Ecological Indicators 13 (2012) 184–188 Contents lists available at ScienceDirect Ecological Indicators journal homepage: www.elsevier.com/locate/ecolind The use of parasites as indicators of ecosystem health as compared to insects in freshwater lakes of the Inland Northwest John Shea ∗ , Gordon J. Kersten, Chris M. Puccia, Andy T. Stanton, Suzi N. Stiso, Erika S. Helgeson, Emily J. Back Biology Department, Gonzaga University, 502 East Boone Ave., Spokane, WA 99258, USA a r t i c l e i n f o Article history: Received 2 March 2010 Received in revised form 24 May 2011 Accepted 10 June 2011 Keywords: Parasites Trematodes Heavy metals Grazing Biodiversity Indicators a b s t r a c t Trematode parasites have complex life cycles, requiring multiple hosts. If these parasites are present in an ecosystem, then one can infer that their respective hosts must also be present. Thus, these parasites may serve as reliable indicators of species diversity in an ecosystem. To test this, we sampled larval trematodes and aquatic insects from three freshwater lakes in Idaho that varied in heavy metal pollution and three lakes in Washington that varied in agricultural use. We hypothesized that if parasites do serve as reliable indicators of ecosystem health, then parasite diversity should be higher at the least disturbed sites and should positively correlate with insect diversity. We found that the Shannon diversity index for parasites was highest at the WA reference lake (1.36) compared to the two WA lakes (0.54 and 0) exposed to cattle grazing. The ID lake with the highest levels of heavy metals experienced the highest insect diversity, but a low trematode diversity. We conclude that parasite diversity indices work as well as insect diversity indices as indicators of ecosystem health, especially in instances of heavy metal pollution. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Parasites with complex life cycles can be ideal indicators of species diversity because they require the presence of both host and parasite in the same time and space (Marcogliese and Cone, 1997; Hudson et al., 2006; Lafferty, 1997; Marcogliese, 2005). For example, the trematode flatworm parasite life cycle usually entails a mollusc host (typically a snail), a second intermediate host (typically an invertebrate or fish), and a vertebrate definitive host (Roberts and Janovy, 2009). If any of these hosts are absent, then the parasite, unable to complete its life cycle, will die. Previous work found that larval trematodes work well as indicators of ecosystem health in estuaries and marine systems (Hechinger et al., 2007; Huspeni and Lafferty, 2004; Hechinger and Lafferty, 2005; Lafferty et al., 2008). Other studies have examined trematode diversity in freshwater systems (Poulin, 1992; Keas and Blankespoor, 1997; Valtonen et al., 2003), especially in regards to heavy metal pollution (Schludermann et al., 2003) and agricultural disturbance (Hernandez et al., 2007; King et al., 2007). Sures et al. (1998) showed that heavy metals accumulate in the trematode Fasciola hepatica thus removing these pollutants from the ecosystem. Since heavy metals impact larval trematodes ∗ Corresponding author. Tel.: +1 509 313 5594; fax: +1 509 313 5804. E-mail address: shea@gonzaga.edu (J. Shea). 1470-160X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecolind.2011.06.001 whose free-swimming stages directly contact water (Griggs and Belden, 2008), their abundance should reflect water quality as suggested by Lefcort et al. (2002) who found lower larval trematode diversity at sites contaminated with heavy metals than at reference sites. Experimental studies suggest that frogs exposed to agricultural pesticides experienced increased infection by trematodes (Kiesecker, 2002) and enhanced infection by lungworms (Gendron et al., 2003). Further, a comparison of frog parasites between forest and clear-cut cattle pastures showed higher abundances of three trematode species from pastures and higher abundances of two trematode species from the forest (McKenzie, 2007). Increased eutrophication also increases trematode infection in frogs, likely because such eutrophication promotes algal growth, which increases the density of the parasite’s herbivorous snail host (Johnson and Chase, 2004; Johnson et al., 2007). Although previous studies used insect diversity to assess freshwater systems (Lenat, 1984; Heatherly et al., 2007; Tomkiewicz and Dunson, 1977; Roy et al., 2003), larval trematode diversity should respond negatively to human impacted freshwater lakes and does so, at least in the instance of acidification (Halmetoja et al., 2000). This study investigates how trematode diversity compares to insect diversity in freshwater lakes that vary either in heavy metal pollution or in agricultural use. We hypothesize that if parasites serve as reliable indicators of ecosystem health, then parasite diversity will (1) be highest at the reference lakes and (2) positively correlate with insect diversity in both pollution regimes. J. Shea et al. / Ecological Indicators 13 (2012) 184–188 Fig. 1. Map of study sites. Idaho consisted of two impacted lakes, ID1 (47◦ 32′ N, 116◦ 27′ W, elevation 655 m) and ID2 (47◦ 32′ N, 116◦ 28′ W, elevation 654 m), and a reference lake, IDR (47◦ 21′ N, 116◦ 41′ W, elevation 651 m). Washington consisted of two impacted lakes, WA1 (47◦ 28′ N, 117◦ 31′ W, elevation 710 m) and WA2 (47◦ 26′ N, 117◦ 30′ N, elevation 704 m) and a reference lake, WAR (47◦ 23′ N, 117◦ 34′ W, elevation 696 m). 2. Materials and methods 2.1. Study areas We sampled aquatic insects and larval trematodes at three sites in Idaho and three sites in Washington in early June of 2009 (Fig. 1). The Idaho reference site was a St. Joe River-fed lake while the other two sites were lakes fed by the Coeur d’Alene River, which is contaminated with heavy metals (Rabe and Bauer, 1977; Sprenke et al., 2000; Lefcort et al., 2004). The Washington reference site was a pothole lake located in Turnbull National Wildlife Refuge while the other two sites were pothole lakes immediately adjacent to the refuge and so experienced agricultural disturbance in the form of grazing. 185 snails. We dissected all snails, identified trematodes to family or, if possible, to species (Schell, 1970, 1985; Pratt and McCauley, 1961). Trematodes that could not be identified (usually because they were immature) were excluded from the diversity (but not prevalence) analysis. For each site, we described the trematode community by calculating species richness, evenness, and the summed prevalence of all trematode species (100×), which accounts for the fact that some snails were infected with two trematode species, especially in high prevalence sites. Thus, the summed prevalence better describes levels of trematode recruitment than does simple prevalence of infected snails. We used these numbers to calculate Shannon diversity indices (H = −pi ln(pi )) for each site. Because of snail mortality, we failed to dissect 100 snails at each site and so corrected for this in the Shannon index calculation. Because of the relatively low infection prevalence found in these snails, we expect low competitive trematode loss and so did not calculate “pre-interactive” prevalence as done in other trematode systems (Hechinger et al., 2007; Lafferty et al., 1994; Kuris and Lafferty, 1994). We calculated a 95% confidence interval for each site to determine if the summed prevalences at two sites were statistically different (Zar, 1999). We identified insects to their lowest possible taxonomic category (Merritt et al., 2008). Dr. Bruce Lang, a local invertebrate expert, confirmed both trematode and insect identifications. For each site, we calculated insect species richness, evenness, abundance, and Shannon indices. 3. Results 3.1. Collection totals We dissected 507 snails and encountered 130 individual trematode infections belonging to eight species (Table 1). In addition, we collected 541 snails to measure total snail density at each lake (Table 2). We collected 771 aquatic insects representing six different orders (Coleoptera, Odonata, Diptera, Hemiptera, Ephemeroptera and Trichoptera). 2.2. Sampling protocol 3.2. Chemical tests Before sampling organisms, we collected water samples to measure biochemical oxygen demand (BOD), conductivity, turbidity, dissolved oxygen (DO), total organic carbon (TOC), pH, hardness, soluble reactive phosphorus (SRP), nitrates, nitrites, chlorides, sulfate and calcium at all six sites. In Idaho, we measured Cd, Pb, Zn and Cu in both the water and substrate. Anatek Labs analyzed water and substrate samples within 5 h of collection. After establishing a 3 × 10 m transect at a random location along the lake’s shore, three researchers collected snails of all species in the transect for 15 min to measure total snail density. Outside the transect, we controlled for snail size and genus by collecting 100 Physa snails (9–14.9 mm in length) from each Idaho lake and 100 Stagnicola snails (19–24.9 mm in length) from each Washington lake (sample size suggested in Huspeni et al., 2005). Because these snail genera were identified by sight, we may have included more than one host species of snail in our analysis. Additionally, three researchers sampled insects for 15 min at each site within the original 3 × 10 m transect, which was allowed to equilibrate for at least 20 min after the initial snail sampling. Both snails and insects were collected using a 27 cm double mesh kitchen strainer (Browne-Halco) with a 2 mm mesh screen. 2.3. Identification and calculations In the lab, we placed each snail in a vial, which was set aside for at least 48 h to allow the cercarial stage to mature and exit the We failed to detect nitrates or nitrites at any of the WA or ID lakes and detected only negligible amounts of SRP at WA1. Heavy metal levels in the water were negligible. Heavy metal levels in the substrate were highest at ID2 and lowest at IDR (Table 3). No unusual patterns were detected among the other chemical tests (Table 4). 3.3. Trematode prevalences In Washington, snails from the reference lake (WAR) experienced the highest trematode summed prevalence at 17% (10.3–25.5%; 95% C.I. in parenthesis) and so differed from the summed prevalence in WA2 at 1.7% (0–8.9%) but not from WA1 at 6.5% (2.4–13.5%). In Idaho, snails from the reference lake (IDR) experienced the highest trematode summed prevalence at 15.6% (7.8–26.9%) and so differed from the summed prevalence in ID1 at 64% (53.6–73.5%), but not from ID2 at 35.6% (25.6–46.1%). 3.4. Shannon diversity indices The Shannon diversity index for trematodes was highest at the reference lakes in both WA (1.36) and ID (1.95, adjusted for sample size) and lowest at WA2 and ID1 (Table 5). The Shannon diversity index for insects was highest at WA2 in WA and ID2 in ID, but lowest at the reference lake in ID (Table 6). 186 J. Shea et al. / Ecological Indicators 13 (2012) 184–188 Table 1 Trematode species sampled from first intermediate host Stagnicola (from WA) or Physa (from ID) snails, their total abundances, their second and definitive host use. Species Total abundance 2nd intermediate host Definitive host Trematodes from Stagnicola snails (WA) Plagiorchis muris Notocotylidae Cotylurus flabelliformis Plagiorchis proximus Ochetosomatidae Immature 1 1 2 2 10 8 Snails, insects, fish Vegetation Snails Insect larvaee Tadpoles NA Cat,a mice,b bat, dogc Mink,d duck, swanc Ducke Muskratf , minkd Snakeg NA Trematodes from Physa snails (ID) Trichobilharzia physellae Echinostoma revolutum Notocotylidae Cotylurus flabelliformis Cephalogonimus salamandrus Immature 2 42 46 4 2 10 None Snails, clams, tadpoles Vegetation Snails Amphibians NA Duckd Mink, duck, swand Mink,d duck, swanc Ducke Amphibiansh NA a b c d e f g h Sohn and Chai (2005). Chai et al. (2007). Yamaguti (1958). Pratt and McCauley (1961). Ulmer (1957). McMullen (1937). Schell (1985). Dronen and Lang (1974). Table 2 Total snails of all species and host snails (Stagnicola or Physa) collected at each site in 15 min by three researchers in a 3 × 10 m transect adjacent to the lakeshore and the respective densities of the host snails. Site WAR WA1 WA2 IDR ID1 ID2 Total snails 303 22 52 21 86 57 Host density (number/m2 ) Host snails 191 19 45 6 85 49 6.37 0.63 1.5 0.2 2.83 1.63 Table 3 Heavy metal concentrations measured from the substrate at the Idaho lakes (mg/kg). Site Cd Cu Pb Zn IDR ID1 ID2 ND 1.07 16.2 8.11 13.7 118 2.90 237 3900 22.6 93.3 2130 Table 4 Select chemical test results for each site in Washington (WA) and Idaho (ID). Site BOD (mg/L) DO (mg/L) TOC (mg/L) pH Conductivity (/cm) WAR WA1 WA2 IDR ID1 ID2 7.65 17 5.19 <5.61 <2.46 <4.23 9.92 2.44 7.19 2.63 4.05 4.08 18.7 19.4 25.5 11.56 7.94 9.82 7.28 6.78 7.05 6.75 6.6 6.73 307 258 336 87.5 161 39.8 Table 5 Calculated trematode species richness, evenness, summed prevalence of all trematode species (100×) and Shannon index for each site. Site Prevalence Shannon index Agriculturally disturbed sites (WA) WAR 5 0.84 2 0.72 WA1 WA2 1 NA 17/100 (0.17) 6/93 (0.06) 1/60 (0.02) 1.36 0.54a 0.0a Heavy metal contaminated sites (ID) 4 0.91 IDR ID1 4 0.64 4 0.66 ID2 10/64 (0.16) 64/100 (0.64) 32/90 (0.36) 1.95a 0.88 1.02a a Richness Evenness Sample size corrections for the Shannon indices. Table 6 Calculated insect species richness, evenness, abundance and Shannon index for each site. Site Abundance Shannon Index Agriculturally disturbed sites (WA) WAR 23 0.67 14 0.65 WA1 18 0.77 WA2 Richness Evenness 156 174 93 2.09 1.72 2.22 Heavy metal contaminated sites (ID) IDR 6 0.46 6 0.61 ID1 0.75 ID2 13 63 108 174 0.83 1.10 1.93 4. Discussion and conclusions The Shannon diversity index for larval trematodes was highest at the reference lakes in both Washington and Idaho, which were impacted by agricultural disturbance and heavy metal pollution respectively (Table 5). Thus, we obtained consistent results for two types of disturbances: heavy metal contamination and agricultural use. Further, we obtained these consistent results using two different host snail species (Physa and Stagnicola). Since most trematodes are trophically transmitted to their final hosts, they serve as a direct indication of functioning food webs (Marcogliese and Cone, 1997; Hechinger et al., 2007). Examination of host use reveals the diversity of animals that must be (or have been) present at the various sites (Table 1). Larval trematodes usually require a mollusc first intermediate host and at least three species in the system examined here also use snails as a second intermediate host. The presence of Cotylurus flabelliformis at both sites suggests a functioning trophic link between snails and ducks. One can also infer the presence of small mammals such as mice, muskrats and cats from the presence of Plagiorchis spp., Notocotylidae and Echinostoma revolutum. Interestingly, the latter trematode was found only in Idaho, but we did collect E. revolutum from WAR later in the summer (unpublished data). Larval trematode diversity failed to directly correlate with aquatic insect diversity in both Washington and Idaho (Tables 5 and 6). In Washington, the highest trematode prevalence and diversity occurred at the reference site (WAR). However, the highest insect abundance occurred at WA1 while the highest insect diversity occurred at WA2, which experienced the lowest trematode diversity. Without a sense of variance, we cannot J. Shea et al. / Ecological Indicators 13 (2012) 184–188 conclude the significance of these differences. In Idaho, the reference site experienced the lowest trematode prevalence, but the highest trematode diversity. In contrast, both insect abundance and diversity was highest at ID2, which had the highest levels of heavy metals in its substrate. These findings suggest that insect taxa, unlike trematode taxa, do not exhibit a differential response to heavy metal pollution, supporting Chadwick and Canton (1984) who found insect diversity inadequate for assessing effects of heavy metal contamination. They describe heavy metal contamination as non-selective stress, which does not target any particular taxon. If correct, then ID2 may have had the highest insect diversity of the three ID sites before it was contaminated with heavy metals. Heavy metal contamination did not selectively eliminate particular insect taxa, but may have done so with larval trematodes. This suggests that larval trematode diversity may outperform insect diversity as a means to assess the health of ecosystems, particularly those impacted by heavy metals. Indeed, larval trematodes seem particularly sensitive to heavy metal pollution as they exhibited lower diversity in heavy metal contaminated lakes (Lefcort et al., 2002). Parasites are used in a variety of other ways to assess environmental pollution, including heavy metals (Sures, 2004). We failed to find any chemical evidence of agricultural disturbance at the WA sites. We know from the landowners that grazing did occur at the two sites in the past, but no grazing occurred during our sampling time. If grazing led to eutrophication in the WA sites, then it may have facilitated the growth of the snail host (Johnson and Chase, 2004; McKenzie, 2007) and so should have resulted in increased trematode prevalence. But the data do not support this. Grazing does impact vegetation, especially seasonally (Lucas et al., 2004) and at least one parasite (Notocotylidae) typically uses vegetation as its second intermediate host. Since it was only found in Washington at the reference lake, over-grazing of aquatic plants along the shore could negatively impact this parasite. Again, the data suggest that larval trematodes can perform as well as aquatic insects as bio-indicators. In this study, snail host density (Table 2) correlated with trematode prevalence in Idaho and trematode prevalence was highest at WAR, which had the highest host snail density. Although these results confirm the logical relationship between trematode infection prevalence and snail density, larger sample sizes at WA2 and IDR may have resulted in different measured trematode prevalences and suggests a limitation to using larval trematodes as an ecosystem indicator. Namely, that snails in sufficient densities must be present at all sites being compared. However, the fact that we obtained similar results using two different host species of snails indicates that this method is robust. Human disturbance in both Washington and Idaho may explain the difference in trematode diversity between the impacted and reference lakes. However, access to a limited number of lakes did not allow for replication so we cannot statistically determine the significance of the observed differences. Additional data on fish and bird diversities at all six sites would shed additional light on the question. Future studies should include these considerations as well as examining other anthropogenic stressors on freshwater systems such as coliform levels, urbanization, persistent organic pollutants and eutrophication. As human activity continues to threaten natural ecosystems, the need for a cheap, quick and reliable method to assess ecosystem health increases, especially in countries that can least afford to allocate limited monetary and human resources to protect their environmental resources. Acknowledgements We thank Dr. Bruce Lang, Dr. Hugh Lefcort, Christy Watson, Mike Rule and the Turnbull Wildlife Refuge, the Gonzaga Student 187 Research Program and the Gonzaga University Jesuits. We also thank Paul Steenman for his help with constructing the map. This study received funding from the Murdock Charitable Trust’s College Research Program for Life Sciences. References Chadwick, J.W., Canton, S.P., 1984. 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