Mating Season, Proportion of Reproductive Females, and Sexual Maturity of the Habu, Trimeresurus flavoviridis

During April 1984 to June 1985, body size, presence of spermatozoa in cloaca. and pregnancy were investigated in 128 female and 198 male habus, Trimeresurus flavoviridis. The mating season of the habu was estimated to be late March to mid-June, especially April and May. The proportion of reproductive females among fully mature females (greater than 110cm in snout-vent length) was 78%. It is quite possible that most fully mature female habus reproduce annually. Although some female habus matured at two years of age (greater than 90-100cm size class in SVL), most females matured at three years of age. Male habus seemed to mature at one year of age (greater than 80cm in SVL). Spermatozoa were also detected from the cloaca of 2 non-reproductive females. Much work has been done on the reproduction of the habu, Trimeresurus flavoviridis (Takara, 1962; Koba et al., 1967; Koba et al., 1970; Nishimura et al., 1983: for more detailed information, see review by Koba, 1971; habu seminar, 1983), but still little is known about the mating season, proportion of reproductive females, and sexual maturity of the habu. Fukada (1959) and Fitch (1960) identified the copulated female snakes by detecting the spermatozoa in their cloaca. In this study, mating season, proportion of reproductive females, and sexual maturity of habu were investigated by identifing the copulated females and gravid females using a technique similar to that of Fukada (1959) and Fitch (1960). MATERIALS AND METHODS One hundred and twenty-eight females and 198 males were examined during the period from May, 1984, to June, 1985 (Table 1 and 4). Most of the specimens (79, 121) collected at the southern area of Okinawa Island were bought from a habn dealer. Two males were collected ou Tokashiki Island, 30km off the west coast of Okinawa Island, and others were collected or trapped in the central and northern areas of Okinawa Island. Snout-vent length (SVL in cm), tail length (cm) and body weight (g) were measured on each specimen. After that, about 2ml of physiological saline was introduced into the cloaca of the specimen using a syringe, and then the saline was deposited on a slide glass by pressing the cloacal region. The deposited fluid (about 0.5ml) was examined for the presence of spermatozoa at 400 power magnification with a microscope. Sex of the specimens was determined by the shape of the cloaca. In the indistinguishable cases, sex was confirmed by inserting a probe into the invaginated hemipenis. In the preoviposition season (May and June), gravid females can be easily identified by their distended abdomens. The females which seemed to be gravid were kept in individual cages in order to confirm the egg-laying. The obtained habus were examined as soon as possible. But the habus bought from the habu dealer were usually kept for several weeks at the dealer until he got enough to sell (15-30 habus). Therefore, those habus might have been examined 1 to 21 days after they were caught in the field. When term of keeping extended over two months, all the speciAccepted 13, Jul. 1987 SHIROMA & KAMURA-HABU REPRODUCTION 17 TABLE 1. Seasonal change in the proportion of females with spermatozoa in the cloaca and gravid females. Size of specimens ranged from 81 to 148cm in SVL. TABLE 2. The proportion of females with spermatozoa in the cloaca (April to July) and gravid females (May and June) in each size class. mens were regarded to be collected in the month that included more days (Table 1). For example, 4 females which were collected during the period from 19th April to 9th May were regarded as collected in April. The specimens less than 80cm (SVL) were excluded from the estimation of mating season because they were immature (Koba et al., 1970; Nishimura and Kamura, 1984). RESULTS Monthly difference in the proportion of the females with spermatozoa in their cloaca and of gravid females is shown in Table 1. Females with spermatozoa in their cloaca appeared during the period from April to July with the highest peak of 26% in May. Gravid females were identified from April to July, with the highest peak of 50% in June. Gravid females with spermatozoa in their cloaca were recognized from April to June (Table 1). The proportion of gravid females with spermatozoa in May was high (83%), but dropped remarkably in June (10%). Active spermatozoa were detected in the cloaca of 5 nonreproductive females (Table 1). The proportions of females with spermatozoa in their cloaca (April to July) 18 Jpn. J. Herpetol. 12 (1). 1987 and gravid females in each size class in the preoviposition season (May to June) is shown in Table 2. Spermatozoa were found in the size classes greater than 9099cm (SVL). Spermatozoa were detected from 13% of the females collected during April to July. Even in the period from April to July, gravid females were not recognized in the size class less than 99cm (SVL). The mean proportion of gravid females larger than 110cm (SVL) was 78%. Compared to the larger class, the proportion of gravid females of the 100-109cm (SVL) size class was extremely low (15%). In the oviposition season, significant difference in mean weightlength coefficient (g/cm3) between reproductive and nonreproductive females was recognized (Table 3). In the case of males, spermatozoa were detected in their cloaca throughout the year, except in January and February when only a few specimens were examined (Table 4). The proportion of males with spermatozoa somewhat decreased from April to June. But it is difficult to estimate the mating season from the investigation. The proportion of males with sperm in their cloaca for each size class is shown in Table 5. Spermatozoa were detected from the size classes greater than 80-89cm (SVL). Therefore, male habus of the 80-89cm size class are already mature. DISCUSSION Fukada (1959) and Fitch (1960) indicated that active sperm of some snake species could remain for several weeks in their cloaca, and that the proportion of dead or not fully active sperm cells rapidly increased with the passage of time. One female habu collected at Chatan town on 11th of April, seemed to have just finished copulation, because its cloacal region was still swollen, and about 5 active spermatozoa were seen per microscopic field. Six days later, the number of active spermatozoa decreased to about TABLE 3. T-test of coefficient of fatness (W/L3) between gravid and non-gravid females (greater than 110cm in SVL) during May and June. TABLE 4. Seasonal change in the proportion of males with spermatozoa in the cloaca. TABLE 5. Proportion of males with spermatozoa in the cloaca in each size classes. 0.5 per microscopic field, and the swelling of the cloacal region had disappeared. After 52 days, active spermatozoa could not be seen, and only five dead spermatozoa were observed within the entire area of the cover glass (24mm×32mm). This indicates the posibility that active spermatozoa in the cloaca of female SHIROMA & KAMURA-HABU REPRODUCTION 19 habus can survive at least 1 week in the cloaca and that dead ones may remain until two months later. The presence of spermatozoa in the cloaca of female habus, therefore, does not imply directly that the female mated just before examination. In this study, a female collected in the period from the 25th of June to the 15th of July contained only a few dead sperm in her cloaca. It seems that she had mated several weeks or months ago. Nishimura et al (1983) reported five cases of mating of habu in the field. The earlist and latest observations were the 28th of March and the 21th of May, respectively. The percentage of spermbearing females in gravid females in June was extremely low compared to May (Table 1). It seems that ovulation of habu occurs during the period from late May to the middle of June (Shiroma, in preparation). These observations suggest the possibility that most females containing sperm in the cloaca in June had mated in an earlier month. We, therefore, conclude that mating of habu occurs mainly in April and May and sometimes in late March and early June. Some snakes in temperate and subtropical zones have two mating seasons, usually spring and fall (Fukada, 1972; Saint-Girons, 1982). There has been no observation of mating of habu in fall (Nishimura et al., 1983), and we also could not find females with spermatozoa in the cloaca in fall. Although the number of specimens examined in fall was not sufficient to drow a firm conclusion, it is quite possible that habus mate in spring only. Koba (1962) reported two reproductive females that were 105.7cm and 108.9cm in SVL, respectively. In this study, the smallest reproductive female was 100cm in SVL. Nishimura and Kamura (1984) reported reproductive female habus of 93, 99 and 95cm in SVL. Kamura and Shimamura (1982) reported the growth record of habus. The mean total length of 1-year-old habus was 76.1cm (SD= 7.0cm) and the mean female tail length was 15.0cm; for 2-year-olds, the values were 110.6cm (SD=11.6cm) and 17.1 cm, for 3-year-olds 131.7cm (SD=10.5 cm) and 19.7cm, and for 4-year-olds 145.4cm (SD=10.8cm) and 21.1cm, respectively. According to this growth record, it seems impossible that in the fourth reproductive season the body size of a 3-year-old female should be less than 109cm in SVL. Also few 1-year-old female habus will grow greater than 90cm (SVL) in their second reproductive season (May and June). Most 90-109cm size class female habus are, therefore, 2year-old individuals. Therefore, it is quite possible that 2-year-old female habus can reproduce in their third reproductive season. But the percentage of reproductive females of the 100-109cm (SVL) class is very low (15%). We, therefore, conclude that some female habus reproduce in their third reproductive season, but most other females in their fourth reproductive season. The size of five sperm-containing male habus (83, 88, 86, 81, and 82cm in SVL) which were collected from July to December coincides with the body size