Int. J. Morphol., 26(2):423-432, 2008. Microscopic Anatomy of the Male Reproductive System in Echinolittorina peruviana (Mollusca: Caenogastropoda) Anatomía Microscópica del Sistema Reproductor Masculino de Echinolittorina peruviana (Mollusca: Caenogastropoda) Viviana M. Castillo & Donald I. Brown CASTILLO, V. M. & BROWN, D. I. Microscopic anatomy of the male reproductive system in Echinolittorina peruviana (Mollusca: Caenogastropoda). Int. J. Morphol., 26(2):423-432, 2008. SUMMARY: Echinolittorina peruviana (Lamarck, 1822), a gonochoric representative of the Littorinidae on the SE Pacific coast, has a male reproductive system adapted for internal fertilization. We describe this system at both macro- and microscopic levels, particularly the compartmentalized organization of the gonad, and the morphology of the penis. The male reproductive system has a variegated conical gonad-digestive gland complex. The gonad presents three compartments, 1) gametogenic acinar among the glandular acini, 2) periacinar with a layer of fusiform somatic cells and, 3) interacinar with glycogen storage cells shared with glandular acini. Spermatogenesis occurs within the acinar gametogenic compartment, with the germinal line organized in centripetal form towards the lumen. The seminal vesicle stores the products of spermatogenesis; in its cephalic region the euspermatozoa are united to the epithelium and the paraspermatozoa are distributed in the lumen. A short duct connects the seminal vesicle to the prostate gland that is open to the pallial cavity over its entire length. The anterior zone of the prostate gland is joined to the cervical spermatic groove that runs along the neck of the snail through the right pallial region; this continues as the penile spermatic groove, ascending from the base to the point of the penis. The penis is acutely conical and unpigmented; towards the anterior and adjacent to its base there is a glandular complex with a mamilliform process and a discoidal glandular region. The secretion from the discoidal region is transformed in a spicule of unknown function, whose histology is described here for the first time. The mamilliform process is formed by the spicular projection and a connective-muscle tissue band which surrounds it and separates it from the discoidal follicular glandular region; the follicular secretion crosses this band and is incorporated into the epithelium which lines the interior of the process. KEY WORDS: Mollusca; Microscopic anatomy; Male reproductive system; Littorinidae. INTRODUCTION The evolution of internal fertilization in the animal morphological and structural diversity among families. kingdom is a reproductive strategy that includes the Among these, the Littorinidae exhibit diverse patterns of organization of a complex reproductive system adapted for organization of the reproductive system among its copulation. The structural complexity associated with the representatives; these patterns have been considered male reproductive system, besides its association with the taxonomically relevant for defining the phylogeny of this habit of internal fertilization, includes the organization of a family (Reid, 1989). The male reproductive systems of the compartmentalized gonad which is able to provide a gonochoric representatives of the Littorinidae include: (1) a specialized environment within which highly species-specific branched gonad among the tubules of the digestive gland, gametes are formed (Brown, 1990); an example of this is (2) a seminal vesicle in which the euspermatozoa are stored, the introspermatozoan, characteristic of the (3) a prostate gland, and (4) a conical penis located under Caenogastropoda. These molluscs have become adapted by the right tentacle of the animal (Fretter & Graham). Glands developing a male reproductive system, which includes that are mamilliform in appearance have been associated with organs of transport that connect the gonad with a conical the penis, located in a row on its ventral surface, as well as penis specialized for copulation (see examples in Fretter, along its length; the number of these glands constitutes a 1941; Fretter & Graham, 1994; Linke, 1933), showing a high taxonomic character for the Littorinidae (Buckland-Nicks Laboratorio de Biología de la Reproducción y del Desarrollo, Departamento de Biología y Ciencias Ambientales, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile. 423 CASTILLO, V. M. & BROWN, D. I. Microscopic anatomy of the male reproductive system in Echinolittorina peruviana (Mollusca: Caenogastropoda). Int. J. Morphol., 26(2):423-432, 2008. & Worthen, 1993; Fretter & Graham; Reid, 1989 and 2002). Histochemical identification of glycogen: PAS-H Penile morphology in the littorinids has been considered to (Periodic acid- Schiff-hematoxylin) staining. Small be its’ most important taxonomic character (Reid, 1989). In portions of tissue representing the gonad -digestive gland spite of the fact that the family Littorinidae is one of the complex and the seminal vesicle were fixed in Gendre (Gabe) most highly studied within the Caenogastropoda, studies at 4ºC for seven hours, and histological preparations were describing the histological organization of the gonad, the made as described above. Two sets of preparations bearing reproductive system, and the body of littorinids from the consecutive sections of each specimen were de-paraffinated southern hemisphere are scarce, and relevant information is and hydrated. One set of preparations was submitted to two limited. hours of 0.1% salivary amylase digestion at 37ºC; and the other remained as an undigested control. Both sets of In the present study we describe the male reproductive preparations were then rinsed in distilled water and stained system of Echinolittorina peruviana, a common inhabitant with PAS-H (Harris’ hematoxylin as a nuclear stain) for the of the SE Pacific coast of South America, providing recognition of glycogen (Gabe). The preparations were finally anatomical observations at macro- and microscopic levels, dehydrated, cleared, and mounted in resin. The histological emphasizing its’ organization and the morphology of the preparations were examined using light microscopy for penis. evidence of the presence of glycogen as noted by a purple red color within the gonadal and seminal vesicle tissue. MATERIAL AND METHOD RESULTS Adult males of Echinolittorina peruviana were collected directly from the rocky intertidal zone at Carvallo General anatomy of the male reproductive system. The beach near Valparaíso, Chile (33º 1’S, 71º 38’O). Freshly gonad-digestive gland complex of the male E. peruviana collected snails were processed without delay for making has a conical-spiral morphology (Fig.1A), with variegated observations using light microscopy. orange-dark brown coloration. The orange coloration represents the gonadal tissue, while the digestive gland tissue Macroscopic analyses. Specimens were anesthetized for 30 is dark brown (Figs. 2A, 2B). The male gonad is branched minutes, at 4°C, in a 1:1 solution of sea water and 7.5% between the digestive gland; it is located above the remainder aqueous Cl2Mg (Pantín, 1967) and then fixed in Backer’s of the visceral mass. The gonad is accompanied by a whitish- marine calcium formol (Pantín) at 4ºC. The specimens were coiled duct, the seminal vesicle, which is clearly visible then dissected in order to prepare drawings and take leading toward the extreme posterior of the columellar photographs. muscle of the shell (Figs.1A; 2A). Toward the anterior, and open to the pallial cavity, lies a voluminous structure with a Histological Analyses. Routine methods for light globular appearance, the prostate gland (Figs. 1A; 2A), which microscopy, entire specimens and transversally sectioned, is located above the columellar muscle of the shell. The cer- and portions of the gonad-digestive gland complex were fixed vical spermatic groove lies anterior to the prostate gland, for 24 hours in Bouin’s solution. Following fixation, the and runs to the neck of the snail, ascending along the dorsal samples were rinsed in 70% EtOH and dehydrated in an side from the base of the penis (penile spermatic groove) to increasing series of ethanol concentrations, followed by clea- its tip (Fig 1A). The penis is located at the base of the right ring in butanol and embedding in Paraplast Plus for eye tentacle (Figs. 1A, B; 2B) and is seen as a white, pointed, histological sectioning (Gabe, 1968). conical structure. Toward the anterior, and adjacent to the base of the penis there is a structure (Figs. 1A, C; 2B) formed Serial histological sections 5 µm in thickness were by a region having a mamilliform appearance (mamilliform taken each 75 µm through the samples and were mounted process), and next to this there is a discoidal region (Fig.1C). on microscope slides. The preparations were processed following routine histological procedure, and stained by a Histological organization of the male reproductive trichrome method to observe topographic coloration, and system. Male gonad. The male gonad has a fundamentally finally dehydrated, cleared, and mounted to obtain permanent acinar organization among the acini of the digestive gland preparations (Valderrama et al., 2004). Transverse and (Fig. 2C). Three compartments can be distinguished within sagittal serial histological sections (Fig.1A) were examined the gonad (Figs. 2D, F, I): a) gametogenic or acinar; b) by light microscopy in order to characterize the perigametogenic or periacinar and c) intergametogenic