Gonzalo A. Collado & Donald I. Brown
Laboratorio de Biología de la Reproducción y del Desarrollo, Departamento de Biología y Ciencias Ambientales, Facultad de Ciencias, Universidad de Valparaíso, Casilla 5030, Valparaíso, Chile.
An Open Access article from Int J. MorphoL, 25(2):315-322, 2007.
SUMMARY: This study describes the microscopic anatomy of the reproductive systems of the keyhole limpets Fissurella latimarginata and Fissurella cumingi to test the existence of a possible isolating reproductive mechanism of morphophysiological type between species, and consequently to determine if the findings provide a valid taxonomic characteristic to distinguish these sympatric species. The results show that in both species the reproductive system is intimately associated with the excretory system. The gonad has a septa organization, and is connected with the distal portion of the right kidney by means of a renopericardial duct which functions as a genital duct. The gametes are evacuated to the mantle cavity through the right kidney opening. Besides transport of the gametes, the genital duct, which connects with the pericardium through the nephrostome, bears a gland attached to its inner wall both in males and females. No marked anatomical differences were noted between the reproductive systems of the two limpets which would allow their unequivocal taxonomic differentiation at the species level.
KEY WORDS: Mollusk; Fissurellidae; Reproductive system; Microscopic anatomy; Taxonomy.
An unusually large number of relatively large species of Fissurella occupy the temperate South American coastline (McLean, 1984). Taxonomic separation of these species has been historically based on the Typological Species Concept, relying mainly on externally visible shell characteristics. Sympatry of some of these species, coupled with individual variability within natural populations (Ramirez, 1974; McLean) has in the past produced some taxonomic confusion with regard to species identification among these limpets. It was resolved to some extent by McLean's synonymy which recognized 13 species from a previous total of 58 names based on characters of the shells, epipodium, and in some cases radulae, complemented by data on their geographic distributions.
The vetigastropods, which include Fissurella latimarginata Sowerby, 1835 and F. cumingi Reeve, 1849, release their gametes into the surrounding seawater at spawning. It is typical within this group that the gametes are transferred from the gonad to the distal portion of the right kidney through a genital duct, and are released to the exterior through a right kidney opening which opens to the mantle cavity. The detailed anatomy of the reproductive system in the Fissurellidae, however, is subject to some controversy. Unresolved discussions have been centered on the location of the opening of the genital duct, on the presence of a gland associated with the genital duct, and on the existence of a renopericardial canal (Boutan, 1885; Erlanger, 1892; Ziegenhorn & Thiem, 1925; and more references below). Until the present, no data have been presented on the microscopic anatomy of the reproductive systems in F. latimarginata and F. cumingi, and consequently has not been available for anatomical analysis of the reproductive system neither to be used in taxonomic differentiation, particularly with regard to structures which may help explain their interspecific reproductive isolation. Among the different barriers of separation between species, a morphophisiologycal isolating mechanism has been proposed in relation to reproductive systems that would prevent the fertilization due to anatomical incompatibilities (Dobzhansky et al, 1980; Savage, 1971), but has been controversial in gastropods because anatomical differences between closely related species are lacking (Graham, 1965; Houston & Hatfield, 1981).
The objective of the present study was to make a comparative microscopic description of the morphology of the reproductive systems ofF. ¡atimarginata andii cumingi to test the existence of a possible isolating reproductive mechanism of morphophysiological type, and consequently to determine if the findings provide a valid taxonomic characteristic to distinguish these sympatric species.
F. latimarginata and F. cumingi are gonochoric species. The reproductive system in both these species is simple, and copulatory organs are absent. The reproductive system is intimately associated to the excretory systems. The right kidney is much more developed than the left kidney in both species and there is a single gonad which is connected only with the right kidney. Upon careful examinations of the dissections, the reproductive systems oiF. latimarginata and F. cumingi were indistinguishable in microscopic organization, and so a single description is presented here for both species. The reproductive system is composed of a) a single gonad, b) a renopericardial duct that transport the gametes to the distal portion of the right kidney, it has a gland attached to its inner wall and is connected by the nefrostome to the pericardium; and c) the distal portion of the right kidney which receives the gametes from the duct. The gametes leave the kidney through its aperture toward the mantle cavity, and from there reaching the exterior (Fig.1). The renopericardial duct which carries the gametes from the gonad to the right kidney is referred to from now on as the "genital duct".
Gonad: This organ is a multilobed sac with variable morphology and size depending on the stage of maturity of the individual. The color in males varies from white-cream to yellow and in females is dark green; in both sexes it is well separated from other organs of the visceral mass. The gonad occupies a ventral position in the body cavity, below the digestive gland and stomach, and is dorsal to the foot. In mature individuals it is swollen, and extends from the posterior portion of the visceral cavity toward its anterior end, occupying all the available space. The gonad is covered with a cubical to low columnar epithelium, under which lies a muscle/ connective tissue layer, from which projects septa of connective tissue toward a common gonadic cavity. The germinal cell line develops around the septa, interacting with the somatic cells in a centrifugal organization towards the gonadic cavity (Figs. 2A, B).
Genital duct and kidney opening: Seen in a transversal section genital duct arises in the upper right mid-zone of the gonad and projects upward in the visceral cavity parallel to the muscular wall of the limpet (Figs. 2C; 3A, C), to which it is anchored by a thin mesentery. A gland is proximally attached to the internal wall of the genital duct lumen (Figs. 2C, G, H; 3A, C). Soon after the gland appears the nephrostome (Figs. 2D, I), which is an aperture bearing a ciliated epithelium joining the duct with the pericardial cavity at the level of the right auricle. Then the duct continues its course below the muscular floor of the pericardium toward the mid-region of the animal to open into the distal part of the right kidney, near the base of the right kidney opening (Figs. 2F, K; 3B, D). Distally, the duct near the kidney shows a widening of the lumen and finger-like epithelial folds that fail to fill the lumen, with apparent difference in length between both sexes (Figs. 2E, J; 3B). The transversal section shows two types of simple epithelia all along the genital duct, one above and one below, supported by a muscular/connective tissue layer. The upper epithelium is observed as a continuation of the epithelium which covers the gland associated with the wall of the genital duct, while the lower epithelium is a continuation of the gonadic wall isolating the gonad from the digestive gland. The upper epithelium is formed of cylindrical cells having a vesicular appearance, generally with basal nuclei, among which are intercalated goblet cells. The epithelial cells release large quantities of cytoplasmic vesicles and secretions into the lumen of the genital duct. Prior to their detachment the vesicles remain connected to the cells by cytoplasmic peduncles. Once the vesicles are released they appear to break open in the lumen and release their contents. The lower epithelium is also secretory, and is formed of flatter cells which are more densely stained. The cells are smaller near the distal portion of the duct and their borders are poorly defined. The histology of the mid-portion of the genital duct in males of F. latimarginata and F. cumingi is shown in Figs. 3G, H.
In both sexes in F. latimarginata and F. cumingi the germ cell line in the gonad presents a centrifugal organization like in the vetigastropod Tegula tridentata (Potiez & Michaud, 1838) (Brown, 1992). The gametes of F. latimarginata and F cumingi travel from the gonad to the right kidney through the renopericardial duct. The similar organization of the reproductive system between the two species does not show differences useful for making unequivocal anatomical distinctions between them. The histological detail of the genital duct (Figs. 3G, H) also fails to reveal interspecific differences in relation to find an isolating reproductive mechanism of morphophysiological type. Consequently, the reproductive system has therefore little value as taxonomic tool at the species level. Anatomical characteristics of the reproductive systems of these species could be useful in making differentiations at higher taxonomic levels or between less closely related species; however, differences in sperm morphology have shown to be useful to distinguish between both species (Collado & Brown, 2003).
Relations between the reproductive and excretory systems of the Fissurellidae were controversial until now. In the Ihering (1877) model, the gonad of Fissurella opens to the right kidney from which there is no renopericardial connection. Boutan (1885) was unable to find a renopericardial connection in Fissurella reticulata (da Costa) [= Diodora apertura (Montagu)], nor did Erlanger (1892) in five species of Fissurella. Boutan however described a "glande annexe" in the internal wall of the genital duct mF. reticulata females. According to Boutan and Erlanger the gonad opened to the right kidney. A renopericardial duct was recognized by Haller (1886, 1906) in a Fissurella species. In the Perrier (1896) model, the Fissurella gonad opened to the right kidney, in which there was a renopericardial connection. Ziegenhorn & Thiem determined that the reproductive system in Fissurella crassa Lamarck, 1822 consisted of a gonad that opened to the right kidney through a gonoduct which contained a gland in its internal wall, and which was also observed in females of Fissurella oriens Sowerby, 1835 (under Fissurella alba Philippi, 1845) and Fissurella limbata Sowerby, 1835. For Ziegenhorn & Thiem the gland was the same as that of Boutan and was also found in Fissurella (= Diodora) nubécula (Linnaeus, 1758) males. Ziegenhorn & Thiem found a renopericardial duct in all the species which they studied. In the Pelseneer (1898) model, the Fissurella gonad opened to the renopericardial duct and the gametes were evacuated through the right renal aperture. More recent studies, and reviews made by Gabe (1951), Fretter & Graham (1962), Delhaye (1976) and Haszprunar (1988) agreed with the Pelseneer observations. The Voltzow (1994) model, based on the literature, shows two renopericardial connections, one for each kidney, with the gonad opening to the right kidney.
The results of the present study show that in F. ¡atimarginata and F. cumingi the gametes are transported to the distal region of the right kidney through the renopericardial duct, which functions as a genital duct, as well as draining the pericardium. In this manner, the renopericardial duct represents a "nephrogonoduct" if we follow the terminology correctly assumed by Pelseneer for this double functional duct. No connection was observed between the gonad and the small left kidney in both F. ¡atimarginata and F. cumingi, which also discharges its contents to the mantle cavity. Andrews (1985), however, described two renopericardial connections in Emarginula reticulata Sowerby, 1813, one for each kidney, the left one revealed ultrastructurally. Fretter (1984) mentioned that the kidney, pericardial cavity, gonad, and gonoduct were coelomic in origin, and that the connections between them remained in some adult prosobranchs, just as in the case of the two species considered in the present study.
The anatomy of the reproductive system in F ¡atimarginata and F. cumingi is similar to that described by Ziegenhorn & Thiem for Fissurella species. A gland of unknown function associated with the genital duct like that described by these authors is also found in F ¡atimarginata and F cumingi, both in male and females. The gland present in other fissurelids seems to be related with the pattern of development of the species; adding extra material to the gametes for clustering the oocytes during spawning in females (Boutan), or packaging the sperms in spermatophores in males (Medem, 1945). In the present study, we were unable to know if the gland adds extra material to the gametes. Another interesting possibility that could not be discarded is that secretions from the gland had a communication role, releasing a chemical like a sex pheromone to mediate an appropriate gregarious response and gamete evacuation from conspecifics. The simultaneous hermaphrodite nudibranch mollusk Aplysia with internal crossed fertilization, produces water-borne attractin pheromones in the oviduct and are released during egg laying (Cummins et al., 2005). Attractins are a family of small proteins in invertebrates that stimulates the formation and maintenance of mating and egg-laying aggregations (Cummins et al.). It is possible that the gland, as a specialization of the gonoduct in Fissurella, could produce substances like attractins acting as chemical signals in sex communication to induce aggregation and release of gametes improving external fertilization. Although no gland has been associated with the gregarious behavior, sexual aggregation associated with gamete release has been described in vetigastropod species (Fretter & Graham, 1962).
Fissurella is one of the youngest genera within the Family Fissurellidae (McLean, 1984). Local species of Fissurella in the Chilean coast appeared in the Pliocene, and increased diversity in the Pleistocene (Herm, 1969). Although these species may have evolved structures adapted for radiating to new habitats in this relatively short geological period, this would not be the case for the reproductive system, which probably requires a longer time period for greater specialization and differentiation.
We thank Drs. Bernardita Campos and Doris Oliva for their valuable comments related to preparation of this MS.
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Figure 1 Fissurella latimarginata and Fissurella cumingi. Common schematic organization of the reproductive system. A. Transverse view. B. Dorsal view. Proportions of structures were not necessarily drawn to scale. Abbreviations: a, anus; c, genital duct (renopericardial duct); ct, ctenidium; g, gland; go, gonad; la, left auricle; Ik, left kidney; lko, left kidney opening; n, nephrostome; p, pericardium; ra, right auricle; rk, right kidney; rko, right kidney opening; v, ventricle.
Figure 2 A-K. Fissurella latimarginata. Photomicrographs of the histological sections of the reproductive system. A. Male gonad showing the centrifugal organization of the gonadal septae. B. Female gonad in vitellogenesis with organization similar to (A). C. Transverse section, showing connection between the male gonad, the gland, and the genital duct. D. Sagittal section of a male showing the nephrostome connecting the genital duct with the pericardial cavity. E. Longitudinal section of a male showing the distal portion of the genital duct opening into the distal region of the right kidney. Note the dendriform epithelial folds which project into the lumen of the duct. F. Sagittal section of a male showing the right kidney opening. Note mass of spermatozoa liberated into mantle cavity G. Sagittal section of a male showing the genital duct directed from the gonad toward the roof of the visceral cavity. Note the gland attached to the internal wall of the duct and a parasite partially obstructing its exit. H. Longitudinal section of a female showing the point of connection between the gonad, the gland, and the genital duct. Note the oocytes at the extreme lower right of the duct. I. Sagittal section in a female showing the nephrostome connecting the genital duct with the pericardial cavity. J. Longitudinal section in a female showing the distal portion of the duct emptying into distal region of the right kidney. Note the dendriform epithelial folds projecting into the lumen of the duct. K. Sagittal section in a female showing the right kidney opening connecting with mantle cavity Abbreviations: c, genital duct; ct, ctenidia in the mantle cavity; e, gonadal epithelium; g, gland; gl, gonadic lumen; go, gonad; h, hemolymph in the hemofymphatic sinus; 1, lip of the renal aperture; mu, muscular roof of the visceral cavity; m, mantle cavity; n, nephrostome; p, pericardium, pa: parasite; ra, right auricle; rk, right kidney; rko, right kidney opening; s, blood sinus; sp, spermatozoa in the mantle cavity; v, visceral cavity; thick arrow, spermatozoa in the gonad organized in sheafs; arrow, oocytes in vitellogenesis; arrowhead, gonadal septae. Scale bar: A, B= 100 µm; C, D, F, G, H, I, K= 500 µm; E, J= 250 µm.
Figure 3 A-D. Fissurella cumingi. Photomicrographs of the constituents of the reproductive system seen in a transversal section. A. Section showing the connecting point between the male gonad, the gland, and the genital duct. B. Section of a male showing the right kidney opening connecting with the mantle cavity. C. Section showing the connecting point between the female gonad, the gland, and the genital duct. D. Section of a female showing the right kidney opening connecting with the mantle cavity. Note an oocyte into the kidney (arrow). E-H. F. latimarginata and F. cumingi. Transverse section of the epithelium of the genital duct (reconstruction). E, G. Transverse section of the upper (E) and lower (G) epithelium of the duct in a male F. latimarginata. F, H. Transverse section of the upper (F) and lower (H) epithelium of the duct in a male F. cumingi. Note the apocrine secretory nature of the duct. Abbreviations: c, genital duct; g, gland; go, gonad; h, hemolymph in the hemolymphatic sinus; 1, lip of the right renal aperture; m: mantle cavity; mu, muscular roof of the visceral cavity; n: nephrostome, p, pericardium; rk, right kidney; rko, right kidney opening; v: visceral cavity; upper epithelium (thick arrow), lower epithelium (arrowhead), spermatozoa in the lumen of the duct (arrow). Scale bar: A-D= 500 µm; E-H= 25 µm.