Evaluation of Sex Ratios of the Olive Ridley Sea Turtle (Lepidochelys olivacea) on the Arribada Nesting Beach, La Escobilla, Mexico

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1 Evaluation of Sex Ratios of the Olive Ridley Sea Turtle (Lepidochelys olivacea) on the Arribada Nesting Beach, La Escobilla, Mexico Oscar E. Hernández-Echeagaray 1, Rubí Hernández-Cornejo 1, Martha Harfush-Meléndez 2 & Alejandra García-Gasca 1 1 Centro de Investigación en Alimentación y Desarrollo. Avenida Sábalo Cerritos s/n, Mazatlán, Sinaloa 82010, Mexico ( ohernandez@estudiantes.ciad.mx; alegar@ciad.mx); 2 Centro Mexicano de la Tortuga. Kilómetro 10 carretera Puerto Ángel San Antonio, Mazunte Santa María Tonameca, Oaxaca 70946, Mexico ( mharfush@conanp.gob.mx) Six species of sea turtles nest in Mexico, four of them on the Pacific coast (Chelonia mydas agassizii, Lepidochelys olivacea, Eretmochelys imbricata, and Dermochelys coriacea), and four species along the Gulf of Mexico and the Caribbean coasts (Lepidochelys kempii, Eretmochelys imbricata, Chelonia mydas, and Caretta caretta). C. caretta is also found in feeding areas on the Pacific coast (Márquez 1990, 1995). Historically, the exploitation of sea turtles, such as fishing and egg consumption, has negatively impacted natural populations. In 1927 the Mexican government prohibited egg consumption and nest destruction, and in 1986 created 17 natural reserves in order to protect nesting beaches. During 1990 the government permanently prohibited fishing or consumption of any sea turtle species nesting along the Mexican coast (Márquez 1995), and in 2002 all nesting beaches were declared Protected Natural Areas (sanctuaries). An important strategy for the recovery of sea turtle populations has been the installation and operation of the so-called turtle camps at several nesting beaches. There are 202 turtle camps for the six sea turtle species in Mexico; 28 are operated by the Ministry of the Environment and Natural Resources (SEMARNAT); eight of these are located on the Gulf of Mexico, three are on the Caribbean coast, and 17 exist along the Pacific coast (all camps on the Pacific coast are olive ridley nesting sites). An additional 174 turtle camps are operated under agreements of nongovernmental Figure 1. States in Mexico in which nesting beaches are located. The star indicates the arribada nesting beach at La Escobilla, in the state of Oaxaca. Marine Turtle Newsletter No. 133, Page 12 organizations, state government agencies, and research centers. Despite having protection status, sea turtles are potentially exposed to other environmental stressors that may affect natural populations. For instance, the temperature-dependent mechanism of sex determination (TSD) in sea turtles makes them particularly sensitive to environmental changes. Sea turtles lack sexually dimorphic sex chromosomes and the differentiation of gonads into ovaries or testes depends on the incubation temperature of the eggs during a critical period of embryonic development known as the thermo-sensitive period (TSP) (Pieau 1996; Wibbels 2003). The TSP has been defined as the time in which the incubation temperature affects sex ratio in embryos (Mrosovsky & Pieau 1991), or the time required to establish an irreversible molecular process that promotes sex differentiation (Merchant-Larios et al. 1997). Generally in sea turtles, lower incubation temperatures (25-28 C) produce males while higher temperatures (30-32 C) produce females (Wibbels 2003). The TSP occurs during the second third of the incubation period, and in the olive ridley, male-promoting temperature (MPT) under controlled conditions occurs at 26 ± 0.5 C whereas female-promoting temperature (FPT) occurs at 33 ± 0.1 C (Merchant-Larios et al. 1997, Torres-Maldonado & Merchant- Larios 2006). In the field, MPT and FPT have been estimated at and C respectively (Sandoval-Espinoza 2011). The pivotal temperature (temperature producing equal proportions of males and females) has been calculated for Mexican populations in the field at C (Sandoval-Espinoza 2011), however it may vary from one population to another, or even within the same population. The olive ridley sea turtle (L. olivacea) was selected for the present study because it is the most abundant of the six species nesting in Mexico. Although most populations are in recovery, this species is still classified as Vulnerable on the Red List of the International Union for the Conservation of Nature (IUCN 2011), and as an endangered species (NOM- 059-SEMARNAT-2001) within Mexico. Its main nesting areas are located in Oaxaca, although other nesting sites are found in Jalisco, Guerrero, Colima, Michoacán, Nayarit, Sinaloa, and Baja California Sur (Fig. 1). Nesting behavior in this species presents two different strategies, 1) solitary, which is the most common and involves 40 to 50 nesting females on the beach (Márquez 1990), and 2) "arribadas,"

2 Figure 2. Histological sections of male (A) and female (B) gonads of L. olivacea differentiated embryos and hatchlings. Medullar cords (Mc) in male gonads and a thickened cortex (Co) in female gonads were used as distinctive characteristics for sex identification. Hematoxylin-Eosin staining. Scale bar = 100 µm. characterized by a large number of females nesting synchronously on the beach (Valverde & Gates 1999). We selected the arribada nesting beach La Escobilla, located in the state of Oaxaca (Fig. 1) to estimate sex ratios of the olive ridley for several reasons: 1) it is a high-density nesting beach with more than 200,000 nests per year, 2) most nests are left to develop in situ, 3) there are no previous records of sex ratios at this location, 4) natural mortality levels provide specimens without sacrifice, and 5) there is a genuine interest in monitoring sex ratios due to the potential effects of climate change. Little information is available regarding sex ratios in the olive ridley. It has been reported that biased hatchling sex ratios are common in species with TSD (Johnston et al. 1995), however, while some variability in sex ratios is expected in natural populations, a sex ratio of 4:1 (females-males) may be disadvantageous because of the feminizing effect on the population, which may affect fecundity (females would lay unfertilized eggs), and therefore viability (Márquez & Jiménez 2010). Nevertheless, multiple paternity has been documented in olive ridleys (Jensen et al. 2006); this may eventually attenuate this feminizing effect. Sandoval-Espinoza (2011) used an indirect method (temperature of the nest) to estimate hatchling sex ratios of the olive ridley on several nesting beaches from the Pacific coast of Mexico (La Escobilla was not included), and found female-biased sex ratios in most locations at the beginning of the nesting season, and male-biased sex ratios at the end of the season. Overall hatchling sex ratios were female-biased. Thus, temperature is a crucial factor for sex determination, yet global warming may affect not only sex ratios, but also reproduction and nesting by modifying environmental conditions in foraging areas, prey availability, and time of migration to breeding sites (Hawkes et al. 2009; Márquez & Jiménez 2010; Mitchell & Janzen 2010; Wapstra et al. 2009; Zhang et al. 2009). Sea turtles do not show dimorphic sexual characters until adulthood, therefore to identify sex in hatchlings, non-lethal methods have been attempted; e.g., metabolites (such as steroid hormones) have been measured from chorio-allantoic fluid or blood (Xia et al. 2011), however in some cases differences between metabolites are not significant, making it difficult to identify sex (Merchant-Larios 1999). Laparoscopy has also been used (Wyneken et al. 2007) but hatchlings must be kept in captivity until they are large enough to survive the procedure, and this is not always possible. Marine Turtle Newsletter No. 133, Page 13 Figure 3. PCR amplification with oligo C6: A) RAPD-PCR; B) MSRF-PCR; C) sodium bisulfite-treated DNA-coupled with RAPD-PCR. DNA was isolated from the skin from ten female (left 1-10) and ten male (right 1-10) hatchlings. M indicates the molecular weight marker (100 bp ladder from Axygen). Lethal techniques include gonad clearing (van der Heiden et al. 1985), urogenital tract morphology (Ceriani & Wyneken 2008), and histology, which requires the dissection of the adrenal-kidney-gonad (AKG) complex to observe distinctive characteristics to identify sex, such as the thickness of the surface epithelium (cortex) in female gonads, and the formation of medullar cords in male gonads (Fig. 2). Histology is the most accurate method for identifying sex in sea turtle hatchlings but it requires sacrifice. One purpose of this study was to develop a non-lethal technique to identify sex in olive ridley hatchlings using molecular markers. Molecular methods to identify sex have been used successfully in birds and mammals (in which sex is established at fertilization by genes present in sex chromosomes) but not in reptile species with TSD. There is one report of a potential sex marker in sea turtles in Oligo RAPD-PCR MSRF-PCR Sodium bisulfite treatment coupled with PCR A * B * B * C * M * Table 1. Genetic similarity coefficients between male and female hatchlings using three different PCR-based strategies and five selected random primers. * indicates low genetic similarity between individuals rather than genders

3 Figure 4. Sex ratios of L. olivacea per group (A) and overall (B) during summer 2010 and winter at La Escobilla beach, Oaxaca. Light grey represents females, dark grey represents males. E: embryos that did not hatch; H1: hatchlings that died in the nest; H2: hatchlings that died after emergence; n: samples size. Column labels indicate percentage of females in each case. which a probe for the non-coding satellite DNA Bkm (banded krait minor) showed sex-specific bands by Southern blot in C. mydas and L. kempi (Demas et al. 1990); nevertheless these results have not been reproduced to date. We developed a PCR-based strategy; DNA was isolated from the skin (and vitelline cord when possible) of male and female hatchlings (previously sexed by histology). We selected these tissues because they are easy to sample and minimally invasive. We collected dead-in-nest hatchlings (n = 20) and differentiated embryos (n = 20) that died of natural causes and failed to emerge. Genomic DNA of 20 males and 20 females was amplified using different methods: 1) by random amplification of polymorphic DNA (RAPD-PCR) (Griffiths & Tiwari 1993), 2) by methylationsensitive restriction fragment analysis (MSRF-PCR) using the methylation-sensitive restriction enzyme BstUI (Davies 2002), and 3) alkaline deamination with sodium bisulfite, using the EZ DNA Methylation kit (ZIMO Research) following the manufacturer s instructions. Methods 2 and 3 were performed to determine if random methylation patterns were different between males and females. In method 2, BstUI is blocked when DNA is methylated, therefore the enzyme will cut only unmethylated DNA. In method 3, unmethylated cytosines will be converted into uracil whereas methylated cytosines remain unchanged; if methylation is sexspecific, banding patterns will be different in males and females after random PCR amplification. Marine Turtle Newsletter No. 133, Page 14 Figure 5. Estimated sand temperatures from the summer nesting season 2010 (A) and two overlapping nesting events during winter (B) in Puerto Angel, Oaxaca, located 25 km (15.5 miles) from La Escobilla. Incubation days are indicated from day 0 (nesting day) to the day of hatching, the square indicates the thermosensitive period. T: mean temperatures, TM: maximum temperatures, Tm: minimum temperatures. Pivotal temperature is around 30 C. In all cases, DNA was amplified with 80 random primers (Operon A to D kits) and four additional oligos suggested by Davies (2002) named M1 to M4. Genetic similarity was calculated according to Bártfai et al. (2003) with the following equation: GS ij = 2N ij /N i +N j where GS ij is the genetic similarity coefficient, N ij are the number of bands present in both males and females, N i are the number of bands present in females, and N j are the number of bands present in males. With these strategies we were not able to find male or femalespecific bands (see Fig. 3 showing oligo C6 as an example, and Table 1 showing the genetic similarity coefficient of samples amplified with five selected oligos), indicating that in the olive ridley sex-specific genetic or epigenetic markers may be absent. With no molecular marker available, we used conventional histology to identify sex in embryos and hatchlings in order to estimate sex ratios for two seasons: summer (incubation period: 30 July to 13 September 2010, n = 219) and winter (two overlapping arribadas, incubation period: 25 November 2010 to 10 February 2011, n = 240). Samples were divided into three groups: 1) embryos that did not hatch (E), 2) hatchlings that died in the nest (H1), and 3) hatchlings that died after emergence (H2). Sex ratios were estimated in all five groups (summer, winter, E, H1, and H2), and a multiple comparison

4 test using a χ 2 (Canal-Díaz 2006) was performed to detect significant differences between groups. No statistically significant differences in sex ratios were found between embryos and hatchlings, or between seasons (χ 2 = 17.25, P = 0.06; Fig. 4A). However, slightly female-biased sex ratios were observed in summer (61% females), whereas no bias was observed in winter (51% females). The overall female ratio was 55% (Fig. 4B). Our summer results are consistent with those from Espinoza- Sandoval (2011) for L. olivacea in Mexico who used an indirect method (environmental and nest temperatures) to estimate sex ratios, finding 69% females during the 2008 and 2010 nesting seasons in Nayarit and Sinaloa respectively. As 2010 experienced a La Niña climatic event (cooler temperatures), we decided to compare the temperatures recorded in the nesting seasons for 2009 and 2010, as warmer temperatures would be expected to produce an increase in females within the sex ratio. We were unable to get temperature records from La Escobilla beach, and therefore we used the air temperature records provided by Tutiempo Network ( from the closest beach of Puerto Ángel (located 25 km (15.5 miles) from La Escobilla). We estimated sand temperatures from air temperatures using the methods of Hays et al. (2003). Summer and winter estimated mean sand temperatures fluctuated around the pivotal temperature range of olive ridleys in Mexico (28-32 C and C respectively) during the TSP (Fig. 5). In summer 2010 (Fig. 5A), minimum temperatures (27.26 ± 2.7 C) during the TSP were slightly above the MPT, and maximum temperatures (32.96 ± 3.85 C) were close to the FPT. The estimated mean sand temperature decline from July- September 2009 to the same period in 2010 was -1.7 C, indicating that 2010 was indeed cooler than In winter (Fig. 5B), maximum temperatures (33.83 ± 0.92 C) were above the FPT, whereas minimum temperatures (25.35 ± 0.97 C) were below the MPT. The estimated mean sand temperature increment was 0.07 C from November February 2010 in comparison with the same period in Temperature data explained (at least in part) why in Oaxaca the nesting season is longer than in other locations as sand temperature is quite stable throughout the year, so embryonic development and hatching take place under favorable conditions. Nevertheless, the sex ratio was female-biased in summer 2010 despite cooler temperatures compared to This may be the result of temperature fluctuations during the TSP, which were close to the FPT. These results do not necessarily reflect a negative effect of global warming, since the overall sex ratio during the season was slightly female-biased (55%) and, according to Márquez & Jiménez (2010) is not even close to a population-feminizing proportion. Nevertheless, this is only a one-year study. To obtain more accurate data regarding sex ratio tendency in this beach, it is necessary to sample several years [as discussed by Mrosovsky & Godfrey (2010)], and to record sand temperatures during the nesting season (planned for future studies) to create a database that will allow us to predict sex ratios through time (population modeling), taking into account climatic events such as El Niño, La Niña, and contemporary climate change. Acknowledgements: A collecting permit was obtained from the Wild Life Department of the Ministry of the Environment in Mexico (DGVS, SEMARNAT) to conduct this work. We would like to thank the staff from Centro Mexicano de la Tortuga for the help provided in sample collection, to Selene Abad-Rosales for technical assistance, to Miguel Betancourt- Marine Turtle Newsletter No. 133, Page 15 Lozano, Itzel Sifuentes-Romero, and Bruno Gómez-Gil for their valuable comments on this work. BÁRTFAI, R., S. EGEDI, G. HUA-YUE, B. KOVÁCS, B. URBÁNYI, G. TAMÁS, L. HORVÁTH & L. ORBÁN Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers. Aquaculture 219: CANAL-DÍAZ, N Sociedad Española de Enfermeria Nefrológica, in 2&Datapageid=4&intInicio=1 Ceriani, S.A. & J. Wyneken Comparative morphology and sex identification of the reproductive system in formalin preserved sea turtle specimens. Zoology 111: DAVIES, C.S Methylation-sensitive restriction fingerprinting. In: K.I. Mills & B.H. Ramsahoye (Eds.). DNA Methylation Protocols. Springer, New York, pp Demas, S., M. Duronslet, S. Wachtel, C. Caillouet & D. Nakamura Sex-specific DNA in reptiles with temperature sex determination. The Journal of Experimental Zoology 253: GRIFFITHS, R. & B. TIWARI The isolation of molecular markers for the identification of sex. Proceedings of the National Academy of Science, USA 90: HAWKES, L.A., A.C. BRODERICK, M.H. GODFREY & B.J. GODLEY Climate change and marine turtles. Endangered Species Research 7: HAYS, G.C., A.C. BRODERICK, F. GLEN & B.J. GODLEY Climate change and sea turtles: a 150-year reconstruction of incubation temepratures at a major marine turtle rookery. Global Change Biology 9: INTERNATIONAL UNION FOR CONSERVATION OF NATURE (IUCN) The IUCN Red List of Threatened Species. JENSEN, m.p., f.a. ABREU-GROBOIS, j. FRYDENBERG & v. LOESCHCKE Microsatellites provide insight into contrasting mating patterns in arribada vs. non-arribada olive ridley sea turtle rookeries. Molecular Ecology 15: Johnston, C.M., M. Barnett & P.T. Sharpe The molecular biology of temperature-dependent sex determination. Philosophical Transactions: Biological Sciences 350: MÁRQUEZ, M.R Sea Turtles of the World. FAO Fisheries Synopsis. FAO, Rome. pp MÁRQUEZ, R Tortugas Marinas. In: W. Fischer, F. Krupp, W. Scheneider, C. Sommer, K.E. Carpenter & V.H. Niem (Eds.). Guía FAO Para la Identificación de Especies Para los Fines de Pesca. Pacífico Centro-Oriental 3: MÁRQUEZ, R. & M.D. JIMÉNEZ El posible efecto del cambio climático en las tortugas marinas. In: A.V. Botello, S. Villanueva, J. Gutierrez & J.L. Rojas-Galaviz (Eds.). Vulnerabilidad de las Zonas Costeras Mexicanas Ante el Cambio Climatico. Universidad Autónoma de Campeche, Mexico. pp MERCHANT-LARIOS, H Determining hatchling sex. In: K.L. Eckert, K.A. Bjorndal, F.A. Abreu-Grobois & M. Donnelly (Eds). Research and Management Techniques for the Conservation of Sea Turtles. IUCN/SSC Marine Turtle Specialist Group. pp MERCHANT-LARIOS, H., S. RUIZ-RAMÍREZ, N. MORENO- MENDOZA & A. MARMOLEJO-VALENCIA Correlation among thermosensitive period, estradiol response, and gonad differentiation in the sea turtle Lepidochelys olivacea. General and Comparative Endocrinology 107: MITCHELL, N.J. & F.J. JANZEN Temperature-dependent sex determination and contemporary climate change. Sexual Development 4:

5 MROSOVSKY, N. & M.H. GODFREY Thoughts on climate change and sex ratio of sea turtles. Marine Turtle Newsletter 128: MROSOVSKY, N. & C. PIEAU Transitional range of temperature, pivotal temperatures and thermosensitive stages for sex determination in reptiles. Amphibia-Reptilia 12: PIEAU, C Temperature variation and sex determination in reptiles. BioEssays 18: Sandoval-Espinoza, S Proporción sexual en crías de tortuga Lepidochelys olivacea en corrales de incubación del Pacífico Mexicano. Doctoral Thesis. Centro Interdisciplinario de Ciencias Marinas, IPN. 80 pp. TORRES-MALDONADO, L.C. & H. MERCHANT-LARIOS Aspectos moleculares de la determinación del sexo en tortugas. Ciencia Ergo Sum 13: Valverde, R.C. & C.A. Gates Estudios de poblaciones en playas de arribadas. In: K.L. Eckert, K.A. Bjorndal, F.A. Abreu-Grobois & M. Donnelly (Eds). Research and Management Techniques for the Conservation of Sea Turtles. IUCN/SSC Marine Turtle Specialist Group. pp van der Heiden, A.M., R. Briseño-Dueñas & D. Rios- Olmeda A simplified method for determining sex in hatchling sea turtles. Copeia 3: WAPSTRA, E., T. ULLER, D.L. SINN, M. OLSSON, K. MAZUREK, J. JOSS & R. SHINE Climate effects on offspring sex ratio in a viviparous lizard. Journal of Animal Ecology 78: WIBBELS, T Critical approaches to sex determination turtles. In: P.L. Lutz, J.A. Musick, & J. Wyneken (Eds). The Biology of Sea Turtles, Vol. 2. CRC Press Inc., Boca Raton Florida, pp WYNEKEN, J., S.P. EPPERLY, L.B. CROWDER, J. VAUGHAN & K.B. ESPER Determining sex in posthatchling loggerhead sea turtles using multiple gonadal and accessory duct characteristics. Herpetologica 63: XIA, Z.R., P.P. LI, H.X. GU, J.J. FONG & E.M. ZHAO Evaluating noninvasive methods of sex identification in green sea turtle (Chelonia mydas) hatchlings. Chelonian Conservation and Biology 10: ZHANG, F., L.Z. GUO & B.R. MURRAY Climate warming and reproduction in Chinese alligators. Animal Conservation 12: Marine Turtle Newsletter No. 133, Page 16