A New Species of Lissorchis (Trematoda: Lissorchiidae) from the Spotted Sucker, Minytrema melanops (Actinopterygii: Catostomidae), in Wisconsin, U.S.A Author(s): Eric J. Gale , Anindo Choudhury , Jennifer L. Bailey and Daniel J. Sutherland Source: Comparative Parasitology, 81(1):61-66. 2014. Published By: The Helminthological Society of Washington DOI: http://dx.doi.org/10.1654/4653.1 URL: http://www.bioone.org/doi/full/10.1654/4653.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Comp. Parasitol. 81(1), 2014, pp. 61–66 A New Species of Lissorchis (Trematoda: Lissorchiidae) from the Spotted Sucker, Minytrema melanops (Actinopterygii: Catostomidae), in Wisconsin, U.S.A. ERIC J. GALE,1 ANINDO CHOUDHURY,1,4 JENNIFER L. BAILEY,2 AND DANIEL J. SUTHERLAND3 1 Division of Natural Sciences, St. Norbert College, 100 Grant Street, De Pere, Wisconsin 54115, U.S.A. (e-mail: anindo.choudhury@snc.edu), 2 Whitney Genetics Lab, La Crosse Fish Health Center, 555 Lester Avenue, Onalaska, Wisconsin 54650, U.S.A. (e-mail: Jennifer_Bailey@fws.gov), and 3 University of Wisconsin-La Crosse, 1725 State Street, La Crosse, Wisconsin 54601, U.S.A. (deceased) Lissorchis nelsoni (Trematoda: Lissorchiidae) is described from the intestines of the spotted sucker, Minytrema melanops (Rafinesque) from the Wolf River in central Wisconsin and from Navigation Pools 8 and 9 of the upper Mississippi River near La Crosse and Genoa, Wisconsin. It is readily distinguished from all other species of Lissorchis by a combination of its surface structures, massive cirrus sac bent at an angle (often almost an inverted ‘‘L’’), bipartite seminal vesicle, large acetabulum, a trilobed ovary, its vitellarium, and a type B uterine configuration, of which the cirrus sac is perhaps the most useful for stained material. It is morphologically similar to Lissorchis mutabile in having a similar size and body shape, similar number and arrangement of vitelline follicles, and a distinctly trilobed ovary. In addition to differences in the cirrus sac morphology, it also differs from L. mutabile in having an extensively coiled uterus that extends on both sides of the hindbody. KEY WORDS: Lissorchis, Lissorchiidae, Minytrema melanops, Catostomidae, spotted sucker, Wisconsin. ABSTRACT: The genus Lissorchis is comprised of 16 nominal species that parasitize the intestinal tract of the catostomid fishes: Lissorchis fairporti Magath, 1917; L. mutabile (Cort, 1918); L. garricki (Simer, 1929); L. translucens (Simer, 1929); L. attenuatum (Mueller and Van Cleave, 1932); L. simeri (Mueller and Van Cleave, 1932); L. hypentelii (Fischthal, 1942); L. polylobatum (Haderlie, 1950); L. crassicrurum (Haderlie, 1954); L. gullaris Self and Campbell, 1956; L. heterorchis (Krygier and Macy, 1969); L. kritskyi Barnhart and Powell, 1979; L. calentinei Christensen, Wellner, and Gleason, 1982; L. minytremi Christensen, Wellner, and Gleason, 1982; L. macropharynx Choudhury and Nelson, 1998; and L. amniculensis Barger, 2010 (Magath, 1917; Simer, 1929; Mueller and VanCleave, 1932; Wallace, 1941; Fischthal, 1942; Haderlie, 1950, 1954; Self and Campbell, 1956; Krygier and Macy, 1969; Barnhart and Powell, 1979; Williams, 1979; Christensen et al., 1982; Choudhury and Nelson, 1998; Hoffman, 1999; Barger, 2010). Two named species, L. fischthali (Aliff, 1973) and L. nanus, are nomina nuda (Barnhart and Powell, 1979; Choudhury and Nelson, 1998). During two separate studies of catostomids in the Mississippi River (Walker, 2007) and in the Wolf River in Shiocton, Wisconsin (this study), numerous 4 specimens of a lissorchiid were collected from the intestines of the spotted sucker, Minytrema melanops (Rafinesque). Upon closer examination and comparison with specimens and descriptions of other Lissorchis spp., it became apparent that these digeneans possessed certain unique morphological characteristics, which led us to describe it as a ‘‘new’’ species, Lissorchis nelsoni. MATERIALS AND METHODS Spotted suckers, M. melanops, were collected from Navigational Pools 8 (near LaCrosse, Wisconsin) and 9 (near Genoa, Wisconsin) of the Mississippi River, by electroshocking in October 2003 and by the use of fyke and hoop nets in April 2003, respectively (Walker, 2007). Spotted suckers were also collected by electroshocking from the Wolf River on 14 October 2009. Fish were kept on ice and examined within 2 to 4 hr of capture. Specimens used for the description were collected live, washed briefly in saline (0.6%), and relaxed and fixed by pouring steaming 10% buffered formalin over the worms in vials and vigorously shaking them for a few seconds. Some live worms were also preserved in 100% ethanol for future molecular studies. Specimens were stained with Semichon’s acetocarmine, and dehydrated, cleared, and mounted in balsam on slides or between coverslips. Once dried, the double coverslip preparations were slid into aluminum holders (Cobb slides, Choudhury and Nelson, 1998). Specimens were examined using ordinary bright-field as well as differential interference contrast (DIC) optics. Drawings were made using a drawing tube attachment. Specimens for scanning electron microsope (SEM) studies were postfixed in osmium tetroxide in phosphate buffer, Corresponding author. 61 62 COMPARATIVE PARASITOLOGY, 81(1), JANUARY 2014 dehydrated through a graded ethanol series, and infiltrated with hexamethyldisilizane (HMDS), after which the HMDS was allowed to evaporate off the specimens; these specimens were then mounted on stubs, sputter-coated with gold, and scanned using a Hitachi S-4800 FE-SEM. Measurements are given in micrometers and are expressed as mean 6 standard deviation followed by range in parentheses. Measurements are from 18 worms unless otherwise mentioned. RESULTS Lissorchis nelsoni n. sp. (Figs. 1–7) Description Body with bluntly rounded ends, tapers to narrower posterior end, forebody more cylindrical than hindbody when fixed, gently arched dorsally, 1,258 6 117 (1,125–1,526) long. Maximum width of forebody 182 6 49 (130–272) with maximum hindbody width of 260 6 25 (200–307). Forebody (measured from anterior end of body to point at which the acetabulum protrudes from the body) 425 6 49 (370–545) long. Forebody-to-hindbody ratio of 0.34 6 0.03. Tegument spinose, dorsal surface spineless, heavy spines present in region of oral sucker set on broad bases, extending ventrally slightly beyond posterior edge of oral sucker, similar spines present in acetabular region; spines decrease in size and abundance ventrally posterior to acetabulum; two rows of small spines occur around acetabular margin, arranged in interlocking zipper-like pattern. Oral sucker almost terminal, 163 6 31 (107–223) long, 172 6 33 (110–234) wide, oral opening directed anteroventrally, 2 conspicuous papillae located on inner margin of oral cavity (roof of cavity), double row of spines present on outer rim extending anterior of midpoint of sucker, fleshy finger-like projections present along anterior surface behind double row of small spines. Acetabulum protrudes, somewhat anteriorly directed, often with shallow lateral grooves that demarcate upper (anterior) and lower (posterior) halves (‘‘lips’’), indicating clamp-like action, 228 6 48 (180–337) long and 228 6 48 (160–306) wide, located at anterior end of middle third of worm with fleshy, finger-like projections along edges (not equally prominent in all specimens), dense spination starting on anterior surface, extending posteriorly along ventral and lateral surfaces, large papillae present on distal inner surface, smaller papillae present on deeper inner surface. Oral sucker:acetabulum length 0.73 6 0.12; oral sucker:acetabulum width 0.76 6 0.10. Pharynx Figures 1, 2. Lissorchis nelsoni. 1. Ventral view, showing mainly internal anatomy. Spination largely omitted for clarity; only anterior-most spines and spines along anterior margin of acetabulum illustrated. Projections along anterior border of acetabulum illustrated. 2. Dorsal view (only the preovarian uterine coils are drawn). muscular, 68 6 13 (53–92) long and 72 6 10 (50– 87) wide. Esophagus short, muscular, occasionally bent, 57 6 11 (35–73) long. Intestinal bifurcation anterior to acetabulum, 140 6 22 (105–190) anterior to forebody margin. Ceca extend posterior to testes, unequal in length, left cecum typically slightly longer than right, terminate 202 6 18 (175–230) from the posterior end of body. Ovary median, immediately pretesticular, distinctly trilobed, 151 6 8 (130–160) long, and 143 6 8 (130–158) wide (total dimensions). Uterus forms loops and coils anterior to GALE ET AL.—DESCRIPTION OF LISSORCHIS NELSONI 63 Figures 3–5. Lissorchis nelsoni. Illustrations of the reproductive complex. 3. Female reproductive complex (dorsal view). 4. Female reproductive complex (ventral view). 5. Cirrus sac. Abbreviations: Lc 5 Laurer’s canal, Sr 5 seminal receptacle, od 5 oviduct, vd 5 vitelline duct, vr 5 vitelline reservoir, ut 5 uterus, sv 5 seminal vesicle, pp 5 pars prostatica, ed 5 ejaculatory duct. ootype and ovary, then winds down left side of hindbody, under testes and upward two-thirds of right side of hindbody, loops and doubles back on itself under testes, and winds up left side once again before crossing over above ovary to metraterm that opens to gonopore; this is a type B uterine configuration (Fig. 7). Eggs small, 15.7 6 1.0 (13.8–17.5) long, 8.6 6 1.0 (6.0–10) wide, operculate, opercular end narrower; mature eggs tanned. Mehlis’ gland inconspicuous, gland cells somewhat scattered. Seminal receptacle small, inconspicuous. Laurer’s canal opens to dorsal medial surface immediately anterior to ovary. Vitellarium follicular, follicles relatively large, few in number, 8.8 6 0.92 (7–11) per side, extends from 64 COMPARATIVE PARASITOLOGY, 81(1), JANUARY 2014 Excretory vesicle dorsal, narrow, tubular, reaching the posterior margin of ovary, excretory pore dorsoterminal. Taxonomic summary Prevalence, intensity, and abundance: Wolf River: 4 of 4 fishes examined (100%, 4–50). Mississippi River Pool 8: 7 of 10 fish examined (70%, 2–30; Pool 9: 5 of 10 fish examined (50%, 4–43). Site of infection: Intestinal tract. Type host: Spotted sucker, Minytrema melanops (Rafinesque). Other reported hosts: None. Locality records: Type locality: Wolf River, stretch of river north and south of Koepke’s Landing, Shiocton, Wisconsin between 44u30910.520N; 88u34940.960W and 44u289940N; 88u34935.810W. Additional localities: Navigation Pools 8 and 9 of the upper Mississippi River near La Crosse and Genoa, Wisconsin. Specimens deposited: Holotype: United States National Parasite Collection (USNPC) 106989, Paratypes: USNPC 106990–106992. Figures 6, 7. Lissorchis nelsoni. 6. SEM. Lateral view. Note the interrupted spination on the ventral surface and spineless dorsal surface. 7. The three uterine configurations seen in Lissorchis spp. showing the schematic position and arrangement of the loops: A 5 type A configuration, most common; B 5 type B configuration in L. nelsoni and L. mutabile; C 5 type C configuration. Arrows indicate orientation of the uterine loops, from proximal to distal, which is also the direction in which fertilized eggs travel from their origin in the ootype. posterior margin of acetabulum to 28 6 9 (15–58) beyond anterior testis, follicles broadly interconnected. Testes 2, tandem, contiguous, ellipsoidal, margins smooth or slightly indented or irregular in places, anterior testes 123 6 16 (102–153) long and 109 6 12 (90–137) wide, posterior testes generally longer, 159 6 20 (115–190) long and 101 6 10 (85–115) wide, posttesticular length 255 6 44 (205–358); testes only marginally obscured by uterus. Cirrus sac large, muscular, thick walled, maximum width 91 6 11 (78–113), reaching posteriorly to level of ovary, extending alongside it, anterior portion overlapping acetabulum dorsally; containing bipartite seminal vesicle, cylindrical, sausage-shaped pars prostatica, winding/folded ejaculatory duct, distal portion muscular, with inverted cirrus. Cirrus spined (minute spines, visible only in everted cirri) and eversible. Specimens examined: The following material was examined for comparison (Canadian Museum of Nature Parasite Collection [CMNPA]; Harold W. Manter Laboratory [HWML]; and USNPC): L. fairporti: HWML 1479, 21994, USNPC 51687; L. mutabile: USNPC 44980; 60456 (both as Triganodistomum mutabile), 76656; L. transluscens: USNPC 51860, 74755; L. garricki (as Alloplagiorchis garricki) USNPC 51376; L. attenuatus: HWML 20373– 20376, 23677 (as L. attenuatum), HWML 23636, 23638, 23639, 38250 (as Triganodistomum attenuatum), USNPC 76654, 76655, 80232; L. simeri: USNPC 8683 (as Triganodistomum simeri); L. hypentelii: USNPC 44974 (as Triganodistomum hypentelii), 74727; L. polylobatus: USNPC 372179 (as Triganodistomum polylobatum); L. crassicrurus: CMNPA1989-0578 (as L. crassicrurum), USNPC 37193 (as Triganodistomum crassicrurum); L. gullaris: CMNPA 1986-0105; 1989-0618; HWML 20377–20382, 22759, 30310, 30314, 30315, 30319, 30439, USNPC 38102, 74754; L. heterorchis: HWML 20702, 23678; USNPC 71345; L. fischthali: USNPC 72602; L. kritskyi: CMNPA1985-0026, USNPC74871; L. calentinei: 76742; L. minytremi: USNPC 76744; L. amniculensis: HWML 49130, 49131; and L. nanus: USNPC 76768. GALE ET AL.—DESCRIPTION OF LISSORCHIS NELSONI Etymology: This species is named after Dr. Patrick A. Nelson, North-South Consultants, Winnipeg, Manitoba, Canada, for his contributions to the ecology of catostomid fishes, boreal fish parasitology, and in grateful recognition of many years of friendship and collaboration with one of the authors (A.C.). Remarks Lissorchis nelsoni places in the genus Lissorchis on the basis of the diagnosis of Bray (2008). It is readily distinguished from all other species in Lissorchis by a combination of its surface structures, massive cirrus sac bent at an angle (often almost an inverted L), bipartite seminal vesicle, large acetabulum, a trilobed ovary, its vitellarium, and a type B uterine configuration (see Fig. 7). The trilobed ovary of L. nelsoni distinguishes it from species that possess multilobed ovaries such as L. calentinei, L. minytremi, L. gullaris, L. fairporti, and L. polylobatum. Species of Lissorchis that have principally trilobed ovaries similar to L. nelsoni are: L. mutabile, L macropharynx, L. heterorchis, L. amniculensis, L. attenuatum, L. hypentelii, L. simeri, L. translucens, L. kritskyi, and L. garricki. A subgroup of these, L. amniculensis, L. simeri, L. kritskyi, L. garricki, and L. mutabile, possess vitelline follicles that are relatively large in size and fewer in number, similar to those in L. nelsoni. However, in L. amniculensis the vitelline follicles extend posteriorly beyond the posterior testis, whereas in L. nelsoni and others in this group the vitellarium extends to just beyond the anterior testis only. Of the remaining species (L. simeri, L. kritskyi, L. garricki, and L. mutabile), L. kritskyi possesses a uterus that forms several short loops immediately anterior to the ovary as it exits the ootype before assuming a ‘‘type A’’ uterine configuration (Fig. 7). The posttesticular length in L. kritskyi is approximately 10% of overall body length, whereas in L. nelsoni it is approximately 20%. Although L. nelsoni shares a type B uterine configuration with L. mutabile, the descendng (sinistral) loop of the uterus of L. mutabile coils at the posterior end but does not extend up the right side of the body, unlike in L. nelsoni. Lissorchis simeri may also possess a type B uterine layout but one that falls between that of L. mutabile and L. nelsoni, with a sinistral uterine loop that encroaches and extends part of the way up the right side of the body. However, we were unable to determine the uterine layout of L. simeri with absolute certainty and the uterus was not described in sufficient detail in the 65 original description to determine its precise configuration. Lissorchis simeri also possesses ova that are larger (24 3 12) than those of L. nelsoni (16 3 9). In any case, the large acetabulum and massive cirrus easily distinguish L. nelsoni from L. simeri. Choudhury and Nelson (1998) suggested that all lissorchiids may have eversible cirri. Had it not been for the very few specimens with everted cirri, we would not be able to determine this fact in L. nelsoni. We were unable to discern inverted cirri or cirrus spines in whole mounts where the cirri were not everted. Overall, L. nelsoni seems most similar to L. mutabile: they both possess a similar body shape and size (comparable length and width as well as being widest in the ovarian region), a prominent acetabulum, similar size and arrangement of vitelline follicles, and a distinctly trilobed ovary. However, the size, shape, and extent of the massive cirrus sac of L. nelsoni and the wider acetabulum set it apart. Choudhury and Nelson (1998) opined that the presence of two papillae on the roof of the oral cavity of lissorchiids may be taxonomically important and possibly phylogenetically informative; L. nelsoni possesses these structures. They are clearly visible using SEM and DIC, but less readily so with ordinary transmitted light. Papillae are typically either more difficult or impossible to discern in flattened or previously frozen specimens. DISCUSSION The morphological characteristics that distinguish L. nelsoni from L. mutabile, as well as all other Lissorchis spp., are present in all specimens of L. nelsoni. Thus, the differential diagnosis of this species is based on consistent, i.e., unvarying, characteristics. All available information points to L. nelsoni being specific to the spotted sucker. Examination of other catostomids from the Wolf River (three species of Moxostoma) did not yield this trematode. The same can be said for the 12 species of catostomids sampled from the Mississippi River drainage, where L. nelsoni was found in only M. melanops (Walker, 2007). Minytrema melanops is also parasitized by L. calentinei and L. minytremi in Kentucky, but they are easily distinguished from L. nelsoni (see Remarks section). No specimens of L. calentniei or L. minytremi were found in samples of M. melanops from the Wolf River and from the Mississippi River drainage (Walker, 2007). Examination of specimens of L. calentinei and L. minytremi from the original description (Christensen et al., 66 COMPARATIVE PARASITOLOGY, 81(1), JANUARY 2014 1982) indicates that the two species are in fact the same; their reported differences disappeared on closer scrutiny (A.C., unpublished) and we use the name L. calentinei for the species. Aliff’s (1973) L. fischthali, described in his unpublished Ph.D. thesis from spotted suckers, also in Kentucky, is very similar to L. calentinei, and may be the same species. The morphological similarity between L. nelsoni and L. mutabile may reflect the fact that their hosts belong to taxa, Minytrema and Erimyzon, that place as sister groups in the phylogenetic analyses of catostomids on the basis of both morphological and molecular data (Smith, 1992; Doosey et al., 2010). We hypothesize, on the basis of the information available, that future phylogenetic analyses of lissorchiids will find L. mutabile and L. nelsoni to be closely related and their cophylogenesis reflected in the relationship between them and their fish hosts. ACKNOWLEDGMENTS We are grateful to Dr. Ron Bruch and personnel of the Wisconsin Department of Natural Resources, Oshkosh, Wisconsin for collecting spotted suckers for this study. We are also grateful to the University of Wisconsin Milwaukee for full access to their SEM facilities. LITERATURE CITED Aliff, J. V. 1973. Digenetic Trematodes from Kentucky Fishes. Ph.D. Thesis. University of Kentucky, Lexington, Kentucky, U.S.A. 292 pp. Barger, M. A. 2010. A new species of Lissorchis (Trematoda: Lissorchiidae) from creek chubsuckers (Erimyzon oblongus) in the Big Thicket National Preserve, Texas, U.S.A. Comparative Parasitology 77: 1–5. Barnhart, M. C., and E. C. Powell. 1979. Lissorchis kritskyi sp. n. (Digenea: Lissorchiidae) from the river carpsucker, Carpiodes carpio (Rafinesque). Proceedings of the Helminthological Society of Washington 46:47–51. Bray, R. A. 2008. Lissorchiidae Magath, 1917. Pages 177– 186 in D. J. Gibson, R. A. Bray, and A. Jones, eds. Keys to the Trematoda. Vol. 3. CAB International, Wallingford, U.K. Choudhury, A., and P. A. Nelson. 1998. Lissorchis marcropharynx n. sp. 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