Temperature-dependent Sex Determination of a Sea Turde, Caretta ...

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148 Shoji Tokunaga, Yuji Iwakiri and Yoshito Nakajima 1991). For sea turdes, high temperatures result in females and low temperatures result in males. The temperature which gives 50% of each sex is called the critical temperature (Pieau, 1976), the pivotal temperature (Mrosovsky and Yntema, 1980), the threshold temperature (Bull, 1980), or SDT50 (Limpus et al, 1985). In this paper, the critical temperature was used to show the temperature giving 50% of each sex, because this was the term first used to represent that temperature. There have been some studies on TSD of Caretta caretta in the U.S.A. (Yntema and Mrosovsky, 1980; Yntema and Mrosovsky, 1982; Mrosovsky, 1988) and Australia (Limpus etal, 1985), but only a few reports are available on TSD inJapanese populations of C. caretta and these were written in Japanese and published in a local journal. Tokunaga (1991) first reported sex determination ofJapanese sea turtles under constant temperatures. Asa preliminary report ofthis study, he reported that nine hatched loggerheads from those incubated at 31°C (no correction for evapora tive cooling) were females. Kamezaki and Kuroyanagi (1991) incubated one clutch ofeggs from Atsumi, Aichi,Japan and reported that the sex ratio (number of hatched turtles, hatching rate) of hatched turdes was 1.0 (34, 0.87) at 26°C, 1.0 (35, 0.90) at 28°C and 0.0 (37,0.95) at 30°C. He concluded that the critical temperature of the clutch of C. caretta exists between 28 and 30°C. This paper deals with TSD of C. caretta from Miyazaki, Japan. The critical temperature of the loggerhead from Miyazaki and other localities in Japan, as well, has not been identified so far. This study also intended to examine sex determina tion underfluctuating temperature. The possible influence ofTSD on the hatchling sex ratio is discussed. Materials and methods Eggs were collected from Miyazaki,Japan. The geography and the turtle nest ing habitat of the beach have been described by Iwamoto etal (1985). Experiments were conducted at the Laboratory of Ecology, Faculty of Science, Kyushu University, in 1989. Three clutches were used for the experiments. Each clutch was removed from its nest by YN after the turtle completed laying and was packed in a Styrofoam box for transport. The eggs were sent to the laboratory by commercial truck delivery with no attempt to control the temperature of the eggs. The number of eggs, date of oviposition and the date of starting incubation ofeach clutch were 127,June 30 and July 4 for the clutch A; 135,July 2 and July 7 for the clutch B; 138,July 2 andJuly 7 for the clutch C. Each clutch was divided into three to five groups (see Table 1). Each group of eggswasincubated in a plasticcontainer (approx. 17 x 31 cm on the bottom) in two layers and the bottom of the container was coveredwithwet vermiculite. A layerof eggs, usually 28 eggs, was set on the vermiculite and covered with a mesh to keep air spacebetween the eggs. Wetvermiculite was put on the mesh,and the secondlayer
Sex determination of a sea turtle 149 of eggs was set, covered with a mesh and a third layer of wet vermiculite. Each plastic containerwas covered with a sheet ofaluminium foil with holes for ventilation. The eggs were incubated in SANYO MIR-150E incubators or Shimadzu BITEC 300 Low-Temp incubators. The air in each incubator was constandy circulated by an interior fan to prevent a temperature gradient. The temperature of each incuba tor wasmonitored dailywith an alcohol thermometer approximately at the middle of the incubator air space. The thermometers had been calibrated againsta standard thermometer before the experiment. Temperature was also monitored by the standardthermometeroncea week. It was confirmed that temperaturedid not vary excessively from the designed temperature by daily reading maximum-minimum thermometer which were set in each incubator. The preliminary experiment showed that the temperature in the wetvermiculite was cooler than the air tempera ture by 0.3°C due to evaporative cooling. Therefore the incubation temperatures were corrected by 0.3°C from the air temperature inside the incubators. The incubation at fluctuating temperature was carried out using a program mable BITEC 300 incubator. The temperature was fluctuated from 27.7 for 12 hours to 30.7°C for 12 hours daily. In the temperature shift experiment, the eggs were incubated at 27.7°C for 19 days and then shifted to 30.7°C for the remainder of the incubation period. Adish ofwater was setin eachincubator to moisten the air. Water was sprayed on the vermiculite when signs of desiccation were observed on the surface of the vermiculite. The humidity of each incubator was monitored daily with a simple psychrometer. The humidity was always >80%, usually >90%. No major differences were detected between incubators. The dateof hatching was defined asthe day when the whole body emerged out side the egg. Hatchlings were killed by injection of about lOmg of Pentobarbital Sodium Salt and fixed in 10% buffered formalin solution after the body cavity was opened. The left gonad, oviductand attached kidneywere removed. The tissues were embedded in Paraplast and cut in 4-8 /xm sections which were stained with haematoxylin and eosinor haematoxylin and the periodicacid-Schiff reaction (PAS); the latter gave better results. All specimens were microscopically sexed using the following histological criteria. Withmalesthe medullawas differentiated into seminiferous tubules,while the cortex was reduced. With females the cortex was well developed and the medulla showed no development of seminiferous tubules. Following sexing of a portion of the specimens byST, mostspecimens were sexed byYI. YI reexamined the sexing ofall specimens at least twice. Statistical calculations were conducted by SAS, SAS Institute (Cary, NC) at the Computer Center, Kyushu University, except the Fisher's exact test and 95% confi dence intervals oftheproportion which were calculated by Stata (Stata Corp.). The level of statistical significance (a) is 0.05.
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