Southeastern Naturalist 155 M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 22001188 SOUTHEASTERN NATURALIST 1V7o(1l.) :1175,5 N–1o6. 51 Diet and Nematode Infection Differ Between Coastal and Inland Populations of Green Treefrogs (Hyla cinerea) Molly A. Albecker1,*, William B. Brantley Jr.1, and Michael W. McCoy1 Abstract - Progressive salinization of freshwater wetlands is likely to trigger significant changes in associated animal communities. Understanding how salinization affects fundamental natural history characteristics, like diet, is necessary to predict consequences of environmental change. We analyzed dietary patterns of Hyla cinerea (Green Treefrog), a generalist frog species known to inhabit freshwater and brackish wetlands. The stomach contents of coastal (e.g., brackish) and inland (e.g., freshwater) H. cinerea differed in both species variety and abundance of prey items. We also observed nematodes, a common anuran gut parasite, in inland individuals but did not observe any nematodes in coastal individuals. Our study shows differences in resource use and parasite load in H. cinerea, suggesting that wetland salinization may impact trophic dynamics and infectious disease in anuran amphibians. Introduction Little is known about how resource-use and trophic dynamics will be affected by changes in habitat-defining characteristics such as salinity, or how ecological relationships will be impacted as habitats are transformed by climate change. As habitats and prey communities change, generalist consumers may demonstrate concomitant shifts in diet (Mahan and Johnson 2007). However, some more discriminant species may maintain dietary preferences despite the changing prey community (Freed 1980). Understanding diet composition and the factors that affect diet across disparate habitat conditions is especially important given that rapid environmental change is expected over the next century. Secondary salinization, or increasing salt concentrations in freshwater wetlands due to anthropogenic factors, is becoming a serious environmental concern worldwide (Herbert et al. 2015). Wetlands along coastal margins are becoming progressively more saline as sea levels rise, inland tributaries are changed, storm surges are magnified, and shipping canals are increasingly dredged, facilitating saltwater flow upstream (Kaushal et al. 2005, Michener et al. 1997, Moorhead and Brinson 1995, Nicholls and Cazenave 2010, Williams 2013). Saltwater segregates organisms and limits species distributions, so species assemblages within saltwater and freshwater wetlands are often distinct, with few salt-tolerant or euryhaline species occurring across both habitat types. Increasing salt concentrations in coastal freshwater wetlands is therefore expected to induce wholesale shifts in species assemblages as freshwater-adapted organisms 1Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC.*Corresponding author - albeckerm09@students.ecu.edu. Manuscript Editor: Cathryn Greenberg Southeastern Naturalist M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 156 emigrate, or are extirpated, and as salt-tolerant organisms immigrate into habitats that become brackish or saline (Guccione 1995, Hunter et al. 2015, Kennish 2001, Morris et al. 2002, Nicholls and Tol 2006, Parkinson 1994, Williams et al. 2003). Indeed, rising salinities may trigger complex shifts in trophic dynamics, potentially impacting the structure of wetland invertebrate and vertebrate assemblages (James et al. 2003). Amphibians are among the putatively salt-sensitive organisms predominantly associated with freshwater wetlands, with only 2% of species documented in saline habitats (Hopkins and Brodie 2015). Amphibians are prominent members of wetland communities and are implicated as key players in the transfer of energy between aquatic and terrestrial habitats, and between trophic levels (Burton and Likens 1975, Gibbons et al. 2006, McCoy et al. 2009). Hyla cinerea (Schneider) (Green Treefrog) is a generalist consumer that inhabits a variety of different wetland types in the Southeastern United States including ponds, drainage ditches, marshes, bogs, and swamps (Engels 1942, 1952; Freed 1980; Lee 2009; Meyers and Pike 2006; Tuberville et al. 2005), and also is recurrently observed in brackish wetlands along coastal regions within its native range (Albecker and McCoy 2017, Johnson and Christensen 1976, Labanick 1976, Oplinger 1976, Wells 2007). We capitalized on the ability of Green Treefrog to inhabit both freshwater and brackish wetland habitats to investigate how salinity of wetlands affects adult frog diets. We did so by comparing the stomach contents of Green Treefrog populations inhabiting brackish wetlands with those of populations inhabiting freshwater wetlands in North Carolina. Understanding how resource use varies with habitat salinity is important when investigating the natural history of an organism, its habitat requirements, and the trophic dynamics within an ecosystem, as well as how each of these may be affected by environmental change. Methods Study area We made field collections in eastern North Carolina (NC) between May and July of 2014. We collected “inland” Green Treefrog from freshwater wetlands in and around Greenville, NC. Greenville is situated on the coastal plains of North Carolina ~190 km east (inland) of the coastal collection sites. Inland, freshwater wetlands contained a wider diversity of plants including sedges, emergent grasses, and hardwood trees such as Taxodium distichum (L.) Rich (Bald Cypress), Liquidambar styraciflua L. (Sweet Gum), Nyssa aquatica L. (Water Tupelo), and Acer rubrum L. (Red Maple). The salinity of the freshwater habitats never exceeded 1 part per thousand (ppt) and were slightly acidic, with pH varying between 5.0 and 6.5. We collected “coastal” Green Treefrogs from salt marsh habitats dominated by Spartina alterniflora Loisel (Smooth Cordgrass) and Phragmites australis (Cav.) Trin. Ex Steud. (Common Reed), which were the only source of shade and structure available in these locations. Individuals collected from coastal populations were calling on emergent vegetation in brackish water with salinities varying between 3 and 24 ppt and a pH of 7.5–8.8. Southeastern Naturalist 157 M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 Field collections We collected 5–7 male Green Treefrogs from each of 4 discrete, freshwater wetlands near Greenville, NC, and 5–7 males from each of 5 discrete, brackish wetlands along North Carolina’s outer banks (Table 1). We placed each captured frog into a small, moistened container for transport back to the laboratory for processing. In total, we sampled the gut contents via stomach dissections from 25 male Green Treefrogs from 4 coastal populations, and 20 males from 4 discrete inland populations (n = 45 individuals; Table 1). At each collection site, we measured salinity (in parts per thousand (ppt)) and pH using a YSI Professional Plus multiparameter meter (Xylem, Inc., Yellow Springs, OH). Dietary analyses The day after collection, we recorded each frog’s weight and snout–vent length (SVL) and then humanely euthanized individuals via 2% MS-222 overdose (all protocols approved by ECU IACUC #D302) (Gentz 2007). Stomachs and intestines were excised and placed in labeled 2-mL micro centrifuge tubes containing 70% ethanol. We identified stomach contents using a Leica Dissection microscop e (Model MDG41). To characterize diets, we sorted and arranged the contents of each stomach and identified contents within the stomach to taxon omic Order, with the exception of Gastropoda, which could only be identified to Class. We limited our taxonomic discrimination to the level of Order, as the majority of the gut contents were partially or mostly digested, leaving only shells or incomplete, small remnants, thereby making a finer resolution impractical. After identification, we conservatively enumerated prey items by determining the minimum number of prey organisms that could be represented by the fragments present (e.g., 8 ant legs would be recorded as 2 ants). We took photos of all contents using Leica Application Suite X software. Statistical methods We excluded nematodes from our diet analyses because nematodes are gut parasites; although they can be transferred via ingestion, they typically are not considered to be prey for adult frogs (Campião et al. 2015). We collected males from chorusing populations, i.e., during reproductively active periods. Because Table 1. Collection sites and dates of coastal and inland Hyla cinerea (Green Treefrog) used for gut contents analysis. Date Area Specific location Latitude Longitude 28-May-14 Coastal Coastal Studies Institute 35º52'26.14"N 75º39'38.54"W 6-June-14 Inland Bellamy Pond 35º33'28.50"N 77º20'55.85"W 10-June-14 Inland Lowe's Retention Pond 35º35'26.49"N 77º19'09.89"W 11-June-14 Inland Oakwood School 35º37'15.80"N 77º26'45.29"W 14-June-14 Coastal Pea Island Highway 12 Ditches 35º41'33.42"N 75º29'07.43"W 25-June-14 Inland Wheat Field 35º37'28.40"N 77º20'32.80"W 28-June-14 Coastal New Inlet Pond 35º41'11.50"N 75º29'03.92"W 3-July-14 Coastal Bodie Lighthouse 35º49'12.48"N 75º33'45.04"W Southeastern Naturalist M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 158 reproductive activities may affect prey consumption (Hirai and Matsui 2000), we compared the individuals whose stomachs held contents against individuals whose stomachs had no contents to determine any differences in probability of containing prey between coastal and inland populations. For this test, we used a general linear mixed-effects model and a binomial family error distribution (Bates et al. 2015). Presence or absence of stomach contents and location (e.g., coastal or inland) were treated as fixed effects, with population treated as a random effect to account for non-target variation due to differences among sites. For those individuals with items in their stomachs, we tested whether total number of prey items for each individual differed according to habitat salinity using a general linear model with a Poisson error distribution (Bates et al. 2015). In this analysis, prey item abundance and location were considered fixed effects, with specific collecting sites treated as random effects. All analyses were conducted in the R statistical programming environment (2014). To assess differences in the gut contents between coastal and inland Green Treefrogs, we compared both the variety and abundance of different prey items between the groups. To compare variety in dietary content, we standardized the data in a binary presence/absence data frame, while abundance analyses were performed on the abundance of prey items present within individuals. We omitted unidentifiable prey items and individuals with empty stomachs from these analyses. We used permutational multivariate analysis of variance (PERMANOVA) to assess differences in prey variety and abundance between the groups. We further explored PERMANOVA results by conducting similarity percentages analyses (SIMPER) to identify which prey orders were most commonly shared between coastal and inland populations. Each of these analyses utilized the R package “vegan” (Oksanen et al. 2016). Results Inland individuals had an average weight of 4.13 g (± 0.75 g) with an average SVL of 43.39 mm (± 3.03 mm). Coastal individuals weighed an average of 4.25 g (± 1.02 g) with an average SVL of 43.07 mm (± 3.86 mm). We observed differences in the number of stomachs with contents vs. stomachs with no contents between inland and coastal locations (Z = 2.23, P = 0.026). Indeed, 85% (95% confidence interval [C.I.] = 0.53–0.96) of inland frogs contained items in their stomachs compared to 56% (C.I. = 0.37–0.74) of the coastal frogs (Fig. 1). However, for the individuals with items in stomachs, there were no differences in the total number of items in the stomachs of coastal and inland Green Treefrogs (Z = -0.03, P = 0.92). On average, coastal frogs had 1.6 (C.I. = 0.94– 2.50) items in their stomachs, whereas inland frogs had 1.55 (C.I. = 0.48–1.93) items in their stomachs. We identified organisms from a total of 10 different Orders in the stomachs of Green Treefrogs across both habitat types, including nematodes (Table 2). We observed that 7 of 22 inland Green Treefrog individuals contained nematodes, a common anuran gut parasite (Bursey and Brooks 2010). Each infected individual Southeastern Naturalist 159 M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 contained an average of 4 (± 3.31 std. dev.) nematodes. No nematodes were observed in any coastal individuals. We found differences in the variety of prey items between coastal and inland populations (F1 = 2.8, P = 0.03; Fig. 2). SIMPER analysis revealed that the prey items most commonly shared across coastal and inland frogs were Coleoptera (shared average = 28%), Aranae (15%), and Orthoptera (15%), wheras Mesostigmata (3%), Gastropoda (3%), and Odonata (3%) contributed the least to similarities (Table 2). Coastal and inland populations contained different abundances of prey items (F1 = 2.8, P = 0.025; Fig. 3). The prey items that contributed the most to observed similarities between in diet abundance between the 2 locations were Coleoptera (38%), Aranae (13%), and Orthoptera (13%), whereas Odonata (3%), Gastropoda (3%), and Mesostigmata (3%) contributed the least to observed similarities (Table 3). Figure. 1. Mean proportion of individuals whose stomachs contained dietary items across coastal and inland Hyla cinerea (Green Treefrog) populations. Error bars represent 95% confidence intervals. Table 2. Prey items present within the stomachs of coastal and inland Hyla cinerea (Green Treefrog). The presence per coastal individual shows the average presence (in percent) that each prey was observed within coastal and inland populations of Green Treefrogs. Contribution to similarities reflects the results of the SIMPER analysis showing average shared contribution to similarities in diet abundance between coastal and inland populations. Average presence (in percent) in Prey Coastal frogs Inland frogs Contribution to similarities 1. Coleoptera 20% 73% 0.283 2. Aranae 30% 9% 0.147 3. Orthoptera 30% 9% 0.147 4. Hymenoptera 10% 0% 0.048 5. Lepidoptera 10% 0% 0.048 6. Mecoptera 0% 9% 0.042 7. Mesostigmata 10% 0% 0.033 8. Gastropoda 10% 0% 0.033 9. Odonata 0% 9% 0.028 Southeastern Naturalist M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 160 Discussion We examined how the stomach contents of male Green Treefrogs differed according to wetland type (e.g., coastal, brackish wetlands or inland, freshwater Figure. 2. Stacked bar plot showing differences in average prey presence (shown in percent) within stomachs of coastal and inland Hyla cinerea (Green Treefrog; see Table 2 for actual values). Figure. 3. Stacked bar plot demonstrating differences in average prey abundance between coastal and inland Hyla cinerea (Green Treefrog; see Table 3 for actual estimates). Although Nematoda abundance is shown here, it was not included in the statistical analyses on these data. Southeastern Naturalist 161 M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 wetlands). This species is considered a dietary generalist, but studies are mixed about whether or not Hylid consumption patterns are opportunistic and non-preferential, or whether individuals maintain dietary preferences across environments (Freed 1980, Leavitt and Fitzgerald 2009). We found that stomach contents of coastal and inland Green Treefrogs differed in both variety and abundance of prey consumed (Tables 2, 3). These findings provide preliminary indications that as environments change, we may expect shifts in trophic dynamics among generalist anuran species that could ultimately impact wetland invertebrate communities. Our study corroborates an investigation of the diets of invasive Green Treefrogs that had been introduced in the Chihuahuan desert in which the authors reported scorpion remnants in frog stomachs, indicating that Green Treefrogs are capable of exploiting novel prey types in new environments (Leavitt and Fitzgerald 2009). One of the most interesting observations that emerged from this study is the complete absence of nematodes (Order Nematoda) from the stomachs of coastal Green Treefrog populations. Nematodes are well-known gut parasites in frogs, so it is likely that their presence in freshwater Green Treefrogs indicates gut-parasite infection (Campião et al. 2015). Infection occurs when nematodes are incidentally consumed via invertebrate vectors, or via direct contact with substrate. In some severe cases, nematode infection can debilitate or kill individuals (Bursey and Brooks 2010, Johnson et al. 2007, Schotthoefer et al. 2003). The lack of nematode infection in individuals inhabiting brackish water may be due to an inability of nematodes, or their vectors, to withstand the increased osmotic stress of brackish environments, resulting in lower gut-parasite infection rates (Thurston et al. 1994). Other pathogens such as Batrachochytrium dendrobatidis (Longcore, Pessier & D.K. Nichols) (Amphibian Chytrid Fungus) and saprolegnia fungal infections are also lower in anurans exposed to salt, which suggests that saltwater may provide a refuge, protecting hosts from certain parasites and pathogens (Karraker and Ruthig 2009, Stockwell et al. 2015). Table 3. Abundance of prey items present within the stomachs of coastal and inland Hyla cinerea (Green Treefrog). The average number per coastal individual represents the average number of prey items of each Order observed within coastal and inland populations of Green Treefrogs. Contribution to similarities reflects the results of the SIMPER analysis showing average shared contribution to similarities in diet abundance among coastal and inland populations. Average abundance per Prey Coastal individual Inland individual Contribution to similarities 1. Coleoptera 0.5 1.080 0.38 2. Aranae 0.3 0.083 0.13 3. Orthoptera 0.3 0.083 0.13 4. Lepidoptera 0.2 0.000 0.06 5. Hymenoptera 0.1 0.000 0.04 6. Mecoptera 0.0 0.083 0.04 7. Mesostigmata 0.1 0.000 0.03 8. Gastropoda 0.1 0.000 0.03 9. Odonata 0.0 0.083 0.03 Southeastern Naturalist M.A. Albecker, W.B. Brantley Jr., and M.W. McCoy 2018 Vol. 17, No. 1 162 We know relatively little about how consumption patterns vary throughout breeding and non-breeding phases in frogs, and our study provides important insights into what adult, male frogs are consuming during reproductively active months. We found that more coastal frogs had empty stomachs, which may impact breeding behavior, fecundity, or the male’s ability to establish and defend breeding territories and calling perches (Li et al. 2009, Martínez et al. 2004). Although frogs are credited as providing important ecosystem services by controlling pest species like mosquitos (Hocking and Babbitt 2014, Premo and Atmowidjojo 1987), little is known about the degree to which this occurs. We did not find any mosquito species in frog stomachs, despite an abundance of mosquitos in all wetlands sampled (M.A. Albecker, pers. observ.). It is possible that mosquitos were consumed by these individuals, but were digested and unidentifiable at the time of collection. Alternatively, previous research has shown that Green Treefrogs preferentially consume larger, more active prey than mosquitos, which may explain their absence (Freed 1980). A wide variety of factors likely influence dietary patterns in adult frogs observed in this study. For example, there may be differences in prey abundances, prey type, and prey availability between brackish and freshwater habitats. Alternatively, habitat type may modify the ability of frogs to effectively navigate through different substrates between habitat types. There may be different metabolic needs due to increased salt stress in coastal wetlands that requires additional energy to process and eliminate excess salts by coastal individuals (Bernabò et al. 2013, Giunta et al. 1984). Additionally, differences in diet may stem from differences in the nutritional value among prey items (Freed 1980), which may be particularly important during the energetically taxing breeding season or in brackish environments that require extra energy to maintain internal ionic balances. As habitats continue to change from anthropogenic modifications and global climate change, research focused on revealing basic biology and natural history of organisms inhabiting both current and impending environments is crucial to accurately predict consequences, and effectively manage impacted communities. Our analyses of dietary patterns of a common, dietary generalist frog species (H. cinerea) documented that abundance and variety of dietary items differed according to habitat salinity. Our results provide valuable insights into how an emerging environmental stressor, secondary salinization, may impact the diets of freshwater species. Acknowledgments We are grateful to R. Trone for assistance with arthropod identification. We also thank A. Stuckert, T. McFarland, C. Thaxton, and J. Touchon for field assistance. Funding for this project was supplied by North Carolina Sea Grant (Project No. 2014-R/14-HCE-3) awarded to M.W. 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