Widespread Infection of Lake Whitefish Coregonus Clupeaformis with The

Journal of Great Lakes Research 36 (2010) 18–28

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Journal of Great Lakes Research

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Widespread infection of lake whiteﬁsh Coregonus clupeaformis with the swimbladder nematode Cystidicola farionis in northern lakes Michigan and Huron

Mohamed Faisal a,b,⁎, Walied Fayed a,c, Travis O. Brenden a,d, Abdelaziz Noor c, Mark P. Ebener e, Gregory M. Wright f, Michael L. Jones a,d a Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA b Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA c Faculty of Agriculture, Alexandria University, Alexandria, Egypt d Quantitative Fisheries Center, Department of Fisheries and Wildlife, Michigan State University, 153 Giltner Hall, East Lansing, MI 48824–1101, USA e Chippewa Ottawa Resource Authority, 179 East Three Mile Road, Sault Ste. Marie, MI 49783, USA f Nunns Creek Fishery Enhancement Facility, HC 47, Box 8100, Hessel, MI 49745, USA article info abstract

Article history: We estimated the prevalence, intensity, and abundance of swimbladder nematode infection in 1281 lake Received 3 August 2009 whitefish (Coregonus clupeaformis) collected from four sites in northern lakes Huron (Cheboygan and DeTour Accepted 11 January 2010 Village) and Michigan (Big Bay de Noc and Naubinway) from fall 2003 through summer 2006. Morphological examination of nematode egg, larval, and mature stages through light and scanning electron microscopy Communicated by Trent M. Sutton revealed characteristics consistent with that of Cystidicola farionis Fischer 1798. Total C. farionis prevalence was 26.94%, while the mean intensity and abundance of infection was 26.72 and 7.21 nematodes/fish, Index words: Lake whitefish respectively. Although we detected C. farionis in all four stocks that were examined, Lake Huron stocks Cystidicola farionis generally had higher prevalence, intensity, and abundance of infection than Lake Michigan stocks. A distinct Swimbladder pathology seasonal fluctuation in prevalence, abundance, and intensity of C. farionis was observed, which does not Lake Michigan coincide with reported C. farionis development in other fish species. Lake whitefish that were heavily Lake Huron infected with C. farionis were found to have thickened swimbladder walls with deteriorated mucosa lining, which could affect swimbladder function. Whether C. farionis infection may be negatively impacting lake whitefish stocks in the Great Lakes is unclear; continued monitoring of C. farionis infection should be conducted to measure responses of lake whitefish stocks to infection levels. © 2010 Elsevier B.V. All rights reserved.

Introduction depends on several factors, including fish age, parasite and intermediate host abundance, and water temperature (Knudsen et al., 2002, 2004). Nematodes of the genus Cystidicola Fischer 1798 (Habronematoidea: Lake whitefish (Coregonus clupeaformis) in North America have Cysticolidae) are parasitic in the swimbladders of physostomus fishes in been found to be susceptible to infection with C. farionis (Lankester the Northern hemisphere. Presently, two species of Cystidicola are and Smith, 1980). However, maturation of C. farionis in lake whitefish recognized: C. farionis and C. stigmatura. C. farionis Fischer 1798 in North America is regarded as atypical, which has led some parasitizes the swimbladders of rainbow smelt (Osmerus mordax)and researchers to theorize that it is a new Cystidicola sp. infecting lake Coregonus, Oncorhynchus,andSalvelinus spp. from Eurasia and North whitefish and not C. farionis (Lankester and Smith, 1980; Dextrase, America, while C. stigmatura Leidy 1886 parasitizes Salvelinus spp. from 1987). In Lake Nipigon, Canada, lake whitefish have been found to be North America (Black, 1983). Through their physical movements and routinely infected with large numbers of immature C. farionis, but no production of toxic metabolites, Cystidicola spp. cause destruction of the mature nematodes have been found in infected individuals (Lankester highly vascularized swimbladder walls, which can affect swimming and Smith, 1980). Immature nematodes are also common in Lake performance and buoyancy control of infected individuals (Lankester Superior lake whitefish, but only small numbers of mature nematodes and Smith, 1980; Black, 1984; Willers et al., 1991; Dzeikonska-Rynko have been found (Lankester and Smith, 1980). Buccal cavity structure et al., 2003). The infection rate of Cystidicola spp. in fish populations and eggs from C. farionis found in lake whitefish exhibit morphological characteristics that are slightly different when compared to eggs collected from other susceptible species in the same environ- ment (Dextrase, 1987; Miscampbell et al., 2004). Despite these ⁎ Corresponding author. S-110 Plant Biology Building, Michigan State University, East Lansing, Michigan 48824, USA. atypical characteristics, extensive genetic studies using samples from E-mail address: [email protected] (M. Faisal). both Canada and Finland have failed to find sequence differences in

0380-1330/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jglr.2010.01.008 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28 19 ribosomal DNA among mature C. farionis nematodes in lake white- (BBN), Naubinway (NAB), Cheboygan (CHB), and DeTour Village fish and other susceptible species, which has led researchers to (DET). The BBN and NAB stocks are located in northern Lake Michigan, conclude that it is indeed C. farionis that is infecting lake whitefish while the CHB and DET stocks are located in northern Lake Huron (Miscampbell et al., 2004). (Fig. 1). Each of these areas has large spawning aggregations of lake In the Laurentian Great Lakes of North America, lake whitefish whitefish, and although b50 km separates some of these locations, is a commercially, ecologically, and culturally important species individuals have been found to display strong fidelity to these areas (Fleischer, 1992; Ebener et al., 2008). The invasion and spread of zebra during the spawning season (Ebener and Copes, 1985; Ebener et al., (Dreissena polymorpha) and quagga mussels (D. bugensis) in the Great 2010). Lakes have been associated with significant declines in lake whitefish Collection of lake whitefish from each of the stocks began in condition, growth, and recruitment (Hoyle et al., 1999; Pothoven fall 2003, and continued seasonally through summer 2006. For the et al., 2001; Mohr and Ebener, 2005). These declines are believed purpose of this study, fall is considered to encompass the months to have been caused primarily from declines in indigenous benthic of October through December, winter encompasses the months of macroinvertebrates, Diporeia spp. in particular, as a result of January through March, spring encompasses the months of April dreissenid invasion in the Great Lakes (Pothoven et al., 2001; Mills through June, and summer encompasses the months of July through et al., 2005; Nalepa et al., 2005). The absence of Diporeia spp. in large September. Additionally, for the purpose of this study, fall 2003 areas of the Great Lakes has resulted in lake whitefish increasing through summer 2004 is identified as the 2004 sampling year; fall consumption of other benthic macroinvertebrates, including dreisse- 2004 through summer 2005 is identified as the 2005 sampling year; nid mussels, gastropods, opossum shrimp (Mysis relicta), ostracods, and fall 2005 through summer 2006 is identified as the 2006 sampling oligochaetes, and zooplankton (Hoyle et al., 1999; Pothoven et al., year. Because of inclement weather conditions, no lake whitefish were 2001; Pothoven, 2005; Hoyle, 2005). Because benthic macroinverte- collected from the CHB stock in fall 2006 or from the NAB stock in brates are known to be immediate hosts for C. farionis, a question has winter 2004. Total numbers of lake whitefish collected and examined been raised whether changes in feeding habits of lake whitefish may for swimbladder nematodes during each sampling period ranged have led to changes in the extent of swimbladder nematode infection from 15 to 35 fish/stock (Table 1). or exacerbated its pathologic impacts. The objectives of this study Sampling locations were typically chosen by contract fishermen were to: (1) identify the species of swimbladder nematodes in lake based on prior commercial catches. Lake whitefish were collected whitefish collected from four lake whitefish stocks in northern lakes using a combination of commercial traps and gill nets. Captured lake Huron and Michigan; (2) measure the prevalence, abundance, and whitefish were transferred (alive or recently dead and shipped on ice) intensity of the swimbladder nematodes in these stocks; (3) evaluate to the Michigan State University-Aquatic Animal Health Laboratory in variations in larval stage development and maturation among the East Lansing, MI for immediate processing. Once at the laboratory, live stocks; and (4) assess the damage to lake whitefish swimbladders fish were sacrificed with an overdose (300 mg/liter) of Tricaine caused by the nematode infection. Methanesulfonate (MS-222, Argent Chemicals, Redmond, WA).

Methods Parasite identiﬁcation and swimbladder pathology

Study area and fish sampling The swimbladder from each lake whitefish was removed intact, dissected, and swimbladder walls examined for the presence of mac- We studied four lake whitefish stocks, two in northern Lake Huron roscopic lesions. Swimbladder nematodes were retrieved manually and two in northern Lake Michigan. For simplicity, we reference these and preserved in 70% ethanol for later identification and enumeration. stocks by the names of their closest fishing ports: Big Bay de Noc Nematodes were cleared in a mixture of glycerol and 70% ethanol

Fig. 1. Map of northern lakes Huron and Michigan indicating the locations of the Big Bay de Noc, Cheboygan, DeTour Village, and Naubinway lake whiteﬁsh spawning locations where lake whiteﬁsh were collected for swimbladder nematode examination. 20 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28

Table 1 was the number of Cystidicola spp. found in a lake whitefish regardless Number of fish examined (Fish), mean prevalence (Prev.), mean abundance (Abu.) and of whether the particular fish is infected or not (zero counts possible). mean intensity (Int.) of Cystidicola farionis in lake whitefish by year and season for fish Intensity was the number of Cystidicola spp. nematodes found in collected from Big Bay de Noc, Naubinway, Cheboygan, and DeTour Village spawning fi stocks. NS = no sampling conducted during that year and season for that spawning infected lake white sh (zero counts not possible). stock. We used generalized estimating equations (GEEs) to test whether prevalence, abundance, and intensity of Cystidicola spp. Year Season Fish Prev. (%) Abu. Int. Prev. (%) Abu. Int. infection differed among stocks, seasons, and years. Given that the Lake Michigan lake whitefish for this study were collected with commercial fishing Big Bay de Noc Naubinway 2004 Fall 28 6.90 0.24 3.50 0.00 0.00 0.00a nets, we felt it was likely that measurements within sampling Winter 24 8.33 0.17 2.00 NS NS NS occasion would be correlated. As a result, we considered fish that Spring 30 53.33 4.73 8.88 0.00 0.00 0.00a were collected together to have an exchangeable correlation a Summer 22 4.55 0.14 3.00 0.00 0.00 0.00 structure, meaning that the correlations among individuals within 2005 Fall 26 26.92 1.88 7.00 10.00 0.27 2.67 a sampling event was the same for all individuals. Because of the Winter 16 6.25 0.08 1.00 0.00 0.00 0.00a Spring 30 20.00 1.23 6.17 0.00 0.00 0.00a very low infection rate of the NAB stock (see Results below), we Summer 30 13.33 0.23 1.75 0.00 0.00 0.00a chose to exclude this stock from the testing of infection parameters 2006 Fall 30 3.33 0.07 2.00 10.00 0.13 1.33 as it was obviously different from the others and the low level of a Winter 30 10.00 0.17 1.67 0.00 0.00 0.00 variability in infection parameters caused problems when fitting the Spring 30 10.00 0.50 5.00 3.33 0.03 1.00 Summer 30 6.67 0.13 2.00 0.00 0.00 0.00a GEEs. We assumed a binomial error structure to test differences in Cystidicola spp. prevalence. To test differences in Cystidicola spp. Lake Huron abundance, we assumed a negative binomial error structure. To test Cheboygan De Tour Village differences in Cystidicola spp. intensity, we first log-transformed the a a 2004 Fall 30 0.00 0.00 0.00 0.00 0.00 0.00 intensity counts and then assumed a normal error structure for the Winter 32 18.75 1.22 6.50 10.00 0.11 1.00 fi Spring 20 85.00 61.65 72.53 36.67 9.87 26.91 GEE. We included rst-order interactions between stocks, seasons, Summer 30 23.33 2.67 11.43 10.00 1.85 18.50 and years in the GEEs to determine if there were significant 2005 Fall 26 11.54 3.15 27.33 30.00 2.87 9.56 interactions among the variables. Differences in prevalence, abun- a Winter 15 0.00 0.00 0.00 43.33 4.17 9.62 dance, and intensity by stocks, years, seasons, or the first-order Spring 30 100.00 85.20 85.20 90.00 27.80 30.89 Summer 28 71.43 33.39 46.75 56.67 9.33 16.47 interactions between these factors were assessed using pairwise 2006 Fall NS NS NS NS 0.00 0.00 0.00a comparisons of least-squares means. Because of the potentially large Winter 30 63.33 7.77 12.26 46.67 2.57 5.50 number of comparisons to be performed, we used a Bonferroni Spring 29 93.10 28.45 30.56 68.97 9.00 13.05 adjustment to control the pairwise test error rate. Summer 30 70.00 5.63 8.05 80.00 24.23 30.29 For infected lake whitefish, we tested differences in maturation of a Intensity was set equal to zero when no infected individuals were collected from a Cystidicola spp. between stocks by modeling the maturation proba- sampling site. bilities for the stocks through multinomial logistic regression and then comparing the odds ratios of being in one maturation stage versus a (1:1) at room temperature and examined microscopically. Mature reference maturation stage between the stocks (Agresti, 2007). We and larval-stage nematodes were identified using the dichotomous modeled the maturation stages through multinomial logistic regres- keys of Ko and Anderson (1969), Smith and Lankester (1979), sion rather than a cumulative logit model because our data did not Lankester and Smith (1980), Black and Lankester (1980), Black meet the proportional odds assumption required for the cumulative (1983), Dextrase (1987), Hoffman (1999), and Miscampbell et al. logit model (Agresti, 2007). We used the earliest development stage (2004). Total numbers of nematodes, as well as maturation stage and as our reference category, thus the calculated odds were that of a lake sex of mature nematodes, were recorded for each lake whitefish. whitefish being infected with a later development stage compared Scanning electron microscopy (SEM) was used to confirm the to the earliest development stage. We chose to test differences in light-microscopical identification of the species within the genus development and maturation levels of Cystidicola spp. in this manner Cystidicola as recommended by Dextrase (1987) and Miscampbell as it was simpler than conducting additional multi-factor tests on et al. (2004). Eggs were extruded from gravid females and their individual maturation stages and because we felt that such tests morphology examined as described by Dextrase (1987). Briefly, the would be redundant with the tests conducted on overall prevalence, mid-sections of female nematodes were excised, covered with a drop abundance, and intensity. We used a similar test to determine of glycerin, and then mounted with a cover slip with gentle pressure whether the stocks differed in the sex ratios of Cystidicola spp. in lake to permit the extrusion of eggs from uteri. Extruded eggs as well as the whitefish infected with adult nematodes. anterior portion of the mature nematodes (including the lips) were Because swimbladder function may be an important factor dehydrated through an ethanol gradient (35–95%), followed by three affecting foraging of lake whitefish, the condition factor (K) of both washes of 100% ethanol, critical point-dried with carbon dioxide, gold infected and non-infected lake whitefish was calculated. Condition coated, and then examined. factor was calculated by dividing a fish's weight (in grams) by the To evaluate the damage to swimbladders caused by nematode cube of its length (in millimeters) and multiplying the resulting infection, swimbladders were examined both visually and histologi- quotient by 100,000 (Anderson and Neumann, 1996). We then cally. Swimbladders from lake whitefish were fixed in 10% buffered evaluated whether prevalence, abundance, and intensity of Cystidicola formalin, dehydrated, and paraffin-embedded. The embedded tissues spp. infection affected lake whitefish condition after accounting for were then sectioned (5 μm thick) and stained with haematoxylin the effects of stock, year, and season of sampling by calculating the and eosin as described by Prophet et al. (1992) and examined residuals from the GEEs described above and then using simple linear microscopically. regression to relate the residuals to K. All analyses were conducted in SAS using the GENMOD or GLM procedures. In most cases, Data analysis we regarded a statistical test with Pb0.05 as statistically significant. The one exception to this was when testing first-order interactions We calculated the prevalence, abundance, and intensity, as defined between stock, year, and season of Cystidicola spp. infection char- by Bush et al. (1997),ofCystidicola spp. for each stock. Prevalence was acteristics in which case a Pb0.10 was considered statistically sig- the percent of lake whitefish infected with Cystidicola spp. Abundance nificant. We made this exception because we were concerned that M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28 21 even weak variable interactions could mask comparisons of main factor levels.

Results

Nematodes were found in the swimbladders of lake whitefish collected from each of the four stocks (Table 1). Total number of infected fish collected during a sampling period ranged from 1 to 16, 0 to 30, 0 to 27, and 0 to 3 for the BBN, CHB, DET, and NAB stocks, respectively. Total number of nematodes recovered from fish for each site ranged from 13 for the NAB stock to more than 6100 for the CHB stock . Most nematodes were found free within the swimbladder cavity, although a few were attached to the bladder walls. Based on morphology, nematodes were identified as larval and adult stages of Cystidicola spp. Additional light and scanning electron microscopy on extruded eggs and mouth parts of collected individuals indicated the Fig. 3. Scanning electron microscopy of buccal cavity of C. farionis mouth showing pseudolabia (p) associated with projecting lip. presence of polar and lateral filaments on eggs (Fig. 2) and the absence of a lip projection in the pseudolabia (Fig. 3). Based on these characteristics, swimbladder nematodes were identified as C. farionis Fischer 1798. Total C. farionis prevalence in lake whitefish was 26.94% (SE= 1.24%). Overall, C. farionis prevalence in the stocks ranged from 2.24% (SE=0.84%) for the NAB stock to 50.00% (SE=2.89%) for the CHB stock. Prevalence in the BBN and DET stocks was 14.68% (SE=1.96%) and 41.44% (SE=2.70%), respectively. Prevalence across all stocks tended to increase during the winter and spring sampling periods, and to decline during the summer and fall sampling periods (Table 1; Fig. 4). Overall mean abundance of C. farionis was 7.21 (SE=0.78) nematodes. For the individual stocks, mean abundance of C. farionis equaled 0.85 (SE=0.20), 20.51 (SE=3.20), 8.18 (SE=1.24), and 0.04 (SE=0.02) nematodes/fish for the BBN, CHB, DET, and NAB stocks, respectively. Abundance generally peaked during the spring sampling period for most of the stocks; the CHB stock in particular had very large increases in C. farionis abundance during the spring (Fig. 4). Of those lake whitefish that were infected with C. farionis, mean intensity of the infection was 26.72 (SE=4.63) nematodes/fish, with stock- level intensity of infection ranging from 1.86 (SE=1.93) and 5.75 (SE=2.92) nematodes/fish for the NAB and BBN stocks to 19.74 (SE=3.82) and 41.04 (SE=5.41) nematodes/fish for the DET and CHB stocks, respectively. C. farionis intensity of infection had greater sampling period variability than did prevalence or abundance; however for most stocks intensity of infection peaked during the spring sampling period and then declined (Fig. 4). When testing differences in C. farionis prevalence among stocks (excluding the NAB stock), seasons, and years, we found that the first- order interaction between year and season (χ2 =18.29, df=6, P=0.006), stock and year (χ2 =8.10, df=4, P=0.088), and stock and season (χ2 =10.91, df=6, P=0.091) were statistically significant. Pairwise comparisons of least-squares means for the stock-by- year interaction indicated that prevalence in 2004 was significantly lower than in 2005 and 2006 for the CHB and DET stocks, but prevalence in 2004 was greater than in 2006 for the BBN stock (Appendix A). Additionally, prevalence in most sampling years was lower for the BBN stock than for the CHB and DET stocks; the exception was in 2004 when there was no difference in the BBN and CHB stocks and the prevalence in the BBN stock was significantly greater than in the DET stock (Appendix A). In 2004, prevalence in the CHB stock was significantly greater than in the DET stock (Appendix A). For the stock-by-season interaction, pairwise comparisons of least- squares means indicated that for the BBN, CHB, and DET stocks, C. farionis prevalence in the spring was significantly greater than in any other season (Appendix B). The only exception to this was the spring-versus-summer comparison for the BBN stock in which no difference was found. For the CHB and DET stocks, prevalence of Fig. 2. Light microscopy (a and b) and scanning electron microscopy (c) of egg extruded fi from females of C. farionis: (a) An egg exhibiting lateral filament arrangement (arrows, C. farionis in the fall was signi cantly less than in summer and winter. 1000×) (b) An egg with polar filaments (400×) and (c) an egg with lateral filaments. During the fall, prevalence in the BBN stock was significantly greater 22 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28

Fig. 4. Cystidicola farionis prevalence, abundance, and intensity (±SE) by sampling occasion for lake whitefish collected from the Big Bay de Noc, Cheboygan, DeTour, and Naubinway spawning stocks. than in the CHB and DET stocks; however, during all other seasons CHB, and DET stocks, and was greater than in the summer for prevalence in the BBN stock was significantly lower than in the CHB the BBN and CHB stocks and greater than in winter for the BBN and DET stocks (Appendix B). Spring prevalence in the CHB stock was and DET stocks (Appendix B).ForboththeCHBandDETstocks, significantly greater than in the DET stock (Appendix B). abundance in the summer and winter was greater than in the For the year-by-season interaction, pairwise comparisons of least- fall.AbundanceinsummerwasgreaterthaninwinterfortheDET squares means indicated that during most years, prevalence of stock (Appendix B). Although there were no differences in fall C. farionis in lake whitefish was significantly greater in the spring abundance of C. farionis among the BBN, CHB, and DET stocks, for than in the other seasons, and that in 2004 and 2006 fall prevalence all other seasons abundance in the BBN stock was significantly was significantly lower than in winter or summer (Appendix C). For lower than in the CHB and DET stocks (Appendix B). There were individual seasons, whether C. farionis prevalence differed among the no differences in abundance between the CHB and DET stocks year of sampling was highly variable (Appendix C). For example, fall in fall, winter, and summer, but the spring abundance in the prevalence in 2005 was significantly greater than fall prevalence in CHB stock was significantly greater than in the DET stock 2004 and 2006, but winter prevalence in 2005 was not different than (Appendix B). in 2004 or 2006 (Appendix C). Differences in abundance for the year-by-season interaction were When testing differences in C. farionis abundance among stocks similar to those found for prevalence. Abundance was greater in the (excluding the NAB stock), seasons, and years, we found that the first- spring than in other seasons, and abundance in the fall was generally order interactions between stock and year (χ2 =11.36, df=4, less than in summer or winter (Appendix C). Additionally, like preva- P=0.023), stock and season (χ2 =11.46, df=6, P=0.075), and lence, differences in abundance in individual years depended on what year and season (χ2 =17.52, df=6, P=0.008) were significant. seasons were tested. Fall abundance in 2005 was significantly greater Pairwise comparisons of least-squares means for the stock-by-year than in 2004 and 2006, but winter abundance in 2005 was not interaction indicated that abundance of C. farionis in the BBN stock in different than in 2004 or 2006 (Appendix C). 2004 was significantly greater than in 2005 and abundance in 2005 When testing differences in C. farionis intensity among stocks was significantly greater than in 2006 (Appendix A 3). For the CHB (excluding the NAB stock), seasons, and years, we found that the first- and DET stocks however, abundance in 2005 was significantly greater order interactions between stock and year (χ2 =10.00, df=4, P= than in 2004 and 2006 (Appendix A). In all sampling years, abundance 0.040) and year and season were significant (Year×Season: χ2 = in the BBN stock was significantly lower than in the CHB and DET 11.82, df=6, P=0.066). However, the interaction between stock and stocks. In 2005 and 2006, there were no differences in abundance for season was not significant (Year×Season: χ2 =9.22, df=6, P=0.16). the CHB and DET stocks, but in 2004 C. farionis abundance in the CHB As a result, we conducted pairwise comparisons of the least-squares stock was significantly greater than in the DET stock. means for the stock-by-year and year-by-season interactions, but we For the stock-by-season interaction in C. farionis abundance, did not test for differences among the levels of the stock-by-season abundance in the spring was greater than in the fall for the BBN, interactions. M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28 23

Compared to prevalence and abundance, there were fewer dif- In general, lake whitefish from Lake Michigan were infected with ferences in intensity among the stock-and-year combinations. For the a larger fraction of adult C. farionis than lake whitefish from Lake BBN and DET stocks, there were no differences in intensity of infection Huron (Table 2). For the NAB stock, which had the lowest levels of between years; however, for the CHB stock intensity of infection was prevalence, abundance, and intensity among the stocks, only adult significantly lower in 2006 than in 2005 and 2004 (Appendix A). For nematodes were collected from infected lake whitefish. For the BBN all sampling years, intensity of infection was significantly lower in the stock, which often had significantly lower infection parameters in BBN stock than in the CHB and DET stocks, but there were no particular seasons and years than the CHB or DET stocks, approxi- differences in intensity of infection between the CHB and DET stocks mately 64% of collected nematodes were in the adult stage. In com- (Appendix A). parison, approximately 43% and 45% of collected nematodes from the For the year-by-season interaction in infection intensity, spring CHB and DET stocks respectively were in the adult stage (Appendix A). intensity in most years was significantly greater than in other seasons Adult C. farionis prevalence, abundance, and intensity of infection (Appendix C). Additionally, in 2004 and 2006 summer intensity exhibited sharp seasonal fluctuations similar to overall prevalence, of infection was significantly greater than in fall and winter. For abundance, and intensity, with peaks generally observed during the both fall and spring, intensity of infection in 2004 and 2005 was spring sampling period (Fig. 2). significantly greater than intensity of infection in 2006 (Appendix C). Sex ratios of C. farionis in infected individuals varied considerably Winter infection intensity was also greater in 2005 than in 2006 by sampling period within the stocks (Table 2). Female to male sex (Appendix C). ratios ranged from 0 to 3 in the BBN stock, 0.23 to 1.78 in the CHB stock, and 0.82 to 3.22 in DET stock. In the NAB stock, female to male Maturation stages and sex of nematodes in infected individuals ratios ranged from 0.33 to 1.00, with an additional sampling in which only one adult female was collected from all sampled lake whitefish Larval stages of C. farionis were identified according to their mor- resulting in an undefined sex ratio for that sampling period. phological criteria. The third larval stage (L3), which is the infective From our multinomial logistic regression model of C. farionis stage, was identified by its dumbbell shaped oral opening, pseudo- maturation stages in infected lake whitefish, we determined that the labia, and the prominent tail protrusion (Fig. 5a). The fourth larval odds of lake whitefish from the BBN stock being infected with L4, SA, stage (L4) showed gonadal primordia, with the tail protrusion starting and adult developmental stages versus the L3 stage were 1.7 (95% CI: its fusion with the nematode body (Fig. 5b). Following the fourth and 1.1–2.7), 2.1 (95% CI: 1.3–3.3), and 4.5 (95% CI: 3.0–6.7) times the final molt, nematodes exhibited fully formed buccal cavity with odds of fish from the CHB stock, and were 2.0 (95% CI: 1.3–3.1), 3.0 circumoral teeth and the tail protrusion disappeared, however, they (95% CI: 1.8–4.9), and 2.0 (95% CI: 1.9–4.4) times the odds of fish from were sexually immature as no gametes (eggs or spermatozoa) could the DET stock. Thus, lake whitefish from the BBN stock were more be seen. We refer to this stage as the sub-adult (SA) stage. Males were likely to be infected with later developmental stage C. farionis than the recognized by their twisted tail and two speculae that were present Lake Huron stocks relative to the L3 stage. The odds of lake whitefish throughout their development (Fig. 5c). Sexually mature males (M) from CHB stock being infected with L4, SA, and adult developmental were identified by the presence of spermatozoa in their vas deference. stages versus the L3 stage were 1.2 (95% CI: 1.0–1.3), 1.4 (95% CI: 1.2– Mature females (F) were recognized by the presence of shelled eggs 1.7), and 0.6 (95% CI: 0.5–0.7) times the odds of those from the DET filling a considerable portion of their bodies (Fig. 5d). stock, meaning that the lake whitefish from the CHB stock were more

Fig. 5. Light microscopy showing larval stages of Cystidicola farionis found in the swimbladder of infected lake whiteﬁsh: (a) Third larval stage (L3), the infective stage, exhibiting a prominent tail papilla (arrow, 400×); (b) Fourth larval stage (L4) with the tail papilla expanding and fusing with the body (arrow, 400×); (c) Male nematode showing spicules (arrow, 200×); (d) Adult gravid female with eggs (arrow, 1000×). 24 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28

Table 2 Proportion of Cystidicola farionis at different maturation stages collected by year and season for ﬁsh collected from Big Bay de Noc (Lake Michigan), Naubinway (Lake Michigan), Cheboygan (Lake Huron), and De Tour Village (Lake Huron) spawning stocks f(L3 =third larval stage, L4 = fourth larval stage, SA = sub-adult stage, AD = adult stage). Also shown is the female to male ratio of C. farionis adults collected from infected lake whiteﬁsh. A “—” in each of the maturation stage categories indicates that no C. farionis were found during that sampling period. A “—” in the F:M category indicates that no C. farionis adults were collected during the sampling period, while a “UDF” in the F:M category indicates that only C. farionis adult females were collected during the sampling period. NS = no sampling conducted during that season.

Year Season L3 L4 SA AD F:M L3 L4 SA AD F:M

Lake Michigan Big Bay de Noc Naubinway 2004 Fall 0.00 0.00 0.00 1.00 1.33 ————— Winter 0.00 0.00 0.00 1.00 0.33 NS NS NS NS NS Spring 0.14 0.44 0.19 0.23 1.75 ————— Summer 0.00 0.00 0.00 1.00 0.50 ————— 2005 Fall 0.02 0.00 0.04 0.94 1.00 0.00 0.00 0.00 1.00 0.33 Winter 0.00 0.00 1.00 0.00 —————— Spring 0.00 0.00 0.16 0.84 1.07 ————— Summer 0.00 0.00 0.57 0.43 2.00 ————— 2006 Fall 0.00 0.50 0.00 0.50 0.00 0.00 0.00 0.00 1.00 1.00 Winter 0.00 0.20 0.20 0.60 0.00 ————— Spring 0.60 0.20 0.00 0.20 0.00 0.00 0.00 0.00 1.00 UDF Summer 0.00 0.00 0.00 1.00 3.00 —————

Lake Huron Cheboygan De Tour Village 2004 Fall —————————— Winter 0.08 0.13 0.15 0.64 1.78 0.00 0.00 0.00 1.00 ∞ Spring 0.18 0.29 0.18 0.35 1.57 0.59 0.28 0.02 0.11 2.00 Summer 0.05 0.10 0.18 0.68 1.08 0.00 0.05 0.00 0.95 2.89 2005 Fall 0.01 0.00 0.00 0.99 1.38 0.08 0.29 0.31 0.31 2.00 Winter —————0.39 0.15 0.15 0.30 3.22 Spring 0.45 0.36 0.08 0.12 1.19 0.38 0.42 0.09 0.11 1.44 Summer 0.09 0.26 0.50 0.14 0.82 0.06 0.17 0.53 0.24 1.79 2006 Fall NS NS NS NS NS ————— Winter 0.17 0.31 0.45 0.07 0.23 0.12 0.27 0.35 0.26 0.82 Spring 0.05 0.45 0.00 0.49 1.24 0.25 0.41 0.00 0.34 1.57 Summer 0.00 0.21 0.00 0.79 1.33 0.03 0.11 0.00 0.87 1.72

likely than the DET to be infected with L4 and SA C. farionis but were less likely to be infected with adult nematodes relative to the L3 stage. As for sex of C. farionis, the odds of lake whitefish from the BBN stock being infected with female versus male C. farionis were 1.19 (95% CI: Fig. 6. Morphological examination of the swimbladder of lake whitefish: (a) Normal 0.8–1.7) times the odds of fish from the CHB stock, meaning the odds swimbladder; (b) Swimbladder infected with a few nematodes (arrow) without for both stocks were relatively equal. Conversely, the odds of lake affecting the transparency of the membrane; (c) Swimbladder with slightly opaque whitefish from the BBN stock being infected with female versus male membrane showing moderate number of nematodes, (arrow); (d) Swimbladder with very thick membrane showing heavy nematodes infection (arrow). C. farionis were 0.6 (95% CI: 0.4–0.9) times the odds of those from the DET stock. The odds of fish from the CHB stock being infected with female versus male nematodes were 0.7 (95% CI: 0.6–0.9) times the odds of those from the DET stock. Histologically, healthy lake whitefish swimbladder walls con- sisted of serosa, tunica fibrosa, tunica muscularis and epithelial Gross and histopathological alterations in lake whitefish swimbladders mucosa with a well-developed vascular system supplying blood to the organ. The mucosal layer was folded with the most outer Clinical and histopathological examination of lake whitefish in- epithelial cells, cuboidal to low-columnar in appearance (Fig. 7a). fected and not infected with C. farionis revealed the presence of In infected lake whitefish, a number of focal lymphocytic and several gross pathological changes due to C. farionis infection. Non- histocytic infiltrates in the subepithelial connective tissue along infected lake whitefish swimbladders had glistening outer mem- with erosion of mucosal lining were observed; intensity of infil- branes and transparent inner membranes with blood vessels apparent trates increased with intensity of infection (Fig. 7b). Blood vessels in within the membranes (Fig. 6a). Conversely, in lake whitefish (252 the deep connective tissues were often congested ( Fig. 7c). In fish) with low to medium infection intensity (1–100 nematodes/fish) heavily infected fish, multifocal lymphocytic and histocytic infil- the swimbladder inner walls were normal appearance and nematodes trates were apparent in the deep connective tissue (Fig. 7d) with could be visualized through the membrane that appeared opaque widespread erosion of the mucosal lining. In a number of heavily and thickened (Fig. 6b). There were 21 lake whitefish with relatively infected fish, the swimbladder and lumen were filled with high infection (N100 nematodes/fish). In terms of gross appearance, nematodes and fibrinous proteinaceous exudate that contained nematode-filled swimbladders walls appeared extremely opaque inflammatory cells (Fig. 7e). Rarely, the swimbladder tunica fibrosa and thickened (Fig. 6c, d), with the lumen often containing yellowish connective tissue was restructured appearing like a granulation turbid fluid. tissue (Fig. 7f). M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28 25

Fig. 7. Light microscopy of lake whitefish swimbladder wall sections stained with hematoxylin and eosin: a) healthy mucosal lining of a non-infected fish, b) focal lymphocytic infiltrates in the subepithelial tissue, c) blood vessels in the deep connective tissues engorged with red blood cells, d) multifocal lymphocytic and histocytic infiltrates (arrows), e) fibrinous proteinaceous exudates (asterisk) that containing inflammatory cells, f) tunica fibrosa of a heavily infected fish taking the appearance of a granulation tissue. All scale bars=25 µm except in a and f=50 µm.

Relationship between K and infection abundance and intensity eggs excluded C. stigmatura as the agent of infection (Lankester and Smith, 1980; Black, 1983; Dextrase, 1987; Miscampbell et al., 2004; We found a positive relationship between K and GEE residuals Hoffman, 1999). Additional confirmation of C. farionis as the swim- for C. farionis abundance (Slope=26.46, SE=9.42) and intensity bladder nematode infecting lake whitefish in northern lakes Huron (Slope=47.17, SE=24.04), suggesting that individuals with greater and Michigan came from examination of the nematode mouth parts, than average C. farionis abundance and intensity had larger condition which showed the absence of a prominent lip projection in the factors than individuals with lower than average abundance and pseudolabia. intensity. The test for whether the model coefficient were different Based on filament arrangement of eggs collected from lake from zero showed statistically significant for C. farionis abundance whitefish nematodes, it appears that the C. farionis collected in our (t=2.81, P=0.005) but was statistically insignificant, although only study are identical to those collected in lake whitefish from lakes marginally so, for C. farionis intensity (t=1.96, P=0.051). It is impor- Superior, Huron, and Ontario (Lankester and Smith, 1980; Dextrase, tant to note, however, that the total amount of variability explained in 1987), but are possibly different from those collected in Finland, Lake the C. farionis GEE residuals by K was low (R2 b0.02) for both models. Nipigon (Ontario), and British Columbia (Miscampbell et al., 2004). Contrary to previous studies that have found lake whitefish to be an Discussion unsuitable host for C. farionis maturation, we clearly found that lake whitefish from lakes Huron and Michigan were able to support the Examination of the morphology of nematodes at both larval and development of C. farionis to adult, sexually mature stages. mature stages through light and scanning electron microscopy Although we detected C. farionis at all four sampling sites, the Lake revealed characteristics consistent with that of Cystidicola spp. Huron spawning stocks generally had higher rates of infection than (Hoffman, 1999). The presence of filaments that were either lateral the Lake Michigan spawning stocks. Although C. farionis has been or polar in arrangement on eggs collected from the nematodes sug- previously reported in lake whitefish from other locations within Lake gested that the nematodes collected in this study were Cystidicola Huron (Lankester and Smith, 1980), this is the first study to have farionis Fischer 1798, while the absence of lateral lobes on the found the nematode in lake whitefish from CHB and DET stocks in 26 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28

Lake Huron, or from any site within Lake Michigan, despite prior explanation to be unlikely. A second explanation could be that the parasitological examination of lake whitefish from these systems heavily infected fish died. This explanation would be in line with the (Amin, 1977; Hoffman, 1999). Therefore, our findings are considered results of Knudsen et al. (2002), who found that Arctic char that were new geographic range expansion of lake whitefish C. farionis in these heavily infected with C. farionis frequently died during the winter and four sites, as well as the first report in Lake Michigan. during spawning when stress levels are high. However, Wagner et al. Considering the relatively short distance separating the collection (this issue) did not find a relationship between stock-level estimates sites, it was somewhat surprising to find significant differences in of natural mortality and indicators of fish health, including C. farionis C. farionis infection parameters among the four stocks. Based on infection intensity, which is contradictory to this hypothesis. Another differences in the L3 infective stage, it would appear that both Lake explanation for the differences in infection parameters among the Huron spawning stocks are continuously exposed to C. farionis, while sites relates to dispersal and movement differences among the stocks. the low number of L3 stage nematodes in lake whitefish from the Lake Analysis of tag-recapture data showed that the stocks were primarily Michigan sites suggests that recruitment is relatively low and that the segregated during the spawning season, which lasts from fall to mid- expansion of C. farionis into lake whitefish in Lake Michigan may be a winter, but that fish from the four stocks (particularly the DET and recent event (Amin, 1977). CHB stocks) were mixed during the remainder of the year (Ebener One explanation for the site-associated differences in C. farionis et al., 2010). While differences in dispersal and movement may help infection parameters that we observed is that feeding habits of lake explain the seasonal fluctuations in infection parameters, it does not whitefish differ between the sites. The benthic invertebrate commu- necessarily explain the much lower C. farionis intensity and nities of lakes Huron and Michigan have undergone drastic changes abundance in the two Lake Michigan stocks, or the lack of C. farionis since dreissenids first invaded the Great Lakes (Pothoven et al., 2001; recruitment in the NAB stock that we observed. Whether the seasonal McNickle et al., 2006; Nalepa et al., 2007, 2009a,b). Many areas of fluctuation of C. farionis in lake white fish is due to one or a com- the Great Lakes are now devoid of Diporeia spp., which has resulted bination of the above-mentioned explanations requires additional in lake whitefish elevating their consumption of other food items. long term monitoring. In Lake Michigan, lake whitefish now primarily consume dreissenid Examination of lake whitefish swimbladders as part of this study mussels, clams and snails (Pothoven and Madenjian, 2008). Con- showed that C. farionis induces pathological effects on swimbladders versely, in Lake Huron, there are still areas where Diporeia spp. are that are commensurate with the degree of infection. With increased still present, and lake whitefish in Lake Huron have been found to infection intensity, swimbladder walls become thickened and lose still consume amphipods, including Diporeia spp. (Nalepa et al., 2007, their transparency, which will affect swimbladder vital functions, 2009a). Amphipods are known intermediate hosts for C. farionis, including gas exchange. Similar signs were described by Snoj et al. which become infected with the nematode by consuming the feces of (1986) in brown trout (Salmo trutta) heavily infected with C. farionis. infected fish containing C. farionis eggs (Smith and Lankester, 1979; Most of the histopathological changes in lake whitefish swimbladders Lankester and Smith, 1980). noticed in this study were in the form of focal to multifocal inflam- The seasonal, often dramatic, fluctuations in C. farionis infection matory cell infiltration, loss of mucosal folding, as well as mucosal parameters in lake whitefish within the study area are rather erosion. These lesions are probably due to mechanical irritation by the puzzling. While seasonal fluctuation in L3 abundance can be related highly mobile nematodes or the lytic enzymes produced by C. farionis to increased abundance of intermediate hosts during the spring and (Zółtowska et al., 2001). Similar effects were described by Willers summer months (Dextrase, 1987; Watson and Dick, 1979; Giæver et al. (1991) in ciscoes infected with C. farionis. et al., 1991; Knudsen et al., 2002), the dramatic fluctuations in the No studies have been performed to determine if the C. farionis- numbers of mature nematodes observed in this study are difficult to induced pathological change can negatively impact its final host at the explain. The experimental study of Black and Lankester (1980), who population level. Extensive studies have been conducted, however, on infected rainbow trout (Oncorhynchus mykiss) with L3 nematodes another swimbladder nematode Anguillicola crassus that infects the extracted from infected lake whitefish, is the only recorded account European eel (Anguilla anguilla). A. crassus caused swimbladder wall for the development of Cystidicola spp. within their final host. Black thickening that affected the ability of eels to migrate to the Sargasso and Lankester (1980) determined that L3 stages undergo two Sea where they develop and mature, and therefore may be con- moltings and reach sexual maturation in the swimbladder within 4 tributing to the worldwide decline of the Eurpoean eel (Kirk, 2003; to 7 months post-infection. Once matured, the nematodes live within Kennedy, 2007). Using advanced radiolabeling methods, Szekely et al. their final host until the host's death (Black and Lankester, 1980, (2005) followed A. crassus infection in captive eels and demonstrated 1981). Although the size of the pneumatic duct in swimbladders of that the swimbladder nematode can cause dramatic deterioration in lake whitefish permits the passage of egg and L3 stage nematodes swimbladder condition of infected eels. (Genten et al., 2009), it is too small to permit passage of adult Our finding of a weak, yet statistically significant, positive rela- nematodes, which frequently were as large as 30 mm in length and tionship between condition factor and C. farionis abundance and 0.2 to 0.5 mm in width. Consequently, we expected to find steady or intensity is likely attributable to several factors that may be increasing increasing levels of infection in lake whitefish overtime like those the weight of infected fish, such as the weight of the nematodes, the observed in Europe in Arctic char (Salvelinus alpinus)(Amundsen accumulation of fluid in the swimbladder of infected fish, or an et al., 2003; Knudsen et al., 2004), and broad whitefish (Coregonus increase in the water content of fish as a result of impaired swim- nasus)(Valtonen and Valtonen, 1978). On the contrary, we observed a bladder functions. In a parallel study to this one, Wagner et al. (2010) dramatic fluctuation in the prevalence, intensity, and abundance of found variations in percent water content among stocks that C. farionis, with abundance of mature nematodes often fluctuating positively correlated with C. farionis intensity of infection, with Lake from high to low levels within a few months, which does not coincide Michigan stocks (BBN and NAB) having lower percent water content with C. farionis development as presented by Black and Lankester and lower C. farionis intensity of infection, and Lake Huron stocks (1980). (CHB and DET) having higher percent water content and higher One explanation for the seasonal difference in C. farionis that we C. farionis intensity of infection. observed is that the C. farionis strain infecting lake whitefish has a In conclusion, this study sheds light on the spread and potential shorter life span than other strains affecting other fish species. Since negative impacts of swimbladder nematodes in lake whitefish stocks the fluctuations, whether increasing or decreasing, involved both in lakes Huron and Michigan, including a range expansion of C. farionis larval and adult nematodes and no dead or lysed nematodes were to four new geographical locations. Whether C. farionis will have a observed in swimbladders of infected lake whitefish, we believe this long term negative impact on lake whitefish stocks in the Great Lakes M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28 27 basin remains unclear, but we emphasize the need for continued Ebener, M.P., Brenden, T.O., Wright, G.M., Jones, M.L., Faisal, M., 2010. Spatial and temporal distributions of lake whitefish spawning stocks in northern lakes monitoring and analysis of C. farionis infection throughout the lakes Michigan and Huron, 2003-2008. J. Great Lakes Res. 36 (Supplement 1), 38–51. and additional studies to determine how infection intensity may affect Ebener, M.P., Kinnunen, R.E., Mohr, L.C., Schneeberger, P.J., Hoyle, J.A., Peeters, P., 2008. stock health. Management of commercial fisheries for lake whitefish in the Laurentian Great Lakes of North America. In: Schechter, M.G., Taylor, W.W., Leonard, N.J. (Eds.), International RANGE EXPANSION REPORT OF CYSTIDICOLA FARIONIS FISHER governance of fisheries ecosystems: learning from the past, finding solutions for the 1798 future. American Fisheries Society Symposium, Bethesda, Maryland, pp. 99–143. 62. Synonyms: Ancynarcanthus cystidicola Schneider 1866; C. canadensis Fleischer, G.W., 1992. Status of coregonine fishes in the Laurentian Great Lakes. In fi Skinker 1930. Biology and management of coregonid shes. In: Todd, T.N., Luczysnki, M. (Eds.), Proceedings of the 4th international symposium on the biology and management Site of infection: swimbladder of coregonid fishes. Quebec City, Quebec, Canada, August 19–23, 1990. Pol. Arch. New Location based on the present study: Lake Michigan (Big Bay de Hydrobiol. 39, pp. 247–259. fi Noc and Naubinway), and Lake Huron (Cheboygan and Detour Genten, F., Terwinghe, E., Danguy, A., 2009. Atlas of sh histology. Université Libre de Bruxelles, Brussels. village). Giæver, A., Klemetsen, A., Halvorsen, A., 1991. Infection of Cystidicola farionis Fischer Other reported hosts: Coregonus spp., Salvelinus spp., Oncorhynchus (Nematoda: Spiruroidea) in the swimbladder of Arctic charr, Salvelinus alpinus (L.), spp., Salmo spp., Osmerus mordax, Prosopium cylindraceum, P. williamsoni, from Takvatn, North Norway. Nord. J. Freshw. Res. 66, 63–71. Hoffman, G., 1999. Parasites of North American freshwater fishes, 2nd edition. Cornell Thymallus articus. University Press, Ithaca, New York. Other reported localities: Other sites in Lake Huron, Lake Ontario Hoyle, J.A., 2005. Status of lake whitefish (Coregonus clupeaformis) in Lake Ontario and (including inland lakes in its watershed), Lake Superior, and British the response to the disappearance of Diporeia spp. In: Mohr, L.C., Nalepa, T.F. (Eds.), Proceedings of a workshop on the dynamics of lake whitefish (Coregonus Colombia (Dextrase, 1987; Miscampbell et al., 2004; Muzzal and clupeaformis) and the amphipod Diporeia spp. in the Great Lakes, pp. 47–66. Great Peebles, 1986); Norway (Knudsen et al., 2002); Green Lake and Lakes Fish. Comm. Tech. Rep. 66. Sparkling Lake, Wisconsin (Willers et al., 1991); Takvatn, north Hoyle, J.A., Casselman, J.M., Dermott, R., Schaner, T., 1999. Changes in lake whitefish (Coregonus clupeaformis) stocks in eastern Lake Ontario following Dreissena mussel Norway (Giæver et al., 1991); and Bothnian Bay (Valtonen and invasion. Great Lakes Res. Rev. 4, 5–10. Valtonen, 1978). Kennedy, C.R., 2007. The pathogenic helminth parasites of eels. J. Fish. Dis. 30, 319–334. Specimen deposited: The Department of Zoology Collection, Michigan Kirk, R.S., 2003. The impact of Anguillicola crassus on European eels. Fish. Manage. Ecol. – State University, accession #: MSUIZ 1361. 10, 385 394. Knudsen, R., Amundsen, P., Klemetsen, A., 2002. Parasite-induced host mortality: indirect evidence from a long-term study. Environ Biol Fish 64, 257–265. Knudsen, R., Curtis, M.A., Kristoffersen, R., 2004. Aggregation of Helminths: the role of Acknowledgments feeding behavior of fish hosts. J. Parasitol. 90, 1–7. Ko, R.C., Anderson, R.C., 1969. A revision of the genus Cystidicola Fischer, 1798 (Nematoda: Spiruroidea) of swimbladder of fishes. J. Fish. Res. Board Can. 26, 849–864. The authors would like to thank current and past members Lankester, M.W., Smith, J.D., 1980. Host specificity and distribution of the swim-bladder of Michigan State University-Aquatic Animal Health Laboratory nematodes, Cystidicola farionis Fischer, 1798 and C. cristivomeri White, 1941 (MSU-AAHL) who assisted in this multi-year effort. Funding was (Habronematoidea), in salmonid fishes of Ontario. Can. J. Zool. 58, 1298–1305. provided by the Great Lakes Fishery Trust Project 2003–06. This is McNickle, G.G., Rennie, M.D., Sprules, W.G., 2006. Changes in benthic invertebrate communities of South Bay, Lake Huron following invasion by zebra mussels manuscript 42 of the MSU-AAHL and 2010-03 of the MSU-Quan- (Dreissena polymorpha), and potential effects on lake whitefish (Coregonus titative Fisheries Center. clupeaformis) diet and growth. J. Great Lakes Res. 32, 180–193. Mills, E.L., Casselman, J.M., Dermott, R., Fitzsimons, J.D., Gal, G., Holeck, K.T., Hoyle, J.A., Johannsson, O.E., Lantry, B.F., Makarewicz, J.C., Millard, E.S., Munawar, I.F., Munawar, M., O'Gorman, R., Owens, R.W., Rudstram, L.G., Schaner, T., Stewart, T.J., Appendix A. Supplementary data 2005. A synthesis of ecological and fish-community changes in Lake Ontario, 1970– 2000. Great Lakes Fish. Comm. Tech. Rep. 67. Supplementary data associated with this article can be found, in Miscampbell, A.E., Lankester, M.W., Adamson, M.L., 2004. Molecular and morphological variation within swimbladder nematodes, Cystidicola spp. Can. J. Fish. Aquat. Sci. the online version, at doi:10.1016/j.jglr.2010.01.008. 61, 1143–1152. Mohr, L.C., Ebener, M.P., 2005. Status of lake whitefish (Coregonus clupeaformis) in Lake Huron. In: Mohr, L.C., Nalepa, T.F. (Eds.), Proceedings of a workshop on the dynamics References of lake whitefish (Coregonus clupeaformis) and the amphipod Diporeia spp. in the Great Lakes, pp. 105–126. Great Lakes Fish. Comm. Tech. Rep. 66. Agresti, A., 2007. An introduction to categorical data analysis, 2nd edition. John Wiley & Muzzal, P.M., Peebles, C.R., 1986. Helminths of pink salmon, Oncorhynchus gorbushcha, Sons, Inc., Hoboken, New Jersey. from five tributaries of Lake Superior and Lake Huron. Can. J. Zool. 64, 508–511. Amin, O.M., 1977. Helminth parasites of some Southwestern Lake Michigan fishes. Proc. Nalepa, T.F., Fanslow, D.L., Messick, G.A., 2005. Characteristics and potential causes of Helminthol. Soc. Wash. 44, 210–217. declining Diporeia spp. populations in southern Lake Michigan and Saginaw Bay, Amundsen, P.A., Knudsen, R., Kuris, A.M., Kristoffersen, R., 2003. Seasonal and Lake Huron. In: Mohr, L.C., Nalepa, T.F. (Eds.), Proceedings of a workshop on the ontogenetic dynamics in trophic transmission of parasites. Oikos 102, 285–293. dynamics of lake whitefish (Coregonus clupeaformis) and the amphipod Diporeia spp. Anderson, R.O., Neumann, R.M., 1996. Length, weight, and associated structural indices, in the Great Lakes, pp. 157–188. Great Lakes Fish. Comm. Tech. Rep. 66. In: Murphy, B.R., Willis, D.W. (Eds.), Fisheries techniques, 2nd edition. American Nalepa, T.F., Fanslow, D.L., Pothoven, S.A., Foley III, A.J., Lang, G.A., 2007. Long-term Fisheries Society, Bethesda, Maryland, pp. 353–383. trends in benthic macroinvertebrate populations in Lake Huron over the past four Black, G.A., 1983. Taxonomy of a swimbladder nematode, Cystidicola stigmatura (Leidy), decades. J. Great Lakes Res. 33, 421–436. and evidence of its decline in the Great Lakes. Can. J. Fish. Aquat. Sci. 40, 643–647. Nalepa, T.F., Fanslow, D.L., Lang, G.A., 2009a. Transformation of the offshore benthic Black, G.A., 1984. Swimbladder lesions associated with mature Cystidicola stigmatura community in Lake Michigan: recent shift from the native amphipod Diporeia spp. (Nematoda). J. Parasitol. 70, 441–443. to the invasive mussel Dreissena rostriformus bugensis. Freshw. Biol. 54, 466–479. Black, G.A., Lankester, M.W., 1980. Migration and development of swimbladder Nalepa, T.F., Pothoven, S.A., Fanslow, D.L., 2009b. Recent changes in benthic macro- nematodes, Cystidicola spp. (Habronematoidea), in their definitive hosts. Can. J. invertebrate populations in Lake Huron and impact on the diet of lake whitefish Zool. 58, 1997–2005. (Coregonus clupeaformis). Aquat. Ecosyst. Health Manag. 12:2–10.Pothoven, S.A. 2005. Black, G.A., Lankester, M.W., 1981. The transmission, life span, and population biology Changes in lake whitefish diet in Lake Michigan, 1998–2001, eds. L.C. Mohr and T.F. of Cystidicola cristivomeri White, 1941 (Nematoda: Habronematoidea) in char, Nalepa. In Proceedings of a workshop on the dynamics of lake whitefish (Coregonus Salvelinus spp. Can. J. Zool. 59, 498–509. clupeaformis) and the amphipod Diporeia spp. in the Great Lakes, eds. L.C. Mohr, and T.F. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on Nalepa, pp. 127–140. Great Lakes Fish. Comm. Tech. Rep. 66. its own terms: Margolis et al. revisited. J. Parasitol. 83, 575–583. Pothoven, S.A., Madenjian, C.P., 2008. Changes in consumption by alewives and lake Dextrase, A.J., 1987. The biology of Cystidicola farionis Fischer 1798 (Nematoda: whitefish after dreissenid mussel invasions in lakes Michigan and Huron. N. Am. J. Cystidicolidae) in salmonid fishes, M.S. thesis, Lakehead University, Thunder Bay, Fish. Manage. 28, 308–320. Ontario, Canada. Pothoven, S.A., Nalepa, T.F., Schneeberge, P.J., Brandt, S.B., 2001. Changes in diet and Dzeikonska–Rynko, J., Rokicki, J., Jablonowski, Z., 2003. The activity of selected body of lake whitefish in southern Lake Michigan associated with changes in hydrolases in excretion–secretion products and extracts from larvae and mature benthos. N. Am. J. Fish. Manage. 21, 876–883. specimens of Cystidicola farionis. Ocean. Hydrob. Studies 32, 117–129. Prophet, E., Mills, B., Arrington, J., 1992. Laboratory methods in histotechnology. Armed Ebener, M.P., Copes, F.A., 1985. Population statistics, yield estimates, and management Forces Institute of Pathology, Washington, DC. considerations for two lake whitefish stocks in Lake Michigan. N. Am. J. Fish. Smith, J.D., Lankester, M.W., 1979. Development of swimbladder nematodes (Cystidi- Manage. 5, 435–448. cola spp.) in their intermediate hosts. Can. J. Zool. 57, 1736–1744. 28 M. Faisal et al. / Journal of Great Lakes Research 36 (2010) 18–28

Snoj, N., Jencic, V., Brglez, J., Vidmar, M., 1986. Cystidiculosis in two-year-old brown clupeaformis) health indicators: linking individual-based indicators to a manage- trout in nursery streams. Zbornik Biotehniska Fakulteta Univerze Edvarda Kardelja ment-relevant endpoint. J. Great Lakes Res. 36 (Supplement 1), 121–134. v Ljubljani, Veterinarstvo 23, 263–268. Watson, R.A., Dick, T.A., 1979. Metazoan parasites of whitefish, Coregonus clupeaformis, Szekely, C., Molnar, K., Racz, O.Z., 2005. Radiodiagnostic method for studying the Mitchill and cisco, C. artedii LeSueur from Southern Indian lake, Manitoba. J. Fish. dynamics of Anguillicola crassus (Nematoda: Dracunculoidea) infection and Biol. 15, 579–587. pathological status of the swimbladder in Lake Balaton eels. Dis. Aquat. Org. 64, Willers, W.B., Dubielzig, R.R., Miller, L., 1991. Histopathology of the swimbladder of the 53–61. cisco due to the presence of the nematode Cystidicola farionis Fisher. J. Aquat. Anim. Valtonen, E.T., Valtonen, T., 1978. Cystidicola farionis as a swimbladder parasite of the Health 3, 130–133. whitefish in the Bothnian Bay. J. Fish. Biol. 13, 557–561. Zółtowska, K., Lopieńska, E., Rokicki, J., Dmitryjuk, M., 2001. The enzymes of Wagner, T., Jones, M.L., Ebener, M.P., Arts, M.T., Brenden, T.O., Honeyfield, D.C., Wright, carbohydrates metabolism from Cystidicola farionis (Cystidicolidae). Wiad. Para- G.M., Faisal, M., 2010. Spatial and temporal dynamics of lake whitefish (Coregonus zytol. 47, 311–315.