Reproductive cycle of the razor clam Solen marginatus (Pulteney 1799) in Spain: a comparative study in three different locations.

ABSTRACT The reproductive cycle of the razor clam Solen marginatus (Pulteney 1799) was studied in three different locations of Spain. Sampling was performed in the natural beds of: Eo Estuary (NW Spain) in 1994 to 1995, Santander Bay (N Spain) in 1998 to 1999 and Terron Estuary (SW Spain) in 1999. In Eo, Santander and Terron, respectively: phase 0 (resting stage) occurred in July to August, September to October and August to September, phase I (proliferation of gonias) happened in: August to October, October to December and September to November, phase II (gametogenesis) was developed in November to April, January to May and December to April and phase III (reproduction) took place in: May to June, June to August and May to July. Two new approaches oriented to the fast monitoring of the gametogenic cycle of the species based on sequential mean drained soft parts weights obtained by simulation and on the macroscopic observation of the gonad are presented.

KEY WORDS: bivalves, Solenidae, Solen, reproduction

INTRODUCTION

The superfamily Solenacea is an infaunal soft bottom dwelling bivalve group consisting of the two marine families Solenidae and Pharidae (= Cultellidae, Cosel 1993).

Razor clams are bivalves whose commercial demand has notably increased in Spain in recent years (Remacha-Trivino 1996). As a consequence of this, the natural beds of these species can be subjected to overexploitation.

Solen marginatus (Pulteney, 1799) is a representative intertidal bivalve of the Spanish coasts. Accordingly, this species is distributed along the west Mediterranean Sea and Atlantic Ocean from Britain to Mauritania (Cosel 1993), comprising all of Spain's coasts. This species can be considered the third most important commercial razor clam of Spain after Ensis arcuatus and E. sillqua.

Previous references dealing with the reproductive cycle of S. marginatus are: Rodriguez-Moscoso et al. (1996), Gaspar (1996), Remacha-Trivino (1996), Tirado et al. (2002), Remacba-Trivino (2002), Martinez (2002) and Lopez et al. (2005). However, there are not precedent studies dealing with the reproductive cycle of this species either in Santander Bay or in Terron Estuary, as well as a comparative survey among these three locations.

Two of the most important strategies oriented to improve the management of the marine medium are a more rational exploitation of the natural resources and the application of aquaculture as an instrument aimed to increase the production of commercial species. Alternatively, artificial culture constitutes a way to compensate the lack of other species, which, because of overharvesting continue to diminish. A thorough comprehension of the reproductive cycle provides: (1) the establishment of close seasons in keeping with the spawning periods; (2) optimization of the breeding conditions oriented towards improving the quality of commercial exploitation and, in a more general framework, with the purpose of expanding sustained development and (3) the genetic selection of varieties of a higher reproductive efficiency and more resistant to pathologies or stress conditions related to reproduction.

The purpose of this study is to draw a comparison among the reproductive cycle of S. marginatus in three separate locations of Spain and to present two new approaches oriented to the fast monitoring of the gametogenic cycle of this species, based on the macroscopic observation of the gonads and on sequential mean drained soft parts weights obtained by simulation. In this way, the understanding of the reproductive biology of S. marginatus is improved, making this information relevant for promoting a future extensive culture for this species, oriented to satisfy its commercial demand and to guarantee the conservation of its natural beds.

MATERIALS AND METHODS

Samples of 50-65 razor clams were collected intertidally in 3 different locations including the Eo Estuary (Asturias, NW Spain), Santander Bay (Cantabria, N Spain) and Terron Estuary (Huelva, SW Spain) (Fig. 1). Specimens from Eo Estuary were collected from June 1994 to May 1995. Specimens from Santander Bay were collected from October 1998 to September 1999. Specimens from Terron Estuary were collected from January 1999 to December 1999.

[FIGURE 1 OMITTED]

Live razor clams were transported to the Invertebrates Laboratory of the Department of Biology of Organisms and Systems (University of Oviedo, Asturias, Spain). Caliper shell lengths and drained weights were measured to the closest 0.01 mm and g, respectively. Next, animals were fixed in 10% formalin in seawater and the following procedures were applied.

Histological Techniques

Twelve animals (six males and six females) per month were used for histology. A sample of approximately 10 x 5 mm visceral mass was dissected from the internal ventral portion of each foot (Remacha-Trivino 1996), starting from a fixed point (i.e., left square) chosen at random, dehydrated in graded ethanols and embedded in paraffin according to Durfort (1993). Seven micrometers serial sections were obtained and stained routinely with Hematoxylin-Eosin. Stages of the reproductive cycle were classified under the maturity scale of Chipperfield (1953) and Lubet (1959) widely used by different authors (e.g., see Villalba 1995), which comprises the following phases: 0 (resting stage), I (proliferation of gonias), II (gametogenesis) and III (reproduction) of subphases: III A (ripeness), III B (early spawning), III C (restoration) and III D (late spawning).

Macroscopic Observation of Gonads

Feet were dissected ventrally along the line of intersection with the sagital plane. Gonads were classified in relation to their macroscopic appearance under the maturity scale used for histology.

Sequential Mean Total Drained Weights

Sequential mean total drained weights were estimated in each month from the arithmetic progression of shell lengths restricted to sizes between 65-130 mm, {[a.sub.L]} = 5 * (12 + L) [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] [65,130], whose elements were obtained by simulation from their corresponding monthly regressions log W | log L.

RESULTS

Extension of the Gonad

The extension of the gonad of Solen marginatus is restricted to the internal portion of the foot where it is enclosed permanently, even in the periods of maximal gonadal development. Follicles line the internal surface of the pedal sinus, adjacent muscular layers and parts of visceras enclosed therein and facing its lumen. The interfollicular connective tissue, common in most bivalve species, is absent.

Histology

Phase 0 (Resting Stage)

Sex Indistinguishable. Spread of follicular tissue limited to minor areas located among the peripheral muscular bundles of the pedal sinus (Fig. 2A). Gonads were not observed in some of the specimens. Hemocytes surrounding the unabsorbed necrotic masses derived from the precedent subphase III D (see below) were frequent. Gametogenic elements reduced to reservoirs of stem cells (Fig. 2B to C) of characteristics: (1) ellipsoidal, spheroidal or polygonal shapes; (2) basophilic to lightly acidophilic staining affinity; (3) bigger size than gonias; (4) high nucleocitoplasmic quotient and (5) boundary cytoplasm-nucleus diffuse.

[FIGURE 2 OMITTED]

Phase I (Proliferation of Gonias)

Intense mitotic activity and spread of gonias, which proliferated by forming disperse poolings of cells located around the internal muscle layers and visceral mass of the foot (Fig. 2D to G). Clusters of gonias were enclosed within the follicular cells to constitute the primary follicles. Oogonias and spermatogonias showed common morphological features. Both cells were roughly ellipsoidal, displaying neutrophyl cytoplasms, slightly acidophilic nuclei, clear boundaries nucleus-cytoplasm and similar sizes, ranging between 3 and 13 [micro]m in diameter (Fig. 2E, G, L).

Phase II (Gametogenesis)

In females, the first previtellogenic oocytes were observed at the beginning of this phase. When compared with oogonias, the former exhibited cytoplasms and nuclei with a similar degree of neutrophilia and a diffuse boundary cytoplasm-nucleus with no visible nucleolus. Some of these first oocytes remained free in the follicular lumen, with no apparent point of attachment to the follicular wall (Fig. 2J). In posterior stages of oogenesis, nuclear membrane and nucleolus became distinguishable and oocytes were found either attached to the follicle wall or free in the follicular lumen, but always connected to aerial follicular cells in the latter case (Fig. 2H, I). In males, spermatocytes were the predominant cellular type in early spermatogenesis (Fig. 2K). These were roughly spheroidal cells of approximately 5 [micro]m in diameter, deep basophilic nuclei and a high nucleocitoplasmic quotient. In posterior phases of early gametogenesis, the gonad gradually invaded the internal muscular and fibrous connective surfaces of the pedal sinus and free portions of the visceras--digestive gland and gut--enclosed within the foot, with the exception of some concrete areas lined exclusively with integumentary musculature. In winter, the progress of the gametogenesis was generally slow or in recession. Late gametogenesis was mainly developed through spring. In females, oocytes nuclei tended to emigrate towards a more apical position within the cytoplasm, whereas the longitudinal axis of the cells was used to reallocate radially towards the follicular lumen. Oocytes were generally attached to the follicular wall by means of stalks that were not as evident as in other bivalve species. Masses of basophilic clusters of atresic oocytes were frequent. In males, proliferation of spermatocytes was increased notably, a process simultaneous with the first manifest masses of spermatozoa. Male gonads were composed mainly of spermatocytes, spermatids and spermatozoa (Fig. 2L). Although spermatocytes continued to be the most frequent spermatogenic stage, densities of the other cellular types were augmented significantly. The first minor releases of gametes took place at the end of this phase.

Phase III (Reproduction)

Subphase III A (Ripeness): Gonads were deeply packed and full of gametes near emission. Follicles were extremely expanded. In most cases, the feet became so dilated that they were hardly retractable. Atresic oocytes were less frequent than in phase II, as indicative of a probable mechanism of resorption. Mature oocytes were detached from the follicle wall, adopting a more spheroid geometry turned to poliedric at highly packed areas (Fig. 2N). Males showed a significant predominance of spermatozoa mainly positioned at the center of the follicular lumen (Fig. 2P).

Subphase III B (Spawning): Phase of spawning was restricted to unspecific areas of the gonad, whose follicles were found to be partially evacuated because of the releases of gametes (Fig. 2M, P).

Subphase III C (Restoration): Intervals among consecutive spawnings were characterized by a restorative activity yielded to a highly variable pattern, depending on sex, number of precedent spawnings and duration of these previous periods of spawning. In females, restoration gave rise to follicles whose oocytes showed a major proportion of immature oocytes. Spreading of oogonias was also observed. In males, the follicles found in coexistence with follicles IIIA and III B, with no spaces in the lumen and without a predominance of spermatozoa, were classified under phase III C, although the pattern of restoration was not clearly observed in males (Fig. 2M, P).

Subphase III D (Last Emissions): Evacuation of all the potentially releasable gametes from the gonad generally happened after a succession of various minor spawnings plus a major one. Nevertheless, gonads examined after the major spawning showed follicles that were not completely evacuated, implying some minor releases after the major spawning to complete this process. In the evacuated follicles, elements of gametogenesis were reduced to small pools of gonias, stem cells and necrotic masses of residual oocytes. A strong parallel resorptive activity took place. Follicles were progressively drawn back down the fibromuscular tissues and reabsorbed later (Fig. 2O, Q). Groups of hemocytes were particularly numerous in this phase by forming aggregates of phagocytosis within the degenerating tissues.

Sequential ratios of phases of the reproductive cycles investigated are presented in Figures 3, 4, 5.

[FIGURES 3-5 OMITTED]

Macroscopic Level

Phases 0 and 1. Gonad not observed macroscopically or reduced to a thin layer of hyaline tissue. Whitish areas corresponding to putative primordia of follicular tissue were detected occasionally over the internal surface of the pedal sinus (Fig. 6A to B). Phase II. Gonad clearly observed macroscopically, extending from discontinuous portions of the pedal sinus to almost all its inner surface area. Internal surface of the gonad mainly distributed in form of small transverse folds of tubular appearance. These folds were whitish in males and brownish in females (Fig. 6C to D). Subphases III A, III B and III C. Pedal sinus almost obliterated by gonad. Internal surface of gonad composed by bigger folds resembling small sacs of fragmented surface in males and wavy surface in females (Fig. 6E to F). Subphase III D. (1) Early stage: Internal surface of gonad arranged in folds similar to the previous ones but less dimensioned. (2) Late stage: Gonadal arrangement analogous to phase II except for the inside of the tubular folds, which are covered in stains, as a probable consequence of the mechanism of resorption (Fig. 6G, H).

[FIGURE 6 OMITTED]

Mean Total Drained Weight

The sequential mean total drained weights of the different reproductive cycles investigated are shown in Figure 7.

[FIGURE 7 OMITTED]

DISCUSSION

Histology

The reproductive cycle of Solen marginatus fits the general pattern observed in other bivalves of temperate waters (i.e., annual cycle, seasonal phases, all the different phases cannot be found simultaneously) being analogous to: Crassostrea virginica, Cerastoderma edule, C. glaucum, Mytilus edulis, M. galloprovincialis, Lithophaga lithophaga, Paphies australis and Venus striatula (Loosanoff 1942, Lubet 1959, Ansell 1961, Boyden 1971, Galinou-Mitsoudi & Sinis 1994, Giguere et al. 1994, Villalba 1995), among other species.

The evolution of the reproductive cycle of S. marginatus is, likewise, globally coincident with other references on this species (Rodriguez-Moscoso et al. 1996, Gaspar 1996, Gaspar & Monteiro 1998, Tirado et al. 2002, Martinez 2002, Lopez et al. 2005) and other Solenidae (Martinez et al. 1997, Casavola et al. 1985, Darriba et al. 2004); with the exception of the well-known expectable differences in duration and occurrence of the different phases of the cycle due, on the one hand, to feeding, temperature, salinity, photoperiod and, in general, other potentially influent environmental conditions; whereas on the other hand, to probable genetic variations, specially from the Southern population of Terron Estuary with respect to the northern ones of Eo Estuary and Santander Bay. The aforementioned deviations are also expected to explain the differences among the three reproductive cycles investigated here.

Spermatogonias and oogonias were analogous to other bivalves (Tranter 1958, Lubet 1959, Darriba et al. 2004). However, some differences with respect to Ensis arcuatus were found. Although protogonias have been described in other bivalve species as cells of similar size to oogonias (De Gaulejac et al. 1995), Darriba et al. (2004) described the protogonias of E. arcuatus as acidophilic cells bigger than oogonias and reported larger and less basophile oogonias than spermatogonias. Nevertheless in S. marginatus, oogonias and spermatogonias showed similar dimensions and degrees of acidophilia.

Distinction between spermatogonias and oogonias in phase I was carried out by the observation of specimens in the transition between the former phase and phase II, in which the subsequent gametogenic stages (i.e., first oocytes and spermatocytes) were clearly observed.

Subphases III A (ripeness), III B (spawning), and III C (restoration) were found simultaneously in only a few animals (Fig. 2, P). In the rest of specimens, two of these three subphases III A to C were always coexisting (Fig. 2M). In consequence, and unlike subphase III D and rest of phases of the reproductive cycle, we did not find razor clams in which the whole follicles were in the same subphase III A to C. Although the decision of classifying the specimens in phase III under one of these three subphases can be based on the state of most of the follicles, subphases III A, III B and III C are not always easy to distinguish in both sexes, as in females: (1) is unusual to find oocytes III A in absence of immature oocytes attached to the follicle wall, and therefore, involved in a simultaneous mechanism of restoration; (2) follicles III A showed frequent intrafollicular spaces, as indicative of a previous spawning. In this way, the loss of the polygonal shape generally observed in the mature oocytes of bivalve mollusks (e.g., Lubet 1959) can be explained as the consequence of a lower intrafollicular compression; (3) follicles III C are also used to show mature oocytes in the lumen. Alternatively, in males: (1) because the production of sperm could be predominant or balanced with production of spermatocytes during subphase III C, it is not clear that in E. arcuatus the process of restoration implies, as affirmed by Darriba et al. (2004), a necessary predominance of spermatocytes against spermatozoa in the follicle; (2) in phase II, we observed follicles with a minor proportion of sperm. If we define the mature male follicles as those with predominance of spermatozoa (e.g., Loosanoff, 1942) then the process of maturation of these follicles could have been reached after the emission of at least a portion of the sperm generated during phase II, giving rise to the frequently observed male follicles with broad luminar spaces, which are potentially classifiable under subphase III B. In synthesis, we define subphases III A to C as substages of the reproductive cycle of frequent coexistence. Therefore, they should be classified more properly at the follicular level than at the individual level, as displayed in Figure 2M, P.

In this study, the gonad of S. marginatus was found to be located exclusively inside the foot during all phases of the reproductive cycle. Therefore, our results contradict the findings of Rodriguez-Moscoso et al. (1996), authors who affirmed that in S. marginatus, the gonad entered the foot during the maturation period.

The function of hemocytes as reserve cells has been investigated by Houtteville (1974) and Medhiboub & Lubet (1988) for the species Mytilus edulis and Ruditapes phillipinarum, who found a set of different transitional stages to reach the final reservory stage termed vesicular cell. Rodriguez-Moscoso et al. (1996) reported a prevalence of glycogen in the muscular tissues and digestive gland of S. marginatus and showed that this polysaccharide was poorly represented in the gonad. On the contrary, contents in lipids ranged between 16% and 30%. Remacha-Trivino (2002) found a parallel disorganization and subsequent reorganization of nephridia and gonads in S. marginatus, which was hypothesized as an indicative of a probable renal cycle simultaneous to the reproductive cycle. Particularly, the number of hemocytes of the distal limb of the nephridia was demonstrated by Stereology to be significantly increased in phase III. Most of these blood cells were observed to form phagocytic aggregates around the degenerating excretory cells. All these findings were interpreted as a probable mechanism of hemocytic transport of reservory substances stored in the kidney to the gonad, where hemocytes would tend to remain accumulated as reservory ceils. Darriba et al. (2004) reported a probable feeding activity of the hemocytes of Ensis arcuatus by supplying nutrients and carrying out the function of reserve cells in the gonad.

Macroscopic Observation of Gonads

The observation of gonads performed at the macroscopic level permits fast approximations of the reproductive condition and to determine the sex of the specimens in the late stages of the reproductive cycle. Although the macroscopic description of gonads is a general complement to the histological approach in studies dealing with reproduction of bivalves, only a few maturity scales have been pursued in establishing a reliable parallelism at both levels of magnification through all the stages of the reproductive cycle in which this relationship was possible. In this context, the absence of structural perifollicular tissues in S. marginatus facilitated the design of a more precise macroscopic maturity scale for the species.

Mean Total Drained Weights

Biometric methods oriented to monitor the reproductive cycle can be classified in: (1) approaches based in one variable (i.e., soft parts weight, shell weight, total weight, soft parts volume, etc.); (2) condition indexes or functions of at least two variables; (3) standardizations derived from the extrapolation of data of (1) and (2) to a concrete value; (4) alternative simple statistical treatments (i.e., descriptive approaches, regressions, etc.); (5) complex statistical or biological treatments (i.e., temporal series or biological models).

Standardizations show the disadvantage of depending on the final value chosen for the extrapolation of data. Thus, results can only be compared in relative terms, unless the same standard value is used for all of the different treatments. In addition, standardizations would be recommended to be based in unbiased estimators, where the sample mean is likely to be the best and simplest election. Also, approaches (1) to (3) present the inconvenience of being range dependant. Therefore, a rational biometrical comparison should be expected to be derived from sequential range-balanced initial data. Although the previous selection of a fixed range for all the samples compared through time is, in practice, statistically questionable for the cases (1) to (3), as it means to lose the information of the discarded data, a common range can be achieved by statistical simulation (e.g., regression), or it can be chosen in terms of a rational criteria of election.

It is well known that one of the aims of condition indexes is to average the differences caused by size or range by dividing the characteristic of interest (e.g., dry soft parts weight) by a fixed variable, which is expected to remain invariant through time (e.g., dry shell weight). However, no effort is generally made in evaluating these variables separately. If it were so, it could be seen that the trends of some condition indexes widely used [e.g., Higgings' (1938), and Walne's (1970)] and the characteristic of interest can show an inverse relationship with respect to size. This fact can be easily verified by comparing the linear regressions between each one of the variables used in these condition indexes against a common characteristic implying dimension, alternative to the fixed one utilized in the condition index (e.g., for Walne's CI, dry soft parts weight versus shell cavity volume and dry shell weight versus shell cavity volume, since the inverse relationship for Higgings' CI). For instance, if we suppose that equations of the previous regressions were: Y = X + 1 and Z = X/2, where Y is the dry soft parts weight, X is the shell cavity volume and Z is the dry shell weight, shell cavity volumes of: 2, 4 and 8 [cm.sup.3] give rise to respective dry soft parts weight of: 3, 5 and 9 g and Walne's CIs of: 3, 2.5 and 2.25. Even for the hypothetical case of parallel slopes (e.g., replacing the second equation Z = X/2 by Z = X - 1), CIs are: 3, 1.66 and 1.29. Because the previous example demonstrates that the variable of interest can be masked in the CI by the influence of the fixed variable, it seems to be more reliable to apply the approach (1) to monitor the reproductive cycle.

Advantages of the approach applied here based in the sequential mean drained soft parts weights are: (1) A common range for the initial data. Grant and Tyler (1983) affirmed that immature invertebrates should not be included in the analysis of gonad index, as they will have smaller gonads than mature animals and their gonad weights, oocyte sizes, etc., are unlikely to follow the adult reproductive cycle. In consequence, the lower limit of our interval of shell lengths was restricted to 65 mm as the smaller specimens were not guaranteed to be mature animals. Alternatively, the upper limit of the interval was established in the maximal shell length of the samples. (2) Subsampled data were balanced by simulation and sequential sample means were estimated from a fixed set of values obtained from the arithmetic progression of shell lengths. Therefore, the final data was equally balanced with respect to the size of the specimens. (3) The standardization was unbiased because it was based on sample means. (4) The present biometrical approach involves only one variable (i.e., total drained weight), avoiding CIs. Dry soft parts weight or drained soft parts weight were probably more precise elections. However, we opted to look for the fastest approach, which reflected a reliable evolution of the reproductive cycle.

Accordingly, growth trends and maximums in mean total drained weight comprised between April and June were coincident with Phases III, followed by the falls and growths between June and August of Phases 0. Phases I started when maximums between August and October were reached and Phases II a month later. Finally, minimums between April and March matched the beginning of Phases III.

ACKNOWLEDGMENTS

The authors thank D. Manuel Fernandez Gonzalez, Montserrat Cayado Lopez, the Shellfishermen Cooperatives of Ambojo (Pedrena, Santander) and Terron Island (Huelva); especially to Pedro Bedia, Felix Ezquerra and Ismael for their help and collaboration during the process of sampling, Dr. Stephane Pouvreau for revision and kind suggestions concerning the introduction of the paper, and Dr. Marta Gomez-Chiarri for the revision of the poster previous to this manuscript. The research in Eo Estuary was supported by the University of Oviedo under the project of asturian thematic FC-TA-96-517-2. A. Remacha-Trivino acknowledges Professor Cruz-Orive for his help and support during his stay at the University of Cantabria (Spain) and for permitting him to carry out the parallel studies of Santander Bay and Terron Estuary to the EC project BMH4-CT97-2437.

LITERATURE CITED

Ansell, A. D. 1961. Reproduction, growth and mortality of Venus striatula (Da Costa) in Kames Bay; Millport. J. Mar. Biol. Assoc. U.K. 41:191-215.

Boyden, C. R. 1971. A comparative study of the reproductive cycles of the cockles Cerastoderma edule and C. glaucum. J. Mar. Biol. Assoc. U.K. 51:605-622.

Casavola, N., N. Rizzi, N. Marano & C. Sarracino. 1985. Ciclo riproduttivo e biometria di Ensis minor (Chenu) (Bivalvia: Solenidae) nel golfo di Manfredonia. Oebalia 11:439-449.

Chipperfield, P. N. J. 1953. Observations on the breeding and on the settlement of Mytilus edulis in the British waters. J. Mar. Biol. Assoc. U.K. 32:449-476.

Cosel, R. 1993. The razor shells of the eastern Atlantic. Part 1: Solenidae and Pharidae I (Bivalvia: Solenacea). Arch. Moll. 122:207-321.

Darriba, S., F. San Juan & A. Guerra. 2004. Reproductive cycle of the razor clam Ensis arcuatus (Jeffreys, 1865) in northwest Spain and its relation to environmental conditions. J. Exp. Mar. Biol. Ecol. 311:101-115.

De Gaulejac, B., M. Henry & N. Vicente. 1995. An ultrastructural study of gametogenesis of the marine bivalve Pinna nobilis (Linnaeus 1758). I. Oogenesis. J. Moll. Stud. 61:375-392.

Durfort, M. 1993. Tecnicas histopatologicas en moluscos. In: Acuicultura marina: fundamentos biologicos y tecnologia de la produccion. F. Castello-Orvay, editor. Barcelona: Univ. of Barcelona. pp. 587-598.

Higgings, E. 1938. Progress in biological inquiries. 1937. Bulletin of the U.S. Bureau of Fisheries Administration Report No. 30. Washington DC: U.S. Government Printing Office, pp. 1-70.

Hooker, S. & R. G. Greese. 1995. The reproductive biology of Pipi, Paphies australis (Gwenlin, 1790) (Bivalvia: Mesodesmatidae). I. Temporal patterns of the reproductive cycle. J. Shellfish Res. 14(1):7-15.

Houtteville, P. 1974. Contribution a l'etude cytologique et exptrimentale du cycle du tissu de reserve du manteau de Mytilus edulis. Pads: Univ. de Caen. These 3er cycle Biologie. 98 pp.

Galinou-Mitsoudi, S. & A. I. Sinis. 1994. Reproductive cycle and fecundity of the date mussel Lithophaga lithophaga (Bivalvia: Mytilidae). J. Moll. Stud 60:371-385.

Gaspar, M. B. 1996. Bivalves do litoral oceanico algarvio. Aspectos da biologia, ecologia e da pescaria dos mananciais de interese economico: aplicacao a gestao dos recursos. Thesis, Univ. de Faro: Portugal. 282 pp.

Gaspar, M. B. & C. C. Monteiro. 1998. Reproductive cycles of the razor clam Ensis siliqua and the clam Venus striatula of Vilamoura, southern Portugal. J. Mar. Biol. Assoc. U.K. 78:1247-1258.

Giguere, M., G. Cliche & S. Brulotte. 1994. Reproductive cycles of the sea scallop, Placopecten magellanicus (Gmelin) and the iceland scallop, Chlamys islandica (O. F. Muller), in Iles-De-La-Madeleine, Canada. J. Shellfish Res. 13:31-36.

Grant, A. & P. A. Tyler. 1983. The analysis of data in studies of invertebrate reproduction. I. Introduction and statistical analysis of gonad indices and maturity indices. Int. J. Invertebr. Reprod. 6:259-269.

Loosanoff, V. L. 1942. Seasonal gonadal changes in the adult oysters, Ostrea virginica, of Long Island, Sound. Biol. Bull. Mar. Biol. Lab. 32:195-206.

Lopez, J., C. Rodriguez & J. F. Carrasco. 2005. Comparativa del ciclo reproductivo de Solen marginatus (Pulteney, 1799) (Mollusca: Bivalvia) en las Rias del Eo y Villaviciosa (Asturias). Relacion con los parametros ambientales. Resumenes X Congreso Nac. Acuicult. pp. 518-519.

Lubet, P. 1959. Recherches sur le cycle sexuel et l'emission des gametes chez les Mytilides et les Pectinides. Paris: Faculte des Sciences de L'Universite de Paris. These Doctoral d'Etat. 162. pp.

Martinez, D., E. Rodriguez-Moscoso, R. Arnaiz, S. Novoa & J. Ojea. 1997. Gametogenesis y composicion bioquimica en una poblacion de Ensis siliqua (Linne, 1758) en la Ria del Barquero (N. Galicia). Actas VI Congreso Nac. Acuicult.

Martinez, D. 2002. Estudio de los Solenidos, Solen marginatus (Pennant, 1777) y Ensis siliqua (Linne, 1758), de los bancos naturales de la Ria de Ortigueira y Ria del Barquero: ciclo gametogenico, composicion bioquimica y cultivo larvario. Santiago de Compostela: Universidad de Santiago de Compostela. Tesis Doctoral. 240 pp.

Medhiboub, N. M. & P. E. Lubet. 1988. Reserches cytologiques sur l'environnement cellulaire ("tissu de reserve") des gonades de la Palourde (Ruditapes philippinarum Adams & Reeve), Mollusque bivalve. Ann. Des Sci. Nat. Zoo. 9:87-102.

Remacha-Trivino, A. 1996. Ciclo reproductivo y biometria de Solen marginatus (Pennant, 1777) (Mollusca: Bivalvia) en la Ria del Eo. Oviedo: Universidad de Oviedo. Disertacion. 26 pp.

Remacha-Trivino, A. 2002. Estereologia Moderna y Anatomia de los espacios de referencia, tejido hemolinfatico y aparato excretor de la especie Solen marginatus (Mollusca: Bivalvia). Interacciones fisiopatologicas del nefridio en relacion al desarrollo del ciclo reproductivo. Oviedo: Universidad de Oviedo. Tesis Doctoral. 308 pp.

Rodriguez-Moscoso, E., E. D. Martinez, R. Arnaiz, G. Mosquera, A. Cervino, A. De Coo, A. Garcia & N. Rua. 1996. Gametogenesis, reservas energeticas y desarrollo larvario en el longueiron, Solen marginatus (Pennant, 1777). IX Simp. Iber. Est. Bentos. pp. 164-166.

Tirado, C., A. Rodriguez, M. A. Bruzon, J. I. Lopez, C. Salas & I. Marquez. 2002. La reproduccion de bivalvos y gasteropodos de interes pesquero en Andalucia. Ed. by Junta de Andalucia. Andalucia: Consejeria de Agricultura y Pesca.

Tranter, D. J. 1958. Reproduction in Australian pearl oysters (Lamellibranchia). IV Pinctada margaritifera (L.). Aust. J. Mar. Freshwater Res. 9:509-523.

Villalba, A. 1995. Gametogenic cycle of cultured mussel, Mytilus galloprovincialis, in the bays of Galicia (NW Spain). Aquaculture 130:269-277.

Walne, P. R. 1970. The seasonal variation of meat and glycogen content of seven populations of oysters Ostrea edulis L. and a review of the literature. Min. Agric. Fish. Food. Fish. Invest. Ser. 2. 26(3).

ANTONIO REMACHA-TRIVINO * AND NURIA ANADON

Departamento de Biologia de Organismos y Sistemas, Area de Zoologia, Universidad de Oviedo, c/Catedratico Rodrigo Uria s/n, E-33071 Oviedo, Asturias, Spain

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