Gonadal development and histochemistry of the tropical oyster, Crassostrea corteziensis (Hertlein, 1951) during an annual reproductive cycle.

ABSTRACT We describe the quantitative and qualitative histology and histochemistry for a tropical oyster, C. corteziensis sampled in a coastal lagoon in Northwest, Mexico. Males were larger than immature oysters, with females presenting intermediate values. In this species, mature organisms were found most of the year, and there were at least two strong spawning periods, one in summer and the second in autumn. The presence of mature oocytes most of the year did not allow for differentiation of an annual reproductive pattern using average oocyte diameter, as has been used in other species. There was a very short resting period in winter, particularly in December 2005, and by January to February 2006 postvitellogenic oocytes can already be found. Both sexes tended to have more lipids in the gonad tissue as maturation advanced, with an inverse correlation to carbohydrate in gonad and in vesicular tissue in females. No differences in lipids or carbohydrates content were found in digestive gland. A negative correlation was found between chlorophyll a content and gonad coverage area in males and females. Maturation occur at sea surface temperatures higher than 20[degrees]C, and spawning when temperature increases above 27[degrees]C.

KEY WORDS: Crassostrea, carbohydrate, female, histology, lipid, male, mollusc, oyster, reproduction, sperm, vitellogenesis

INTRODUCTION

In Mexico, oyster aquaculture has been focusing principally in two oyster species, Crassostrea virginica in the Gulf of Mexico and C. gigas in the Pacific Coast. However, the recently increase of summer mortalities of C. gigas have encouraged new alternatives for the growing oyster farming industry, mainly of species adapted to semi and tropical waters, like C. corteziensis. This species has been proposed as viable option for oyster farmers in Mexico (Ramirez & Sevilla 1965, Chavez-Villalba et ai. 2005). The annual reproductive cycle for C. corteziensis has been described in relation to its gonadosomatic index (GSI), calculated as gonad weight in relation to body weight (Frias-Espericueta et al. 1997, 1999). However, these authors specified that measuring GSI was difficult in oysters because gonad develops within the mantle. When comparing the annual reproductive pattern in C. corteziensis using light microscopy and paraffin histology differences in relation to when spawning starts and how much it lasts have been observed; from June to October 1972 (Stuardo & Martinez 1975) and May to November 1973 (Cuevas-Guevara & Martinez-Guerrero 1979), both studies done in Nayarit. In Sonora, a northern locality C. corteziensis began spawning from April to September 1964 (Ramirez & Sevilla 1965). A study of Ruiz-Dura (1974) generally cited as a histological description of C. corteziensis, refers to Ostrea corteziensis, and presents larvae in the mantle. In contrast to a resting phase in winter and a short spawning period in summer in C. gigas (Lubet & Mann 1987), there are probably several spawning events and they last longer in C. corteziensis. Nevertheless, the culture methods that are now being applied in ah attempt to induce maturation in C. corteziensis are based on those that have been proven adequate for C. gigas (Mazon-Suastegui et al. 2002, Chavez-Villalba et al. 2005, Caceres-Puig et al. 2007). A description of the reproductive cycle of wild C. corteziensis is needed to achieve maturation in captivity and for management of fisheries resources. The aim of this study is to analyze gametogenesis in male and female gonad, detailed gamete structure using resin histology and lipid and carbohydrate content by histochemistry over a one-year period in a wild C. corteziensis population from Ceuta Lagoon System, Mexico.

MATERIALS AND METHODS

Site, Temperature, Chiorophyll a

Oysters used for this study were sampled from Ceuta Lagoon System located in the mainland side of the Gulf of California, on the coast of the state of Sinaloa in the Northwest of Mexico. We used satellite-derived estimates of sea surface temperature and chlorophyll a (a proxy of phytoplankton) using full resolution (1 km monthly dataset were made available by Dr. Mati Kahru, Scripps Institution of Oceanography) merged products (chlorophyll a from SeaWiFS and MODIS-Aqua 2002-2006; SST from MODIS-Aqua and MODIS-Terra 2002 to 2006). Time series were made in the coastal area close to the mouth of Ceuta Lagoon System (Fig. 1). A comparison between in situ and satellite data of sea surface temperature was possible (February to August 2007) and a high correlation was found ([r.sup.2] = 0.97), suggesting that variations between the coastal area and the areas close to the mouth of the Ceuta Lagoon System--where the sampling site is located--are similar.

Oyster Sampling and Morphometry

Thirty oysters were sampled monthly from Ceuta Lagoon System from April 2005 to April 2006 from roots of mangrove in the intertidal zone. Oysters were packed in plastic containers and transported to Centro de Investigaciones Biologicas del Noroeste (CIBNOR) laboratory at La Paz, B.C.S., Mexico in less than 24 h. On arrival, organisms were washed and scrubbed to eliminate epibionts, and total weight was recorded for each organism, the oyster meat was removed from the shell and a 2-mm cross-section of at the mid visceral level that included a portion of digestive gland was dissected, placed in cassettes (Leica Jet II cassettes, Leica Microsystems, Wetzlar, Germany) and fixed in either Davidson's solution for 48 h (Shaw & Batle 1957) or Karnovsky solution for 24 h (Kamovsky 1965) for histological analyses.

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Paraffin Sections

Tissue samples were dehydrated in ethanol series of progressive concentration (70%, 80%, 95%, and 100%), cleared in xylene and embedded in paraffin (Paraplast X-Tra, Mc Cormick Scientific, San Diego, CA, USA). Slides were prepared in 4-[micro]m thick sections using a rotary microtome (Leica RM 2155, Leica Microsystems) and mounted on glass slides. Each slide was stained after removing the paraffin (Leica ST5020 Multistainer, Leica Microsystems). To analyze gonad structure, each slide was stained 5 min with Harris's hematoxylin (SJV Grupo Bioquimico Medico, Mexico) and counterstained for 12 min with eosin-phloxine. For histochemistry, slides were stained with Alcian Blue PAS (ABPAS, alcian blue 5 min, Schiff's reagent 15 min) to show neutral mucopolysaccharides that are colored in a range of magenta (Sheehan & Hrapchak 1980), or Sudan Black B (SBB) to show lipids colored in black (Rodriguez-Moscoso & Arnaiz 1998). Cover slips were applied with Entellan mounting media (Sheehan & Hrapchak 1980).

Resin Sections

The samples fixed in Karnovsky solution were rinsed and progressively dehydrated in ethanol baths (70%, 95% and 100%) and embedded in resin (JB-4 Plus, Polysciences, Warrington, PA, USA). Semithin (0.5 [micro]m) sections were prepared with a rotary microtome with tungsten carbide disposable blades (Leica TC65) and stained 2 min with polychromatic staining (Tolivia et al. 1994) for analysis under light microscopy at x 100 (Olympus BX50, Olympus Optical, Japan).

Quantitative Histology

Gonad Coverage Area

The area occupied by the gonad was determined using an image system analyzer (Image Pro Plus v. 4.) at x 4 (7.9 [mm.sup.2]) from a mean of three different slices from each specimen. The image analysis was based on the intensity of the tissue-specific color and the gonad and visceral area were automatically calculated in pixels and expressed in [micro][m.sup.2]. The area reported as the gonadal coverage area (GCA) was calculated as: GCA = (gonad occupation area/total area) x 100.

Oocyte Frequency

Frequency was calculated by counting each type of oocyte in three regions of the ovary of each female that appeared within an area of 1.44 [mm.sup.2] at x 20.

Oocyte Area and Diameter

The diameter and area of oocytes was determined with digitalized images at x40 (Image Pro Plus software v. 4.5, Media Cybernetics, Silver Spring, MD, USA). Approximately 40 50 oocytes in three randomly selected regions of a slide were observed ir the nucleus was visible. Because oocytes deviate markedly from a sphere, the theoretical diameter (TD) was calculated from the total area (A) of each oocyte using the following formula: TD = [square root of 4A/[pi]] (Briarty 1975, Saout et al. 1999). Gonia were not measured.

Histochemistry

The images obtained from the microscope were digitalized (Image Pro Plus v. 4.5) and ah image signal processing technique was applied for the automatic identification of pixels. The technique is based on the conversion of the image in color to an intensity level image to enhance a determined dye (Otsu 1979). The proportion of required pixels from the total on the resulting image represents a measure proportional to the lipid or carbohydrate present in the sample. For each organism, the total amounts of pixels were calculated for the three tissues analyzed and the proportions of lipids or carbohydrates were reported as percentage per area per tissue per oyster.

Statistical Analysis

Oocyte measurements of each female were analyzed with a nested ANOVA (40-50 oocytes per female when possible, 10 oysters per month). Morphometric analyses, GCA, oocyte frequencies, and lipid and carbohydrate content per oyster were analyzed by one-way ANOVA, followed by Newman-Keuls post hoc analysis to assess the significant differences (P < 0.05) between either months (14 levels) or maturation stage (5 levels). All statistical analyses used Statistica version 8.0. Where specified, sexes were analyzed separately. Percentage results wcre transformed to arcsine before ANOVA but only untransformed means are presented. All data are reported as mean [+ or -] standard error, except where indicated.

RESULTS

The sea surface temperature and chlorophyll a during the sampling period are reported in Figure 2A. Temperature fluctuated just below 20[degrees]C from December 2005 to February 2006 and above 30[degrees]C in August to September 2005. Temperatures above 30[degrees]C are common in this locality. Chlorophyll a showed an inverse relationship to temperature, with lower values between June and September 2005 and higher values in winter, with a descent in January 2006.

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Sex Ratio and Morphometry

A larger proportion of females were found in most of the sampled months, with the exception of the end of April 2005 and January to February 2006 (Fig. 2B). Immature oysters were found in most sampled months, except in May and August through September 2005 and March through April 2006. No differentiated males were found in November 2005.

Total oyster weight (with shell) is reported in Table 1. Females in vitellogenesis, were signifieantly larger than undifferentiated and previtellogenic females, with intermediate values for the other stages. In contrast, larger males were found in the spawned stage. Males were larger than immature oysters, with females presenting intermediate values (Females = 92.9 [+ or -] 4.3ab; Undifferentiated : 80 [+ or -] 4.7 b; Males = 103 [+ or -] 6.1a, P < 0.05, results not shown).

Wet tissue weight followed a similar trend, with females in vitellogenic stage larger than those in postvitellogenic stage, followed by spawned females and females in previtellogenic stage, and smaller weight for indeterminate oysters. Males in spawned stage were larger, followed by males in late and early gametogenesis, with a similar wet weight in mature males and indeterminate oysters.

Description of Developmental Stages

The description of the developmental stages are summarized in Table 2 for females and Table 3 for males, with photos of each stage presented in Plate 1 for females and in Plate 2 for males. A total of 3,070 oocytes were used to determine average oocyte size.

The polychromatic staining applied by Tolivia et al. (1994) in semithin sections of epoxy resin, produced positive results in resin type glycol methacrylate used in this study. The type of resin used apparently does not affect the staining method and the same polychromatic results were obtained.

Frequency of Developmental Stages

Females of C. corteziensis in previtellogenic, vitellogenic, and spawned stages were observed during most of the year, but no spawned females were found at the end of April 2005 and from January to April 2006. From December 2005 to February 2006, a large proportion of undifferentiated individuals were present (Fig. 3A). By Match 2006, 75 % of females were either in the vitellogenic or postvitellogenic stages. During Match and April 2006, there were no indeterminate oysters.

Spawned males were found during most of the year, with the exception of July 2005 and November 2005 to March 2006. In November 2005 only undifferentiated oysters were found, and in winter approx. 20% of the oysters were in previtellogenesis and the rest were undifferentiated indicating a resting phase (Fig. 3B).

Gonad Coverage Area (GCA)

When the GCA was analyzed in relation to developmental stages, the highest values were found in mature or postvitellogenic females, followed by females in spawned and vitellogenic stages, with the lowest GCA values for previtellogenic and undifferentiated stages, although not significantly different between them (Table 1). The GCA in males was similar among late gametogenesis, mature and spawned males. Males in early gametogenesis had similar GCA than indeterminate oysters.

When using GCA to indicate the annual reproductive pattern of females, higher values were found at the beginning of April 2005, with a decrease by the end of April and till June and an increase in July 2005 (Fig. 4A). Another decrease occurred from August 2005 until February 2006 and gradually increased during Match and April 2006.

In males, no significant differences were found for GCA in relation to the annual reproductive pattern, although the GCA in males followed a similar trend as in females.

Number of Oocytes and Types

The frequency of each type of oocytes in females is shown in Figure 4B, with the largest number of cells occurring in April 2005, and mostly postvitellogenic oocytes. By June 2005, the oocyte number was less than hall. The number of oocytes increased during July to August 2005, with another marked reduction in September 2005, followed by gradual reduction until only atresic oocytes remained until December 2005. In January 2006, there were some mature oocytes, with the gradual increase in numbers by spring 2006, indicating the beginning of vitellogenesis.

Oocyte Diameter

Average oocyte diameter by stage is presented in Table 1. Postvitellogenic females had the largest oocytes, followed by females in spawned and vitellogenesis stages. The lowest values were found in previtellogenic stages.

Average oocyte diameter was not significantly different in relation to the annual reproductive pattern (results not shown).

Histochemistry

Lipid content was affected by the developmental stage in males and females (Table 1, Plate 3). In females, there were more lipids in the gonad in postvitellogenic stage and lower in immature oysters. No differences were found in lipid content in digestive gland or vesicular connective tissue. Carbohydrate content in gonad were higher in immature oysters and in females in previtellogenic stages, followed by females in vitellogenic stages, and lower content in postvitellogenic and spawned stages. Carbohydrate content in vesicular connective tissue was higher in immature oysters and females in previtellogenic stages, intermediate values in vitellogenic and postvitellogenic stages and lower values in spawned females. No differences in carbohydrate content were found in digestive gland of females.

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In males, lipid content was similar in late gametogenesis, mature and spawned stages, and lower in early gametogenesis and undifferentiated oysters. No differences for lipid content were found in digestive gland or vesicular connective tissue in males. Carbohydrate content in gonads was higher in undifferentiated oysters and males in early gametogenesis stages compared with males in late gametogenesis, mature or spawned stages. No differences were found for carbohydrate content in digestive gland or vesicular connective tissue.

DISCUSSION

The sex distribution observed for C. corteziensis here was similar to that observed by Cuevas-Guevara and Martinez-Guerrero (1979) in the same species collected in a southern locality (Nayarit, Mexico), with in general a larger proportion of females along the year and an absence of immature oysters during peaks of reproduction. C. corteziensis has been described as being protandric hermaphrodite, predominantly male when small and female when large (Baqueiro-Cardenas 1991). However, we found that males were slightly but significantly bigger than females of C. corteziensis. In C. gigas females tend to be bigger than males (Baghurst & Mitchell 2002), but males predominate when the environment is less favorable (Steele & Mulcahy 1999) or when temperature is low (Fabioux et al. 2005). Alternatively, the population might be subject to periodic harvest of bigger individuals that artificially modify the size and sex structure, as no strong mortalities have been reported in this locality for this species.

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In contrast to C. gigas and C. virginica, that present a unimodal gametogenic cycle with spawning in summer (Heffernan et al. 1989; Lango-Reynoso et al. 2000), C. corteziensis presented mature females with oocytes that were ready to spawn or in the process of spawning during most of the year, except in winter when no females in vitellogenic or postvitellogenic stages were found, although some females were already in previtellogenic stage. Males had a similar behavior, but no mature males were found on November 2005 and only some males in early gametogenesis were found from December 2005 to February 2006. Males and females must mature in the same period to ensure that gametes will be available at the same time for fertilization. A lack of matute males during November, with a large proportion of females in spawned stages, indicates that November can be considered as the end of the reproductive period. The small proportion of females that still remain in November in postvitellogenic stage had mostly remnant and atretic oocytes that will probably be reabsorbed. A decrease in the number of cells that occurred in June 2005 with the presence of atretic oocytes probably indicates a partial spawning event. Thereafter, there were atretic oocytes present till December 2005, indicating continuous spawning events or degenerating of unspawned oocytes. In accordance, the larger proportion of females in spawned stage found in May 2005 and June 2005 and particularly in September and November 2005 indicates partial spawning of cohorts with stronger spawning in autumn. Because oocytes in different stages of development were present most of the year, no significant differences were found in the annual reproductive pattern of the average oocyte diameter, which has been proposed as a reproductive scale in C. gigas (Lango-Reynoso et al. 2000). However, the frequency of each oocyte type differed significantly throughout the year, and it is evident that the biggest contribution to total number of oocytes per female is given by postvitellogenic oocytes. The biggest quantity of gonad was found in April 2005 that consisted mostly of postvitellogenic oocytes. A decrease was observed in June with a second peak in July to August and a gradual decrease from September to December, at least of postvitellogenic oocytes. Atretic oocytes, probably indicating spawning activity, were found continually from June to December 2005, but were higher in August to September and November 2005. In November the number of total oocytes is similar to September, but most oocytes are atretic or are being reabsorbed, contrary to September. The presence of a big number of atretic oocytes in August to September and November 2005 partially coincides with female maturation frequency, where only a small proportion of spawned females were found in August, and a larger proportion was found in April and June 2005.

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Stuardo and Martinez (1975) found that C. corteziensis sampled in a more southern locality (Nayarit) were in gametogenesis from January to November, and had a high proportion of mature or spawning females from April and into December, with bigger peaks in June and August, associated with partial spawns. Frias-Espericueta et al. (1997) found that the GSI in C. corteziensis in the same southern locality peaked in April, decreased in May, and had a second smaller peak in July after which it decreased in autumn and that these decreases were a result of spawning activity. In contrast, C. corteziensis sampled from a more northern locality (Sonora) had a shorter reproductive period, with spawned organisms present from July to September (Ramirez & Sevilla 1965). A prolonged spawning period in a tropical species is concordant with results obtained with other species of tropical oysters of other localities (Velez 1977, Joseph & Madhyastha 1984, Angel 1986), whereas a more condensed spawning period is typical of template species, which is probably regulated by temperature and food availability, among others (Sastry & Blake 1971). In fact, C. virginica cultured in subtropical or tropical zones also presented an extended gametogenesis activity throughout the year and the spawning period extended over several months (Baqueiro-Cardenas et al. 2007). In C. gigas the reproductive period has been extended or shortened in relation to temperature and other environment factors (Chavez-Villalba et al. 2002, Fabioux et al. 2005).

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GCA in females revealed an annual maturation pattern that closely matches the reproductive cycle asserted by the maturation stage frequency for females. Frias-Espericueta et al. (1997) proposed that a reduction in the GSI in C. corteziensis in Nayarit was a result of spawning activity that occurred when the temperature increased in 4[degrees]C, although they did not find a correlation between GSI and temperature. We did find a correlation with temperature and the GCA in males (r = 0.52; P < 0.05). This result suggest that in C. corteziensis, maturation occurs at temperatures higher than 20[degrees]C, and spawning when temperature increases above 27[degrees]C, similarly to C. gigas where spawning occurs when temperature rises above 19[degrees]C (Mann 1979), suggesting a temperature threshold (Ruiz et al. 1992). Higher GCA in males and females were observed when chlorophyll a levels were lower, whereas higher chlorophyll levels corresponded to the resting period in winter. The biochemical composition analyzed by histochemistry revealed that oysters accumulated more lipids in gonad in more advanced stages of maturation, and that this proportion decreased in spawned females, but not in males. In females, a significant correlation was found between GCA and lipids in gonad (r = 0.65; P < 0.01) and inverse but low correlations with GCA and carbohydrates in gonad (r = [+ or -] 0.38; P < 0.05) and vesicular tissue (r = -0.25; P < 0.05). This indicates that carbohydrates diminish in vesicular tissue and in gonad as maturation proceeds, and are probably used for lipogenesis, as well known for molluscs during gametogenesis (Gabbott 1975, Barber & Blake 1991). In males, a similar tendency was observed in gonads, with a significant positive correlation between lipids in gonad and GCA (r = 0.75; P < 0.01) and an inverse correlation between carbohydrates in gonad and GCA (r = -0.48; P < 0.05). Although males accumulate lipids in advanced gametogenic stages, this accumulation is not as strong as in females, and thus probably transference from other tissues is not as necessary. Paez-Osuna et al. (1993) found that protein and lipid content were lowest in winter in C. corteziensis and a decline of carbohydrates was coincident with the period when gametogenesis was occurring. Mobilization of reserves from vesicular tissue during gametogenesis is concordant to this tissue being a reserve in oysters and that its carbohydrate content decreases during maturation (Berthelin et al. 2000a), whereas the role of the digestive gland during maturation is minor, as lipid content remains stable (Berthelin et al. 2000b).

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In conclusion, C. corteziensis has a larger proportion of females along the year and an absence of undifferentiated oysters during peaks of reproduction, with males slightly but significantly bigger than females, probably a result of continuous harvesting of the wild population that has affected the sex proportion and size of the population in this location. This species had mature females with oocytes ready to spawn or in the process of spawning during most of the year, with several spawning events throughout the year and with a short resting phase in winter, with males following a similar trend. The presence of mature oocytes most of the year did not allow for differentiation of an annual reproductive pattern using average oocyte diameter, as has been used for C. gigas. A negative association between chlorophyll a and GCA in females and males is probably a result of transference of lipids and carbohydrates between gonad and reserves stores in other tissues. Maturation occurs at temperatures higher than 20[degrees]C, and spawning when temperature increases above 27[degrees]C.

ACKNOWLEDGMENTS

The authors thank the local fishermen for their hospitality and assistance during collection of wild oysters at aquaculture facilities at Ceuta Lagoon System, Sinaloa; J. Guevara (Ostricola Guevara); to Eulalia Meza, Monica Reza, Olivia Arjona, Gabriel Gonzalez, and Juan M. Mackliz for their technical assistance, and A. M. Ibarra for comments on the manuscript. This research was funded by grants from SAGARPA-CONACYT 2003-02-035 to A.M. Ibarra and SEP-CONACYT 2006-24333 to E. Palacios.

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Stuardo, J. & A. Martinez. 1975. Relaciones entre algunos factores ecologicos y la biologia de poblaciones de Crassostrea corteziensis Hertelein, 1951, de San Blas, Nayarit, Mexico. pp. 1-49.

Tolivia, J., A. Navarro & D. Tolivia. 1994. Polychromatic staining of epoxy semithin sections: a new and simple method. Histochemistry 101:51-55.

Velez, A. R. 1977. Annual Reproductive Cycle of the Oyster Crassostrea rhizophorae (Guilding) from Bahia de Mochima. Boletin del Instituto Oceanografico. Universidadde Oriente. 16:87-98.

C. RODRIGUEZ-JARAMILLO, (1) M. A. HURTADO, (1) E. ROMERO-VIVAS, (1) J. L. RAMIREZ, (1) M. MANZANO (1) AND E. PALACIOS (1,2) *

(1)Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico; (2) UMR/CNRS Universite de Bretagne Occidentale, Institut Universitaire Europeen de la Mer LEMAR- Laboratoire des sciences de l'environnement marin (UMR 6539) Technopole Brest Iroise, Place Nicolas Copernic, 29280 Plouzane-France

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

TABLE 1.
Female and male morphometry and histochemistry classified by
stage of gonad development of Crassostrea corteziensis were
sampled from Ceuta Lagoon System, Sinaloa, Mexico from
April 2005 to April 2006.

                                  Stage 0               Stage I
                              Undifferentiated     Previtellogenesis

Females
  Total oyster weight (g)   79.7 [+ or -] 4.4 b   69.9 [+ or -] 6.7 6
  Wet tissue weight (g)     14.1 [+ or -] 0.9 c   14.9 [+ or -] 1.7 bc
  GCA (%)                    1.3 [+ or -] 0.2 d    1.6 [+ or -] 0.4 d
  Average diameter (gm)               -            6.1 [+ or -] 0.1 c
Lipids (% area)
  Gonad                      7.3 [+ or -] 0.3 c    9.0 [+ or -] 1.4 bc
  Digestive gland           11.9 [+ or -] 0.3 a   12.0 [+ or -] 1.5 a
  Vesicular tissue           6.7 [+ or -] 0.3 a    7.7 [+ or -] 0.2 a
Carbohydrates (% area)
  Gonad                     18.8 [+ or -] 0.9 a   19.4 [+ or -] 1.9 a
  Digestive gland           17.6 [+ or -] 1.0 a   17.4 [+ or -] 1.6 a
  Vesicular tissue          20.0 [+ or -] 1.0 a   21.6 [+ or -] 0.6 a

                                   Stage 0               Stage I
                                   Immature           Early gametog.

Males
  Total oyster weight (g)   80.7 [+ or -] 4.7 b   79.8 [+ or -] 9.2 b
  Wet tissue weight (g)     14.4 [+ or -] 0.9 b   15.8 [+ or -] 3.7 ab
  GCA(%)                     1.3 [+ or -] 0.2 b    0.9 [+ or -] 0.4 b
Lipids (% area)
  Gonad                      7.3 [+ or -] 0.3 b    8.1 [+ or -] 1.2 b
  Digestive gland           11.9 [+ or -] 0.3 a   10.7 [+ or -] 0.4 a
  Vesicular tissue           6.7 [+ or -] 0.3 a    7.2 [+ or -] 0.4 a
Carbohydrates (% area)
  Gonad                     18.8 [+ or -] 0.9 a   19.6 [+ or -] 0.9 a
  Digestive gland           17.6 [+ or -] 1.0 a   20.6 [+ or -] 0.7 a
  Vesicular tissue          20.0 [+ or -] 1.0 a   23.0 [+ or -] 0.7 a

                                   Stage II             Stage III
                                Vitellogenesis      Postvitellogenesis

Females
  Total oyster weight (g)    111 [+ or -] 11.0 a   89.6 [+ or -] 4.9 ab
  Wet tissue weight (g)     22.8 [+ or -] 2.1 a    18.8 [+ or -] 1.5 ab
  GCA (%)                   15.9 [+ or -] 2.5 c    28.6 [+ or -] 3.2 a
  Average diameter (gm)     27.1 [+ or -] 1.4 b    31.6 [+ or -] 0.8 a
Lipids (% area)
  Gonad                     10.6 [+ or -] 1.3 6    13.9 [+ or -] 0.7 a
  Digestive gland           11.4 [+ or -] 0.8 a    11.9 [+ or -] 0.5 a
  Vesicular tissue           6.5 [+ or -] 0.4 a     6.6 [+ or -] 0.3 a
Carbohydrates (% area)
  Gonad                     15.4 [+ or -] 1.4 ab   14.3 [+ or -] 0.9 b
  Digestive gland           14.7 [+ or -] 1.5 a    17.5 [+ or -] 0.9 a
  Vesicular tissue          17.5 [+ or -] 1.9 ab   18.9 [+ or -] 0.7 ab

                                   Stage II              Stage III
                                Late gametog.              Mature

Males
  Total oyster weight (g)   92.2 [+ or -] 7.5 b    85.0 [+ or -] 12.9 b
  Wet tissue weight (g)     17.3 [+ or -] 1.9 ab   13.2 [+ or -] 2.3 b
  GCA(%)                    19.9 [+ or -] 2.9 a    28.4 [+ or -] 7.0 a
Lipids (% area)
  Gonad                     12.6 [+ or -] 1.6 a    12.8 [+ or -] 2.3 a
  Digestive gland           12.0 [+ or -] 0.4 a    11.6 [+ or -] 0.8 a
  Vesicular tissue           5.9 [+ or -] 0.2 a     7.5 [+ or -] 0.7 a
Carbohydrates (% area)
  Gonad                     13.8 [+ or -] 5.4 b    10.9 [+ or -] 1.6 b
  Digestive gland           16.4 [+ or -] 1.5 a    19.2 [+ or -] 0.3 a
  Vesicular tissue          19.6 [+ or -] 1.6 a    18.2 [+ or -] 1.4 a

                                  Stage IV
                                  Spawned

Females
  Total oyster weight (g)   94.3 [+ or -] 7.3 ab
  Wet tissue weight (g)     15.1 [+ or -] 1.3 bc
  GCA (%)                   18.3 [+ or -] 1.9 b
  Average diameter (gm)     30.1 [+ or -] 1.0 ab
Lipids (% area)
  Gonad                     11.3 [+ or -] 0.5 b
  Digestive gland           11.6 [+ or -] 0.3 a
  Vesicular tissue           7.0 [+ or -] 0.3 a
Carbohydrates (% area)
  Gonad                     15.1 [+ or -] 0.9 b
  Digestive gland           16.9 [+ or -] 1.0 a
  Vesicular tissue          16.8 [+ or -] 0.9 b

                                  Stage IV
                                  Spawned

Males
  Total oyster weight (g)    124 [+ or -] 9.7 a
  Wet tissue weight (g)     22.3 [+ or -] 1.6 a
  GCA(%)                    22.2 [+ or -] 3.2 a
Lipids (% area)
  Gonad                     13.1 [+ or -] 0.5 a
  Digestive gland           10.7 [+ or -] 0.6 a
  Vesicular tissue           6.4 [+ or -] 0.4 a
Carbohydrates (% area)
  Gonad                     13.7 [+ or -] 2.4 b
  Digestive gland           17.1 [+ or -] 1.5 a
  Vesicular tissue          17.1 [+ or -] 1.7 a

(a) Data were analyzed using stage as the independent variable
(5 levels) in a unifactorial ANOVA (P < 0.05). Results are reported
as mean [+ or -]SE. Different letters indicate significant
differences (P < 0.05).

TABLE 2.
Description of female developmental stages for C. corteziensis.

                                            Paraffin-HE-40 X

Stage 0     Undifferentiated     Clutches of gonial cells can be found,
                                   particularly near the gonoducts
                                   and surrounded by vesicular
                                   connective tissue. This stage could
                                   represent a period of recuperation
                                   with some reabsorbed or destructed
                                   follicles, with most of the tissue
                                   being vesicular and sometimes,
                                   oocytes that have not been
                                   reabsorbed can be observed at
                                   this stage and that remain till
                                   the next rematuration.
Stage I     Previtellogenesis    Elongated and interconnected follicles
                                   or gonadal tubules are surrounded
                                   by abundant vesicular connective
                                   tissue. Oogonias are round (6.2
                                   [+ or -] 0.1 [micro]m diameter).
                                   Previtellogenic oocytes (14.4
                                   [+ or -] 0.2 gm) have scarce
                                   basophilic cytoplasm. Some
                                   vitellogenic oocytes can be found.
Stage II    Vitellogenesis       The follicles are large and full of
                                   polygonal or pedunculated
                                   vitellogenic oocytes (25.7 [+ or -]
                                   0.2 [micro]m) attached to the
                                   follicles walls and presenting an
                                   acidophil cytoplasm; previtellogenic
                                   oocytes can still be found.
Stage III   Postvitellogenesis     The follicles are full of
                                   postvitellogenic oocytes of
                                   homogeneous size (38.0 [+ or -]
                                   0.1 [micro]m) that are either
                                   polygonal or round, are not longer
                                   attached to the follicles
                                   wall and have an acidophil
                                   cytoplasm. Some vitellogenic and
                                   previtellogenic oocytes that are
                                   still attached to the follicles
                                   walls are present. There is a
                                   considerable reduction of vesicular
                                   connective tissue.
Stage IV    Spawned              Spawning might be partial, with
                                   spaces in the follicles left
                                   by spawned  mature oocytes.
                                   Previtelogenic and vitellogenic
                                   oocytes can be found attached
                                   to the follicles walls and there
                                   are abundant hemocytes. Ciliated
                                   pithelium can be observed
                                   in the spawning ducts with
                                   postvitellogenic, and degenerating
                                   oocytes (overmaturated and atretic
                                   oocytes) The area occupied by the
                                   gonad is usually smaller compared
                                   with the early spawned stage. Some
                                   large, round oocytes detached from
                                   the follicle wall can be found (40
                                   [+ or -] 0.5 [micro]m).

                                        Resin-Polychrome-100 X

Stage 0     Undifferentiated     Pre-meiotic stages of oogenesis
                                   can be found.
Stage I     Previtellogenesis    Oogonia are present in groups of
                                   2-4 cells attached to follicles
                                   walls; they possess a large nucleus
                                   and a single nucleolus. The
                                   previtellogenic  oocytes are
                                   elongated and are in  contact
                                   with auxiliary cells and
                                   follicle cells.
Stage II    Vitellogenesis       Early vitellogenic oocytes are
                                   pedunculated and still attached
                                   to the follicle. Late vitellogenic
                                   oocytes  are longer. Cytoplasmic
                                   inclusions are present, the nucleus
                                   is round and a vitelline coat
                                   can be observed. Previtellogenic
                                   oocytes  and oogonia can still
                                   be found.
Stage III   Postvitellogenesis   The nucleolus is in close contact
                                   with nuclear envelope and there are
                                   cortical granules at the pariphery
                                   of the oocyte.
Stage IV    Spawned              Two kind of non spawned oocytes can
                                   be observed: 1. atresic oocytes
                                   with nuclear and cytoplasmic
                                   membrane breakdown and vacuolation
                                   of the ooplasm and 2. Overmatured
                                   oocytes with distension of
                                   endoplasmic reticulum.

                                              References

Stage 0     Undifferentiated     Stage I (Cuevas-Guevara and
                                   Martinez-Guerrero, 1979).
                                   Phase I(Stuardo and Martinez, 1975)
Stage I     Previtellogenesis    Stage II (Cuevas-Guevara and
                                   Martinez-Guerrero, 1979).
Stage II    Vitellogenesis       Phase II (Stuardo and Martinez, 1975)
Stage III   Postvitellogenesis   Stage III (Cuevas-Guevara and
                                   Martinez-Guerrero, 1979) Phase II
                                   (Stuardo and Martinez, 1975)
Stage IV    Spawned              Equivalent to stage IV and V
                                   (spawning and postpawning)
                                   described by Cuevas-Guevara and
                                   Martinez-Guerrero (1979). Phase III
                                   and IV(Stuardo and Martinez, 1975)

TABLE 3. Description of male developmental stages for C. corteziensis.

                                          Paraffin-HE-40 X

Stage 0     Undifferentiated   Clutches of gonial cells can be
                                 found near the gonoducts and
                                 surrounded by vesicular
                                 connective tissue. In this stage,
                                 it is not possible to distinguish
                                 between male and female gonad.
Stage I     Early              Spermatogonia are attached to the
            gametogenesis        follicle walls in the male gonadal
                                 tubules. The vesicular connective
                                 tissue is abundant.
                                 Spermatocytes are distributed in
                                 centripetal pattern from the
                                 acinus walls to the lumen.
Stage II    Late               The follicles are filled with all the
              gametogenesis      sexual cell categories including
                                 spermatozoa. The vesicular
                                 connective tissue is reduced.
Stage III   Mature             The follicles are larger and full of
                                 spermatozoa with tales oriented
                                 towards the lumen. Some
                                 spermatocytes can still be
                                 observed. The follicles are
                                 surrounded by vesicular
                                 connective tissue which is
                                 significantly reduced and the
                                 follicles unite.
Stage IV    Spawned            The interconnected follicles full
                                 of spermatozoa can be observed
                                 liberating sperm to the
                                 gonoduct. Residual
                                 spermatozoids can be observed
                                 in the lumen of empty follicles
                                 that also have abundant
                                 hemocytes.

                                      Resin-Polychrome-100x

Stage 0     Undifferentiated   Premeiotic stages of
                                 spermatogenesis can be found
Stage I     Early              Spermatogonia are about 5-6 pm
              gametogenesis      in size, with a spherical nucleus
                                 that contains one big nucleolus.
                                 In the primary spermatocyte
                                 (3.1-3.6 [micro]m) is possible to
                                 see 4 stages of early prophase:
                                 leptotene, zygotene, pachytene
                                 and diplotene. Secundary
                                 spermatocytes (2.3-2.9) are
                                 spherical in shape.
Stage II    Late               The spermatocytes in metaphase
              gametogenesis      are characterized by the presence
                                 of synaptonemal complexes in
                                 the nucleus. Follicles containing
                                 all the male cells categories.
Stage III   Mature               The spermatids are spherical in
                                 shape (1.5-2 [micro]m). There is
                                 a progressive reduction of the
                                 nucleus and condensation of
                                 heterochromatin. Mature sperm
                                 is abundant.
Stage IV    Spawned            The head of the spermatozoon are
                                 around 1.5 gm, has a nucleus
                                 that is wider than long. In the
                                 basal part of the nucleus, two
                                 spherical mitochondria can be
                                 distinguished. The sperm
                                 flagellum is evident.

                                       References

Stage 0     Undifferentiated   Equivalent to Phase I
                                 (Cuevas-Guevara and
                                 Martinez-Guerrero, 1979)
Stage I     Early              Equivalent to Phase II
              gametogenesis      (Cuevas-Guevara and
                                 Martinez-Guerrero, 1979)
Stage II    Late
              gametogenesis
Stage III   Mature             Equivalent to Phase III
                                 (Cuevas-Guevara and
                                 Martinez-Guerrero, 1979).
Stage IV    Spawned            Equivalent to Phase IV
                                 (Cuevas-Guevara and
                                 Martinez-Guerrero, 1979).