Olive ridley sea turtles (Lepidochelys olivacea) are a vulnerable species with declining wild populations.1 One reason for this decline is habitat loss, which can lead to decreased food availability. Poor nutrition in turn can lead to reproductive consequences such as skipped breeding years and fewer eggs.2,3 If undernourished turtles are producing fewer eggs, hatchling numbers will decline, making species recovery more difficult. Therefore, assessing nutrient storage and utilization would also be helpful to better understand how environmental disturbances are impacting turtles’ ability to fuel the energetically expensive physiological process of reproduction.
TRADITIONAL METHODS FOR ASSESSING NUTRIENT INTAKE IN WILD TURTLES HAVE SERIOUS LIMITATIONS
Traditional methods for assessing nutrient intake in wild turtles have serious limitations. One traditional technique called stomach flushing consists of prying a turtle’s mouth open, inserting tubes down the esophagus, and pumping salt water through to force the stomach contents up a retrieval tube. This is a highly invasive process that can harm the turtle and only provides information about what a turtle has eaten immediately prior to capture, providing little information on long term food intake.4 In an effort to develop less invasive means to assess nutritional state, the feasibility of applying two recently-developed, less invasive techniques to turtles in the wild were evaluated.
The first method is ultrasonography of subcutaneous fat, the tissue layer below the epidermis and dermis. Using ultrasonography to measure subcutaneous fat has been validated with necropsy in leatherback sea turtles but not in other sea turtle species. If a turtle is not eating this layer should thin as fat is redistributed to other tissues for energy. Well nourished, consistently eating turtles should therefore have thicker layers of subcutaneous fat than poorly nourished turtles.5 Subcutaneous fat measurement may thus provide information about a turtle’s long-term access to food. The second method is blood β-hydroxybutyrate (BHB) measurement. BHB is a ketone body that is produced from stored fat in the liver during fasting as an alternative energy source to glucose.6 Price et al. conducted a feeding study in captive juvenile green sea turtles and concluded that BHB is an effective indicator of fasting status in this particular species since levels of BHB increased rapidly during fasting. BHB levels may therefore reflect mobilization of fat in fasting turtles. The first objective of my project was to determine whether subcutaneous fat and blood BHB measurement could also be applied as assessment methods for fat storage and mobilization in other sea turtle species like olive ridleys. To evaluate the applicability of these techniques in non-captive turtles, wild reproducing olive ridleys were used in this study. Whereas ultrasonography of subcutaneous fat may provide information about nutrient storage in this species, BHB may serve as a fasting indicator.
Understanding differences in fat storage and utilization between sexes would also be helpful for assessing the energetic and nutritional strategies used to maximize reproductive output of olive ridleys. The second objective of my project was to determine whether there were differences in subcutaneous fat and blood BHB between sexes. Female sea turtles are capital breeders who fast during reproduction.7 Capital breeders build up energy reserves at foraging grounds, cease feeding, and then migrate to nesting beaches, fueled by fat stores.8 During this migration, females deposit yolk in their eggs. These eggs are contained in ovarian follicles. At nesting beaches, olive ridley females mate and lay multiple nests over a four-month period either during monthly mass-nesting events or solitarily. After a female has laid her last nest for her nesting season the remaining unfertilized eggs are absorbed in a process called follicular atresia.9 This reproductive strategy suggests females will retain substantial fat stores at the beginning of their respective nesting seasons which they subsequently draw upon to fuel the energetic demands of nesting. Males are not as commonly studied as females, but in contrast, they typically do not fast during migration. Therefore, females utilizing a capital breeding strategy are expected to have thinner subcutaneous fat layers and higher BHB levels than males when they arrive at nesting beaches and post-mating (i.e. immediately after mating).
Sampling was conducted at Ostional Wildlife Refuge in Ostional, Costa Rica from June to September in the years 2016 through 2018. Ostional Beach is a unique site where thousands of olive ridleys migrate to mate and nest. Mating olive ridleys float on the surface of the ocean making sampling of both males and females possible. After copulation males and females were separated by our team and brought onboard a boat for sampling. A 10 mL blood sample was taken from the turtle’s dorsal cervical sinus within five minutes of capture and placed immediately on ice to maintain integrity of the sample (Figure 1). Blood samples were later centrifuged at the field station to separate out the plasma, which was immediately frozen to prevent sample degradation.
In addition to blood sampling, three ultrasound images were taken in the dorsal shoulder region of the subcutaneous fat using the shoulder joint as a landmark to keep the measurements consistent. Images were taken in this region because a turtle’s shell blocks the ultrasound signal, limiting measurements to soft tissue regions. This location also consistently yielded the clearest images of tissue layers with multiple ultrasound measurements in leatherbacks.10 ImageJ software was then used to measure the depth of the subcutaneous fat from sonogram images collected in the field. The three measurements were averaged for total fat depth (Figure 2).
To categorize females based on reproductive state, females were placed on their shell in a car tire and gonadal ultrasounds were taken. Females with follicles were categorized as early season females and females in atresia were categorized as late season females. Gonadal ultrasounds were also used to detect if the intestines contained food. Intestines were only discernible in the sonograms if they were full.11 Ultrasounds were not taken of the males.
Blood samples were returned to Texas A&M University where BHB was measured with a commercial BHB kit (Sigma Aldrich, St. Louis, MO) and read using a microplate spectrophotometer at 450 nm. The BHB standard curve provided with the commercial kit measured a range of concentrations from 0–10 mM. Because undiluted blood BHB concentrations exceeded this range, olive ridley blood samples were first diluted to 1:16 to allow them to be measured with the kit.
For the statistical analysis, two-way analyses of variance (ANOVAs) were used to compared means of subcutaneous fat and BHB between 26 females and 16 males. Next, the same series of ANOVAs was run contrasting 12 early versus 14 late season females. Lastly, a two-sample t-test was run assuming unequal variances for visible intestines and BHB to determine the relationship between intestinal state and BHB levels. The BHB concentrations of nine turtles with no visible intestines and of 17 turtles with visible intestines were compared in the t-test.
FEMALES...ARE EXPECTED TO HAVE THINNER SUBCUTANEOUS FAT LAYERS AND HIGHER BHB LEVELS THAN MALES
Subcutaneous fat was distinguishable from surrounding tissues in sonograms (Figure 2) and easily measurable using image analysis software. There was no significant difference in subcutaneous fat between the sexes or in early versus late season females (Figure 3). A 1:16 dilution of blood gave measurable concentrations of BHB, which likewise did not differ between the sexes or over time (Figure 3). There was a significant difference in the comparison between BHB levels and intestinal state (P < 0.001). Turtles with no visible intestines had a BHB average of 98.3 (± 18.5) mM and turtles with visible intestines had a BHB average of 85.7 (± 5.7) mM, suggesting that BHB levels are elevated in fasting animals.
The results of this study indicate that subcutaneous fat and blood BHB are measurable in wild olive ridleys; therefore, ultrasonography and blood BHB measurement appear to be practical non-invasive techniques worth further analysis. In future studies, however, ultrasonography measurements of subcutaneous fat will need to be validated by comparing fat layers in olive ridleys at necropsy to ultrasonographic measurement of subcutaneous fat in the dorsal shoulder region as was done in leatherbacks.12 Additionally, further studies should monitor how subcutaneous fat and blood BHB levels change during the four-month nesting period. Females are expected to be fasting during this period, so blood BHB is expected to increase as subcutaneous fat decreases.13,14 Additional blood samples and ultrasounds can be taken from females previously sampled at post-mating during the monthly mass-nesting events to track changes in nutrient storage and utilization.
Ultrasonography was also used to detect intestinal contents in sonograms of females, a technique not previously described. BHB was elevated when intestines were empty and was lower when intestines were full. This result matched expectations that BHB should increase during fasting, a period when the intestines should be empty. The result also suggests that BHB may be functioning as an energy source in sea turtles in the same capacity as in mammals.
In comparing subcutaneous fat and BHB between sexes, females were expected to have thinner subcutaneous fat layers and higher BHB concentrations; however, there was no difference between sexes in either of these categories. If females are fasting, a difference is expected in these measurements because females should be fueling the deposition of yolk in eggs with fat reserves while males are fueling reproductive effort with food intake before arrival at nesting beaches.15 Therefore, results suggest that females may be feeding at mating grounds. To further test this possibility, these same parameters were compared in early and late season females at post-mating. If females are fasting during the reproductive season, late season females should have thinner subcutaneous fat layers and higher BHB concentrations than early season females. However, there was no difference between early and late season females in any of these categories, providing further support for the possibility of females fueling reproductive costs with food intake at mating and nesting grounds.
NEW CONSERVATION POLICIES... SHOULD BE CONSIDERED TO INCREASE FOOD AVAILABILITY
Once fully validated, ultrasonography of subcutaneous fat and blood BHB measurement will provide less invasive and potentially more informative methods for assessing sea turtle feeding than stomach flushing. Expanding the application of these novel techniques to other sea turtle species should help us understand how food availability and nutrition impact sea turtle reproduction, as well as the physiological differences between males and females during reproduction. It will be particularly important to determine if both sexes have access to the nutrients needed to maximize egg production and population recovery. Currently, there are no sea turtle prey monitoring or management conservation policies in place in Costa Rica, which hosts two mass-nesting beaches and several solitary nesting beaches. If females are feeding during the reproductive season as our results suggest, new conservation policies, including monitoring and management of sea turtle prey species, should be considered to increase food availability, nutritional health, and reproductive output of sea turtles, thus helping to sustain these sea turtle populations for many years to come.
This work was supported in part by a Texas Sea Grant (NA18OAR4170088). I would like to thank Dr. Duncan MacKenzie and Brianna Myre for their extensive help and guidance with this project. Their countless hours have helped me become a better researcher and made this project possible. Thank you also to Dr. Wayne Versaw for use of the spectrophotometer.
Jeffrey D. Miller, “Reproduction in Sea Turtles,” in The Biology of Sea Turtles, ed. Peter L. Lutz and John A. Musick (Boca Raton: CRC Press, 1997) 51–81.
Annette C. Broderick, Brendan J. Godley, and Graeme C. Hays, “Trophic Status Drives Interannual Variability in Nesting Numbers of Marine Turtles,” Proceedings of the Royal Society B 268, no. 1475 (July 2001): 1481–87, http://doi.org/10.1098/rspb.2001.1695.
Andrew R. Solow, Karen A. Bjorndal, and Alan B. Bolten, “Annual Variation in Nesting Numbers of Marine Turtles: The Effect of Sea Surface Temperature on Re-Migration Intervals,” Ecology Letters 5, no. 6 (November 2002): 742–46, https://doi.org/10.1046/j.1461-0248.2002.00374.x.
Gregory A. Forbes and Colin J. Limpus, “A Non-Lethal Method for Retrieving Stomach Contents from Sea Turtles,” Wildlife Research 20, no. 3 (1993): 339–43, https://doi.org/10.1071/WR9930339.
Heather S. Harris et al., “Validation of Ultrasound as a Noninvasive Tool to Measure Subcutaneous Fat Depth in Leatherback Sea Turtles (Dermochelys coriacea),” Journal of Zoo and Wildlife Medicine 47, no. 1 (February 2016): 275-79, https://doi.org/10.1638/2015-0023.1.
Edwin R. Price et al., “Serum Triglycerides and β-Hydroxybutyrate Predict Feeding Status in Green Turtles (Chelonia mydas): Evaluating a Single Blood Sample Method for Assessing Feeding/Fasting in Reptiles,” Journal of Experimental Marine Biology and Ecology 439 (January 2013): 176–80, https://doi.org/10.1016/j.jembe.2012.11.005.
Miller, “Reproduction in Sea Turtles,” 51–81.
Virginie Plot et al., “Leatherback Turtles Are Capital Breeders: Morphometric and Physiological Evidence from Longitudinal Monitoring,” Physiological and Biochemical Zoology 86, no. 4 (2013): 385–97, https://doi.org/10.1086/671127.
David Wm. Owens, “The Comparative Reproductive Physiology of Sea Turtles,” American Zoologist 20, no. 3 (1980): 549–63, https://doi.org/10.1093/icb/20.3.549.
Harris et al., “Validation of,” 275–79.
A. L. Valente et al., “Ultrasonographic Imaging of Loggerhead Sea Turtles (Caretta caretta),” Veterinary Record 161, no. 7 (2007): 226–32, http://dx.doi.org/10.1136/vr.161.7.226.
Harris et al., “Validation of,” 275–79.
Harris et al., “Validation of,” 275–79.
Edwin R. Price et al., “Serum Triglycerides and β- Hydroxybutyrate,” 176–80.
Miller, “Reproduction in Sea Turtles,” 51–81.
Nicole Guentzel ‘19
Nicole Guentzel ‘19 is a graduating senior Biology major with a minor in Spanish from Baytown, Texas. Nicole participated in the 2018–2019 class of the Undergraduate Research Scholars where she completed her thesis, which culminated in this article, under the guidance of Dr. Duncan MacKenzie. After graduation, she hopes to pursue an advanced degree and encourage other students to do undergraduate research.