Tissue and Cell 44 (2012) 32–46 Contents lists available at SciVerse ScienceDirect Tissue and Cell j our na l ho me p age: www.elsevier.com/locate/tice The ultrastructure of the ejaculatory duct in the springtail Orchesella villosa (Geoffroy) (Hexapoda, Collembola) and the formation of the spermatophore Pietro Paolo Fanciulli, Zaira Valentina Zizzari, Francesco Frati, Romano Dallai ∗ Department of Evolutionary Biology, University of Siena, via A. Moro 2, I-53100 Siena, Italy a r t i c l e i n f o Article history: Received 24 June 2011 Received in revised form 7 October 2011 Accepted 10 October 2011 Available online 5 November 2011 Keywords: Collembola Reproduction Ejaculatory duct Spermatophore a b s t r a c t The initial part of the ejaculatory duct of Orchesella villosa contains a “valve” and a “sorter” avoiding respectively the reflow and allowing the separation of the secretion for the spermatophore stalk from the sperm fluid. For most of its length, the ejaculatory duct lumen is divided into two parts: in the dorsal part the sperm fluid flows while in the ventral district the secretion for the stalk occurs. Laterally, on both sides of the duct, longitudinal muscle fibers are present. The epithelium of the dorsal region consists of two types of long secretory cells; the most peculiar of them are those provided with extracellular cisterns flowing directly into the duct lumen as it occurs in 1st type of epidermal cells. These cells could be involved in the control of the viscosity of the sperm fluid. The second type of cells produce a secretion probably involved in the formation of the outer coat of the apical sperm droplet. The ventral epithelium consists of short cells contributing to the enrichment of the secretion for the spermatophore stalk and perhaps also to the viscosity of the secretion flowing in the lumen. In the distal part of the ejaculatory duct, the ventral district is provided with a thick layer of muscle fibers and with 3 + 3 cuticular laminae dividing the lumen into a series of slits through which the secretion of the stalk is squeezed out into filaments. This organization allows the twisting and hardening of these filaments. A drop of sperm fluid is laid on top of the long and rigid spermatophore stalk. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Collembola are basal hexapods that perform reproduction by indirect sperm transfer through spermatophores laid by the males on the soil and then picked up by the females (Schaller, 1953; Mayer, 1957; Proctor, 1998; Hopkin, 1997), sometimes following a very elaborated courtship behaviour (Bretfeld, 1970, 1974, 1976; Betsch-Pinot, 1977). The spermatophore consists of a sperm droplet at the top of a stalk (Schaller, 1971). The sperm droplets may (Orch- esella villosa) or may not (Allacma fusca) be protected by a coat (Blancquaert and Mertens, 1977; Döring, 1986; Hopkin, 1997) and contains a variable number of sperm cells, about 1700 in O. vil- losa (Entomobryidae), while only about 600 in the Symphypleona A. fusca (L.) (Dallai et al., 2009). The sperm of Collembola, at the end of spermiogenesis, are dis- coidal in shape, provided with a long apical cylindrical appendage and are rolled up around an extracellular dense mass of material; in this shape, they are accumulated in the distal region of the male genital duct (Dallai et al., 2003, 2004). After spermatophore uptake ∗ Corresponding author. Tel.: +39 577 234412; fax: +39 577 234476. E-mail address: dallai@unisi.it (R. Dallai). by the female the sperm become straight and lose their apical extra- cellular cylinder. The preliminary study by Döring (1986) on the ejaculatory duct of O. cincta described some structural peculiarities somewhat simi- lar to those observed in A. fusca (Dallai et al., 1999). The ejaculatory duct of a sexually mature male of O. cincta shows a complex organi- zation allowing the secretions of the spermatophore stalk and of the sperm fluid in two distinct and overlapping longitudinal districts (Döring, 1986). In this paper we have investigated with electron microscopy such organization, in O. villosa in order to clarify how the sper- matophore of this species is built. 2. Materials and methods Several males of O. villosa (Geoffroy, 1762), collected in the neighbourhood of Grosseto and Siena (Italy), were dissected under a light microscopy, using thin needles, in a 0.1 M phosphate buffer pH 7.2 to which 3% of sucrose was added (PB). After removing the male genital system, the distal part of testes, deferent ducts and ejaculatory duct were isolated and fixed overnight in 2.5% of glutaraldehyde in PB ad 4 ◦ C. After rinsing in PB, the material was post-fixed in 1% O s O 4 for 1 h, then rinsed in PB and dehydrated in a graded series of alcohol and finally embedded in a Epon–Araldite 0040-8166/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2011.10.003