Japanese Journal of Herpetology 16(2): 42-48., Dec. 1995 (C)1995 by The HerpetologicalSociety of Japan

Mating Season of the Japanese Mamushi, blomhoffii blomhoffii (: Crotalinae), in Southern Kyushu, Japan: Relation with Female Ovarian Development

KIYOSHI ISOGAWA AND MASAHIDE KATO

Abstract: Mating in the southern Kyushu population of the Japanese mamushi, Agkistrodon blomhoffii blomhoffii, occurred in August and September. The largest ovarian follicles of mated females were 6mm or more in length, whereas those of unmated females were usually less than 6mm in length. In addition, body mass was significantly greater in mated females than in unmated ones. These suggest that in females of the Japanese mamushi, mating is strongly associated with ovarian follicle development and body mass. Key words: Mamushi; Agkistrodon blomhoffii blomhoffii; Mating season; Follicle development; Sexual receptivity

Many reports have been published on the the southern Kyushu, Japan and to examine the mating season of of the genus relation of the occurrence of mating with Agkistrodon (Allen and Swindell, 1948; Behler ovarian development, body size, and nutritional and King, 1979; Burchfield, 1982; Fitch, 1960; conditions in females. Gloyd, 1934; Huang et al., 1990; Laszlo, 1979; Li et al., 1992; Wright and Wright, 1957). MATERIALSAND METHODS Fukada (1972) surmised that mating in the . -Forty-five mature females and the Japanese mamushi, Agkistrodon blomhoffii same number of mature males of Agkistrodon blomhoffii, as well as in many of the colubrids of blomhoffii blomhoffii were used. All were cap- Japan, occurs in May and June. No data, tured in Kagoshima Prefecture, Japan during however, have been obtained for the mating the summer of 1989. Until the beginning of the season of the Japanese mamushi. experiment, the females and males were kept In snakes, male courtship is generally evoked separately from the other sex in outdoor by an attractiveness stimulus from a female, and enclosures. Mice were supplied as food. In mating occurs when the female is sexually recep- advance of the experiment, the posterior ends tive (Garstka and Crews, 1982; Ross and Crews, of the female reproductive tracts were examined 1977; Whittier and Crews, 1986). Thus, it seems to confirm the absence of spermatozoa by the to be possible to define female sexual attractivity smear method (Gloyd, 1934; Rahn, 1940), by the occurrence of male courtship, and female because spermatozoa deposited in females of A. sexual receptivity by the incidence of actual b. blomhoffii through copulation are stored in copulation (Crews, 1976; Whittier and Tokarz, these regions (Isogawa, 1986). 1992). In addition, it has been reported that in Experiment and housing. -The experiment female snakes, sexual attractivity seems to be was conducted from 10 April to 20 November induced by heavy feeding which results in the 1990. At the beginning of the experiment, all increase of liver and fat-body masses (Garstka snakes were removed from the artificial hiber- and Crews, 1982; Macartney and Gregory, 1988), nacula where they had hibernated, and were and sexual receptivity seems to be dependent on separated into three breeding groups, each con- ovarian follicle development, which is related to sisting of 15 females and 15 males. Snakes in body size (Hawley and Aleksiuk, 1976). The each group were housed together in an outdoor purpose of the present study is to clarify the enclosure (5m x 3m), in which a shelter and a mating season of the Japanese mamushi from basian for water were provided and weeds were allowed to grow. As food, as many mice as Accepted 29, Oct. 1995 there were snakes were dropped into each ISOGAWA AND KATO-MATING OF MAMUSHI 43

TABLE 1. Number of female Japanese mamushi, Females which did not mate in the course of Agkistrodon b. blomhoffii, possessing spermatozoa the experiment were killed for control data at the in the posterior ends of the reproductive end of the experiment. Measurement data from tracts in each month during the three breeding groups were combined and the experiment. compared between mated and unmated females. Statistical analyses. -Differences between means of measurements in mated and unmated females were analyzed for statistical significance by Student's t-test when variances were homogeneous, and by Cochran t-test (Gad and Weil, 1982) when variances were heteroge- neous. Analysis of covariance (ANCOVA) was also applied using SVL as a covariate for body mass, and body mass for the other mea- surements.

RESULTS enclosure once a week throughtout the experi- Six females died during the experiment. At ment period. necropsy, no spermatozoa were observed in At the end of each month except October and these specimens. on the last day of the experiment, all females Number of females with spermatozoa. -Nine were examined for the presence or absence of females had numerous spermatozoa in the spermatozoa in the posterior ends of the posterior ends of their reproductive tracts on 31 reproductive tracts by the method described August, and 18 females on 30 September. These above. When a given female possessed sper- females were considered to have mated during matozoa (mated), the individual was either August and September, respectively. No other immediately isolated into an outdoor enclosure females possessed spermatozoa throughout the where it was kept until the late autumn of the study period (Table 1). following year for the observation of its parturi- Comparisons in measurements. -Four of the tion, or killed and measured for snout-vent females which mated in August and 11 in length (SVL; cm) and body mass (g). Liver and September were randomly selected and killed on abdominal fat-body masses (g) and the number 31 August and 30 September, respectively. and length of ovarian follicles of the killed Twelve females which did not mate throughout specimens were also measured as criteria for the experiment period were also killed on 20 nutritional and ovarian conditions, respective- November. Then the following comparisons ly. Measurements of the follicle length were were made between the 15 mated and 12 made to the nearest half-millimeter with a clear unmated females. plastic ruler. Data from both ovaries were Means of the measurements were compared combined for each specimen. Atretic follicles between the mated and unmated females using (Isogawa et al., 1989) were excluded from the t-tests (Table 2). SVL exhibited no significant data. difference between the mated and unmated

TABLE2. Comparisons of snout-vent length, body, liver, and fat-body masses, length of the largest follicle, and the total number of follicles between mated and unmated females of the Japanese mamushi, Agkistrodon b. blomhoffii. Data are presented as mean±standard deviation. n. s.: not significant at p=0.05. 44 Jpn. J. Herpetol. 16 (2). 1995

TABLE 3. Analysis of covariance (ANCOVA) of body mass on SVL and of liver and fat-body masses, length of the largest follicle, and the total number of follicles on body mass between mated (N=15) and unmated females (N=12) of the Japanese mamushi, Agkistrodon b. blomhoffii. Values in table are F- ratios.

FIG. 1. Relationship between snout-vent length (SVL) and body mass in mated (N=15, closed circles) and unmated females (N=12, open circles) of the females. The other measurements were Japanese mamushi, Agkistrodon b. blomhoffii. significantly greater in the mated females than in Regression lines are shown by solid (mated females) the unmated ones. and broken lines (unmated females). ANCOVA show- The results of ANCOVA are shown in Table 3 ed significant difference in elevation, but not in slope and Figs. 1 and 2. Regression equation, correla- (Table 3). tion coefficient, and probability for each com- bination in the mated and unmated females were Reproduction of mated females in the subse- as follows: SVL vs. body mass (Fig. 1), mated: quent year. -The 12 females which had mated in Y=4.82X-123.99, r=0.84, p<0.001; unmated: August and September were housed in an out- Y=2.07X-17.17, r=0.47, p>0.05. Body mass door enclosure until late autumn of the follow- vs. liver mass (Fig. 2A), mated: Y= ing year. Of these, one died soon after 0.03X+1.96, r=0.64, p<0.05; unmated: Y= emergence from hibernation, and two did not 0.05X-0.31, r=0.73, p<0.01. Body mass vs. reproduce in 1991. The remaining nine gave fat-body mass (Fig. 2B), mated: Y=0.13X- birth to 5-13 (x=7.9) young and four of them 1.96, r=0.67, p<0.01; unmated: Y=0.10X- also produced one or two (five in total) stillborn 0.77, r=0.43, p>0.05. Body mass vs. length young and two to five (13 in total) unfertile eggs of the largest follicle (Fig. 2C), mated: in the period of 27 August through 20 September Y=0.02X+5.17, r=0.40, p>0.05; unmated: 1991. Y=0.04X+1.48, r=0.74, p<0.01. Body mass vs. the total number of follicles (Fig. 2D), DISCUSSION mated: Y=0.23X+0.67, r=0.47, p>0.05; Mating season. -Marzec (1980) observed unmated: Y=0.02X+14.84, r=0.06, p>0.05. copulation of A. b. blomhoffii on 7 June 1964. There were no significant differences in slopes However, his specimens, the locality of which of any combinations between the mated and was not described, had been kept under com- unmated females. Elevations of SVL vs. body pletely artificial conditions all year round. In mass and body mass vs. length of the largest the present study, females possessing sper- follicle differed significantly between the mated matozoa in the posterior ends of the reproduc- and unmated females, whereas those of the other tive tracts were found only in August and three combinations did not differ significantly September. Considering that all specimens used (Table 3). That is, the adjusted values of body in the present study were housed in outdoor mass and length of the largest follicle were enclosures throughout the experiment period, it significantly greater in the mated females than in is reasonably assumed that mating in the the unmated ones, whereas those of liver and southern Kyushu population of the Japanese fat-body masses and the total number of follicles mamushi occurs in August and September. exhibited no significant differences between the Occurrence of summer-autumn mating is mated and unmated females. known in other Agkistrodon such as A. The largest follicles in the mated females were bilineatus (Burchfield, 1982; Laszlo, 1979), A. 6mm or longer, whereas those in the unmated piscivorus (Allen and Swindell, 1948; Wright females were shorter than 6mm except one folli- and Wright, 1957), and A. shedaoensis (Jiang cle (6.0mm in length, Table 4). and Zhao, 1980; Zhao, 1980). On the other ISOGAWA AND KATO-MATING OF MAMUSHI 45

FIG. 2. Relationships between body mass and liver mass (A), fat-body mass (B), length of the largest follicle (C), and the total number of follicles (D) in mated (N=15, closed circles) and unmated females (N=12, open circles) of the Japanese mamushi, Agkistrodon b. blomhoffii. Regression lines are shown by solid (mated females) and broken lines (unmated females). ANCOVA showed significant difference in elevation only in Fig. 2C, but not in slope in any combinations (Table 3). hand, mating in A. contortrix (Gloyd and Con- has been reported that A. ussuriensis from Jilin, ant, 1990) and A. blomhoffii brevicaudus from China mates mainly in late May to early June, Zhejiang, China (Huang et al., 1990) is common occasionally in July to August (Li et al., 1992). in spring with a occasional occurrence in The mating season of snakes of the genus autumn. Richard C. Goris (Gloyd and Conant, Agkistrodon seems to vary with the localities as 1990: 253) observed two matings of A. ussurien- well as the species. Thus, further study is sis (=A. caliginosus) from Korea in the outdoor necessary to ascertain whether the Japanese terraria in late August and deduced that mating mamushi mates also in spring or not. in this species occurs in autumn. However, it Relation between the occurrence of mating

TABLE4. Length distributions (as combined at one-millimeter intervals) of the largest ovarian follicle the total number of follicles in mated and unmated females of the Japanese mamushi, Agkistrodon b. blomhoffii. in each size class is also presented in parentheses. Data from both ovaries are combined for each . See Tables 2 and 3 for statistical significance. 46 Jpn. J. Herpetol. 16 (2). 1995 and the measurements. -Comparisons by t-tests also likely that males of the Japanese mamushi exhibited statistically significant differences in can visually or olfactorily distinguish females liver and fat-body masses between the mated and that will be able to reproduce in the following unmated females of the Japanese mumushi. year those that will not, as assumed for the rat- However, ANCOVAs have revealed no signifi- tlesnake, Crotalus viridis (Macartney and cant differences between them. These results Gregory, 1988) and the garter snake, Tham- indicate that the larger masses of liver and nophis sirtalis parietalis (Garstka et al., 1982). fat-body in the mated females are due to the However, to prove these assumptions, observa- larger body mass per se. tions of actual mating behavior of the Japanese Comparisons of body mass and length of the mamushi are required. largest ovarian follicle between the mated and In the garter snake, Thamnophis sirtalis unmated females of the Japanese mamushi also parietalis, both positive (Hawley and Aleksiuk, revealed significant differences in the t-tests. 1976) and negative correlations (Whittier and These differences were retained even when SVL Tokarz, 1992) have been reported between the or body mass was removed as the covariate in the degree of ovarian development and the occur- ANCOVAs. SVL and the total number of rence of female sexual behavior. For the ovarian follicles did not differ between the mated Japanese mamushi, we obtained results similar and unmated females in the t-test and the AN- to those of Hawley and Aleksiuk (1976). COVA, respectively. These results suggest that In Crotalus viridis oreganus (Macartney and the mated females of the Japanese mamushi Gregory, 1988) and Thamnophis sirtalis were more plump than the unmated ones in body parietalis (Bona-Gallo and Licht, 1983; Garstka proportions and that the female mating success et al., 1985; Mendonca and Crews, 1990; Whit- was strongly associated with both body mass and tier and Crews, 1986; Whittier et al., 1987), the size of ovarian follicles in this snake. mating has been thought to be a prerequisite for In the Japanese mamushi, it has been ascertain- activating female ovarian functions. In these ed experimentally that body mass loss of adult snakes, mating precedes ovarian recrudescence. females is relatively small (less than 5% of body In the Japanese mamushi, however, mating does mass) in the period of several weeks of late not seem to be a prerequisite for follicular autumn when their feeding activities are usually development, because females of this snake low (Isogawa, unpubl.). Thus, body mass of isolated from males have been ascertained the unmated females of the Japanese mamushi is experimentally to become vitellogenic (Isogawa, considered to have been already small in August unpubl.). Mating in the Japanese mamushi or September of 1990, probably because of poor appears to occur at a time when female ovarian feeding during the summer of the year. follicles reach a certain size, as described below. In addition, it has been ascertained that In the mated females of the Japanese females of the Japanese mamushi with a large mamushi, the largest ovarian follicles were 6mm body mass usually have several ovarian follicles or more in length, whereas those in the unmated of 6mm or more in length in late autumn even females were less than 6mm in length. These when they are isolated from males (Isogawa, un- results suggest that the threshold length of publ.); that is, in the female Japanese mamushi, ovarian follicles for mating of the females is the non-occurrance of mating does not cause 6mm in this snake. This is supported by the follicular atresia. Therefore, it can be said that finding that in females of the Japanese mamushi, the difference in length of the largest ovarian ovarian follicles of 6mm or more in length can follicle between the mated and unmated females become vitellogenic after the administration of of the Japanese mamushi was not due to occur- exogenous gonadotropins (Isogawa et al., 1989). rence of follicular atresia in the latter. We assume that in the female Japanese Regarding the relation of body mass of female mamushi, mating is mainly dependent upon the snakes with mating success, the following ovarian follicle development and the increase of possibilities can be considered: 1) males court body mass due to heavy feeding during the sum- any female, but only females with a large body mer immediately before mating. In addition, mass are sexually receptive, 2) only females with we assume that the female Japanese mamushi a large body mass are receptive and males court mates at a time when the largest ovarian follicle such females, or 3) every female is receptive, but reaches 6mm in length in late summer to early males court only females with a large body autumn. mass. In mating of the Japanese mamushi, we Effectiveness of the summer-autumn mat- consider case 2) to be the most probable. It is ing. -The effectiveness of the summer-autumn ISOGAWA AND KATO-MATING OF MAMUSHI 47

mating on the following year reproduction FUKADA, H. 1986. Delayed fertilization in the of the female has been ascertained in Japanese mamushi. Jpn. J. Herpetol. 11(3): 156- Agkistrodon contortrix (Schuett, 1982) and 157. Thamnophis sirtalis (Blanchard, 1943; Blan- GAD, S. C. ANDC. S. WEIL. 1982. Statistics for tox- chard and Blanchard, 1941). In the present icologists. In: A. W. Hayes (ed.), Principles and Methods of Toxicology. p. 273-320. Raven Press, study, nine females of the Japanese mamushi New York. which had mated in August through September GARSTKA,W. R. AND D. CREWS. 1982. Female con- of 1990 gave birth to young in the autumn of trol of male reproductive function in a Mexican 1991. It was not clear whether these females snake. Science 217: 1159-1160. had already possessed spermatozoa deposited GARSTKA, W. R., B. CAMAZINE,AND D. CREWS. through a previous copulation in a region other 1982. Interactions of behavior and physiology dur- than the posterior ends of the reproductive ing the annual reproductive cycle of the red-sided tracts. Nonetheless, it is evident that sper- garter snake (Thamnophis sirtalis parietalis). matozoa deposited in the female Japanese Herpetologica 38 (1): 104-123. mamushi through a copulation can overwinter in GARSTKA,W. R., R. R. TOKARZ,M. DIAMOND,A. its reproductive tracts to be used effectively for HALPERT, AND D. CREWS. 1985. Behavioral and physiological control of yolk synthesis and deposi- fertilization of ova in the following year, as has tion in the female red-sided garter snake (Tham- been suspected in a few previous studies nophis sirtalis parietalis). Horm. Behav. 19: 137- (Isogawa, 1986; Fukada, 1986). 153. GLOYD,H. K. 1934. Studies on the breeding habits ACKNOWLEDGMENTS.-We are grateful to R. C. and young of the copperhead, Agkistrodon Goris and H. Ota for reviewing an early draft of the mokasen Beauvois, Pap. Michigan Acad. Sci., Arts, manuscript. Special thanks are due to E. -M. Zhao Letters 19 (1933): 587-604+3 pl. for providing useful literature. LOYD,H. K. ANDR. CONANT. G1990. Snakes of the Agkistrodon Complex: A Monographic Review. LITERATURE CITED Soc. Stud. Amph. Rep., Oxford, Ohio. 614 p. +52 ALLEN,E. R. AND D. SWINDELL.1948. The cotton- pls. mouth moccasin of Florida. Herpetologica 4 (1st. HAWLEY,A. W. L. ANDM. ALEKSIUK.1976. Sexual Suppl): 1-16. receptivity in the female red-sided garter snake BEHLER,J. L. ANDF. W. KING. 1979. The Audubon (Thamnophis sirtalis parietalis). Copeia 1976 (2): Society Field Guide to North American and 401-404. Amphibians. Alfred A. Knopf, New York. 720 p. HUANG, M. -H., Y. -M. CAO, F. -X. ZHU, AND Y. -F. BLANCHARD,F. C. 1943. A test of fecundity of the QU. 1990. Female reproductive cycle and em- garter snake Thamnophis sirtalis sirtalis (Linnaeus) bryonic development of Chinese mamushi in the year following the year of insemination. Pap. (Agkistrodon blomhoffii brevicaudus). In: E. -M. Michigan Acad. Sci. Arts, Letters 28 (1942): 313- Zhao (ed.), From Water onto Land; issued to com- 316. memorate the Ninetieth Birthday of the Late Pro- BLANCHARD,F. N. ANDF. C. BLANCHARD.1941. The fessor Cheng-Chao Liu. p. 173-177. Chinese Soc. inheritance of melanism in the garter snake Tham- Stud. Amph. Rep., Chengdu. (in Chinese, with nophis sirtalis sirtalis (Linnaeus), and some evidence English abstract) of effective autumn mating. Pap. Michigan Acad. ISOGAWA,K. 1986. Delayed fertilization in reptiles. Sci. Arts, Letters 26 (1940): 177-193. Newsletter, Jap. Soc. Comp. Endocrinol. (40): 4-7. BONA-GALLO,A. AND P. LICHT. 1983. Effects of (in Japanese) temperature on sexual receptivity and ovarian ISOGAWA,K., S. HIGASHIZONO,A. MORIYA,AND T. recrudescence in the garter snake, Thamnophis OKADA. 1989. Effects of mammalian gonadotropins sirtalis parietalis. Herpetologica 39 (2): 173-182. on follicular development in the Japanese mamushi BURCHELD,P. M. 1982. Additions to the natural Agkistrodon b. blomhgffii: Relationship between history of the crotaline snake Agkistrodon bilineatus follicular size and hormone sensitivity. In: M. Mat- taylori. J. Herpetol. 16(4): 376-382. sui, T. Hikida and R. C. Goris (eds.), Current CREWS, D. 1976. Hormonal control of male court- Herpetology in East Asia. p. 54-58. Herpetol. Soc. ship behavior and female attractivity in the garter Jpn., Kyoto. snake (Thamnophis sirtalis parietalis). Horm. JIANG,Y. -M. ANDE. -M. ZHAO.1980. Ecological data Behav. 7: 451-460. of Snake-Island pit-viper and Pallas' pit-viper. Acta FITCH, H. S. 1960. Autecology of the copperhead. Herpetol. Sinica 1(5): 1-5. (in Chinese, with English Univ. Kansas Publs., Mus. Nat. Hist. 13 (4): 85- resume) 288+8 pl. LASZLO,J. 1979. Notes on reproductive patterns of FUKADA, H. 1972. Growth and maturity of some reptiles in relation to captive breeding. Int. Zoo Japanese snakes (Review). Snake 4 (2): 75-83. (in Yearbook 19: 22-27. Japanese, with English abstract) LI, J. -L., M. -P. HUANG, Y. DIAO, AND L. -X. SUN. 48 Jpn. J. Herpetol. 16 (2). 1995

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要 旨 南九州 におけ るニホ ンマムシの交尾期、 及び交尾 と雌の卵巣発達 との関係 について 五十川清・ 加藤正秀 南九州産 ニホ ンマムシの交尾期を屋外放飼場 結果か ら,雌 ニホ ンマムシの交尾行動は,卵 巣 で調べた.そ の結果,8月 と9月 に交尾が確認 発達 と体重に密接に関連 してい ることが示唆 さ された.交 尾 した雌の最大卵胞の長 さは6mm れた. 以上であ ったが,交 尾 しなか った個体 の卵胞 は, ほ とん どが6mm未 満 であった.ま た,体 重 は, (896鹿 児島県串木野市下名2248養 命酒製 交尾 した雌の方が著 しく大 きか った.こ れ らの 造(株)中央研究所串木野支所)