in the primary lamellae of the gill (arrow) of Fundulus heteroclitus. (H&E stain; primary lamellae of the gill in Fundulus heteroclitus. (H&E stain; magnifica- magnification, ×20). tion, ×100).

Figure 4. Possible digenetic trematode life cycles demonstrating intermediate and final hosts (adapted from ref. 2). productive organs typical of both sexes in a single individual (1). intermediate host (a fish or invertebrate) (1). If the cercariae infect a Figure 4 illustrates a typical life cycle of a digenetic trematode (2). second intermediate host, then migration to the host’s target tissue Digeneans are oviparous, and their eggs, usually large and opercu- occurs, and the cercariae differentiate into metacercariae (4). The lated, pass in the feces of the definitive host (a fish, bird, or mammal) metacercariae encyst in various tissues, depending on the type of (1, 3). A ciliated, free-swimming miracidium hatches in about 21 digenean (4). The metacercaria-infected tissues are ingested by the days after the eggs enter water. The miracidium infects the first definitive host (1, 3, 4). Once present in the definitive host, the intermediate host (a mollusk, frequently a snail) (1, 3, 4). Asexual metacercaria develops into an adult digenean (3). The life cycle is reproduction occurs, and sporocysts are formed (1, 4). After ap- complete once the adult digenean reaches sexual maturity in the proximately 6 weeks, free-swimming cercariae are released into the definitive host (1, 3, 4). The species of trematode and various envi- water from the first intermediate host (4). The released cercariae ronmental factors (e.g., host availability and air and water penetrate or are ingested by either the definitive host or a second temperatures) lead to variations in the length of the life cycle. Ap-

Volume 43, No. 2 / March 2004 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science 33 proximately 1700 species of adult digeneans commonly infect wild trematodes as well as other parasites and bacteria (4). Although the fish and, although infections are frequent, they are generally asymp- presence of metacercariae are prevalent throughout fish populations, tomatic (3). the infections rarely are lethal (3). However, if the affected fish Metacercariae usually have the same characteristics as adult become stressed by capture and transport, immune function may be digeneans, except for mature reproductive organs. Because the size compromised, leaving them more susceptible to the effects of resi- and shape of the reproductive organs are used to determine the dent parasites. species of digeneans and only the larval stage was observed in this Because a definitive host is needed to complete the life cycle, it is case, it was impossible to speciate the infesting trematode in these unlikely that parasite burdens would increase in the laboratory, killifish (3). thereby calling into question the value of specific control measures The effects of larval digenetic trematodes differ from those of (4). However, praziquantel is considered an effective agent against adult digenetic trematodes. Larval digeneans can cause pronounced digenean metacercariae. One management option considered for tissue damage to the host during cercarial migration (4). Migration newly arrived fish was to treat for digenean metacercariae prophylac- can generate hemorrhage, inflammation, and necrosis in the affected tically with praziquantel. Prolonged (24 h) immersion in aquaria host tissues (3). Once the cercariae differentiate and become en- containing 2 to 10 mg of praziquantel/liter for 24 h likely would cysted, they generally cause minimal damage to the host, unless large reduce metacercariae numbers (9). Because the fish presented were numbers accumulate and interfere with physiologic processes (1, 3, on a short-term renal study and no other fish in the tanks exhibited 4). Like those of encysted larval digenetic trematodes, the effects of signs, treatment for the parasites was not elected. The morbidity and adult digeneans are not serious as long as infections are not severe mortality associated with this shipment of fish has not been ob- (1). Adult digeneans generally are observed in the gastrointestinal served in any other shipments. tract, but they can localize in the circulatory system, urinary bladder, The skin lesions noted at necropsy may have been due to trauma, peritoneal cavity, swim bladder, or ovary (1, 3). as skin bacterial culture findings were negative for pathogenic bacte- Although Diplostomum and Neascus are common genera, there are ria. It is probable that the presence of the encysted metacercariae and many others that are seen with regularity. Diplostomum (eye fluke) the stress of shipment led to the illness and mortality in the fish. metacercariae infect the lens or vitreous body of fish (4). Once infec- tions become severe, the metacercariae can cause blindness and References subsequent death of the fish due to predation or starvation (1, 3, 4). 1. Smith, S. A. and E. J. Noga. 1993. General parasitology, p. 139-141. Unlike most larval digenetic trematodes, Diplostomum metacercariae In M. K. Stoskopf (ed.), Fish medicine. W. B. Saunders Co., Philadel- often are not encysted (1). Neascus metacercariae infect the skin and phia. sometimes the gills and muscle of fish (4). These metacercariae cause 2. Roberts, R. J. 1978. Fish pathology. Bailliere Tindall, London. the formation of black cysts, which are often surrounded by black 3. Noga, E. J. 2000. Fish disease, p. 13-25 and 163-166. Iowa State pigment secreted by the host (black spot disease) (1, 3, 4). University Press, Ames, Iowa. Heavy larval trematode infection causes behavioral changes in fish 4. Flynn, R. J. 1973. Parasites of laboratory animals, p. 688-693. Iowa that facilitates parasite transmission by making it easier for predators State University Press, Ames, Iowa. to capture infected prey (5, 6). Some examples of these adverse 5. Barber, I., D. Hoare, and J. Krause. 2000. Effects of parasites on fish behavior: a review and evolutionary perspective. Rev. Fish Biol. Fisher- behavioral modifications include changes in foraging efficiency, habi- ies 10:131-165. tat selection, competitive ability, predator–prey relationships, 6. Lafferty, K. D. and A. K. Morris. 1996. Altered behavior of parasitized swimming performance, sexual behavior, and mate choice (5). Com- killifish increases susceptibility to predation by bird final hosts. Ecology pared with unparasitized fish, parasitized fish spent less time shoaling 77:1390-1397. and more time foraging solitarily as well as exhibited conspicuous 7. Krause, J. and J.-G. J. Godin. 1994. Influence of on the behaviors more often, thereby increasing their chances of being con- shoaling behavior of , Fundulus diaphanus. Can. J. Zool. sumed by a predator (6, 7). Olsen and Pierce reported an occurrence 72:1775-1779. of larval trematode-induced gill cartilage proliferation in a school of 8. Olson, R. E. and J. R. Pierce. 1997. A trematode metacercaria causing gill cartilage proliferation in steelhead trout from Oregon. J. Wildlife steelhead trout and, although the proliferation was insufficient to Dis. 33:886-890. cause mortality in the trout, performance was compromised (8). 9. Krum P., D. Gillette, and G. A. Lewbart. 1992. Pathology and treat- Fish acquired from their natural that are subsequently ment of encysted digenean metacercaria in the catfish, Corydoras schwartzii, housed in laboratory facilities frequently are infected with digenetic p. 118. Proceedings of the 23rd Annual International Association for Aquatic Animal Medicine Conference, Hong Kong.

34 CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science Volume 43, No. 2 / March 2004