Tissue sampling for the yellow mud turtles (YMT) for this study took place in May 2018 on Gimlet Lake at our long-term research site on the Crescent Lake National Wildlife Refuge (CLNWR), in Garden County, Nebraska, USA (41°45.24′N, 102°26.12′W). The Gimlet Lake marsh complex is a shallow (average depth 0.8 m), sandhill lake with marsh habitat [88 ]. YMTs exhibit temperature-dependent sex determination (TSD) with females produced under warm incubation conditions and both males and females produced under cooler conditions [59 (link)]. Mark-recapture and nesting ecology studies were ongoing here from 1981 through 2018. At this site, YMTs typically overwinter terrestrially buried in upland sandhills adjacent to wetlands, emerge in April and May, and migrate to the water, and then most females return to the same sandhills to nest in June, although some do not reproduce every year [67 , 87 ]. By July all turtles begin leaving the wetlands to estivate in the sandhills for the remainder of the summer (see also [89 (link)]). During field seasons, drift fences were constructed parallel to the shore between three overwintering sites and the lake, and monitored continuously each day.
During years (including 2017) when the fences were in place during the nesting season, each captured female was x-rayed to determine clutch size, and the width of each egg on each x-ray was measured. A regression equation relating mean clutch x-ray width with actual mean egg mass from a subset of nests that were subsequently excavated allowed us to estimate egg mass and clutch mass (both in g) for each gravid female in 2017 (n = 85). The equations for these relationships and fit are as follows: Actual Egg Width = 0.98(Estimated Egg Width) + 1.52, R2 = 0.89; Actual Egg Mass = 0.64(Estimated Egg Mass) – 6.07, R2 = 0.85; and in a sample of N = 1795 YMT eggs collected over several years egg width can be reliably used to estimate egg mass (and therefore total clutch mass) Egg Mass = 0.68(Egg Width) – 6.77, R2 = 0.85 (Iverson, unpublished data). This enabled us to examine the effects of those measures of reproductive output on immunity in the spring of 2018 as these 85 female turtles emerged from brumation.
Once captured, turtles were transported back to the field laboratory where morphometric data were recorded, including maximum carapace length (CL in mm), maximum plastron length (PL in mm), and body mass (BM in g). Up to 0.5 ml of whole blood was collected from the cervical sinus via 26 gauge heparinized syringe, centrifuged (7000 rpm) in a cryotube for 5 min to separate blood components. Blood plasma was pipetted to a separate cryotube. Both plasma and packed red blood cells were immediately flash frozen in liquid nitrogen until transport to Iowa State University for storage at − 80 °C. Sampled turtles (98 males and 102 females) were transported back to their initial capture location and released on the opposite side of the fence to proceed to the lake.
During years (including 2017) when the fences were in place during the nesting season, each captured female was x-rayed to determine clutch size, and the width of each egg on each x-ray was measured. A regression equation relating mean clutch x-ray width with actual mean egg mass from a subset of nests that were subsequently excavated allowed us to estimate egg mass and clutch mass (both in g) for each gravid female in 2017 (n = 85). The equations for these relationships and fit are as follows: Actual Egg Width = 0.98(Estimated Egg Width) + 1.52, R2 = 0.89; Actual Egg Mass = 0.64(Estimated Egg Mass) – 6.07, R2 = 0.85; and in a sample of N = 1795 YMT eggs collected over several years egg width can be reliably used to estimate egg mass (and therefore total clutch mass) Egg Mass = 0.68(Egg Width) – 6.77, R2 = 0.85 (Iverson, unpublished data). This enabled us to examine the effects of those measures of reproductive output on immunity in the spring of 2018 as these 85 female turtles emerged from brumation.
Once captured, turtles were transported back to the field laboratory where morphometric data were recorded, including maximum carapace length (CL in mm), maximum plastron length (PL in mm), and body mass (BM in g). Up to 0.5 ml of whole blood was collected from the cervical sinus via 26 gauge heparinized syringe, centrifuged (7000 rpm) in a cryotube for 5 min to separate blood components. Blood plasma was pipetted to a separate cryotube. Both plasma and packed red blood cells were immediately flash frozen in liquid nitrogen until transport to Iowa State University for storage at − 80 °C. Sampled turtles (98 males and 102 females) were transported back to their initial capture location and released on the opposite side of the fence to proceed to the lake.
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