JOURNAL OF MORPHOLOGY 230:79-97 (1996) Sperm Storage in Spermathecae of the Great Lamper Eel, Amphiuma tridacfyhm (Caudata: Amp hi u midae) DAVID M. SEVER, J. SEAN DOODY, COURTNEY A. REDDISH, MICHELLE M. WENNER, AND DON R. CHURCH Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 (D.M.S., C.A.R., M.M. W.); Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana 70402 (J.S.D., D.R.C.) ABSTRACT The spermathecae of ten female Amphiuma tridactylum were examined by light and electron microscopy during the presumed mating and ovipository seasons (March-August) in Louisiana. Spermathecae were simple tubuloalveolar glands in the dorsal wall of the cloaca. Six of the ten specimens were vitellogenic, and all of these specimens contained sperm in their sperma- thecae and had secretory activity in the spermathecal epithelium. Two nonvitel- logenic females also had sperm in their spermathecae and active epithelial cells, whereas the other nonvitellogenic females lacked stored sperm and secretory activity in the spermathecae. In specimens storing sperm from March-May, the sperm were normal in cytology, and secretory vacuoles were contained within the epithelium. In the August sample, however, evidence of sperm degradation was present, and secretory material had been released into the lumen by an apocrine process. We therefore hypothesize that the spermathecal secretions function in sperm degeneration. Q 1996 Wiley-Liss, Inc. The Amphiumidae consists of three spe- of A. tridactylum and described these glands cies of Amphiuma, elongate, fossorial sala- in A. means and A. phloleter. In addition, manders with reduced limbs and one exter- Sever ('gla, '94) reported on the phylogeny nal gill slit, a paedomorphic character of the spermathecae and other cloacal glands (Duellman and Trueb, '86). Two of the spe- in the Amphiumidae. cies are aquatic and often exceed 70 cm snout- Kreeger ('42) stated that female A. tridac- vent length (SVL); A. means occurs in the tylum could store sperm in their spermathe- coastal plain from Virginia to Louisiana, and cae for 7 or 8 months, and Baker ('62) found A. tridactylum occurs from western Alabama that isolated females could retain living sperm to Texas and up the Mississippi River valley for 15 months. In females of other sala- to western Kentucky. A. phloleter is a dwarf manders, the longest period that Sever ('95) (20-25 cm SVL), terrestrial species limited to felt was reliable for duration of sperm stor- the Florida panhandle and southern Ala- age was 6 months. The best way to ascertain bama (Conant and Collins, '91). In the Missis- the cytological condition of sperm during stor- sippi River valley, A. tridactylum is com- age is by use of electron microscopy, but no monly called the "great lamper eel" (Parker, ultrastructural studies of the spermathecae '37; Baker, '45; Dundee and Rossman, '89). of Amphiuma have heretofore existed. We A number of reports exist on the reproduc- recently had the opportunity to cytologically tive biology of Amphiuma, but most of these examine the spermathecae of ten females are anecdotal and some are contradictory sacrificed during the mating and ovipository (Table 1). The majority of the literature con- period, and herein we present our findings on cerns A. tridactylum. Sperm in the female the ultrastructure of sperm storage in this cloaca was first observed by Davison (1895). species. These results are then compared with The only report of mating behavior is an those on the ultrastructure of the sperma- observation of copulation by cloacal apposi- thecae of other salamanders (for recent re- tion (Baker et al., '47). Sperm storage glands, views see Sever, '95; Sever et al., '96). spermathecae, in the female cloaca were first described by Kreeger ('42), and Sever ('92a) Address reprint requests to David M. Sever, Department of extended her observations on spermathecae Biology, Saint Mary's College, Notre Dame, IN 46556. o 1996 WILEY-LISS, INC. 80 D.M. SEVER ET AL MATERIALS AND METHODS light microscopy (LM) or for embedding in Great lamper eels were collected from road- epoxy resin for thin (LM) or ultrathin sec- side ditches at three sites, each within 10 km tions for transmission electron microscopy of Hammond, Tangipahoa Parish, Louisiana. (TEM). Testes and vasa deferentia were re- We obtained specimens monthly from Febru- moved from males, and cloacae were dis- sected from females. For all specimens, tis- ary-May 1995. We captured most individuals sues were initially fixed in 10% neutral by hand but also used a dipnet, and a few individuals were caught in funnel traps baited buffered formalin (NBF) (for LM) or in a 1:l with chicken necks. We concentrated collect- solution of 2.5% glutaraldehyde in Millonig's phosphate buffer at pH 7.4 and 3.7% formal- ing efforts at night after rain, when lamper dehyde buffered to pH 7.2 with monobasic eels were more active; many were obtained and dibasic phosphate (TEM). Carcasses of from ditches that were dry prior to rainfall. all specimens are stored in NBF in the re- Because females become scarce in sum- search collections at Saint Mary's College. mer, presumably as a result of nesting (Cagle, For paraffin infiltration prior to sectioning '48; Fontenot, '901, we placed 22 presumed for LM, the tissue was rinsed in water after adult females captured between 9 and 13 fixation, dehydrated in ethanol, cleared in May into two outdoor cattle tanks (2.0 m Histosol (National Diagnostics, Inc., Man- diameter, 0.7 m sidewall height) in Hammond, ville, NJ), and embedded in paraffin. Sections Louisiana. Mature females were considered (10 km) were cut with a rotary microtome, those >33 cm total length (Fontenot, 'go), and a&ed to albuminized slides, and alter- and females were identified by black pigmen- nate slides were stained with hematoxylin- tation in the cloaca1 wall (Baker, '37). Cattle eosin (HE) (for general cytology) or alcian tanks were filled with water to a level of blue 8GX (AB) (for primarily carboxylated approximately 15cm and contained mud from glycosaminoglycans) at pH 2.5 followed by a ditch at one of the collecting sites. Eight the periodic acid-Schiff method (PAS) (neu- plastic nesting boxes (25 x 50 x 20 cm) con- tral carbohydrates and sialic acids). Proce- taining mud were placed at staggered heights dures followed Kiernan ('90). in each tank, and some individuals entered After initial fixation, tissues prepared for the boxes. Plywood sheets were placed over plastic infiltration prior to sectioning for LM the cattle tanks to prevent further filling due and TEM were trimmed into 1.5 mm blocks, to rainfall. Tanks were examined for eggs rinsed in Millonig's buffer, postfixed in 2% every few days until 25 July when other osmium tetroxide, dehydrated in ethanol, circumstances dictated that the observations cleared in propylene oxide, and embedded in must end. an epoxy resin (EMBED-812; Electron Mi- Table 2 presents the data on the specimens croscopy Sciences, Fort Washington, PA). from which spermathecae were examined by Semithin sections (0.5-1 km) for LM were light and electron microscopy. Those lulled cut with glass knives, placed on microscope on 22 August were specimens that had been slides, and stained with toluidine blue. Ultra- maintained in the cattle tanks and subse- thin sections (70 nm) for TEM were collected quently kept in a living stream (180 cm x 50 on uncoated copper grids and stained with cm x 60 cm; Frigid Units, Inc., Toledo, Ohio) solutions of uranyl acetate and lead citrate. at Saint Mary's College until time of sacri- These sections were cut with RMC XLlOOO fice. The number of yolky ovarian oocytes and RMC MT7 ultramicrotomes, and thin was counted in each female, and 11 such sections were viewed with a Hitachi H-300 oocytes from each female were measured to transmission electron microscope. Terminol- the nearest 0.1 mm with a dial caliper under ogy for sperm ultrastructure follows Picheral 5 x magnification. In addition to the females, ('79). single males were respectively collected (sac- rificed) on 19 February (3 March), 6 March RESULTS (14 March), 21 April (5 May), and 9-13 May Specimens maintained in cattle tanks (18 October). During the time lamper eels were kept in Specimens were killed by immersion in cattle tanks, seven individuals died. One of 10% MS-222, and snout-vent length (SVL) the specimens retrieved from the cattle tanks was measured from the tip of the snout to the later proved to be a male (and was sacrificed posterior end of the vent. Tissues were ex- on 18 October so the testes and vasa deferen- cised from freshly killed specimens and fixed tia could be prepared for histological examina- for preparation by paraffin infiltration for tion). None of the females kept in the cattle TABLE 1. Literature on the reproductive biology ofAmphiuma Species State Spermiogenesis Vitellogenesis Mating Sperm storage Oviposition Nesting Hatching Reference Not specified October Midsummer McGregor (1889) Winter Sturdivant ('49) A. means AL Nov Ultsch and Arce- neaiix...... ~, ('88) . NC July Brimley ('39) FL July Brimley ('10) FL January-February Weber ('44) FL October-May 15 months Baker ('62) sc July Hildebrand ('10) A. tridactylum AK August-September Hay (1888) LA August-March NovemberJuly February-April July-September Wilson ('40) LA January-March Wilson ('41) LA March-December Kreeger ('42) LA January-May MayJune Nov Cagle ('48) LA October-May 15 months April-May Fall Baker ('62) LA December-February November-April February-winter Rose ('67) LA September-May June June-November November- Fontenot ('90) December TN May August- Davison (1895) September TN August-winter Parker ('37) TN July August August-September Baker ('37) TN July Baker et al.