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. 2021 Sep 7;9(1):coab072. doi: 10.1093/conphys/coab072

Biomarkers of reproduction in endangered green sea turtles (Chelonia mydas) nesting at Tortuguero, Costa Rica

Renato Saragoça Bruno 1,2,✉, Jaime Alberto Restrepo Machado 3, Gilberto Rafael Borges Guzman 1, Jorge Iván Ramos Loria 3, Roldán Arturo Valverde 2,3
Editor: Steven Cooke
PMCID: PMC8422948  PMID: 36082195

Abstract

Understanding the timing of vitellogenesis is essential for identifying threats to the reproductive success of endangered oviparous vertebrate species, such as sea turtles. We measured concentrations of testosterone (T) and vitellogenin (VTG) in green sea turtles (Chelonia mydas) nesting at Tortuguero, Costa Rica, as biomarkers of ovarian development. Testosterone concentration increased from the first to second month and VTG concentration increased at the third week of sampling. These results show that Tortuguero green sea turtles were still producing both biomarkers early into the nesting season. VTG concentration was negatively correlated with female weight, suggesting that larger females start nesting earlier at Tortuguero and that we may have sampled larger females further into their reproductive cycle.

Keywords: Conservationendocrinologyreproductive biologysea turtlesTortuguero

 

Testosterone concentration increased on the second month and vitellogenin (VTG) concentration increased on the third week of the green sea turtle nesting season at Tortuguero, Costa Rica, presumably as both biomarkers were being produced post-nuptially. Heavier female green sea turtles showed lower VTG concentration, likely because those individuals started nesting earlier.

Introduction

Vitellogenesis is a similar process in most oviparous reptiles, during which lipids and proteins are mobilized from the body’s stores and progressively added to developing ovarian follicles, forming the egg yolk that nurtures growing embryos (Ho et al. 1980, 1982; Hamann et al. 2003; Polzonetti-Magni et al. 2004). The series of physiological processes occurring during vitellogenesis are regulated by specific gonadal and non-gonadal hormones (Urist and Schjeide 1961; Callard and Klotz 1973; Licht et al. 1980; Ho et al. 1980, 1982; Ho 1987; Wibbels et al. 1990; Heck et al. 1997; Mosconi et al. 1998; Hamann et al. 2003; Polzonetti-Magni et al. 2004).

Oestrogens induce growth and structural modifications in the liver of snakes and lizards that are associated with the production and secretion of proteins (reviewed by Ho 1987). Evidence indicates that estradiol 17β (E2) induces the production of vitellogenin (VTG), which is the main protein sequestered into growing ovarian follicles during vitellogenesis and cleaved into the main proteinaceous components of egg yolk (Bergink and Wallace 1974; Ho et al. 1981; Ho 1987; Hamann et al. 2003; Polzonetti-Magni et al. 2004).

Because yolk deposition into ovarian follicles is crucial to reproductive success of oviparous vertebrates (Polzonetti-Magni et al. 2004), the importance of studying endocrine mechanisms that control vitellogenesis has been highlighted by several studies, many of which focused on threatened and endangered sea turtle species (Licht et al. 1980; Owens and Morris 1985; Wibbels et al. 1990; Rostal et al. 1998; Hamann et al. 2003; Polzonetti-Magni et al. 2004; Sifuentes-Romero et al. 2006; Smelker et al. 2014; Myre et al. 2016). However, there are significant gaps regarding the hormonal regulation of the onset and completion of vitellogenesis in reptiles (Hamann et al. 2003; Polzonetti-Magni et al. 2004). Research is lacking on the variation of such hormones, associated proteins and requisite body condition during the reproductive cycle of free-ranging migratory reptiles, such as sea turtles (Hamman et al. 2003).

Sea turtles start depositing yolk into follicles 8–12 months prior to the nesting season (Wibbels et al. 1990; Miller 1997; Hamann et al. 2003). A pre-migratory surge of E2 4–6 weeks prior to migration is followed by a significant and gradual decline in the concentration of this hormone 1–2 weeks before migration, declining gradually towards the end of the nesting season (Wibbels et al. 1990; Smelker et al. 2014; Myre et al. 2016). Although E2 induces the production of VTG, these two biomarkers of reproduction are usually not correlated, given the long-lasting inductive effects of E2 (Heck et al. 1997). Thus, E2 peaks in advance of VTG, and VTG concentration then decreases significantly but gradually throughout the nesting season of female loggerhead sea turtles (Caretta caretta) (Smelker et al. 2014; Myre et al. 2016). In this way, pre-ovulatory follicles may act as a sink for VTG.

Testosterone (T) concentration in marine turtles significantly increases 1–2 weeks prior to the reproductive migration (Wibbels et al. 1990), and a general decreasing trend in the concentration of T has been shown for loggerhead sea turtles towards the end of the nesting season, following a trend akin that of VTG (Wibbels et al. 1990; Rostal et al. 1998; Smelker et al. 2014; Myre et al. 2016). Concentration of T is also thought to be responsible for mating behaviour (Wibbels et al. 1990; Hamann et al. 2003; Polzonetti-Magni et al. 2004) and for termination of the reproductive cycle (Ho et al. 1981; Wibbels et al. 1990; Myre et al. 2016).

The fluctuations of these biomarkers—which control ovarian recrudescence, as well as trigger sea turtle reproductive migration—are poorly understood (Hamann et al. 2003; Polzonetti-Magni et al. 2004). The objective of this study was to investigate concentration of T and VTG in green sea turtles (Chelonia mydas) nesting at Tortuguero, Costa Rica. We also investigated the effects of body size and condition on concentrations of reproductive biomarkers in female green sea turtles nesting at Tortuguero.

Materials and methods

Tortuguero National Park (TNP), on the Caribbean coast of Costa Rica, was created in 1970 to protect the 29 km of what is now the most important mating and nesting ground for green sea turtles in the Atlantic basin (Troëng and Rankin 2005). From 1986 to 2019, an estimated yearly average of 98 137 (± 44 633) green sea turtle nests were laid along the 29 km of Tortuguero’s nesting beach (Bruno et al. 2020).

Green sea turtles arrive in Tortuguero in late April and early May to reproduce (Carr 1954). Nesting significantly increases in July, peaks in September and occurs through late October (Garcia-Varela et al. 2014). Our study was conducted in July and August 2018 on the northernmost 8 km of Tortuguero Beach, near the Sea Turtle Conservancy (STC) station.

Under Southeastern Louisiana University’s IACUC and the Costa Rican Ministry of the Environment’s permits (001/2018 and 002/2018, respectively), we used 18-gauge, 2.5-inch heparinized needles to collect up to 15 ml of blood from the cervical sinus of adult female green sea turtles nesting at Tortuguero. All blood sampling was conducted following Owens and Ruiz (1980) protocols. We sampled blood from nesting females during oviposition and placed the samples on ice until centrifugation, and blood plasma was stored in liquid nitrogen prior to analysis.

We exported the samples to a −80o C freezer at Southeastern Louisiana University, Hammond, LA, USA (CITES 18US75808C/9). Following procedures by Myre et al. 2016, we extracted steroid hormones twice from 10 to 30 μl of plasma with 5 ml of diethyl ether in each extraction. After steroid extraction, we reconstituted samples with 300 μl of assay buffer and plated it in duplicate in a 96-well microtiter plate coated with Anti-T antibodies of a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA) kit (ENZO Life Sciences, Farmingdale). This assay was fully validated to measure T in green turtles (Allen et al., 2015). Subsequently, we performed ELISAs according to manufacturer’s specifications and read the plate in a microplate reader at 405 nm. To find the concentrations of T in each sample, ELISA results were analysed using the four-parameter logistic equation in SigmaPlot v14.0.

For the VTG assay, we followed the same protocol that was described by Smelker et al. (2014). For this, a solution of purified sea turtle VTG was used to create a standard curve for the indirect ELISA. We diluted the samples 1:30000 to ensure that optical density fit into the linear portion of the standard curve (between 20% and 80% of absorbance). We plated the samples in duplicate into a 96-well flat bottom polysterene microtiter plate (Thermo Fisher Scientific, Pittsburg, PA, USA), which were sealed and incubated overnight at 4oC. The next day, we washed the plates three times with 150 μl of a phosphate buffered saline (PBS) solution and a non-ionic detergent (Tween-20). To maximize specificity, we pre-incubated rabbit anti-VTG antibody with 5 μl of male sea turtle plasma in a solution of PBS with 5% non-fat dry milk (blotto) for 1 hour. Pre-adsorbed anti-VTG antibody (1:40000) was then added to the plates, which were sealed and incubated for 1 hour at room temperature on a shaking platform. After three more washes, we added 100 μl of goat anti-rabbit antibody coupled with horseradish peroxidase diluted in blotto to the plates (BIORAD, Hercules, CA, USA) and incubated for 2 hours at room temperature on a shaking platform. After another washing cycle, we added 100 μl of tetramethybenzidine peroxidase for enzyme-immunoassay to each well of the plate and incubated for 10 minutes at room temperature to promote colour development. To stop the colorimetric reaction, we added 100 μl of sulphuric acid (1 N H2SO4) to each well. The plates were then read in a Bio-Rad Model 680 Microplate Reader with a 450-nm wavelength filter.

In the field, we measured curved carapace length (CCL) with a flexible, 1.5-m measuring tape on the centre of the carapace from the top of the nuchal scale to the inner part of the notch between the supracaudal scales. We measured straight carapace length (SCL) with a metal calliper between the most anterior and the most posterior part of the turtle’s carapace (Wyneken 2001). Both CCL and SCL measurements were taken at least three times in cm, until three measurements did not differ by more than 1 cm. In addition to body measurements, we weighed (kg) and calculated the condition index (CI) of a sub-group of 16 female green sea turtles nesting at TNP using the following formula by Bjorndal et al. (2000)Inline graphic. Finally, we marked turtles with metal tags for individual identification.

STC’s research personnel has monitored sea turtle nesting activity at TNP since 1955. This included the collection of morphometric data of nesting females at the northernmost 8 km of nesting beach (Bjorndal and Carr 1989, Troëng and Rankin 2005). Since 1986, to account for the total number of nests laid at TNP during one nesting season, STC staff has conducted a weekly survey of the entire 30 km of nesting beach (Troëng and Rankin 2005; Garcia-Varela et al. 2014). The estimation of green sea turtle clutches laid at TNP yearly was described in detail by Troëng and Rankin (2005). For this study, we accessed STC’s long-term database to extract biometric data of Tortuguero green sea turtles.

For all statistical analyses, we used SYSTAT 13.0 and we used ggplot2 package in R 3.6.3 to create the figures. To assess the normality of the data, we visually analysed the histogram of the studentized residuals and carried out One Sample KS tests. We used linear regression analyses to investigate the relationship between T and VTG and the morphometric parameters of individual female green sea turtles (such as SCL, CCL, weight and CI). To analyse temporal fluctuation of T and VTG early in the nesting season and to investigate differences in SCL and CI per month, we used analysis of variance (ANOVA).

Results

We measured circulating concentrations of T and VTG in 66 blood samples of green sea turtles nesting at Tortuguero. Two of these samples were collected from the same female and in consecutive nesting events (11 days apart). Mean T concentration was 1100 (± 1000 pg ml−1). Our average intra-assay coefficient of variation (CV) for the T assays was 8% and the inter-assay CV was 12.9%. Mean VTG concentration in the samples was 27.9 (± 6.6 mg ml−1). Validation and specificity testing of VTG ELISA was conducted by Smelker et al. (2014). Our intra-assay CV for these VTG assays was 5.1% and the inter-assay CV was 12.8%.

We found a positive relationship between T and VTG (linear regression, r2 = 0.315, F1,63 = 28.995, P < 0.005) (Fig. 1). VTG concentration was negatively correlated with green sea turtle weight (linear regression, r2 = 0.278, F1,13 = 5.017, P < 0.05) (Fig. 2). SCL of female green sea turtles nesting at TNP between 1955 and 2017 varied per month, and significantly larger females nested in June and July than in August and September (ANOVA, F1,5542 = 39.974, P > 0.001) (Fig. 3).

Figure 1. VTG concentration (n = 66) was positively correlated with testosterone concentration (r2 = 0.315). Scatter plots shown with the regression line and 95% confidence interval.

Figure 1

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Figure 2. VTG concentration (n = 16) was negatively correlated with green sea turtle weight (r2 = 0.278). Scatter plots shown with the regression line and 95% confidence interval.

Figure 2

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Figure 3. SCL of turtles nesting in June and July was significantly greater (asterisk) than that of turtles nesting in August and September. Boxplot shown with standard deviation and median. Whiskers showing highest and lowest observation (n = 5547).

Figure 3

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Mean T concentration was significantly lower (ANOVA, F1,63 = 6.426, P = 0.014) in July than in August (Fig. 4). From 20 July to 22 August, mean VTG concentration (n = 56) was significantly higher during the third week of sampling (ANOVA, F1,52 = 7528, P = 0.008) (Fig. 5). The only nesting green sea turtle we recaptured in this study showed a similar pattern of increasing T and VTG circulating concentrations over 11 days. Both VTG and T were higher in the second nesting event than in the first. However, because of the small sample size, no significance could be attributed to the change in the concentration of endocrine correlates between the two nesting events. CI was significantly lower in July than in August (ANOVA, F1,14 = 7.656, P > 0.05) (Fig. 6). Neither SCL, CCL nor body weight varied significantly from July to August 2018.

Figure 4. Testosterone concentration (n = 66) was significantly lower in July than in August. Boxplot shown with standard deviation, median and data points. Data points displayed with jitter to avoid overlap. Whiskers showing highest and lowest observations.

Figure 4

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Figure 5. Concentration of VTG (n = 52) was significantly higher during the third week of sampling. Boxplot shown with standard deviation, median and data points. Data points displayed with jitter to avoid overlap. Whiskers showing highest and lowest observations.

Figure 5

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Figure 6. CI of Green sea turtles nesting at Tortuguero was significantly lower in July than in August. Boxplot shown with standard deviation, median and distribution of data points. Data points displayed with jitter to avoid overlap. Whiskers showing highest and lowest observations.

Figure 6

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Finally, there was positive relationship between weight and SCL (linear regression, r2 = 0.613, F1,226 = 358.103, P < 0.005) and CCL (linear regression, r2 = 0.599, F1,13 = 19.430, P < 0.005) (Figs 7 and 8). However, CI was not significantly correlated to any of the other body measurements nor to the concentrations of T and VTG.

Figure 7. Green sea turtle weight (n = 14) was positively correlated to SCL (r2 = 0.613). Scatter plots shown with the regression line and 95% confidence interval.

Figure 7

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Figure 8. Green sea turtle weight (n = 15) was positively correlated to CCL (r2 = 0.599). Scatter plots shown with the regression line and 95% confidence interval.

Figure 8

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Discussion

We sampled blood from female green sea turtles during four weeks at the start of the nesting season at Tortuguero, which lasts up to 4 months. Concentration of Testosterone increased significantly from July to August, and VTG concentration, which was elevated during the whole sampling period, significantly increased during the third week of sampling. Interestingly, we sampled blood twice from the same female with an 11-day interval and observed that both T and VTG concentrations almost doubled between the first and second encounter. VTG concentration in Tortuguero green sea turtles shown in our study were up to 40 000 times greater than basal VTG concentrations in juvenile Green (Herbst et al. 2003) and adult female loggerhead sea turtles that were not preparing to breed (Smelker et al. 2014). Moreover, VTG concentration in nesting Tortuguero green sea turtles were akin to that of juvenile Green (Herbst et al. 2003) and Kemp’s Ridley (Lepidochelys kempii; Heck et al. 1997) sea turtles injected with E2 and of nesting Loggerheads (Smelker et al. 2014; Myre et al. 2016). As reptilian VTG estimated half-life is between 15 and 25 days (Bast and Gibson 1985), the results of our study showed that one of the components of vitellogenesis, the production of VTG (Ho 1987), was still taking place early into the nesting season of Tortuguero green sea turtles. Whether VTG produced early in the nesting season of Tortuguero green sea turtles was still being incorporated in ovarian follicles requires further research.

Elevated concentrations of gonadal steroids in loggerhead sea turtles early in the nesting season (Wibbels et al. 1990), the follicular hierarchy observed in necropsied nesting green sea turtles (Limpus et al. 2003) and pre-ovulatory surges in estradiol concentration in captive loggerhead sea turtles (Kakizoe et al. 2010) led authors to suggest that there may be continued follicular development early into the nesting season. In contrast, leatherback sea turtles appeared to arrive at the nesting beach with fully developed ovaries, as no hierarchy in follicular size was detected via ultrasonography (Rostal et al. 1996). Likewise, ultrasonography of captive Kemp’s Ridleys concluded that the entire complement of ovarian follicles was fully developed by the time of mating (Rostal et al. 1990, 1997; Rostal 2005). However, leatherback sea turtles travel long distances from their foraging areas to their nesting grounds (Shillinger et al. 2008; Benson et al. 2011), presumably having ample time to complete ovarian development prior to arrival at nesting beaches. Kemp’s Ridley sea turtles have lower clutch frequency and size than Tortuguero green turtles (Van Buskirk and Crowder 1994; Rostal et al. 1997; Bjorndal 1999; Rostal 2005; Shaver et al. 2016) and may afford the space in the coelomic cavity to hold the full complement of mature follicles for the nesting season all at once. In summary, due to distinct constraints for reproduction of different sea turtle species and populations, timing of completion of yolk deposition into ovarian follicles may vary within the sea turtle superfamily Chelonioidea.

Concentration of VTG in our study was negatively correlated with female green sea turtle body weight, which may be due to sampling larger turtles earlier into their nesting effort, when their VTG concentrations were higher. Relationships between body size and reproductive phenology are found throughout Reptilia. For example, smaller female viviparous lizards reproduce later than their larger counterparts, presumably due to having to divide energy between growth and reproduction (Bauwens and Verheyen 1985). Additionally, larger female diamond-backed terrapins (Malaclemys terrapin) nest earlier than their smaller counterparts, which may allow them to have access to the fittest males in the population (Wolfe et al., 2021, in review). Measuring a different metric of the sea turtle reproductive output, Hatase and Tsukamoto (2008) found that the size of female loggerhead sea turtles influences reproductive frequency, with larger individuals having shorter remigration intervals than smaller ones. Larger female green sea turtles grow slower than their smaller conspecifics (Bjorndal et al. 2000) and may be able to reach the energetic threshold necessary for reproduction sooner and nest earlier in the season than smaller individuals. Additionally, if larger female green sea turtles swim faster, they may arrive earlier at Tortuguero than smaller females.

Our data are consistent with previous studies that have examined the morphometry of green sea turtles. Mean SCL of female Tortuguero green sea turtles was similar to values reported by Bjorndal et al. (2000) for adult green sea turtles throughout the Caribbean Sea. The mean CCL of our study was close to that reported for adult green sea turtles nesting in the Southern Great Barrier Reef (Limpus and Chaloupka 1997) and on Cyprus (Broderick et al. 2003). Green sea turtle CI in our study was similar to other populations (Bjorndal et al. 2000; Thomson et al. 2009; Labrada-Martagón et al. 2010). Moreover, in agreement with Bjorndal et al. (2000), CI was not significantly correlated with SCL, CCL or weight in our study. Mean body weight was unsurprisingly strongly correlated to green sea turtle SCL and CCL. From the standpoint of morphometry, our results suggest that our findings with reproductive biomarkers of gonadal function may be applicable to other green sea turtle populations.

Mean concentration of VTG was higher and mean concentration of T was lower than previously reported for loggerhead sea turtles (Wibbels et al. 1990; Smelker et al. 2014; Myre et al. 2016). Owens (1980) and Wibbels et al. (1990) reported that green sea turtles have an overall lower concentration of gonadal steroids than other sea turtle species, and dietary intake has previously been cited to affect concentrations of gonadal steroids and overall reproductive output in female reptiles (Limpus and Nicholls 1988; Chaloupka et al. 2008; Lovern and Adams 2008). Finally, both Wibbels et al. (1990) and Smelker et al. (2014) used a radioimmunoassay to measure T in their samples, whereas we used ELISAs, which complicate direct comparisons. Radioimmunoassay and ELISA have been reported to yield slightly different, but correlated results when used to measure concentration of gonadal and adrenal steroids (Lewis et al. 1986; Khatun et al. 2009).

Understanding the reproductive physiology of endangered species is crucial for conservation, and ours was the first study investigating reproductive biomarkers of endangered green sea turtles at their most important breeding ground in the Atlantic Basin. The information provided here can be used to guide further studies regarding the fluctuation of reproductive biomarkers during the nesting cycle and the timing of completion of yolk deposition into the ovarian follicles of free-ranging sea turtles.

Funding

This work was supported by the National Geographic Society [EC-51596-18], Sea Turtle Conservancy, Sigma Xi Grant in Aid of Research, American Museum of Natural History, IDEAWILD, CORBANA and Varcli Pinares S.A. Kenneth (Major) Dyson Professorship to R.A.V.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors thank the community and national park rangers of Tortuguero, Costa Rica, for their support. The author thanks all STC research assistants and staff and all the personnel from ASVO Tortuguero and ASOPROTOMA.

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