Reproductive Physiology in Eastern Snapping Turtles (Chelydra serpentina) Exposed to Runoff from a Concentrated Animal Feeding Operation

Animal manure is commonly applied to croplands as a fertilizer. Runoff from these fields can contaminate water bodies with sex steroids, nutrients, and other pollutants (Lee et al., 2007). The eastern snapping turtle (Chelydra serpentina) is a freshwater species found in a variety of aquatic environments, including those adjacent to agricultural fields, and has been successfully used as a bioindicator species (e.g., Bishop et al., 1998). However, and despite their high abundance and wide distribution throughout the eastern and central United States, no study has evaluated the impact of animal manure on this species. Our main objective was to evaluate the reproductive condition of turtles sampled from a pond receiving runoff from land-applied animal manure from concentrated animal feeding operations (CAFOs) in relation to those sampled from a reference pond.

Turtles were sampled during the breeding season (May–July 2008 and 2009). Trapping sites included ponds at the CAFO site as previously described (Leet et al., 2012) at Purdue's Animal Science Research Farm (40°30′17″N, 87°0′47″W, 0.8 ha, average depth 1.5 m) and a reference site at Martell Forest (40°25′48″N, 87°2′27″W, 0.4 ha, average depth 2.5 m) in Indiana, USA. Distance between sites was 8 km. Martell Forest consists of 150 ha of forestland, with the pond at the center of the property, isolated from agricultural runoff. Turtles were captured with the use of baited (corn and canned tuna) hoop nets placed at the edges (<1.0-m depth) of ponds. Traps were checked daily. Sex was determined by the ratio of the precloacal tail length to the posterior plastron lobe length; turtles with values >1.20 were considered males (Ernst and Lovich, 2009). Turtles were weighed with the use of a spring scale and bled from the caudal vein with the use of 1-mL tuberculin syringes fitted with 1.3-cm, 26-G needles. Blood (<1 mL) was placed in 1.3-mL heparin tubes, centrifuged at 3,000 × G for 5 min, and plasma stored at −80 C. Animals were marked on their marginal scutes with a file to prevent resampling the same individuals.

Water temperature (±0.2 C) was measured with the use of a portable YSI meter (Model 55, Yellow Springs, Ohio, USA) daily between 8:00 and 10:00 am at a middle depth (∼0.75–1.25 m) and <5 m from the edge. Water samples were collected for quantification of sex hormones as described previously (Leet et al., 2012).

Enzyme-linked immunosorbent assay (ELISA) is the most commonly used method of quantifying vitellogenin (VTG), but in the absence of an antibody specific to C. serpentina VTG, we used a phosphoprotein staining assay to semiquantify plasma VTG. The method is cost effective (<$3/sample), fast (52 samples run in <12 hr), and sensitive. Plasma aliquots containing 1 µg of total protein (determined as described by Bradford, 1976) were fractionated with the use of 4% stacking/8% resolving SDS-PAGE, gels incubated in Pro-Q Diamond Phosphoprotein Gel Stain (Molecular Probes, Eugene, Oregon, USA), and bands detected with the use of ultraviolet light at 365 nm (Fig. 1A). Purified rainbow trout (RT) VTG (250–700 ng, Cayman Chemical, Ann Arbor, Michigan, USA) was used as a positive control to semiquantify relative levels of VTG with a detection limit of 12 µg/mL. Confirmation of putative VTG bands was accomplished by peptide sequencing (MALDI-TOF-MS/MS, ABI 4800 Plus, Applied Biosystems, Inc., Foster City, California, USA) after excision of bands from gels and trypsin digestion. Sequences were blasted against the National Center for Biotechnology Information (NCBI) databases with the use of Mascot (Matrix Science Ltd., Boston, Massachusetts, USA) with ion scores >63 utilized to determine significant homology. Sequencing resulted in a 13–amino-acid peptide, SPQLEEYNGIWPR, homologous to VTG from the Chinese pond turtle Mauremys reevesii (gi|192384655). To semiquantify relative plasma VTG concentrations, a RT standard curve was created by plotting concentration versus pixel densities calculated as integrated density values (IDV) with the use of Automatic Image Capture Software (Alpha Innotech Corp., San Leandro, California, USA), similar to that described by Lattier et al. (2002). Coefficient of variation across gels ranged from 0.04 to 0.14. Images of turtle VTG bands were compared against RT-VTG bands by fitting their IDVs calculated from the putative VTG band in the gel picture to the RT-VTG standard curve (Fig. 1B, C). Plasma T concentrations were quantified with the use of radioimmunoassay (Jensen et al., 2001). Antiserum was obtained from Fitzgerald Industries (Concord, Mississippi, USA; 20-TR05; 20-ER06).

Mean and ranges in water temperature did not differ between ponds (Table 1). Concentrations of sex steroids in the water were higher at the CAFO site (Table 1), but still low compared to other agricultural ditches sampled within this same CAFO (Leet et al. 2012).

Forty turtles were sampled, 21 from the CAFO pond (10 males, 5 each year; and 11 females, 7 in 2008), and 19 from the Martell pond (10 males, 5 each year; and 9 females, 5 in 2008). Both years, CAFO turtles had greater mass compared to controls (mean±SD; 4.4±1.0 and 6.2±1.6 kg vs. 2.9±1.0 and 3.2±1.0 kg, for females and males, respectively; F = 28.6, P<0.0001). Vitellogenin was detected in 17 of 20 females and none of the males. The three negative females were likely juveniles (average weight, 1.5 kg). Female VTG and male T concentrations were greater in CAFO turtles (Fig. 2). When all animals were combined, body weight was positively correlated with VTG in females (VTG = −37.07+196.6*body weight (kg)−24.95*(body weight−3.70)², R² = 0.31, P = 0.01) and T in males (19.09+6.81*body weight+2.00*(body weight−4.7)², R² = 0.40, P = 0.02).

Ours is the second study to evaluate the impacts of runoff from manure-treated farm fields on aquatic turtles. Irwin et al. (2001) also reported increased VTG in female painted turtles (Chrysemys picta) from a pond receiving runoff from manure-treated fields and contaminated with 0.05 to 1.80 ng/L E2, and no VTG in males. However, CAFO turtles were of comparable size to those from the reference site. These authors speculated the increase in female VTG resulted from exposure to exogenous estrogens and could lead to the production of larger clutches/eggs.

Although we did not measure nutrients, the CAFO pond was likely highly enriched, judging by its green color, dense aquatic vegetation, and high water concentrations of nutrients from adjacent ditches (19.7±3 and 1.5±0.4 mg/L for total nitrogen and total phosphorous) in previous studies (Leet et al., 2012). This eutrophication could explain why turtles from the CAFO site were heavier (e.g., Naimi et al., 2012). We have also observed this in fish sampled from this site (Leet et al., 2012). Because both VTG and T were related to body mass, our results suggest that nutrient pollution indirectly resulted in higher plasma VTG in females and T in males because of an increase in body mass. We do not know the population-level implication of this increased body mass, but increased clutch sizes are likely (e.g., Elgar and Heaphy, 1989).

We thank Brett Lowry for helping with turtle sampling. Rod Williams assisted with trapping and blood collection. Linda Lee and Steve Sassman conducted sex steroid analysis from water samples. Jenna Cavallin and Leah Wehmas assisted with plasma hormone analyses. Whit Gibbons provided helpful comments. This study was funded by the US Environmental Protection Agency STAR program (RD833417).