Intestinal damage in locally occurring game fish infected with the acanthocephalan, Leptorhynchoides thecatus F. B. Reyda 1, C. Lange 2, J. Sheehan 2, U. Habal 2, D. Willsey 2, L. Laraque 2, & M. O Rourke 2 INTRODUCTION Recent survey work on the intestinal parasites of Otsego Lake fishes (Reyda, 2010; Szmygiel and Reyda, 2011) has demonstrated that the acanthocephalan Leptorhynchoides thecatus is common in several fish species of potential economic importance in Otsego Lake and nearby waters. Leptorhynchoides thecatus appears to have a very low level of host specificity and has been found in the locally occurring centrarchid species, Largemouth bass, Smallmouth bass, Pumpkinseed, Redbreast sunfish and Rock bass. In addition, L. thecatus has been encountered in Yellow perch, Eurasian carp, Chain pickerel, White sucker, and Walleye in Otsego Lake. The low level of host specificity in L. thecatus has been previously reported in the literature (DeGiusti, 1949). Acanthocephalans attach to the host intestinal wall using its hooked proboscis, causing extensive tissue damage and potential fatality in various vertebrates (Nickol and Crompton, 1983). The pathological effects of L. thecatus can be seen microscopically at the point of direct attachment between the proboscis and host intestine. Extent of pathology depends on the depth of penetration and the amount of worms present in an infected intestine, but has only been documented in a single previous study (Venard and Warfel, 1953). Given its common local occurrence, the current histological study was undertaken to address the potential pathology of L. thecatus to its hosts by focusing on infections in Smallmouth bass (Micropterus dolomieu), and Yellow perch (Perca flavescens). METHODS The fish specimens used for this study were collected as follows: Ten Yellow perch were collected in February 2011 in Canadarago Lake Otsego County, New York, by ice fishing. A single Smallmouth bass was collected in September 2011 from Brookwood in Otsego Lake, Otsego County, New York, by hook and line. Fish were immediately returned to the Biological Field Station Hop House Lab in Springfield, New York and stabilized in aquaria. Fish were sacrificed via prolonged anesthetization with FinQuel, or via pithing. The fish were then necropsied immediately following measurement of total length in centimeters. During each necropsy the intestine and the pyloric cecae (if present) were isolated from the rest of the abdominal cavity with an anterior incision at the stomach and a posterior incision at the anus. The intestine was then opened with a single longitudinal incision and the pyloric cecae were opened with several incisions. The intestines and pyloric cecae were subsequently fixed in 10% formalin, or, in the case of the Smallmouth bass specimen, in 10% neutral buffered 1 Assistant Professor of Invertebrate Zoology and Researcher, Biology Department and Biological Field Station, SUNY Oneonta 2 SUNY Oneonta Undergraduate Student, Biology Department, SUNY Oneonta
formalin. Pieces of intestinal or cecal tissue with attached L. thecatus were cut into ~3 4 mm square plugs. Histological sections were prepared by conventional methods. First, tissue plugs with L. thecatus were dehydrated in a graded ethanol series, followed by xylene, xylene mixed with paraplast, and ultimately paraplast at 56C for a minimum of eight hours for maximum infiltration of paraplast into the tissue. Tissue plugs were embedded in pure paraplast and sectioned at 10 micron intervals using an Olympus CUT 4060 microtome. Sections were dried onto warmed slides using warmed Sodium silicate. Once dry, slides were stained with Delafield s Hematoxylin and counterstained with Eosin, and subsequently mounted under long coverslips with Canada balsam. Once dry, slides were examined with an Olympus CX 41 compound microscope and images were captured with the attached Luminera Infinity 2 Digital Microscope Camera. Figure 1. Light micrographs of histological sections of Yellow perch intestine. 1A, Uninfected intestine. 1B, 1C, Proboscis of Leptorhynchoides thecatus embedded into intestinal layers.
RESULTS Although many of the Yellow perch and Smallmouth bass we encountered during survey work (e.g., Szmygiel and Reyda, 2011) were infected with L. thecatus, a single Yellow perch, 32 cm in length from Canadarago Lake, and a single Smallmouth bass, 44 cm in length from Otsego Lake, were selected for intensive histological analysis in which multiple tissue plugs were sectioned. These two specimens (Yellow perch, FR11_1; Smallmouth bass, FR11_60) were chosen because they were heavily infected, containing at least 30 individual attached L. thecatus. A portion of uninfected intestinal tissue of Yellow perch is shown in Figure 1A for comparison. Figure 1A illustrates the tissue layers of the intestine, including the mucosal layer, consisting of microvilli containing epithelial and goblet cells and underlying lamina propria; the submucosal layer; the muscular coat; and the serosa of connective tissue. This can be compared with Figures 1B and 1C in which the proboscis of L. thecatus is visible. Damage to the mucosal wall is visible in Figures 1B and 1C. Epithelial cells are torn and disrupted, and the proboscis is embedded within the underlying lamina propria. The gap in Figures 1B and 1C is thought to be an artifact (see discussion), but surrounding the gap a proliferation of erythrocytes and leukocytes are visible. It seems that in these infections the proboscis only penetrates the muscosal layer; it does not reach the submucosa. Figure 2. Light micrographs of histological sections of Smallmouth bass digestive system. 2A, Uninfected pyloric cecum, sagittal section. 2B, Proboscis of Leptorhynchoides thecatus embedded into intestinal layers, with trunk of worm also visible.
Figure 2 (continued). 2C, Higher magnification view of L. thecatus proboscis shown in 2B. 2D, Another plane of section of same L. thecatus specimen pictured in 2B, 2C.
A portion of uninfected pyloric cecum of Smallmouth bass is shown as a sagittal section in Figure 2A, illustrating the extent of folding and ridges that characterize the surface of the pyloric cecum, a pattern also observed in the intestine. The remaining figures are cross sections of the intestinal wall with a proboscis of L. thecatus attached. In Figures 2B, 2C and 2D the proboscis is embedded deep into the muscosal wall, disrupting the microvilli and penetrating the lamina propria. In Figures 2B and 2D the actual trunk of the worm is also visible, nestled between microvilli. Again, a gap is visible in each of these figures, but this is thought to be an artifact. The high magnification view of the proboscis in Figure 2C illustrates a proliferation of erythrocytes and leukocytes surrounding the area of proboscis attachment. Figure 2C also clearly illustrates the individual hooks on the proboscis. These hooks are the namesake of the Phylum Acanthocephala, the thorny-headed worms. Figures 2B and 2D illustrate the overall extent of proboscis penetration. In both examples, the proboscis has fully penetrated the mucosal layer, and barely reaches, but does not penetrate, the submucosal layer. DISCUSSION One of several technical hurdles encountered in this study was the presence of gaps in the histological sections of both Yellow perch and Smallmouth bass intestine. These gaps were observed between the proboscis and the host tissue to which it was attached. This apparent retraction of the proboscis from the host tissue has been observed previously (Venard and Warfel, 1953; Bullock, 1963) and is considered an artifact of fixation that occurs when the parasite is killed at the time of preservation. That is, there would be no gap in an actual infection of L. thecatus in a living Yellow perch or Smallmouth bass because the parasite by definition attached to the intestinal wall with its hooks. Evidence of this is apparent in that the tissue surrounding the gaps in Figures 1B, 1C, 2B, 2C, and 2D is damaged, presumably from contact with the proboscis. The intestinal damage observed in this study is extensive at the level of the mucosal layer. Although no cases of full intestinal perforation were observed, the tearing of epithelial cells and the disruption of microvilli and the underlying lamina propria raises the possibility that secondary infections with bacteria could result. Although parasitic attachment may not be the sole contributor to host animal death (Nickol and Crompton, 1983), the effects based on the histopathology validate that acanthocephalans can impair host health (Bullock, 1963, this study), though this may be difficult to measure in wild populations. CONCLUSION The documented damage caused by L. thecatus to the intestines of Yellow perch and Smallmouth bass raises the possibility that acanthocephalans play a role in the variability of populations of economically important game fish species in Otsego Lake and nearby waters.
ACKNOWLEDGEMENTS Timothy Pokorny (SUNY BFS), Michael Schallert (Ridgefield Springs, New York), Daniel Henning (Chicago, Illinois), and Dr. Allan Green (SUNY Oneonta) kindly provided several fish specimens for this study. Thanks to Stephen Daniels, Janine Caira (University of Connecticut), and to Allen Anderson (SUNY Oneonta) for offering technical advice. We are grateful to Allison Schulman for providing some of the sections for this study, and to Janine Caira and her students for hosting us during some of the histological work of this project. This project was supported with funds from a Student Research Grant from the SUNY Oneonta Research Foundation awarded to U. Habal., D. Willsey., L. Laraque., and M. O Rourke, and with a Teaching, Learning and Technology Center Fellowship awarded to F. B. Reyda. REFERENCES Bullock, W. L., 1963. Intestinal Histology of some salmonid fishes with particular reference to the histopathology of acanthocephalan infections. Journal of Morphology. 112: 23-44. DeGiusti, D. L. 1949. The life cycle of Leptorhynchoides thecatus, an acanthocephalan of fish. Journal of Parasitology. 35: 437-460. Lincicome, D. R., and H. J. VanCleave 1949. Distribution of Leptorhynchoides thecatus, a common acanthocephalan parasite in fishes. American Midland Naturalist. 41(2): 421-431. Nickol, B. B., and D. W. T. Crompton. 1985. Biology of the Acanthocephala. Cambridge University Press, Cambridge, England. Reyda, F. B. 2010. Parasitic worms of fishes of Otsego Lake and nearby water bodies, 2009. In 42 nd Annual Report of the SUNY Oneonta Biological Field Station. Pp. 276 281. Szmygiel, C., and F. B. Reyda. 2011. A survey of the parasites of Smallmouth bass (Micropterus dolomieu) In 43 rd Annual Report of the SUNY Oneonta Biological Field Station. Pp. 235 240. Venard, C. E., and J. H. Warfel. 1953. Some effects of two species of Acanthocephala on the alimentary canal of the largemouth bass. Journal of Parasitology. 39: 187-190.