Reproductive pattern of the squalid callista Megapitaria squalida from Northwestern Mexico.

ABSTRACT The annual reproductive cycle of the squalid callista Megapitaria squalida living in the Ojo de Liebre lagoon was analyzed from January to December 2001. The reproductive activity was analyzed qualitatively (through histological analysis), defining five gonad development stages (undifferentiated, developing, ripe, spawning and spent). A quantitative analysis was also conducted (through the gonadic index and oocyte diameter) to correlate the reproductive activity with water temperature and salinity. Male and female gonads developed and spawned in parallel through time. M. squalida displays a clearly seasonal reproductive cycle in the Ojo de Liebre lagoon, with a long reproductive activity period (January to August) that coincides with the rise in temperature, plus a well defined inactivity period (September to December) with 100% of undifferentiated or spent organisms and the onset of which coincides with the temperature decrease. M. squalida's reproductive cycle does not seem to be influenced by salinity variations in the Ojo de Liebre lagoon. The spawning size of M. squalida was determined at 92 mm SL at an approximate age of 2.2 y. However, some individuals started spawning at 64 mm SL. Marked differences were found in some aspects of the reproductive pattern of M. squalida compared with studies conducted in other sites, and the likely causes are discussed here.

KEYWORDS: reproduction, bivalve, Megapitaria squalida, histology, Mexican Pacific.

INTRODUCTION

The squalid callista Megapitaria squalida (Sowerby, 1835) is one of the most abundant bivalves in northwestern Mexico. It is distributed from the Ojo de Liebre lagoon in Baja California Sur, (including the Gulf of California) to Macora in Peru (Keen 1971). M. squalida lives in muddy and sandy bottoms at depths ranging from one to 120 m, and can reach up to 120 mm in body length (Singh et al. 1991).

Studies on growth (Castro-Ortiz et al. 1992), ecological aspects (Baqueiro & Stuardo 1977, Anguas-Velez & Castro-Ortiz 1990, Singh et al. 1991), mantle anatomy (Garcia-Gasca & Garcia-Dominguez 1995), and reproductive aspects (Baqueiro & Stuardo 1977, Singh et al. 1991, Villalejo-Fuerte et al. 1996a, Baqueiro & Aldana 2000, Villalejo-Fuerte et al. 2000) have been conducted in M. squalida.

Although M. squalida is usually considered as a species with a low commercial value, in northwestern Mexico the fishery of this clam has intensified recently, resulting in an increase in production from 31.4 tons in 1996 to about 972 tons in 2004 (SAGARPA 2006). The above derives from the fact that the species is been captured, more and more, as an alternative resource when the main commercial species are unavailable because of the enforced fishing restrictions. M. squalida is captured throughout the year from the main water bodies along the state of Baja California Sur, including the Gulf of California and the Mexican coasts of Pacific Ocean. The capture regimen of M. squalida is based on previous studies of some populations from the Gulf of California (Singh et al. 1991, Villalejo-Fuerte et al. 1996a, 2000); however, the reproductive cycle varies with the geographic location, according to the species' specific phenotypic response to the particular environmental conditions in each location (mainly water temperature and food availability) (Porter 1964, Hesselman et al. 1989, Cruz & Villalobos 1993, Rodriguez et al. 1993).

Hence, a basic understanding of the reproductive cycle of M. squalida is needed for an appropriate timing of the fishing effort and the development of a sustainable fishery. Thus, the objective of this study is to determine the reproductive cycle of M. squalida in the Ojo de Liebre lagoon, located in the Pacific coast, and compare it with studies conducted in other localities.

MATERIALS AND METHODS

The Ojo de Liebre lagoon (24[degrees]02'N, 110[degrees]24'W) is located within the Sebastian Vizcaino bay, in the west coast of the Baja California peninsula, Mexico (Fig. 1). There, 30 M. squalida adult specimens were collected per month by a scuba diver at 3- to 6-m deep from January to December 2001. The bottom water temperature and salinity were recorded at the time of sampling by means of a U-10 HORIBA water-quality analyzer. In the laboratory, total and soft body weights and shell length (SL) were recorded for each clam. To determine the extent of gonad development, the visceral mass of each clam (gonad included) was dissected and fixed in 10% formalin. Later, a section of tissue from the dorsal area of each visceral mass was dehydrated in alcohol and embedded in paraplast X-tra. Sections (7 [micro]m) were placed on slides and stained with haematoxylin and counterstained with eosin (Humason 1979).

[FIGURE 1 OMITTED]

Because M. squalida does not exhibit sex dimorphism, specimens were sexed through histological analysis. Sex ratios were analyzed by month, testing the null hypothesis of a 1:1 sex ratio, using a chi-square test ([alpha] = 0.05) (Zar 1996).

Qualitative characteristics were used to sort specimens into one of five arbitrary gonad stages previously established for the same species: undifferentiated, developing, ripe, spawning and spent (Baqueiro & Stuardo 1977, Villalejo-Fuerte et al. 2000). The relative frequencies of gonad-development stages throughout the year were obtained. This enabled the description of the reproductive cycle.

To obtain quantitative information on the reproductive activity, the oocyte diameter and a monthly gonad index were determined. Oocyte diameter was measured from digitalized images of histological sections by means of the SCAN PRO software (Version 5.0, Systat Software, Inc., Point Richmond, CA, USA). Oocytes were individually traced by hand with the pointer, and the software automatically measured the major- and minor-axis lengths. Then, both dimensions were averaged and this was considered as the estimated diameter. In this way, a monthly mean oocyte diameter was calculated. At least 100 oocytes were measured from each slide. Only oocytes showing a nucleolus were sampled, assuming that this structure is located at the cell center (Laruelle et al. 1994). Individuals with few measurable oocytes and extensive phagocytosis (i.e., "spent" specimens) were excluded following the criteria of Grant and Tyler (1983). A one-way ANOVA, followed by a Tukey posthoc mean comparison test, was used to assess significant differences between months. The monthly gonad index was computed using a numerical grading system (Heffernan et al. 1989). Four categories were established according to the degree of gonad development, with 0 = undifferentiated and spent, 1 = developing, 2 = partially spawned and 3 = ripe. The monthly gonad index was determined by multiplying the number of specimens assigned to each category by the category score, summing all such values and dividing this amount by the total number of clams analyzed. The monthly values of oocyte diameter and gonad index obtained, enabled to carry out correlation analyses of reproductive activity with temperature and salinity, using a Spearman-rank correlation analysis (Zar 1996). The STATISTICA (version 6.0) software (StatSoft, Inc. 2001) was used for all statistical analyses. The significance level ([alpha]) was set at 0.05.

The size at spawning in the population is defined as the smallest length at which the 50% of the cumulative frequency of sampled females and males are spawning (Somerton 1980). To establish the size at spawning in the M. squalida population, the relative cumulative frequency of ripe and spawning organisms was fitted to a logistic model. Additionally, to have a gross estimation of the age at spawning in the population, the Bertalanfy growth equation (Lt = 92[1-[e.sup.-0.545] (t - 0.07)]) obtained for M. squalida by Castro-Ortiz et al. (1992) was used. However, for this study the L[infinity] value (92) was replaced by a L[infinity] value (128) calculated with the FISAT software (Gayanilo et al. 1995) using length frequencies of individuals collected in the Ojo de Liebre lagoon.

RESULTS

A total of 384 specimens were collected, including 128 females (33.3%), 156 males (40.6%), and 100 (26%) undifferentiated specimens. The monthly sex-ratio analysis shows that a significant difference (P < 0.05) from the expected 1:1 ratio occurred in August, September and October (Table 1). However, the sex ratio for the total sample (0.83F:1M) did not differ significantly (P > 0.05) from the expected 1:1 ratio. Clams ranged in SL from 64 mm to 121 mm (mean = 91.3 mm, standard deviation = 9.5 mm).

The reproductive cycle of M. squalida in Ojo de Liebre showed a clear seasonal pattern (Fig. 2). The relative frequencies of gonad development stages throughout the year were obtained pooling females and males together, because no differences in the temporality pattern between sexes were observed. The development stage occurred from January to March, with a peak frequency in January (92.8%). Ripe clams were observed from February to August with a variable frequency. The spawning stage was observed from February to August, reaching the peak frequency in April, June, July and August (78% to 94%). From September to December 100% clams were inactive (undifferentiated and spent stages).

[FIGURE 2 OMITTED]

Oocyte diameter showed significant differences (one-way ANOVA, P < 0.01) throughout the year (Fig. 3a). Small oocytes (30.6-33.4 [micro]m) were observed from January to March. Oocyte diameter increased considerably from April to July and August, when significantly larger oocytes (44.2-45.2 [micro]m; P < 0.01) were present. The above was consistent with histological observations, which revealed that the highest reproductive activity took place from April to August. From September to December all clams were spent or undifferentiated, so that oocyte diameter values could not be obtained.

Gonad index displayed a seasonal tendency throughout the year (Fig. 3b). From these data, a clear resting period from September to December was also apparent, whereas gonad index values were higher from April to August, coinciding with the ripe and spawning stages.

[FIGURE 3 OMITTED]

Seawater temperature showed a clearly seasonal variation in the Ojo de Liebre lagoon (Fig. 3c). The cold months (January to April and December) had temperatures under 18[degrees]C. The lowest temperatures were registered in January and February (15.9[degrees]C). The warmer months (July to October) had temperatures above 22[degrees]C. Water temperature reached its peak value (23.4[degrees]C) in August. Salinity in the Ojo de Liebre lagoon displayed a slight fluctuation (Fig. 3d). The minimum salinity occurred in January (33.4 [per thousand]), and the maximum in March and May (34.3 [per thousand]). The mean oocyte diameter and gonad index were positively correlated with temperature (R = 0.86; P = 0.006 and R = 0.76; P = 0.03, respectively), but not with salinity (R = 0.24; P = 0.57 and R = 0.54; P = 0.17, respectively).

To establish the size at spawning in the M. squalida population studied, relative cumulative frequency data of ripe and spawning organisms were fitted to the logistic model (r = 0.99, Fig. 4). The size at spawning in the M. squalida population was 92 mm SL, which may correspond to an age of 2.2 y old (according to our gross estimation). However, individual organisms may start spawning at 64 mm SL (1.2 y old).

[FIGURE 4 OMITTED]

DISCUSSION

Slight differences in seasonal gametogenesis and spawning patterns between males and females have been reported in some bivalves (Goodwin 1976, Sloan & Robinson 1984, Baron & Ciocco 2001). In our study, male and female gonads developed and underwent spawning in parallel through time, as reported in other bivalves (Villalejo-Fuerte et al. 2002, Choi & Chang 2003). This synchronization seems to be related to changes in water temperature, as reported by Gribben et al. (2004) for P. zelandica, and it is characteristic of species living in temperate zones.

Many studies point out that temperature is the most important environmental factor in the regulation of reproduction in bivalves (gametogenesis and spawning) (Giese & Pearse 1974, Sastry 1979, Barber & Blake 1981, Cruz & Villalobos 1993, Rodriguez et al. 1993, Wada et al. 1995), which may trigger or synchronize the "temporality" of the different development stages (Lubet 1983, Gallardo 1989). Thus, the rise in water temperature may stimulate gonad ripening (Giguere et al. 1994). In Ojo de Liebre, the reproductive activity of M. squalida (measured through the variation in the mean oocyte diameter and gonad index) was significantly and positively correlated with water temperature. The relationship between temperature and reproductive activity is well documented for other bivalves living in the Mexican Pacific coasts, including Chione californiensis (Garcia-Dominguez et al. 1993), Glycymeris gigantea (Villalejo-Fuerte et al. 1995) and Laevicardium elatum (Villalejo-Fuerte et al. 1996b).

In Ojo de Liebre lagoon, M. squalida shows a clearly seasonal reproductive cycle. This includes an extended period of reproductive activity (January to August) coinciding with the period of temperature increase, followed by a well-defined reproductive inactivity (September to December) where 100% of organisms are either undifferentiated or spent, the onset of which coincided with the drop of temperature. Likewise, a seasonal reproductive pattern has already been reported in the same location for other bivalves such as Nodipecten subnodosus (Arellano-Martinez et al. 2004a).

However, although temperature undoubtedly affects reproduction, other environmental factors may seem to interact with it to determine the annual reproductive activity pattern in any given geographic area (Sastry 1970). In this sense, the variation in the onset of gonad proliferation in Ruditapes decussatus has been found to be caused by interannual salinity fluctuations (Shafee & Daoudi 1991), and spawning in A. ventricosus coincides with minimum and maximum salinity values (Luna-Gonzalez et al. 2000). In other cases, extreme salinity values have been found to lead to a steady reduction in the duration of gonad development (Muranaka & Lannan 1984, Utting 1993). To this respect, salinity shows hypersaline gradients in the Ojo de Liebre lagoon (Lankford 1977), with salinities over 47 [per thousand] recorded in some areas (Contreras, 1985), being either shallow zones or as a result of human activities (brine discharges by the salt-producing company into some areas of the lagoon). However, in this investigation salinity in the study area (El Borbollon) was found to be relatively constant throughout the whole study period, likely caused by its closeness to the lagoon mouth. Thus, the reproductive cycle of M. squalida in this locality seems to be unaffected by salinity variations, because no significant correlation was found with this variable. This does not rule out the possibility that another clam bank located farther into the lagoon (where greater salinity variations occur) might be affected by this environmental factor. However, further research is required to obtain concluding results to this respect.

Currently, there is available information on reproductive aspects of M. squalida from four additional localities of the Mexican Pacific coast (Fig. 1): in the Gulf of California, from Bahia Concepcion (Villalejo-Fuerte et al. 1996a), Bahia Juncalito (Villalejo-Fuerte et al. 2000) and Bahia de La Paz (Singh et al. 1991), and outside the Gulf of California, from Bahia Zihuatanejo (Baqueiro & Stuardo 1977), a site located in the southern Mexican Pacific coast. Our findings reported here differ substantially in some aspects with the studies mentioned earlier (Table 2). The most obvious difference refers to the temporality of the reproductive cycle, which might be associated with water temperature and/or food availability in each locality.

As mentioned earlier, in Ojo de Liebre lagoon M. squalida displays a marked interruption in the reproductive activity (high percentage of undifferentiated or spent organisms) from September to December. However, this complete interruption of the reproductive activity during the annual cycle was absent in any other locality, whereas the undifferentiated stage occurred only at low levels in some months (Singh et al. 1991, Villalejo-Fuerte et al. 1996a, Baqueiro & Stuardo 1977, Villalejo-Fuerte et al. 2000). This clear resting period at the Ojo de Liebre lagoon might derive from having included immature specimens. However, the range of sizes analyzed (64 mm to 121 mm SL) is larger than the size of the smallest specimen (42 mm SL) reported as having undergone spawning (Villalejo-Fuerte et al. 1996a), so that this finding is valid. In this sense, Baqueiro and Aldana (2000) point out that the undifferentiation or lack of reproductive activity periods is not species-specific requirements, but responses to environmental conditions. Villalejo-Fuerte et al. (2000) mention that the absence of a reproductive inactivity period in Bahia Juncalito may result from food being abundant throughout the year, so that the clam population produces gametes continuously. This situation might also occur in the other localities of the Gulf of California, as well as in Bahia Zihuatanejo. In contrast, the Ojo de Liebre lagoon has been regarded as an area with low food availability (Millan et al. 1987, Delgadillo-Hinojosa et al. 2002, Arellano-Martinez et al. 2004b) and low food quality (Arellano-Martinez et al. 2004b). This situation of low food availability and quality in Ojo de Liebre might lead to the marked resting period and foster a marked energy storage/use cycle, as reported by Arellano-Martinez et al. (2004b) for N. subnodosus in the same locality. Nevertheless, further studies are required on this aspect to corroborate it for M. squalida.

On the other hand, in the case of the populations living in the Gulf of California and Bahia de Zihuatanejo, the ripe and spawning stages were present (although at different times and proportions) throughout the whole study period (Table 2), so that it is assumed that M. squalida from these localities displays a continuous reproductive cycle. This reproductive behavior has also been observed in other bivalves from tropical latitudes, including Laevicardium elatum (Villalejo-Fuerte et al. 1996b), Periglypta multicostata (Garcia-Dominguez et al. 1998), Pinna rugosa (Ceballos-Vazquez et al. 2000) and Spondylus calcifer (Villalejo-Fuerte et al. 2002). In contrast with the above, in the Ojo de Liebre population the ripe and spawning stages are restricted to the period January to August, which indicates that the reproductive cycle of M. squalida in this locality seems to be seasonal. This lagoon is considered as a temperate geographic zone (Briggs 1974), whereas the Gulf of California is regarded as a tropical-subtropical transition zone (Hendrickx et al. 2005) and Bahia de Zihuatanejo, a tropical one (Briggs 1974). Differences in the reproductive pattern of any given species are known to occur along a latitudinal range, given that critical temperatures (cold or warm) occur in different months or prevail all the year round, facilitating a continuous reproduction (Hesselman et al. 1989). The earlier mentioned coincides with reports by Porter (1964) and Hesselman et al. (1989), who mention that the differences in the reproductive cycle among various populations seem to result from phenotypical responses to variations in environmental conditions, mainly food availability and temperature. To this respect, Baqueiro and Aldana (2000) state that some mollusc species display different gonad-recovery and spawning patterns associated with the microenvironmental conditions of their habitat.

Furthermore, this study revealed that the mean oocyte diameter of M. squalida living in the Ojo de Liebre lagoon was larger (30.6-45.2 [micro]m) than the one reported by Quinones-Arreola (2003) for M. squalida living in Bahia Juncalito, Gulf of California (25-40 [micro]m). Oocyte growth depends on an adequate energy supply available for gametogenesis (Barber & Blake 1983). The availability of enough energy depends on both, food availability and the animal's metabolic rate, which to some extent is influenced by environmental temperature (Barber & Blake 1983). As mentioned earlier, food availability is high all the year round in Bahia Juncalito, which should favor a larger oocyte size, something that does not occur. This could be explained assuming that the metabolic rate in Bahia Juncalito might be higher than in Ojo de Liebre, given that temperature in the former is 5[degrees]C higher (Quinones-Arreola 2003). Additionally, the constant oocyte production (continuous reproductive cycle) in Bahia Juncalito involves more accelerated periods of gonad recovery and, consequently, smaller oocytes compared with oocytes from Ojo de Liebre are produced. For its part, food is not abundant in the Ojo de Liebre lagoon but there is a marked resting period, which would favor nutrient accumulation. Afterwards, nutrients would be available for gonad ripening, favoring a larger oocyte size.

This study found that the sex ratio in the population studied was 1:1. This finding coincides with reports for this same species in other localities (Table 2). This indicates that sex ratio is a species-specific characteristic unaffected by environmental conditions. Villalejo-Fuerte et al. (2000) infer, from a 1:1 sex ratio, that mortality in M. squalida (either natural or by fishing) does not differ between sexes, that no sexual dimorphism occurs in terms of size (because clams are caught only in the case of large specimens), and that growth rate seems to be similar for males and females. Nonetheless, further research is required to confirm the above.

As regards the size at spawning of M. squalida (92 mm SL) no comparisons are possible, given that no background information is available. However, smaller spawning organisms (42-50 mm SL) were reported in Bahia Concepcion (Villalejo-Fuerte et al. 1996a), which are even below the smallest size reported in this study for a spawning organism (64 mm SL). According to the earlier mentioned, the size at spawning in Ojo de Liebre might be overestimated in this study. To corroborate this hypothesis, further research is required including smaller organisms.

ACKNOWLEDGMENTS

The authors acknowledge the fellowships granted by the Programa Institucional de Formacion de Investigadores and CONACyT to M. Quinones-Arreola, and thank the Instituto Politecnico Nacional for funding this work; the Comision de Operacion y Fomento de Actividades Academicas and EDI for the fellowships granted to M. Arellano-Martinez, B. P. Ceballos-Vazquez and M. Villalejo-Fuerte. The authors also thank Maria Elena Sanchez-Salazar, MSc. for her editorial contribution to the English manuscript.

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MARCIAL ARELLANO-MARTINEZ, (1) * MARCOS F. QUINONES-ARREOLA, (2) BERTHA PATRICIA CEBALLOS-VAZQUEZ (1) AND MARCIAL VILLALEJO-FUERTE (1) (1) Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional, Apartado Postal 592, La Paz, B.C.S. 23000, Mexico. (2) Centro de Investigaciones Biologicas del Noroeste, Apdo. Postal 128, La Paz, Baja California Sur 23000, Mexico.

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

TABLE 1.
Female and male frequencies, Chi square ([chi square]) values and sex
ratios of Megapitaria squalida by month. Numbers in bold indicate a
statistically significant differences from a 1:1 ratio (P [less than
or equal to] 0.05).

Month       Female   Male   Total   [chi square]   Sex Ratio F:M

January       13       9      22        0.73          1.44:1
February      15      17      32        0.13          0.88:1
March          9      14      23        1.09          0.64:1
April         17      12      29        0.86          1.42:1
May           18      11      29        1.69          1.64:1
Jun           15      13      28        0.14          1.15:1
July          13      16      29        0.31          0.81:1
August        10      21      31        3.90          0.48:1
September      0       9       9        9.00          0.11:1
October        1       8       9        5.44          0.13:1
November       1       5       6        2.67          0.20:1
December       3       3       6        0.00          1.00:1
TOTAL        115     138     253        2.09          0.83:1

TABLE 2.
Available information on reproductive aspects of Megapitaria squalida.

                  Sex Ratio         Oocyte
Locality             F:M           Diameter        Resting Period

Zihuatanejo Bay    0.88:1             --           Not defined

La Paz Bay           --               --           Not defined

Juncalito Bay      1.02:1       25-40 [micro]m     Not defined

Concepcion Bay        1:1             --           Not defined

Ojo de Liebre      0.83:1     30.6-45.2 [micro]m   September to
  lagoon                                             December

                     Spawning Period
Locality              (Main Period)               Source

Zihuatanejo Bay   All months except July   Baqueiro & Stuardo 1977
                  (October to May)         Baqueiro & Aldana 2000
La Paz Bay        All months               Singh et al. 1991
                  (August to October)
Juncalito Bay     All months               Villalejo-Fuerte et al. 2000
                  (July to August)         Quinones-Arreola 2003
Concepcion Bay    All months               Villalejo-Fuerte et al.
                  (June to August)           1996b
Ojo de Liebre
  lagoon          February to August       This work