Particular aspects of gonadal cycle and seasonal distribution of gametogenic stages of Mytilus galloprovincialis cultured in the Estuary of Vigo.

ABSTRACT The Mytilus gonadal cycle in Vigo Estuary follows the histological pattern proposed by Lubet (1957, 1959). We show details not previously described, due to sexual differences, environmental characteristics and high periodicity samplings: an out of phase between reserve tissue and gonadal cycles; a "bridge stage" determined by intermediate characteristics among the last spawnings, gonad resorption and regeneration of the reserve tissue; a moving forward of the initial stages and different duration by sexes; and clear-cut differences between spawnings and restorings: in winter, they are a slow and less intense process, with abundant reserve tissue, high atresia and they do not end in effective spawnings, whereas in spring are very rapid and intense processes causing massive spawnings. The atresia present two phases: cytoplasmatic structures self-lysis and digestion by hemocytes. Winter atresia is related to unfavorable conditions to stimulate spawning. Temperature and food availability determine the temporal evolution of the gametogenic cycle in male and female differently. The mantle tissue index varies depending on the number of gametes and is suitable as a gonadal index in this species.

KEY WORDS: atresia, chlorophyll a, gametogenic stages, Mytilus galloprovincialis, seasonal distribution, temperature, mussel 

INTRODUCTION

Since 1987, Galicia (NW Spain) ranks as the second world producer of the mussel, Mytilus galloprovincialis Lmk, producing approximately 260,000 MT/year (Perez et al. 1991). The high production is due to the high primary production in the Galician estuaries associated with the intermittent presence of marine upwellings rich in nutrients from the Atlantic water (Tenore et al. 1982). The main aspects of mollusk culture are growth and reproduction, both closely linked. In-depth studies of changes at histological, biochemical and metabolic levels would lead to greater knowledge of bivalves reproduction and better control and optimization of their production.

The reproductive cycle is a genetically controlled response to the environment (Sastry 1975). Thus, the reproductive cycle of Mytilus, although it is characteristic of this species, timing and duration are determined by the interaction between endogenous and exogenous factors, varying with the geographical area and different annual environmental conditions (Rodhouse et al. 1984, Seed & Suchanek 1992, Villalba 1995). The most important environmental parameters affecting the bivalves reproductive process include temperature and food availability (Lubet 1981, Seed & Suchanek 1992, Pazos et al. 1997, Ceballos-Vazquez et al. 2000).

In species of the genus Mytilus, the anatomic proximity of the genital papilla to the mantle tissue is used for the expansion of the gonadal structure and the gametogenic development that takes place mainly in this tissue. Mantle tissue is defined as a laminar anatomic space, of variable thickness, surrounded by an epithelium, comprising different types of cells subject to fluctuations in the population following a seasonal rhythm. Mantle basically constitutes reserve tissue formed by vesicular cells (VC) where glucogen is stored and adipogranular cells (ADG) that accumulate protein granules, including lipids and small amounts of glucogen (Pipe 1987a, Peek et al. 1989); the gonad is comprised of different germinal cells, gonoducts, and auxiliary cells and the circulatory system formed by hemolymph, hemocytes, sinuses, veins, and channels.

The mantle tissue of Mytilus has 2 interrelated physiologic functions: accumulation of reserve substances in the VC and ADG cells and development of the gonad that invades the mantle, proliferating at the expense of the reserve tissue. An inverse seasonal development has been described between the reserve tissue cycle, especially glycogen and the gametogenic cycle (Bayne et al. 1982, Lowe et al. 1982), and their control by same endogenous factors, especially neuroendocrine factors (Mathieu et al. 1991, Danton et al. 1996),

This work provides a description in detail of the reproductive cycle of the mussel cultured in the Vigo Estuary (Galicia, NW Spain), and its seasonal distribution determines the spawning periods in this leading production area and their relation to temperature and amount of food available. The results also show a possible control of both reserves tissue and gametogenic cycles, by some different endogenous factor.

MATERIALS AND METHODS

Biometric Analysis

A total of 22 samples were collected fortnightly from June 1993 to August 1994 to cover a complete annual and reproductive cycle. In each sampling, 30 adult individuals from 8-10 cm in length were randomly collected, all from the same floating mussel bed (raft) at depths of 5 m.

The individuals were immediately conveyed to the laboratory in insulated tanks with seawater and processed the same day. To determine the gonadal condition index (GCI) we calculated the ratio between the mantle fresh weight (Wm) and the fresh weight of the meat (difference between the total weight -Wt- and the weight of the shell -Wc-), as used previously by Aguirre (1979) for this species: GCI = [W.sub.m]/[W.sub.t]-[W.sub.c]) x 100.

Histologic Preparations

Each individual was examined histologically to determine the phase or gametogenic stage. A small section (0.5 x 1 cm) of the central part of one hemimantle was fixed in Bouin solution for 4 h, dehydrated and embedded in paraffin. Sections 5-[micro]m thick were cut and stained with Harris hematoxylin and eosin and mounted on a microscopic slide. For classification of each stage of the gametogenic cycle, initially the model and nomenclature proposed by Lubet (1959) were followed.

Environmental Parameters

Temperature ([degrees]C) and chlorophyll a concentration ([micro]g/L), at depth of 5 m in the water column were registered weekly by Centro de Control do Medio Marino-Xunta de Galicia (CCMM). The data belonged to the station nearest to the sampled cultivation area. Temperature was measured by using a CTD and chlorophyll with spectrofluorimetric methods.

Data Presentation and Statistical Analysis

GCI data are shown using the average and the standard deviation as estimators of central trend of the sample. Statistical analysis used a statistical software package on Windows (SPSS Inc, 1989-1999). Distribution types and their homocedasticity were analyzed by Kolmogorow-Smimov and Levene tests, respectively. Seasonal comparisons of biometric parameters by gametogenic stage and sex were conducted by applying nonparametric contrasting (2-tail) Kruskal-Wallis and Mann-Whitney tests. Correlation between temperature and chlorophyll a was estimated by Pearson coefficient ([r.sub.s]).

RESULTS AND DISCUSSION

Biometric Analysis

A total of 660 individuals was sampled, 45.2% males, 54.2% females and 0.06% was impossible to determine the sex; no cases of hermaphroditism were found. The ratio between sexes was 1.19, similar to those found for Mytilus edulis and M. galloprovincialis (Lubet 1959, Villalba 1995).

Average size of the individuals analyzed was 9.26 [+ or -] 0.3 cm, total average fresh weight was 46.8 [+ or -] 6.89 g, and the average shell weight was 19.92 [+ or -] 3,16 g. No significant differences were noted between sexes for these two parameters (P < 0.01, Mann-Whitney test), but there were for the Wm (P < 0.01, Mann-Whitney test; 5.6 [+ or -] 2.21 g in males and 4.42 [+ or -] 1.58 g in females). This was possibly due to the different characteristics of gametogenic development in both sexes and to the larger number of gametes formed in males.

Gametogenic Cycle and Their Temporal Evolution

To histologically describe of the gonadal cycle in Mytilus galloprovincialis in the Vigo Estuary we followed the model proposed by Lubet (1959). However, we observed important characteristics not described previously, probably due to the environmental conditions in the area and the high periodicity with which the samples were taken.

The start of the gametogenic cycle is more or less synchronous in both sexes, with the appearance of a small percentage of individuals at the end of June in stage 0. This stage extends to August in males and to October in females (Fig. 1A, Fig. 2), characterized by a rapid regeneration of the reserve tissue (mainly VC cells) and the absence of gonad tissue (except for some residual gamete from the previous cycle). Prior to stage 0 a period of resorption of all the mantle tissue occurs in Mytilus, in which mantle tissue is only constituted of cells from connective tissue (stage IIID) (Lubet 1959, Villalba 1995). We found no male individuals with these characteristics, and only 6% of females showed degradation of the follicular structure accompanied by a considerable accumulation of hemocytes inside and outside the follicles and in the gonoducts, probably with phagocytic function (Figs. 1B, 2).

[FIGURES 1-2 OMITTED]

The number of individuals in these two initial stages of the gonadal cycle is very low. In its place, from July to October, we found a high percentage of individuals (70% to 100%) with intermediate characteristics among the spawnings of the last gametes of the ending cycle (stage IIIB-end), follicular degrading and resorption by hemocytes and regeneration of the reserve tissue. We have termed the overlapping of these three stages (IIIB final, IIID and 0) as the "bridge stage" (Figs. 1C, D, 2). These results show a gametogenic cycle out-of-phase with the reserve tissue cycle opposite to a close and inverse relationship as described in the bibliography (Lubet 1959, Lowe et al. 1982, Gabbott 1983, Mathieu et al. 1991, Mathieu & Lubet 1993, Danton et al. 1996); we assume that they may be regulated and determined by some different factors.

Along with the regeneration of reserve tissue, proliferation of gonias (spermatogonias and oogonias) commences, forming new gonadal follicles or laying on the empty residual follicle walls of the previous cycle (stage I) (Fig. 3A, B). Proliferation of gonias is restricted to the terminal ends of the follicles, from where they migrate to periphery. In males, their number is higher than in females, and in the latter the oogonias migration occurs at the same time as their differentiation into previtelogenic oocytes that anchor to the follicular wall for growth. The percentage of individuals at this stage in time (14% of males between July and August, and 24% of females between July and November; Fig. 2) appears to indicate that, as in previous cases, it is slower in females. At this stage, the reserve tissue abounds mainly in the ADG cells arranged around the VC cells and the walls of the follicles in development. This arrangement seems to suggest the mediation of the ADG cells in the mobilization of the glucogenic reserves of the VC cells and in its transportation to the germinal line cells for their development and maturation, either directly or via other cellular types of the follicular wall, as hypothesized by Pipe (1987a, 1987b).

[FIGURE 3 OMITTED]

Differentiation and meiosis of the gonias to give rise to the whole germinal line sequence (stage II) commences in August and continues into October to November when 85% of males and 34% of females are at this stage (Fig. 2). This seems to point to the fact that this stage is very rapid in females. The follicles are small and, in males, a thick cortex of spermatogonias, spermatocytes I and spermatides arranged centripetally is observed, and spermatozoids are organized in radial rows with the flagella towards the follicular light (Fig. 3C). As in other species, the second meiotic division is extremely rapid and it is difficult to observe spermatocytes II under optical microscopy. In females, small groups of oogonias, previtelogenic oocytes at different phases of maturity anchored on the follicular wall, some vitelogenic pedunculated or free oocytes in follicular lumen and atresic oocytes are observed (Figs. 3D).

These two initial stages of gametogenesis show a certain asynchrony between sexes. Furthermore, they take place 2 mo before that described by Villalba (1995) in the same and in other areas of the Galician estuaries, shown an asynchrony between different populations, probably influenced by environmental factors.

Gametogenesis continues throughout winter, the number and volume of follicles increases as the reserve tissue decreases, until stage IIIA is reached in both sexes in December. This is described as a stage for the physiologic and cytologic maturity of the gametes prior to spawning from December to March, where the gonadal follicles take up the whole mantle tissue, full of mature gametes, and the reserve tissue has now been fully consumed. We observed this in females (Fig. 4A); nevertheless the males reach this stage with relatively small follicles and with a large amount of reserve tissue, mainly ADG cells (Fig. 4B), which again suggests a different regulation of reserves and reproductive cycles. From this point onwards and until April there are similar images, prior to spawnings, with an increase in the volume of follicles and a gradual decrease in the reserve tissue in males. We prefer, however, to restrict naming stage IIIA to the mature stage attained between November and December, prior to the first emission of gametes, since after this gametogenesis occurs at an increasingly rapid and intense rate and is accompanied by numerous gonial mitoses that mark "gonadal restoration," thus making it difficult to classify each individual. The higher number of males than females at this stage from November to the end of December may indicate a slow down of spermatogenesis during winter.

[FIGURE 4 OMITTED]

The first emissions of gametes (stage IIIB), starts at the end of autumn, are noted by the presence of spermatozoids and mature oocytes in the gonoducts, the loss of radial arrangement of spermatozoids in the follicular lumen in males (Fig. 4C) and an increase of follicular light and predominance of pedunculated vitelogenic oocytes in females (Fig. 4D). These autumn-winter spawnings are also characterized by a large amount of reserve tissue, mainly ADG cells, unlike that described by other authors (Lubet 1957, Villalba 1995), the spawn does not affect all the follicles or the gametes formed, which in addition present high atresia and degradation mainly in females.

Consecutive restoration of the germinal cells occur after these spawnings (stage IIIC), once again noting numerous gonial mitoses and the subsequent formation of new cohorts of gametes (Fig. 5A, B), which are evacuated through the gonoduct. Gametogenesis continues and increases until spring, with the number and size of follicles gradually increasing, in the same manner as the number of divisions of germinal cells and of gametes formed and emitted into the medium. Likewise, the volume and number of reserve tissue cells decreases until only a few VC cells remain among the follicles. By mid March, the first spring spawning takes place. This affects all the follicles and total expulsion of all the mature gametes occurs. In males, a narrow, discontinuous belt of spermatogonias and some spermatocytes I remain (Fig. 5C), whereas in females small groups of oogonias, some very young previtelogenics and a variable number of pedunculated vitelogenic oocytes are noted (Fig. 5D). Following this, another intense, rapid period of gametogenesis (stage IIIC) takes place and extends for 15-20 days (Fig. 6A, B). At the initial moment of this restoration, the follicles are once again compressed due to the small recovery of the reserve tissue. Numerous hemocytes also appear located around the follicles and between the VC cells and beside small isolated ADG cells.

[FIGURES 5-6 OMITTED]

As restoration progresses, the proliferation of spermatogonias and oogonias is observed, with an increase in spermatocytes I, some spermatides and spermatozoids in males, and an increase in previtelogenic oocytes, some pedunculated vitelogenic oocytes and a few mature oocytes in the follicular lumen in females. These intense spawnings and restoration occur continually through April and May (Fig. 2).

In summer, the potentiality of the gonad gradually decreases and spawnings and restorations become slower and less intense. Smaller, partially empty follicles are observed, with very few mature gametes and no gonias on the walls to withstand another restoration. For this reason, we have named these final spawnings as stage IIIB final (Fig. 6C, D), and in the majority of individuals this runs parallel to the formation of reserve tissue and the reabsorption of gonadal tissue at the end of the gametogenic cycle, as noted earlier.

Gametogenic development in Mytilus is, then, a dynamic, continuous process, and from the first emission of gametes we may distinguish between spawnings and restorations in winter from those occurring in spring. In winter it is a slow, less intense process with abundant reserve tissue and a high atresia with degradation of gametes. This leads us to assume that they do not end in effective, viable spawnings. Whereas the spawnings and restorations in spring are very rapid and intense processes, giving rise to massive synchronic spawnings in both sexes, it leaves practically empty follicles and the reserve tissue completely exhausted.

Temperature and Food Effects on Temporal Evolution of Gametogenic Cycle

We have already described the temperature and availability of food as decisive factors for somatic growth and gonadal development in bivalve mollusks (Sastry 1975, Seed & Suchanek 1992, Pazos et al. 1997, Ceballos-Vazquez et al. 2000), which also appear to determine the duration of the different phases of gametogenesis (Hilbish & Zimmermann 1988), spawning (Starr et al. 1990, Pazos et al. 1997) and subsequent larval development (Sastry 1975, Bayne et al. 1976). Although an inter-annual variation for these factors has been described, they also vary seasonally (Fig. 7) correlated in a significant manner ([r.sub.s] = 0.9; P < 0.01): the winter minimum temperatures coincide with the minimum chlorophyll a values; both parameters increase from spring onwards, reaching their maximum values in summer.

[FIGURE 7 OMITTED]

The male-female ratio at each gametogenic stage shows a different seasonal evolution (Fig. 2). Between June and September, the high rate of spawning males (36% to 70%) and of females at what we have termed the bridge stage (80% to 100%), where we include Stage IIID (6%), would suggest that the high summer temperatures completely inhibit oogenesis but not spermatogenesis, which may continue until early autumn. Also, the high concentration of phytoplankton seems to influence the development of reserve tissue. Both factors determine the paralyzing of one gonadal cycle and the start of another, as also noted by Lubet (1981). On the other hand, the higher percentage of males at Stage II (70%) and of spawning and restoration females in October and December would suggest that the fall in temperature and nutrients delays spermatogenesis in relation to oogenesis. Throughout the winter, consecutive spawnings and restorations occur synchronically in both sexes, with fewer overlapping of previous stages, until reaching the main spawnings in spring.

To date, numerous works have attempted to explain the effect of environmental parameters on gonadal development and spawning in diverse bivalve mollusks. Some of these works lay greater importance on temperature (Sastry 1975, Newell et al. 1982, Gaspar & Monteiro 1999), whereas others focus on food availability (Emmett et al. 1987, Starr et al. 1990, Jaramillo & Navarro 1995). Our results seem to point towards the existence of a temperature above and below which oogenesis and spermatogenesis are affected differently. The slowness of the gametogenic process and the small emissions of gametes during the winter, gonadal reactivation and massive spawnings in spring and the paralyzing of the gametogenic cycle in females in summer, when nutrients are still abundant, may only be explained by a effect of both factors, as noted by Janzel and Villalaz (1994) and by Claereboudt and Himmelman (1996) for pectinides.

Atresia Phenomena

In the description of the gametogenic cycle, we have indicated the existence of atresia phenomena and ovocytary degeneration, mainly during spawnings and restorations in autumn-winter and at the end of the gametogenic cycle (bridge stage and IIID). This phenomenon is frequent in bivalve mollusks (Pipe 1987b, Dorange & Le Pennec 1989, Motavkine & Varaksine 1989, Beninger & Le Pennec 1991), although there is no precise knowledge of its physiologic significance. According to Motavkine and Varaksine (1989), this may be due to the limited capacity of the follicle to maintain germinal cells, to self-cleaning processes at the end of the gametogenic cycle such as the preparation for the following one and to stress situations (environmental contamination, nutritional deficit, low temperatures). Due to the intense oocytary atresia observed in winter and its lessening nearer to spring spawnings, we considered that this may also be related to a high permanence of mature oocytes in the follicular lumen, despite the lack of favorable conditions in the medium to stimulate spawning.

This phenomenon has been related to oocytes lysosomal activity (Pipe & Moore 1985), and the products deriving from this lysis could be reabsorbed by the auxiliary cells, hemocytes and epithelial cells in the gonoducts, as pointed different authors (Pipe 1987a, Lubet et al. 1987, Dorange & Le Pennec 1989, Le Pennec et al. 1991). We observed this. phenomenon within the follicle and throughout the gonoduct (Fig. 8A) and this seems to present two clearly differentiated phases. Initially, there are lysis of the oocytary membrane and the cytoplasmatic structures, giving rise to large masses of disperse cytoplasmatic material with nuclei without nucleoli and more translucent in comparison with normal oocytes nuclei. (Fig. 8B). This is followed by digestion of the lisated material by hemocytes (Fig. 8C). At the same time as oocytes atresia, in males we observed masses of hemocytes in the interior of follicles and gonoducts (Figs. 8D), which also suggests degradation of spermatozoids, as also noted by Bayne et al. (1978).

[FIGURE 8 OMITTED]

Gonadal Condition Index

Gonadal condition indices are also widely used in the growth and reproduction study of marine invertebrates (Grant & Tyler 1983), and are the simplest, most efficient way to make an initial approximation to the state of sexual development in an individual. Some authors use all the meat (somatic and gonadic tissue) to calculate this index (Guillou et al. 1990) or parts of the visceral mass such as the gonad (Emmett et al. 1987, Jaramillo & Navarro 1995). Taking into account that the gonad in Mytilus develops by invading the mantle tissue during the reproductive cycle, Aguirre (1979) identifies the condition index of this tissue as the gonadal condition index.

In this work, the condition index of the mantle or gonadal condition index (GCI) shows a clearly defined seasonal variation (Fig. 9A) and is statistically significant because of the gametogenic stage (Fig. 9B). Seasonally, both in males and in females, the minimum values occur at the end of spring and early summer, after the main spawnings and at the end of the gonadal cycle. From summer onwards, this increases until attaining its maximum value in early spring, prior to the spring spawnings. In terms of the stages of the gametogenic cycle described, this index presents significant differences between males and females (P < 0.01, Mann-Whitney test). In females this reaches a maximum at Stage IIIA and a minimum after the spring spawnings; whereas in males the maximum value is attained in the winter restorations with the minimum at Stage I. This confirms the suitability of the mantle tissue index as a gonadal index, because in both sexes it seems to vary more depending on the number of gametes rather than the amount of reserve tissue.

[FIGURE 9 OMITTED]

ACKNOWLEDGMENTS

The authors thank Jose and Antonio Antepazos and the fishermen of the "Antepazos I", who kindly provided the mussels used in this work, and the Centro de Control de Calidad do Medio Marino for providing temperature and chlorophyll a data. We are also grateful to Ian Emmett for correcting the English of the manuscript. This research was supported by a grant from Autonomous Galician Government (Xunta de Galicia -PGIDT01PXI30112PR-) and University of Vigo.

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M. P. SUAREZ, (1) C. ALVAREZ, (2) P. MOLIST (2) AND F. SAN JUAN (1), *

(1) Dpto. Bioquimica, Genetica e Inmunologia, Facultad de Ciencias, Universidad de Vigo, Lagoas Marcosende, 36200 Vigo (Pontevedra) Spain; (2) Dpto. Fisiologia y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Lagoas Marcosende, 36200 Vigo (Pontevedra), Spain

* Corresponding author. E-mail: [e-mail]