Vol. 101: 51–60, 2012 DISEASES OF AQUATIC ORGANISMS Published October 10 doi: 10.3354/dao02508 Dis Aquat Org Experimental infection of yellow stingrays Urobatis jamaicensis with the marine leech Branchellion torpedinis David P. Marancik1,*, Alistair D. Dove2, Alvin C. Camus1 1Department of Pathology, The University of Georgia College of Veterinary Medicine, Athens, Georgia 30602, USA 2Department of Veterinary Services and Conservation Medicine, Georgia Aquarium Inc., Atlanta, Georgia 30313, USA ABSTRACT: Infestations of elasmobranchs by the marine leech Branchellion torpedinis can be problematic in aquaria and negatively affect host health. To better characterize the extent and pathogenesis of disease, 12 yellow stingrays Urobatis jamaicensis were infected with 1 or 3 leeches for 14 d. Leeches were associated with anorexia, extensive cutaneous ulceration, de - creased host packed cell volume (PCV) and serum total solids (TS), and mortality in 3 rays. Aver- age decrease in host PCV positively correlated with ulcer size and parasite:host ratio. Average decrease in host serum TS positively correlated with parasite:host ratio. Blood chemistry and total white blood cell counts revealed no significant trends. Additional necropsy findings included gill and splenic pallor, pericardial edema, perirenal edema, and decreased hepatocellular lipid deposits. Microscopic evaluation of leeches demonstrated host erythrocytes and proteinaceous fluid within parasite intestines, confirming active blood feeding. Results indicate B. torpedinis has the potential to cause significant disease in elasmobranchs, including death in as few as 5 d, and identifies ulcer size and parasite:host ratio as risk factors for disease. Elucidation of this host-par- asite interaction helps characterize host response to parasites and facilitate care of parasitized elasmobranchs in aquarium and wild settings. KEY WORDS: Branchellion torpedinis · Leech · Urobatis jamaicensis · Stingray · Infection Resale or republication not permitted without written consent of the publisher INTRODUCTION Phillipines describes hundreds of leeches per fish but no associated host mortality (Cruz-Lacierda et Leeches are uncommonly implicated as significant al. 2000). parasites of fish (Burreson 2006), with sporadic re - In contrast, upwards of 100 Austrobdella translu- ports of infestations in the literature. Epizootics in - cens parasitizing Australian sand whiting Sillago cil- volving wild and cultured fish describe large num- iate in stocked saltwater ponds resulted in the deaths bers of leeches attached to host skin and within the of dozens of fish during successive years, presumably buccal cavity, with variable host disease and mor- from exsanguination (Badham 1916). Loss of blood tality. Severe oral ulcerations in Atlantic menhaden was also suggested in death of pink salmon Onco- infested with Myzobdella lugubris in North Car- rhynchus gorbuascha fry parasitized by Pisicola olina appeared to have little overall effect on fish salmonicitica in hatchery trays and Macrobdella dec- health (Sawyer & Hammond 1973). A reported Zey- ora and Haemopis grandis infection of wild brook lanicobdella arugamensis infection in tank-reared trout in Maine (Earp & Schwab 1954, Rupp & Meyer orange-spotted grouper Epinephelus coioi des in the 1954). *Email: [email protected] © Inter-Research 2012 · www.int-res.com 52 Dis Aquat Org 101: 51–60, 2012 Although host mortality is often presumed to be There is little information published on Branchel- from blood loss, there may be additional factors lion torpedinis beyond its ecology and anatomy. We affecting fish health during leech infections. Ectopar- have been able to gain insight into its life history by asites can represent stressors for fish and have been observing the leech in aquaria under constant, 21°C associated with decreased foraging (Milinski 1984), environmental conditions. Unlike many leech spe- impaired anti-predator behavior (Milinski 1985), and cies that are semi-permanent and dislodge from reduced fecundity and growth (Iwama et al. 1999). hosts after taking a blood meal to deposit cocoons For example, shorthorn sculpin Myxocephalus scor- onto vegetation and bottom substrate (Kearn 2004), pius infected with the leech Malmiana brunnea B. torpedinis appears to remain attached to the host showed significantly lower energetics than unin- for the duration of its life. This prolonged, close con- fected sculpin: Energy deficiencies attributed to tact with elasmobranch hosts may result in more leech feeding, tissue damage, and loss of metabo- extensive disease compared to leeches that act as lites, accounted for an energy loss of ~750 cal wk−1 temporary parasites (Burreson 2006). Cocoons are per gram of leech (Mace & Davis 1972). shed from the host into the bottom substrate and Leeches also play a role as vectors of disease in hatch in ~30 d. When removed from the host, B. tor- fish. Leech transmission of hematozoa including Try- pedinis are short-lived in laboratory settings with panosoma spp. and Trypanoplasma spp. has been survival lasting <5 d. demonstrated in elasmobranchs and teleosts (Kruse To better understand the pathogenic potential of et al. 1989, Burreson 2007). There is evidence impli- Branchellion torpedinis and to characterize risk fac- cating leeches as mechanical vectors of viruses in tors for disease, yellow stingrays Urobatis jamaicensis teleosts (Ahne 1985, Mulcahy et al. 1990, Faisal & were used as an infection model. As non- schooling Schulz 2009) and speculation that attachment site and sessile fish, these relatively small elasmo branchs ulcers (Faisal et al. 2011), and perhaps transmission adapt well to captivity and serve as suitable experi- by leeches themselves (Bragg et al. 1989), may pre- mental models. Behavioral, biochemical and hemato- dispose hosts to bacterial infections. Further physio- logic changes were monitored for 14 d during infec- logic and pathologic effects may exist in the leech- tion. Rays were necropsied on Day 14 and gross and host relationship, but are yet unidentified. microscopic lesion development was examined at Branchellion torpedinis is a marine leech that attachment and feeding sites. Data were correlated exclusively parasitizes elasmobranchs. Inadvertent with cutaneous damage and parasite:host weight introduction of B. torpedinis into closed aquarium ratios. Investigating these processes will enhance settings is problematic, as parasites thrive under the understanding of this host-parasite interaction and low environmental stress and high host availability elasmobranch responses during disease states. there. High leech fecundity and a lack of adequately studied, safe, and effective chemotherapeutics often make management difficult. Observations at the MATERIALS AND METHODS Georgia Aquarium, Atlanta indicated that B. torpedi- nis can negatively impact host health. Parasitism of Experimental system multiple demersal and pelagic elasmobranch species often results in ulceration of skin, oropharynx, gill Twelve wild-caught yellow stingrays were main- slits, and cloaca. Severe infestations have been asso- tained individually in partitioned sections of a 300 l ciated with lethargy, anorexia, and death of the host. aquarium with sand substrate and recirculating arti- Branchellion torpedinis belongs to the order Rhyn- ficial sea water (Instant Ocean® Sea Salt, Aquarium chobdellida, which is comprised of the families Glos- Systems) at 21°C, 30 ppt salinity. The rays received siphoniidae and Piscicolidae that parasitize freshwa- alternating 12 h periods of light and dark and were ter, and freshwater and saltwater fish, respectively. fed a daily, rotating diet of shrimp, fish, and clam ad These jawless leeches have a large caudal sucker for libitum, with amounts recorded. Physical exam and 3 attachment and feed on host fluids through a straw- repetitive skin scrapes prior to experimental trials like proboscis inserted from a smaller, cranial sucker revealed no ectoparasitic infections. (Kearn 2004). Feeding is believed to be aided by sali- Rays were assigned to 2 experimental groups and vary gland enzymes released into the tip of the pro- allowed a minimum of 3 wk to acclimate. Experimen- boscis, which enable penetration of host tissue and tal group ‘L1’ consisted of 6 rays (2 males and 4 fe- prevent hemostasis (Sawyer et al. 1991, Moser & males, weight range 380 to 550 g), each infected with Desser 1995). 1 leech (weight range 632 to 763 mg). Experimental Marancik et al.: Infection of yellow stingrays with Branchellion torpedinis 53 group ‘L3’ consisted of 6 rays (3 males and 3 females, potassium, chloride, bicarbonate, calcium, phospho- weight range 315 to 475 g), each infected with 3 rus, and magnesium. Serum osmolality was calculated leeches (combined weight range 1.831 to 2.145 g). as 2 Na (mEq l−1) + (Urea [mg dl−1])/2.8 + (Glucose [mg Leeches collected from captive elasmobranchs were dl−1])/18 (Carlson 1997), as sufficient blood volumes transported directly to the laboratory for infection tri- for measured osmolarity could not be collected. als and manually placed on the dorsum of the rays. When rays were infected with 3 leeches, the leeches were placed approximately 8 cm apart. Leukocyte count Twenty µl of heparinized whole blood was added Sampling regimen to 1980 µl of Natt-Herrick stain (ENG Scientific) adjusted for use in elasmobranchs by adding 0.3 g Three randomly selected rays were subjected to NaCl and 0.2 g urea to 10 ml filtered Natt-Herrick’s the hematologic sampling regimen to ensure