The golden mussel Limnoperna fortunei (Dunker, 1857) is a medium-sized bivalve mollusk belonging to the family Mytilidae (Darrigran & Damborenea, 2006). Mytilidae is a family of small to large saltwater mussels (Soot-Ryen, 1955). Some of these species are edible and have high commercial value. However, L. fortunei is the only freshwater representative of Mytilidae (Darrigran & Damborenea, 2006). It is recognized as a successful invader of aquatic environments in Asia, South America and North America. An extensive revision was recently made by several authors about the biology, distribution, reproduction, ecology and other aspects of the life history of the golden mussel (Boltovskoy et al., 2015 for review). Special attention has been paid to its environmental impact (Darrigran, Penchaszadeh, & Damborenea, 2000; Darrigran, 2002; Brugnoli, Clemente, Boccardi, Borthagaray, & Scarabino, 2005; Boltovskoy, Sylvester, Otaegui, Leites, & Cataldo, 2009; Santos et al., 2012; Boltovskoy & Correa, 2015). L. fortunei is native to the rivers of China and Southeast Asia. However, this mollusk has been expanding its distribution into various parts of the world, as a consequence of increasing international trade and the great flow of vessels (Campos et al., 2014). Associated with this information, L. fortunei is capable of surviving fully in saline waters, lakes, humid areas and other water courses (Campos et al., 2014), showing various important features of a successful invader, such as a cycle of short duration, capacity of rapid growth and high fertility, in addition to wide physiological tolerance to several abiotic factors (Morton, 1977; Oliveira, Calheiros, Jacobi, & Hamilton, 2011; Campos et al., 2014). Therefore, this species has great potential for establishing itself on all continents, since any environmentally suitable area can become a potential habitat (Campos et al., 2014).

Limnoperna fortunei is a filter-feeding (Morton, 1973) opportunist with a short life span (lives on average three years) and reaches between 3 and 4 cm when adult (Darrigran & Damborenea, 2006; Campos et al., 2014). It is also gonochoric with external fertilization, and rare hermaphroditism has been recorded (Darrigran, Damborenea, & Penchaszadeh, 1998; Darrigran, Damborenea, Penchaszadeh, & Taraborelli, 2003; Callil, Gomes, & Soares, 2012). It has gonads that develop inside the visceral mass and the pallial tissue (Morton, 1982; Darrigran & Damborenea, 2006), and a high reproductive potential (Morton, 1977).

Several environmental and economic impacts are caused by Limnoperna fortunei, as recorded by Darrigran (2002), Takeda, Fujita and Fontes-Junior (2007), Santos et al. (2012) and Oliveira, Ayroza, Castellani, Campos and Mansur (2014). The control and management of the species depends on knowledge of its reproductive aspects. Thus, the reproductive cycle phases and period of spawning events of L. fortunei have been the focus of several studies (Morton, 1982; Darrigran, Penchaszadeh, & Damborenea, 1999; Darrigran et al., 2003; Damborenea & Penchaszadeh, 2006; Callil, Gomes, & Soares, 2012).

The gametogenic cycle of Limnoperna fortunei is regulated by environmental factors, especially temperature (Morton, 1982; Darrigran, Penchaszadeh, & Damborenea, 1999; Darrigran et al., 2003). Gonadal development, changes in the germinal epithelium and the duration of each reproductive cycle vary according to climatic conditions. In subtropical regions, there is usually no definite and synchronic sequence of the gonadal development stages during the reproductive cycle. As a result, there may be continuous production of gametes, accompanied by numerous spawning events throughout the year (Darrigran & Damborenea, 2006). Thus, in order to contribute to a better understanding of the reproduction of L. fortunei, we described the gonadal organization and the cellular dynamic of the male and female germinal epithelium during each reproductive cycle over its life history, using high resolution histology associated with quantitative data.

MATERIALS AND METHODS

Sampling area: A total of 1 228 specimens of the golden mussel Limnoperna fortunei were collected quarterly from March 2010 to December 2012 (during autumn, winter, spring and summer) in rivers of the upper Paraná River floodplain, Brazil (22o45’39.96” S - 53o15’7.44” W), being 315 individuals (138 males, 177 females) in 2010, 465 individuals (186 males and 279 females) in 2011 and 448 individuals (195 males and 253 females) in 2012. A subsample of 688 reproductively active individuals (52 females + 90 males in winter; 23 females + 45 males in autumn; 83 females + 99 males in spring; and 97 females + 199 males in summer) varying from 1.0 to 3.86 cm valves length, were measured with a pachymeter, and was used to classify the reproductive phases. Sampling was carried out using a Petersen grab (modified for benthic sampling) or manually (when they were on submerged branches or on aquatic macrophytes). The valves of the golden mussel were separated, opened and the visceral mass was removed by cutting the adductor and retractor muscles. The visceral mass of these 688 individuals was removed and fixed by immersion in Bouin´s fluid. A sample of gonads from males and females was photographed using a digital camera (Fujifilm HD 14MP Finepix S2800) and processed for light microscopy.

 

Histology and light microscopy (LM): The visceral mass fixed in Bouin´s fluid was dehydrated in ethanol and embedded in historesin (Leica historesin). Serial sections (3 µm thick) were stained with Periodic-Acid-Schiff’s (PAS)/Haematoxylin/Metanil Yellow (Quintero-Hunter, Grier, & Muscato, 1991) and 0.5 % Toluidine Blue (Vidal, 1987; Mello & Vidal, 1980). These sections were stained using the Reticulin Method, which enhances basement membranes, and resulted in a black hue of reticulin fibers (modified from Puchtler & Waldrop, 1978; Vidal, 1988). Gonad sections were evaluated using a computerized image analyzer (Leica LAS Interactive Measurements).

 

Alterations of the germinal epithelium during the adult reproductive life history: The reproductive cycle of the golden mussel was classified in phases based on the alterations of the germinal epithelium (Boltovskoy et al., 2015) considering the stages (and steps) of the germ cell differentiation, predominant type of germ cell, repletion of gonadal tubules, and the presence of mature germ cells in the gonoduct. Therefore, reproductive phases of males were recognized according to the presence of spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. As regards females, the presence of oogonial proliferation, early prophase oocytes, previtellogenic oocytes, vitellogenic (early vitellogenic oocytes, middle vitellogenic oocytes) and full-grown oocytes, empty ovarian tubules and atretic oocytes were considered.

 

Quantitative analysis: The frequency of each reproductive phase throughout the seasons of the year between females and males was calculated from the number of adults analyzed (n = 688).

The size of the vitellogenenic oocytes was measured using a computerized image analyzer (Leica LAS Interactive Measurements), in order to check the variation between the minimum and maximum diameter of these oocytes.

RESULTS

Gonadal morphology: The gonadal tissue from the golden mussel L. fortunei was located throughout the visceral mass and was dispersed by the pallial tissue. The testis (Fig. 1 A, Fig. 1B) consisted of a white tissue, while the ovary (Fig. 2 A, Fig, 2B, Fig. 2C) was formed by a tissue with orange clusters, the oocytes.

In serial histological sections (longitudinal and transversal), the testis (Fig. 1C, Fig. 1D, Fig. 1E) and ovary (Fig. 2D) of the golden mussel L. fortunei showed a system of branched tubules (Fig. 2E, Fig. 2F) with an arborescent aspect that opened in a ciliated gonoduct (Fig. 2G, Fig. 2H, Fig. 2I). With gonadal development, the gonadal tubules increased in number and protruded into the pallial tissue. The tubules in the testis and ovary were delineated by an epithelium that contained somatic and germ cells. The germinal epithelium rested on the basement membrane (Fig. 1F, Fig. 1G, Fig. 1H and Fig. 2J, Fig. 2K, Fig. 2L). Developing male and female germ cells protruded into the lumen of testicular and ovarian tubules.

 

Spermatogenesis: Male germ cells in distinct steps of spermatogenesis were found in the testis of the golden mussel L. fortunei throughout the reproductive cycle (Fig. 3A, Fig. 3B, Fig. 3C, Fig. 3D, Fig. 3E, Fig. 3F, Fig. 3G, Fig. 3H, Fig. 3I). Among the germ cells were the Sertoli cells (Fig. 3I). They rested on the basement membrane of the germinal epithelium that lined the tubular testis and extended toward the lumen, coming into contact with spermatogonia, spermatocytes and spermatids. With the proliferation of the germ and somatic cells, the testicular tubules lengthened and, consequently, the whole testis enlarged. As regards spermatogenesis, the morphological characterization of the male germ cells that were present in the testis of L. fortunei was summarized in Table 1.

 

Oogenesis: Female germ cells in distinct steps of oogenesis were found in the ovary of the golden mussel Limnoperna fortunei throughout the reproductive cycle (Fig. 4A, Fig. 4B, Fig. 4C, Fig. 4D, Fig. 4E, Fig. 4F, Fig. 4G, Fig. 4H, Fig. 4I). In the germinal epithelium that lined the ovarian tubules, oogonial proliferation formed clusters of germ cells (nests) that projected into the ovarian lumen (Fig. 4A, Fig. 4B, Fig. 4C, Fig. 4D). In the nests, oogonia entered into meiosis, and gave rise to oocytes (Fig. 4E, Fig. 4F). Concomitant with oogonial proliferation, somatic epithelial cells also proliferated. Resulting from both germ and somatic cell proliferation, the epithelium and the ovarian tubules lengthened. As a consequence, the whole ovary enlarged. During their entire development, the oocytes remained attached to a short extension of the epithelium of the ovarian tubules. As the oocytes increased, they protruded into the tubular lumen and, consequently, the diameter of the tubules also enlarges. Early vitellogenic oocytes presented a minimum diameter of 40.16 µm (Fig. 4F) and with the progression of vitellogenesis, the oocytes reached a maximum diameter of 142.13 µm (Fig. 4I), when they were called full-grown oocytes (number of oocytes measured = 83). Oogonial proliferation and progressive morphological changes in the ooplasm of the oocytes were used to classify oogenesis in distinct stages that included oogonial proliferation, chromatin nucleolus, primary growth and secondary growth (vitellogenesis). As regards the oogenesis stages, the morphological characterization of the female germ cells that were present in the ovary of L. fortunei was summarized in Table 2.

 

Reproductive phases: During the adult reproductive life history of males or females of L. fortunei, the renewal of the germ cells, their differentiation, development, maturation and release resulted in gonadal alterations that characterized different phases over each reproductive cycle.

Thus, considering the types of germ cells, as well as their predominance in the gonad, four phases were described over the reproductive cycle of L. fortunei males: regenerating, developing, sperm releasing capable and regressing (Table 3; Fig. 5A, Fig. 5B, Fig. 5C, Fig. 5D, Fig. 5E, Fig. 5F, Fig. 5G, Fig. 5H, Fig. 5I, Fig. 5J, Fig. 5K, Fig. 5L).

As regards, the females and the types of germ cells, as well as their predominance in the gonad, four phases were described over the reproductive cycle of L. fortunei: regenerating, developing, spawning capable and regressing (Table 4; Fig. 6 A, Fig. 6B, Fig. 6C, Fig. 6D, Fig. 6E, Fig. 6F, Fig. 6G, Fig. 6H, Fig. 6I, Fig. 6J, Fig. 6K, Fig. 6L).

 

Reproductive parameters: The valve lengths of sexually active (mature) individuals varied from 1.72 cm to 3.72 cm (n = 255) and 1.01 cm to 3.86 cm (n = 433) for females and males, respectively. Parameters of immature individuals, i.e. with valves having a total length below 1.0 cm were not considered.

The frequency of individuals in different reproductive phases showed that the majority of females (36.54-86.96 %) and males (50.0-80.40 %) were in the development phase (Fig. 7). Females presenting full-grown oocytes, i.e. belonging to the spawning capable phase, were found only in summer (Fig. 7). In contrast, males in this same phase were found throughout the year (Fig. 7).

DISCUSSION

The serial longitudinal sections from the whole gonad of the adult male or female of L. fortunei showed that, in reproductively active individuals, the testis or ovaries are formed by a continuous system of branched tubules. The continuous system of tubules is confirmed by the reticulin stain that enhances the basement membrane components. The basement membrane is synthesized by the somatic cells from the germinal epithelium, as is usual in epitheliums (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2008). Thus, the germinal epithelium rests upon a basement membrane that separates it from the pallial tissue. Recent studies by Franco, Heude-Berthelin, Goux, Sourdaine and Mathieu (2008), Franco et al. (2010) and Franco, Kellner, Goux, Mathieu and Heude-Berthelin (2011) also recognized that bivalve gonads are formed by a continuous system of branched tubules.

As seen in L. fortunei, except for the gonoduct, in both males and females, the epithelium, bordering the gonadal tubules, houses germ cells and is active in gamete production. Thus, the proliferation of the germ and epithelial cells is responsible for the increase in the number and length of the gonadal tubules throughout the reproductive cycle. Other reproductive characteristics of bivalves, including L. fortunei, are that in the female the oocytes protrude toward the ovarian lumen, are not surrounded by a layer of somatic cells and remain connected to the germinal epithelium throughout their development. This connection, referred to as a stalk, is broken when the mature oocytes are released into the luminal compartment of the ovary (Pipe, 1987; Shafee, 1989; Garton & Haag, 1993; Darrigran & Damborenea, 2006; Saucedo & Southgate, 2008). Therefore, it is reasonable to assume that the nutrition of the oocytes occurs through the stalk.

In the mollusk, reproduction is a cyclical event during its life history. Thus, various scales (i.e. inactive, developing, maturing, mature, and spent) categorizing gonadal alterations throughout the reproductive cycle are reported in the bivalve literature (see Lubert, 1959; Shafee, 1989; Saucedo & Southgate, 2008; Franco et al., 2011; Boltovskoy et al., 2015 for review). Despite reporting the same event, reproductive cycle scales vary depending on the author (Darrigran & Damborenea, 2006; Callil, Gomes, & Soares, 2012; Boltovskoy et al., 2015).

Most authors (Boltovskoy et al., 2015 for review) consider the initial stage of the reproductive cycle as inactive or resting. However, in this stage there is intense cell proliferation and restocking of the gonad. Thus, the stage or phase previously called “inactive” becomes “regenerating” in this study. Herein, we proposed new terminology based on the dynamic of the germinal epithelium, to recognize phases over the reproductive cycle of L. fortunei, i.e. Developing, Spawning Capable, Regressing and Regenerating.

The characterization of the reproductive phases in the female L. fortunei takes into account the presence and predominance (in the gonad) of: oogonial proliferation and entrance into meiosis; morphological changes in the ooplasm of the oocytes from the previtellogenic to vitellogenic stages; full-grown to mature oocytes and the spawning event. As regards the males, the characterization of the reproductive phases takes into account the presence and predominance (in the gonad) of: spermatogonial proliferation and entrance into meiosis; differentiation of the gametic cells from spermatids to spermatozoa; and the release and presence of sperm in the gonoduct.

When analyzing the distribution of the reproductive phases of individuals by seasons, L. fortunei was observed to reproduce throughout the year. This is based on the presence of individuals belonging to the regressing phase in the four seasons. This means that they were actively spawning. These data on the life history, herein shown, are similar to studies on the reproductive cycle conducted on other species of mussels (Çek & Sereflisan, 2006), including the golden mussel (Callil, Gomes, & Soares, 2012).

Despite that other studies showed that the golden mussel may have one to five spawning periods throughout the year (Callil, Gomes, & Soares, 2012), depending on the geographical area inhabited, Morton (1982) has stated that it is quite difficult to establish a pattern of spawning for opportunistic species, including L. fortunei, which may have various reproductive strategies, in order to occupy new environments. This opportunistic species spawns throughout the year (Darrigran, Penchaszadeh, & Damborenea, 1999).

The morphological data and reproductive parameters shown here confirm how informative the cellular dynamic of the germinal epithelium is for the understanding of the cyclic gonadal events during the adult reproductive life of the mollusk in general. This knowledge about the gametogenesis and reproductive cycle of invasive or opportunist species (e.g. Limnoperna fortunei) can become a fundamental tool for the development of control strategies and implementation of programs to decrease their proliferation in natural environments (Darrigran & Damborenea, 2006). Therefore, the data presented herein can contribute to new knowledge about the life history of the golden mussel.

ACKNOWLEDGMENTS

We thank the following: John J. Stanley Jr for the grammatical review; Angelo A. Agostinho (PELD-Sítio 6/CNPq) and the Center for Research in Limnology, Ichthyology and Aquaculture-Nupélia Universidade Estadual de Maringá-UEM for financial and logistical support; Alice M. Takeda (Nupélia/DBI/UEM) for the opportunity to work with the golden mussel; Laboratório de Bentos/Nupélia; Laboratório de Patologia - HU/UEM; and Departamento de Morfologia – IBB – UNESP.

RESUMEN

El mejillón dorado Limnoperna fortunei (Dunker, 1857) es una especie invasora que se ha dispersado rápidamente y ha colonizado diferentes hábitats potenciales distribuidos por todo el mundo, causando impactos ambientales y económicos. El control de esta especie depende del conocimiento de los aspectos reproductivos. Entre marzo 2010 y diciembre 2012 fueron muestreados periodicamente alrededor de 1 200 especímenes de L. fortunei en la planicie de inundación del Alto Rio Paraná, Brasil. Con el fin de contribuir con una mejor comprensión de los aspectos reproductivos de L. fortunei, se describió la dinámica celular del epitelio germinativo masculino y femenino durante la vida reproductiva anual de esta especie, mediante histología de alta resolución basada en la actividad del epitelio germinal y el consecuente desarrollo de las células germinales. En este estudio se reconocieron las siguientes fases reproductivas: desarrollo, capacidad de desove, regresión y regeneración. En los machos se describieron las siguientes células germinales: espermatogonias, espermatocitos primarios y secundarios, espermátidas y espermatozoides. Mientras que en las hembras se estudiaron: nidos celulares, ovogonias, ovocitos profásicos tempranos, ovocitos pre-vitelogénicos y ovocitos vitelogénicos (ovocitos vitelogénicos tempranos, ovocitos vitelogénicos y ovocitos completamente desarrollados). Los datos morfológicos y parámetros reproductivos que se muestran aquí confirman cómo la dinámica celular del epitelio germinal permite la comprensión de los acontecimientos cíclicos gonadales durante la vida reproductiva de adultos del molusco, en general. El conocimiento de la gametogénesis de esta especie invasora puede convertirse en una herramienta fundamental para el desarrollo de estrategias de control y aplicación de programas para disminuir su proliferación en ambientes naturales.

 

Palabras clave: Bivalvia, Mytilidae, ovogénesis, espermatogénesis, ovario, testículo, Limnoperna fortunei.

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