Annual Cycle of Sperm Storage in Spermathecae of the Red-Spotted Newt, Notophthalmus viridescens (Amphibia:

Female sperm storage was studied in a population of Notophthalmus viridescens from South Carolina. Spermathecae initiate production of a glycoprotein secretory product in October. At this time ovarian follicles are immature (0.5-0.9 mm dia), and mating does not occur despite spermiation in males. Six of the 10 females collected in December had sperm in their spermathecae, indicating onset of mating. Unmated females collected in October and sacrificed in February and March possessed mature ovarian follicles (1.3-1.4 mm dia), and the spermathecae contained large secretory vacuoles 2-3 km dia. Release of secretory product is concomitant with the appearance of sperm in the spermathecae. Thus mated females lack secretory vacuoles in the spermathecal epithelium, and additional synthesis of secretory product does not occur. All females collected in February and March have mated. Sperm are embedded in the spermathecal epithelium and are undergoing degradation in February. Degradation of sperm in the lumen and epithelium is evident in specimens examined from May and June. Atresia of ovarian follicles begins in April in captive specimens, and specimens captured from the bay in May are spent. A general postbreeding emigration from the pond occurs in summer. Fourteen females collected 7 March were injected with human chorionic gonadotropin (hCG) on 9 March and laid fertile eggs 10-18 March. Two of these females were sacrificed each month from April-September; all retained some sperm in their spermathecae, but further oviposition did not occur. Four females were kept 1 year after oviposition of fertile eggs, and oviposition again was induced with hCG; these eggs were infertile, and spermathecae lacked sperm. Spermathecae are inactive from June-September in captive and wild-caught specimens. Sperm may be stored effectively up to 6 months (December-May), and no evidence was found for retention of viable sperm from one breeding season to the next. Q 1996 Wiley-Liss, Inc. The red-spotted newt, Notophthalmus viridescens Rafinesque, is widespread in eastern North America (Mecham, '67). Like other salamanders in the seven families comprising the suborder Salamandroidea, males produce a spermatophore that becomes lodged in the female's cloaca during courtship (Verrell, '82). Sperm from the spermatophore cap migrate into exocrine glands, spermathecae, in the cloaca of the female (Hardy and Dent, '86). These glands are simple tubuloalveolar glands in all families except Plethodontidae, in which spermathecae are compound alveolar glands (Sever, '94). During oviposition, eggs are fertilized internally during their passage through the cloaca by sperm released from the spermathecae (Jordan, 1893; Boisseau and Joly, '75). Dent ('70) used transmission electron microscopy to examine the spermathecae of immature N. uiridescens and those of mated and unmated mature females from various populations in central Virginia, middle TenAddress reprint requests to David M. Sever, Department of Biology, Saint Mary's College, Notre Dame, IN 46556. o 1996 WILEY-LISS, INC. 156 D.M. SEVER ET AL nessee, and western Massachusetts. However, he did not follow the annual cycles of spermathecal cytology and sperm storage in any single population. These annual cycles are of interest due to the considerable amount of intraspecific variation that has been reported in the reproductive biology of N. viridescens. For example, in a population from Massachusetts, Adams ('40) found sperm in the spermathecae of females examined in every month of the year, although sperm were especially abundant in the fall and spring. Matings in Adam's population occurred in fall during a "false breeding season" (Gage, 1891; Jordan, 1893; Pope, '24) as well as spring; males had sperm in their vasa deferentia from September through May, and some males possessed sperm during the midsummer months (Adams, '40). Thus, Adams found a prolonged mating season, which could account for the presence of sperm in spermathecae throughout much of the year. In contrast, Massey ('88, '90) reported matings from mid-March to late June in newts from the mountains of Virginia, and no fall breeding season existed. An August-September emigration to terrestrial hibernacula occurs, and adults do not migrate back to the ponds until the following March-April. A year after breeding, eight marked females were recaptured in pit traps around a partially fenced pond (Massey, '90). Seven of the females had sperm in their spermathecae, although they were not abundant, indicating no recent insemination. Massey ('90) concluded that the sperm were retained from a previous breeding season, and these females had stored sperm for at least 10 months. Thus, Massey ('90) found a relatively short mating period, but a prolonged period of sperm storage. In this report, we present the first cytological observations on the annual cycle of sperm storage and spermathecal secretions in a single population of Notophthalmus viridescens. We extend the observations of Dent ('70) and compare our findings to those on the cytology of spermathecae in other salamanders (Pool and Hoage, '73; Boisseau and Joly, '75; Brizzi et al., '89, '95; Sever, '91a, '92a, '94, '95; Sever and Brunette, '93; Sever and Kloepfer, '93; Sever et al., '95). MATERIALS AND METHODS All newts were collected from Castor Bay (Wood Duck Bay), a 3.5 ha Carolina bay on the Department of Energy's Savannah River Site (Aiken County, SC). The bay held water throughout the period of the study. Collections were made on 24 October 1993, and 18 February, 10 March, 17 May, 20 June, 27 June, 2 August, and 3 December 1994 (Tables 1-3). Some specimens were sacrificed within 2 days after capture, whereas others were maintained in the lab for various periods of time prior to sacrifice, as described below. Females from the October and March collections were separated from males based upon their swollen bodies, indicating presence of enlarged ovarian follicles, and/or the TABLE 1. Branchiated (Br) and metamorphosed (Me) specimens collected in October a n d December 1993 a n d Februaw 1994 Specimens collected 24 October 1993 Ovarian follicles > 0.5 mm