High prevalence of buccal ulcerations in largemouth bass, Micropterus salmoides (Centrarchidae) from Michigan inland lakes associated with Myzobdella lugubris Leidy 1851 (Annelida: Hirudinea)

Widespread mouth ulcerations were observed in largemouth bass collected from eight inland lakes in the Lower Peninsula of Michigan during the summer months of 2002 and 2003. These ulcerations were associated with, and most likely caused by, leech parasitism. Through the use of morphological dichotomous keys, it was determined that all leeches collected are of one species: Myzobdella lugubris. Among the eight lakes examined, Lake Orion and Devils Lake had the highest prevalence of leech parasitism (34% and 29%, respectively) and mouth ulcerations (53% and 68%, respectively). Statistical analyses demonstrated that leech and ulcer prevalence varied significantly from one lake to the other. Additionally, it was determined that the relationship between the prevalence of ulcers and the prevalence of leech attachment is significant, indicating that leech parasitism is most likely the cause of ulceration. The ulcers exhibited deep hemorrhagic centers and raised irregular edges. Affected areas lost their epithelial lining and submucosa, with masses of bacteria colonizing the damaged tissues. Since largemouth bass is a popular global sportfish and critical to the food web of inland lakes, there are concerns that the presence of leeches, damaged buccal mucosa, and general unsightliness may negatively affect this important sportfishery.


INTRODUCTION
T he largemouth bass (Micropterus salmoides) is an important species in the food web of inland lakes in the Laurentian Great Lakes basin (Bettoli et al., 1992). Due to its key predator-prey relationship with other aquatic organisms, largemouth bass plays a central role in the trophic cascades in the Great Lakes environment. In the state of Michigan, one-half million anglers predominately fish for largemouth bass, representing about 30 % of all state licensed anglers (State of Michigan, 2004). Therefore, there is a need to better understand disease conditions, including parasitism, which can have an impact on this popular recreational fishery. Leeches are known to feed upon a variety of terrestrial and aquatic animals, including fish (Sawyer, 1986). Leeches can be semi-permanent, attaching to only one host for the greater part of their adult life, or their attachment can be intermittent, attaching to several hosts throughout their lifetime (Sawyer, 1986). Adult leeches generally detach themselves from their fish hosts to deposit egg cocoons, from which juvenile leeches emerge after a few months to find their own fish hosts on which to feed, and grow into adults (Sawyer, 1986). The piscicolid leech Myzobdella Original contribution Parasite, 2011, 18, 79-84 lugubris is an intermittent blood-sucking parasite that parasitizes on a number of freshwater and estuarine fish species, including the largemouth bass, channel catfish (Ictalurus punctatus), white catfish (I. catus), mottled sculpin (Cottus bairdi), and burbot (Lota lota) (Daniels & Sawyer, 1975;Sawyer et al., 1975;Amin, 1981;Schramm et al., 1981;Muzzall et al., 1987;Morrison & Fox, 1993;Choudhury et al., 2004;Schulz et al., 2011). Interestingly, the life cycle of M. lugubris can involve a crustacean host on which this piscicolid leech deposits its egg cocoons, such as the blue crab, Callinectes sapidus (Daniels & Sawyer, 1975). The preferred site of attachment for M. lugubris is primarily on the fin soft rays, causing erosion and epithelial hyperplasia (Dechtiar, 1972;Schulz et al., 2011). However, Noga et al. (1990 reported the presence of both ulcerations and attached M. lugubris in the mouth of four adult largemouth bass from Currituck Sound, North Carolina, though the relationship between the leech presence and the mouth ulcerations was never established. Herein, we report on widespread mouth ulcerations in adult largemouth bass caught from eight inland lakes of Michigan with a significant association with the presence of M. lugubris.

FISH AND SAMPLING SITES
A total of 326 adult largemouth bass, Micropterus salmoides, with a mean total length of 30.2 cm ± 5.1 cm and mean total weight of 399.9 g ± 279.3 g were collected in summer months from eight inland lakes in Michigan's Lower Peninsula between July 2002 and September 2003. The lakes sampled were Jordan Lake, Randall Lake, Norvell Lake, Devils Lake, Eagle Lake, Lake Orion, Union Lake, and Independence Lake ( Fig. 1, Table I). Fish were collected primarily by electro-fishing, hook and line angling, and trap nets by biologists from the Michigan Department of Natural Resources. Fish were transported alive in tanks with aerators to the Aquatic Animal Health Laboratory at Michigan State University, East Lansing, Michigan. The number of fish samples from each lake is given in Table I.

LABORATORY AND SAMPLE PROCESSING
Fish were anesthetized with 250 mg/liter Tricaine Methanesulfonate (MS-222, Argent Chemicals, Redmond, WA) and their buccal cavity thoroughly examined for the presence of ulcers and leeches. Leeches, when present, were collected, their numbers/fish recorded, and kept separately in vials containing saline (0.6 % NaCl solution). Leeches were allowed Lake Latitude Longitude

Number of fish with mouth ulcerations
Prevalence of mouth ulceration (%)

STATISTICAL ANALYSES
A one-way analysis of variance (ANOVA) was conducted to determine the significance of mouth ulcerations and leech prevalence. Additionally, Tukeyadjusted multiple comparisons were used to compare the prevalences of mouth ulcerations and leeches among lakes. A P-value less than 0.05 was considered significant. Calculations were performed using the SAS version 9.2 software (SAS Institute, Inc., Cary, North Carolina).

E xamining the buccal cavity of largemouth bass
revealed the presence of leeches crawling freely, attached to buccal mucosa, or buried in furrows within the mucosa. The overall prevalence of leeches in largemouth bass' buccal cavity in the eight lakes combined was relatively high (20 %, 64 out of 326 fish examined). Most fish harbored more than three leeches, but as many as six or seven leeches were common in fish from Lake Orion and Devils Lake. There were significant differences in leech prevalence among lakes with Lake Orion and Devils Lake being the highest at 34 % and 29 %, respectively (Table I).
On the contrary, no leeches were attached to the buccal mucosa in fish from Union Lake. All leeches attached to the buccal mucosa of largemouth bass shared the same morphological criteria. Leech bodies were grayish to dark red, often contained scattered pigment cells, and measured 24-36 mm in length when relaxed (Fig. 2). All leeches exhibited the cylindrical body morphology; divided at the 13 th segment into a narrow anterior and a wider posterior region; had one pair of eyes; and a concave caudal sucker.
Clinical examination revealed the presence of widespread ulcerations of the buccal mucosa at the roof of the mouth as well as on the tongue mucosa. The prevalence of buccal ulcerations was relatively high since it was found in 105 out of 326 largemouth bass examined (32 %). In several fish, leeches were found embedded in the buccal mucosa, particularly at the roof of the mouth (Fig. 3A). Even though leeches were within the mucosa, they were in continuous movement, a matter that caused detachment of the mucosal layer. Most of the ulcers exhibited deeper hemorrhagic centers with inflamed irregular edges (Fig. 3B). The size of ulcers varied from minute to relatively large. In advanced cases, ulceration covered a considerable portion of the buccal mucosa, often with yellowish-white material (Fig. 3C). In some instances, ulcers were found on the tongue with larger hemorrhagic areas surrounding the leech attachment sites (Fig. 3D).
Similar to leech prevalence, buccal ulceration prevalence was highest in Lake Orion (53 %) and Devils Lake (68 %) and did not exist in the leech-free Union Lake. Moreover, ulcer prevalence varied significantly among lakes. For example, the prevalence of ulcers in fish from Devils Lake was significantly higher than Eagle, Independence, Randall, and Union lakes (P < 0.01). The difference in ulceration prevalence between Randall Lake and Jordan and Norvell lakes was statistically significant (P < 0.05). Additionally, the prevalence of ulcers in fish from Lake Orion was greater than the prevalence of ulcers in fish from Randall, Union, Eagle, and Independence lakes, and was statistically significant (P < 0.05). When the individual fish data of the eight lakes was combined, statistical analysis revealed that the interaction between the prevalence of ulcers and the prevalence of leeches was statistically significant (P < 0.05).

DISCUSSION
M orphological criteria indicated that the leeches collected in this study were all Myzobdella lugubris Leidy 1851 Khan & Meyer, 1976;Barta, 1991;Hoffman, 1999). It was surprising that M. lugubris was noticed only in the buccal cavity, since it is known also to attach to skin (Miller & Olson, 1973;Muzzall et al., 1987;Morrison & Fox, 1993;Choudhury et al., 2004), fins (Daniels & Sawyer, 1975;Schramm et al., 1981;Muzzall et al., 1987;Morrison & Fox, 1993), and isthmus (Schramm et al., 1981;Morrison & Fox, 1993). Moreover, statistical analyses revealed that M. lugubris prevalence is significantly related to the buccal ulceration, suggesting that the leech feeding habits and ability to burrow into the mucosa are most likely the cause of these ulcers. Indeed, most of the recent ulcers observed took the size and shape of the M. lugubris contour. The ulceration is probably exacerbated by the intermittent nature of M. lugubris parasitism, which is associated with continuous attachment, detachment, and movement into other parts of the mucosa. This study and that of Noga et al. (1990) in the largemouth bass are the only reports of M. lugubris having a tendency toward buccal mucosa. Myzobdella lugubris produces two types of secretions that lyse host epithelium and aid in the attachment to host surfaces (Michel & Devillez, 1978;Appy & Cone, 1982). The tissue damage caused by this leech compromises the integrity of bass buccal mucosa and allows for potential invasion by pathogenic bacteria and viruses. In this context, recent studies from the authors' laboratory have demonstrated that M. lugubris carries the Viral Hemorrhagic Septicemia Virus (Faisal & Schulz, 2009), a disease that has devastated important fish stocks in the Laurentian Great Lakes; and Flavobacterium psychrophilum, the causative agent of both Bacterial Coldwater Disease and Rainbow Trout Fry Syndrome, which can cause high mortalities in salmonids (Schulz & Faisal, 2010). Other species of leeches are well known for functioning as vehicles for transmission of infectious agents in fish (Dombrowski, 1952;Ahne, 1985;Cusack & Cone, 1986;Kruse et al., 1989;Jones & Woo, 1992;Zintl et al., 2000;Kikuchi et al., 2002;Hemmingsen et al., 2005;Kikuchi & Fukatsu, 2005). Whether or not M. lugubris transmits pathogens to the largemouth bass is currently unknown and deserves further investigation. Prevalence of M. lugubris varied greatly from one lake to the other. Many biological, chemical, and physical components of each waterbody determine the presence and infection parameters of a certain parasite in a locale, including the presence and density of susceptible hosts, the prevailing temperature, the com- (A) burrowing into the buccal mucosa of a largemouth bass (arrow) from Orion Lake; (B) ulcerations and leeches (arrows) in the roof of the mouth of a largemouth bass from Orion Lake; (C) severe ulceration associated with a leech infestation in the roof of the mouth of a largemouth bass from Devils Lake with suppurative inflammation and subsequent pus formation (arrow); and (D) M. lugubris (arrow) attached to the tongue of a largemouth bass from Devils Lake. Original contribution Parasite, 2011, 18, 79-84 position of the fish community, and the presence of other parasite species (Kennedy, 2009). Unfortunately, the information available on the hydrobiology of each of the lakes is not enough to allow identification of the factors that led to high leech prevalence in lakes Orion and Devils or its total absence in Union Lake. The presented data represents the most comprehensive account of M. lugubris prevalence in largemouth bass in Michigan's inland lakes. The inland lakes from which largemouth bass samples were collected were never examined previously for leeches of largemouth bass. Therefore, the findings from this study are considered to be new geographical range extensions for M. lugubris.  , 1981;Becker & Dauble, 1979;Choudhury et al., 2004;Font & Tate, 1994;Klemm, 1972;Klemm, 1982;Klemm, 1991;Miller & Olson, 1973;Sawyer, 1972; . Specimen deposited: AMNH, in submittal process.