Performance of diploid and triploid Crassostrea gigas (Thunberg, 1793) grown in tropical versus temperate natural environmental conditions.
KEY WORDS: oyster, tropical, temperate, growth, sex, gonad, chlorophyll-", sea surface temperature, Crassostrea gigas
Originally from Japan, the Pacific oyster Crassostrea gigas has been introduced and cultured in different countries since the 1920s (Mann 1979). Worldwide production of this species reached 625,925 tons in 2014. whereas capture by fishery of all oyster species was just 130,754 tons (FAO 2016). In Mexico, it was introduced in the 1970s after a favorable diagnostic given when evaluated at the Pacific Coast of the Baja California Peninsula (Islas-Olivares 1975), a temperate area. Later, it was also introduced into tropical Sonora state coasts in the Gulf of California as an alternative to the native oyster Crassostrea corteziensis (Hertlein, 1951), whose population size had diminished on a large scale (Chavez-Villalba et al. 2005). After the introduction of C. gigas and until the 1990s, production of this species had been increasing but since the 1990s production has decreased, especially in Sonora State, where production fell to practically zero by 1999. Presently, practically all of Mexico's production of this species comes from aquaculture performed in the Pacific Coast of the Baja California Peninsula, where environmental conditions are temperate rather than tropical. Environmental conditions in the Gulf of California are tropical, and aquaculture sites usually record high sea surface temperatures (SST; >30[degrees]C) and low productivity (chlorophyll a <1 mg/[m.sup.3]) for extended periods of time during the warmest summer months (July-September). On the other hand, conditions on the Pacific Coast of the Baja California Peninsula are more temperate because of a strong influence of the California Current System from the North Pacific (Zaytsev et al. 2003). Sea temperatures reach a maximum of 28[degrees]C during short time periods in the fall, and the highest productivity occurs in the spring and early summer (Garate-Lizarraga et al. 2000).
Environmental conditions of productivity, temperature, and salinity are known to influence growth of marine bivalves such as Crassostrea gigas. In temperate areas, Brown (1988) found that for regions on the Pacific Coast of Canada, growth rates were highly site-specific, and depended on food supply and salinity conditions. Differences in growth for C. gigas diploids and triploids between culture environments have been found associated to temperature and natural productivity. For example, Davis (1988) showed that triploids reared in two sites of similar productivity grew faster where temperature was higher (20[degrees]C versus 16[degrees]C on average); and in France, Garnier-Gere et al. (2002) found that triploid C. gigas also grew faster at a site with higher productivity than a site with poor productivity.
Inasmuch as increasing temperatures might have a beneficial effect on growth of oysters in temperate areas as Canada, United States, and France, temperatures above the optima not only limit the natural distribution of this and other species (Somero 2002), but also the exploitation of some species through aquaculture. That is, high temperatures as those present in tropical areas might act as a selective or negative force for Crassostrea gigas as this species is known to live and grow at temperatures between 4[degrees]C and 24[degrees]C (Walne 1979, cited by Shatkin et al. 1997), with an estimated lethal thermotolerance of 30[degrees]C (Le Gall & Raillard 1988, cited by Bougrier et al. 1995), or 32.5-37.5[degrees]C, depending on the previous acclimation temperature (Carvalho-Saucedo 2004). Physiological effects of increasing temperatures on C. gigas have been studied, finding that oxygen consumption increases with temperature (Bougrier et al. 1995, Tran et al. 2008) and metabolic costs also increase with temperature (Hawkins 1995), resulting in negative energy balances at increasing temperatures, which if continually sustained could finally result in high levels of mortalities (Bougrier et al. 1995). Gonad maturation is also accelerated with increasing temperatures (Chavez-Villalba et al. 2002).
This work was undertaken to assess the effects that different environmental conditions at tropical and temperate aquaculture sites have on growth, survival, and reproduction of diploid and triploid Crassostrea gigas. Previous studies in Mexico have evaluated growth or reproduction for this species in diploid condition either at the Pacific Coasts of the Baja California Peninsula (Islas-Olivares 1975) or at different sites within the Gulf of California (Ochoa-Araiza & Fimbres-Pena 1984, Chavez-Villalba et al. 2007, Chavez-Villalba et al. 2010), but there are no reports as far as the authors are aware in which growth, survival, and reproductive development of the Pacific oyster have been simultaneously compared between tropical and temperate areas, nor does there exist a comparison between diploid and triploids under these conditions.
MATERIALS AND METHODS
Experimental Diploid and Triploid Crassostrea gigas
The type of triploids used in this study was "biological triploids" derived from mating tetraploid and diploid oysters (Guo et al. 1996). Diploids and biological triploids eyed-larvae were bought directly from Whiskey Creek Hatchery, Oregon (United States) in September 2006. On their arrival to the Aquaculture Genetics and Breeding laboratory (AGB laboratory) at CIBNOR, La Paz Mexico, they were certified for their triploid condition using a sample of the eyed-larvae by flow cytometry (Ploidy Analyzer; Partee, Germany) following Allen (1983) and Allen and Bushek (1992). After settling and growing them to 2-mm spat size in the AGB laboratory, and before transporting the groups to the field, further certification of the triploid condition was done of 100 spat following the same flow cytometry procedures as before. The number of spat found to be triploids was 94. which translate into a 94% triploidy before deployment to the preculture area.
Spat were transported to the Estero de Rancho Bueno in Baja California Sur (Fig. 1) for preculture until they reached the size to be freed in Nestier trays (above 2 cm). Culture density of spat sizes was two spat per cm" mesh bag (1-mm opening). The long-line culture method, with five trays of oysters per module, was used for both preculture and grow out of the culture at each site evaluated. Once they reached the size required, they were transferred to the grow-out sites as described below. The same culture method of suspended modules in a longline was used for each of the evaluation sites. Culture densities were 300 oysters/tray for the first 30 days of grow out, and was decreased monthly as follows: 200/tray from day 30 to 60, 100/tray from day 61 to 90, 80/tray for 90-120 days, 80/ tray from 121 to 150 days, 50/tray from 151 to 180 days, and 30/tray from 181 to 210 days.
Three aquaculture sites were evaluated: (1) Bahia de Ceuta (24[degrees] 06' to 24[degrees] 15' N; 107[degrees] 11' to 107[degrees] 24' W) in Sinaloa State, (2) Estero de Bacorehuis (26[degrees] 05' to 26[degrees] 30' N; 109[degrees] 05' to 109[degrees] 20' W), which locates in the border of the states of Sonora and Sinaloa, and (3) Estero Rancho Bueno at Bahia Magdalena (24[degrees] 15' to 25[degrees] 20' N; 111[degrees] 30' to 112[degrees] 15' W), located on the Pacific Coast of the Baja California Peninsula (Fig. 1). The last site is characterized by recording temperate environmental conditions (Garate-Lizarraga & Siqueiros-Beltrones 1998), whereas the first two are located in the Gulf of California and record tropical conditions.
Experimental Groups and Sampling
Diploid and triploid oysters were simultaneously deployed to the three grow-out sites in early January 2007. A total of 4,500 juveniles (2.26 cm average shell height) of each ploidy were deployed in each site. After an initial and unexpected mortality of approximately 66% of both diploid and triploid oysters that occurred in January in Ceuta, the survivors were redistributed in two trays to achieve the same stocking densities per tray in each replicate than those used at the other two sites. Samples were taken back to the AGB laboratory monthly for evaluations from February to September 2007. a common grow-out time in Mexico to reach 9-cm shell height oysters.
For each culture site evaluated, three (pseudo)replicates (one culture module = replicate = stocked initially with 1,500 oysters) per ploidy were evaluated monthly for survival [(numbs stocked per replicate at time 1-numbs death per replicate at time 2)/numbs stocked at time 1], Grow-out traits were evaluated (shell height, shell length, total weight, and tissue weight) from random-sampled oysters by selecting 30 individuals/ replicate/ploidy/site and transported back to CIBNOR in La Paz where all the biometrical data were taken.
Oysters were first sampled by dissecting a small piece (~2 mm) of their mantle tissue and preserved at--80[degrees]C until processing for ploidy verification by flow cytometry. Then the posterior part of the whole organism that includes the gonad was sampled for histological analyses, and fixed with Davidson's solution. After ploidy verification, individual's gonad samples were randomly selected within verified ploidy groups for histological analyses. Each of the 30 triploid oysters sampled monthly per replicate and a random sample of 10 per replicate of the diploids were individually analyzed for their ploidy by flow cytometry. Procedures for flow cytometry of mantle tissue were those standardized in our laboratory, and described in Maldonado-Amparo et al. (2004).
Gonad Developmental Stages and Genders
Monthly samples of gonads before deployment to the culture areas and from each replicate within each diploid and triploid groups and at each site were fixed in Davidson's solution, and preserved in 70% alcohol. All samples were taken from the middle part of the gonad area. Histological analyses were done on 30 diploid and 30 triploids oysters before deployment, and from each site thereafter by selecting all diploids verified for their ploidy, and randomly selecting 30 triploids after ploidy verification of those sampled monthly per site. Tissues were processed by conventional histology, and stained with Harris's hematoxylin and contrasted with eosin-phloxine (Sheehan & Hrapchak 1980). Gametogenic stages were determined after a qualitative classification adapted from Lango-Reynoso et al. (2000) and Enriquez-Diaz (2004) for Crassostrea gigas, divided into six categories for diploid oysters and modified into seven categories for triploid oysters (Table 1). For each oyster, microscopic observation of slides obtained from the posterior region of the gonad were used for assigning each to a gametogenic stage (0-V) group, and to classify individuals as males, females, hermaphrodites, or undifferentiated.
Finally, the numbers of developing oocytes (vitellogenic and postvitellogenic) were counted in randomly selected triplicate areas of the female gonad slides, each with an area of 0.322 [mm.sup.2] using a microscope at a 10X magnification for all oysters classified as females and hermaphrodites in both ploidy classes, and for the experimental period from April to September. The use of this methodology or variations from it have been previously reported for other mollusc and crustacean species (Rodriguez-Jaramillo et al. 2008, Gomez-Gutierrez et al. 2010, Arcos-Ortega et al. 2015). March was not included because the only site with vitellogenic or postvitellogenic oocytes was Ceuta, with the other two sites only showing initial oogenesis in the few females found.
Sea Surface Temperature and Chlorophyll-a Per Site
Satellite-derived datasets (1 -km resolution) of SST (merged data from MODIS-Aqua and MODIS-Terra) and chlorophyll-a (hereafter referred as Chl-a) as a proxy of phytoplankton (multi-sensor merged Chl-a, from SeaWiFS, MERIS, MODIS-Aqua, and MODIS-Terra) were used. Monthly time series and anomalies were estimated for coastal areas immediately adjacent to the study sites as an approximate to the environmental conditions that may influence the performance of Crassostrea gigas. SST anomalies were expressed as the difference from the long-term mean (2002-2015), whereas monthly anomalies of Chl-o were calculated as the ratio of a current value to the corresponding long-term monthly mean (1997-2015) and then expressed as percentage anomaly, that is. 100 X (anomaly-1). A comparison between time satellite data and the Multivariate El Nino Index (MEI, http://www.esrl.noaa.gov/psd/enso/mei/ index.html) was performed.
Because food and temperature are considered the most important factors influencing energy budgets of bivalves (Grant 1996), a rough estimate of accumulated thermal degrees (ATD) and Chl-a was calculated per site with the purpose of understanding possible differential stress effects (Brown 1988, Bevelhimer & Bennett 2000) of temperatures and Chi-a at each site during the summer months (from June to September). Monthly mean SST and Chl-a at each site were multiplied by the number of days in that month, and added to the total accumulated during the summer months.
Growth and survival: For all growth traits, any individual within the triploid group that was shown by flow cytometry to be other than triploid, such as mosaics having mixed cells for diploid and triploid (Allen et al. 1996, 1999) or were diploid, was eliminated from the data for further analysis. A three-factor analysis of variance was used to establish the effects of culture site (three sites), age-month (February to September 2007), and ploidy (diploids and triploids), as well as all their interactions, on survival, meat index, and growth traits. Survival data were transformed to arcsine before the analyses. Post hoc analyses of means were performed using Tukey comparisons.
Triploids: The percentage triploidy per site and month was analyzed with a two-factor (sites and months) analysis of variance with interactions to evaluate the percentage changes during the experiment. Percentages were transformed to arcsine before analysis (Sokal & Rohlf 1981).
Gametogenic stage: To evaluate if differences in gonad maturation stages (0, I, II, IIIA, IIIB, IV, and V) occurred between ploidies, sites, and months evaluated, a logistic model (normal distribution, logarithmic-link function) was used assigning each increasing gametogenic developmental stage an increasing numerical value (0 = 1, I = 2, II = 3, IIIA = 3.5, IIIB = 4. IV = 5, and V = 6) for the analyses. The interpretation of this numeric value is that the higher the mean value found (for a ploidy, site, or month), the more advanced maturation stages can be inferred. This permitted to analyze statistical differences between sites and ploidies. The interpretation of the estimated mean gametogenic value (GV) is straightforward: larger means indicate more advanced gametogenic stages were present and vice versa for smaller means.
Number of oocytes (vitellogenic and postvitellogenic): Were first analyzed using two models, one for diploids and another for triploids. The first one was a two-factor (month and site) with interaction model for number of oocytes in diploids for which there were females or hermaphrodites for all months. The second one was a two-factor (month and site) main effects model used for evaluating triploids because there were no females or hermaphrodite present for several months for one or more sites. Post hoc analyses of means were performed using Tukey comparisons for unequal sample sizes. In addition, differences among diploids and triploids per site were analyzed with a one-factor (ploidy) model using the means obtained for the different months and ploidy groups described earlier. Only in the months in which both diploid and triploids had females or hermaphrodites was it possible to count oocytes numbers that could be used for the analysis.
To establish if statistically significant changes in genders and more specifically in the proportion of males and females occurred during the experimental time that would indirectly prove that more than one maturation cycle occurred at the tropical sites, a reduced logistic model (binomial distribution, logit-link function) was used, allowing for modeling males and included categorical variables of ploidy, site, and month.
All analyses were done using Statistica v.8.
Percentage of Triploids
Figure 2 shows the results of this analysis (back transformed to percentage for clarity). The percent of triploids found was significantly affected by "month-age" and "culture site" (P < 0.0001 for both), but not by their interaction (P = 0.55). The percent of triploidy decreased during the culture months, with significantly less triploids from April (93%) to September (84%) than at the beginning of the evaluation. February (99%). The site with the lowest mean percent triploids was Rancho Bueno (85%), followed with a significantly higher percentage in Ceuta (96%) and Bacorehuis (94%), which did not show significant differences between them. For Rancho Bueno, the lowest percentage of triploids was observed during April (74%), whereas at the tropical sites they were seen in May (86%) and September (88%). The number of mosaic oysters found within the triploid group were three in Ceuta (June), two in Bacorehuis (August), and one in Rancho Bueno (September).
All main effects, "ploidy," "month-age," and "culture site" were significant (P < 0.0001) for all growth traits, and all interactions between those effects were also significant (P < 0.001).
Figure 3 shows the growth means per ploidy group and the monthly growth patterns from February to September of diploids and triploids for each trait, and at each culture site. On average for the ploidy groups and culture months regardless of trait, the best growth was observed for oysters grown at the temperate site, Rancho Bueno, followed by those at the two tropical sites. The site with the least growth was Bacorehuis (Fig. 3). Except for shell length, triploid oysters grown in Rancho Bueno grew significantly better than diploids (Fig. 3. mean comparisons).
The interactions between month-age, ploidy, and culture site for all traits was as a result of diploids and triploids growth rate becoming largely different from April onward for oysters grown at the tropical sites of Ceuta or Bacorehuis, whereas monthly differences between ploidy groups differed only in July and September at the temperate site of Rancho Bueno (Fig. 3), where triploids had a greater tissue weight than diploids.
In terms of the three main effects, month-age and culture site there were significant differences (P < 0.001), whereas ploidy showed no differences (P = 0.94). On the other hand, the interactions between culture site and ploidy, and between month-age and culture site, were significant (P < 0.001). Mean survival during the culture months are shown in Figure 4.
On average during the culture months and ploidy groups, monthly survival of oysters at Rancho Bueno (98.1%) was significantly higher than that at Bacorehuis (94.8%), with the lowest survival occurring at Ceuta (92.0%). The last site lower survival was a consequence of the high mortality seen after planting in February. Survivals of diploid and triploid oysters were significantly different only when grown at Rancho Bueno, with diploids survival being greater than triploids. No differences in survival between diploid and triploids were found at Ceuta or Bacorehuis (Fig. 4, mean comparisons).
Because of the low survival rates observed at Ceuta after first planting, samples taken from diploid and triploid survivors during February were sent for pathology analyses to test for the presence of the Oyster Herpes Virus type 1 (OsHV-1). The results demonstrated that sampled oysters were polymerase chain reaction-positive for the virus (R. Vazquez-Juarez, CIBNOR, personal communication).
Qualitative Description of Proportion of Oysters in Different Gametogenic Stages
Proportions of gametogenic stages during the culture months are depicted in Figure 5. At deployment to the culture areas in January 2007, 30 juveniles were sampled for histological analyses from each ploidy group finding that they were undifferentiated (data not shown).
For diploids, after transfer to the grow-out sites, Ceuta showed the highest number of oysters reaching maturity and spawning stages by April; new gonad development was observed in May, with mature and spawned diploid oysters being observed again in June and July; by August and September oysters had spawned, or were in resting stages or inactive. At Bacorehuis, a delayed beginning of the gametogenic cycle was observed when compared with Ceuta, with diploid oysters undifferentiated or in early gametogenesis in February, and mature oysters not present until April. At this site, the proportion of mature diploid oysters remained constantly high from April to July; by August most oysters had spawned and by September they were in a resting stage, or undifferentiated, or had spawned. At Rancho Bueno--the temperate site--, the gametogenic cycle was even more delayed than at Bacorehuis, with large numbers of undifferentiated diploid oysters in February until March, but by April about the same proportion of mature oysters were found as in Bacorehuis. Also at Rancho Bueno, the proportion of mature diploid oysters decreased by May and the proportion of spawned oysters increased from May to June. At the same time and contrary to what was found at the tropical sites, a large increase in undifferentiated diploid oysters also occurred from May to June. Mature oysters increased again from July to August. Two large separate spawning events are apparent at this site, one in July and a second one in September.
For triploids, a similar pattern as that observed for diploids between sites was seen for the most mature gametogenic stages (IIIA and HIB), with Ceuta presenting the most mature triploid oysters by April, and Bacorehuis in June. The maturation stage IIIB was not observed for triploid oysters grown at Rancho Bueno, the temperate site, and fewer mature triploid oysters were found during the culture months when compared with the tropical sites. Spawning of a large percentage of triploids was evident in July for Ceuta, whereas no evidence of triploid spawning was observed at the other tropical site, Bacorehuis. Spawning during July in Ceuta was followed by a large percentage of triploid oysters reaching the gametogenic stage II during August indicating that a new gametogenic cycle had begun, and by September spawned triploid oysters were seen again. At the temperate site of Rancho Bueno, some triploids spawned in August. The spawning of triploids in Rancho Bueno was 2-3 mo behind diploids, but in Ceuta was only 1 mo behind. Although no direct evidence of triploids spawning was found at Bacorehuis, the proportions of mature triploids decreasing from June to July, and then increasing again from August up to September indicates that a spawning event had occurred, which was followed by a new gametogenic cycle. This is further evidenced by the increase in the proportion of early gametogenic stage II triploid oysters from June to July, and the decrease in August and September when mature triploid oysters were observed again at this site.
Quantitative Analyses of Gametogenic Stages
The generalized linear logistic model assumes a normal distribution and a mean (numeric) gametogenic stage value that is indicative of a more or less advanced cycle. Deviance (0.77) and the scaled deviance (1.04) values were similar and close to one, indicating an adequate goodness of fit for this model assuming normality. All effects (site, ploidy, and month), and all interactions between them were significant (P = 0.000). For the following results differences were significant (P < 0.05). The site effect resulted from differences between all three sites: on average of ploidy groups and culture months, the lowest mean GV was found for Rancho Bueno (2.5), which was significantly smaller than that at Bacorehuis (3.10), and both were significantly smaller than that at Ceuta (3.56). The ploidy effect resulted from triploid oysters having, on average across sites and months, a significantly lower GV (2.64) than diploids (3.47). The interaction between sites, ploidy, and month resulted from different GV between sites for diploid and triploids during the culture time (Fig. 6). At Bacorehuis diploids and triploids did not differ in GV up to June, but from July to September diploids had increased GV, whereas in triploids it decreased. At Ceuta diploids had significantly higher GV than triploids in February and March, but triploids GV increased from May to July and were not different from those found for diploids until August, when triploids GV decreased significantly, but those in diploids remained high. Gametogenic values at Rancho Bueno for diploids and triploids did not differ in February and March, but the GV for triploids decreased significantly from a high GV in April to June, and although it increased significantly from June to July, still was significantly lower than the GV from diploids for the remaining culture time.
Number of Vitellogenic and Postvitellogenic Oocytes
The mean number of oocytes for diploids (female and hermaphrodite oysters) showed an interaction between month and site that was a consequence of the significant decrease in oocytes numbers at the tropical sites of Ceuta and Bacorehuis from July to August, whereas the number of oocytes at the temperate site Rancho Bueno remained high during those same months (Fig. 7). The mean number of oocytes for diploids for the complete culture period was 134.26 for Rancho Bueno, 131.68 for Ceuta, and 134.82 for Bacorehuis. For triploids (Fig. 7), the mean number of oocytes was significantly larger at Bacorehuis (81.28), followed by Ceuta which was significantly less (79.65) and Rancho Bueno with the lowest number of oocytes (24.44). Also, the numbers of oocytes in triploids decreased between June and July, caused by a decrease in the numbers found for oysters grown at Ceuta and Bacorehuis, which at least for Ceuta agreed with a spawning event that occurred in those months (see Fig. 5).
In regard to the analyses evaluating the number of oocytes between diploid and triploid females carried out independently for each culture site, the results indicated that the number of oocytes between diploids and triploids were not significantly different for Ceuta and Bacorehuis. At Ceuta the diploids and triploids had 108 and 73 oocytes, respectively, with a P value of 0.21, whereas at Bacorehuis diploids and triploids had 123 and 74 oocytes, respectively, with a P value = 0.08. The temperate site Rancho Bueno had significant differences in number of oocytes between diploids and triploids (P < 0.01), with triploids having less oocytes (n = 24) than diploids (n = 130).
Qualitative Description of Sexes
The proportion of sexes (undifferentiated, male, hermaphrodite, female) found for diploids and triploids within each culture site and during the culture months are shown in Figure 8. Ceuta was the only site where oysters were found to have sexually differentiated during February and March. At Rancho Bueno and Bacorehuis, more than 70% diploids and 90% triploids were not yet differentiated during those same months.
At Ceuta, similar proportions of diploid males and females were found during February, together with a small proportion of hermaphrodites, and by March, the proportion of females increased. Qualitative comparisons of proportions of sexes (Fig. 8) indicated that a change in sex from more females to more males occurred for diploid oysters in Ceuta between April and June. Diploid hermaphrodites at this site had the highest proportion in February with lower proportions thereafter. Triploids at this site also indicated changes in sex during the culture months: in February, only males and undifferentiated oysters were found, but by March, the percentage of undifferentiated oysters decreased, and the first females were observed. By April, the proportion of females was large and the first hermaphrodites were observed, but by May, there were only males. Then the percentage of males decreased, and the numbers of females and hermaphrodites increased in June, a trend that continued up to July with more females present. By August, more males were again present than females and hermaphrodites.
In Bacorehuis--the other tropical site--, the dominant sex observed during the culture months for diploids was consistently male, and the percentage of hermaphrodites during the culture months was similar to Ceuta. For triploids on the other hand, a larger proportion of males in March decreased by April, when the proportion of females increased. The first hermaphrodite triploids were observed during April at this site, increasing by May and remaining present during the culture months. In June, the proportion of males increased, whereas that of females and hermaphrodites decreased. From July to September, proportions of sexes including hermaphrodites were similar to those for June.
Diploid males and females in Rancho Bueno were present at approximately the same frequencies during the culture months, with the exception of August and September. In August, there were more females than males, whereas in September there were more males than females. The percentage of hermaphrodite diploids at this site, when present (April, June, July, and September), was low. On the other hand, undifferentiated oysters were abundant in February and March, and as a difference from the two tropical sites, were present during May and June. For triploids within this last site during the culture months, more males and undifferentiated oysters were identified than females or hermaphrodites. As with diploids, the largest percentages of undifferentiated triploid oysters were observed in February, March, May, and June. On the average of culture months, triploid males were more abundant than females, and there were fewer triploid hermaphrodites during the culture months when contrasted with the other culture sites.
Quantitative Analyses of Proportion of Males versus Females A reduced logistic model comparing only proportions of males and females (by modeling males) during the culture months for both ploidies and at each site indicated significant effects of ploidy (P = 0.01) and month (P = 0.00), but not site (P = 0.90). Among the interactions, all those with site (site by ploidy, site by month, and site by ploidy by month) were significant (P = 0.00). Only the interaction between ploidy by month was not significant (P = 0.63). The ploidy effect (P = 0.01) resulted because on the average for culture sites and months, there were more males among triploids than diploids. The interaction between ploidy and sites was a result of Rancho Bueno and Ceuta diploids having significantly less males than those seen within triploids, but Bacorehuis did not, and instead had a tendency for the inverse (more males in diploids than triploids). The cause of the ploidy, month, and site interaction is better explained by presenting the time pattern for the proportion of males within each site and ploidy (Fig. 9). For the following results, means were compared through confidence intervals, where significant differences were defined at P < 0.05.
When contrasting the mean numbers of males and females between ploidies, no significant differences were found for Bacorehuis, but significant differences were found at Rancho Bueno and Ceuta, where on the average, more males were present within triploids than diploids (Fig. 9). On the other hand, the proportion of diploid or triploid males found during the culture months at Rancho Bueno did not show any significant differences, but at Ceuta, diploid males decreased slightly and not significantly from February to April, and then significantly increased up to June, remaining constant until September. Triploids at Ceuta had a similar pattern in the proportion of males than diploids from February to April, but the changes occurring during the culture months in the proportion of males were more accentuated and reached significant differences. For triploids, a significant decrease in numbers of males was observed from May to July that increased significantly again by August, remaining high in September. At Bacorehuis, a large proportion of diploid males found initially during March decreased slightly and not significantly by April remaining at 60%-80% males during the remaining culture months. For triploids on the other hand, the initially large proportion of males in March decreased significantly by April and May, and then increased significantly by July, where it remained stable up to September (Fig. 9).
Sea Surface Temperatures and Chlorophyll-a Concentration
Sea surface temperature at Rancho Bueno during preculture was 26.5[degrees]C in November, decreasing in December to 24.6[degrees]C and in January 2007 to 22.4[degrees]C (Fig. 10A and B). matching with warm conditions associated to a weak El Nino event (see SST anomalies and the MEI. Fig. 10B). Temperatures at Bacorehuis and Ceuta during the transfer in January 2007 were 18.7[degrees]C and 21.6[degrees]C, respectively. Sea surface temperature at Rancho Bueno showed a decreasing trend from January to May 2007 followed by an inverse pattern until October with a maximum of 26.4[degrees]C. Sea surface temperature records were superior to 30[degrees]C during August-September in the tropical sites.
Chl-a concentration was higher at the two tropical sites than at the temperate site from January to March (Fig. 10C), although Chl-a anomalies (%; Fig. 10D) indicated poor productivity conditions in all sites that might be linked to the ENSO conditions mentioned earlier. On the other hand, the lowest mean values were recorded during July for Bacorehuis (0.49 mg/[m.sup.3]) and Ceuta (0.92 mg/[m.sup.3]), whereas this low productivity condition was recorded in Rancho Bueno from June to December, with the lowest mean values on November (0.3 mg/[m.sup.3]). Chi -a in Rancho Bueno showed an increasing trend from January to June with the highest concentration occurring in April (6.3 mg/[m.sup.3]). Low Chl-a condition was similar in all sites during August (Chl-a <1.5 mg/[m.sup.3]). Time series of Chl-a anomalies (i.e., deviations from the expected mean values) showed dominant poor productivity conditions for Bacorehuis and Ceuta during late 2006-2007.
Similarly, estimates of the ATD from June to September at each site (Table 2) indicated that, when compared between culture sites, Bacorehuis and Ceuta recorded similarly high ATD values, and the lowest value was observed for Rancho Bueno. Accumulated Chl-a concentrations (ACHL-a) followed an inverse pattern to that of ATD: lowest ACHL-a for Bacorehuis, followed by Ceuta, with the highest values for Rancho Bueno (Table 2).
When both, Chl-a and SST are combined to obtain a ratio between their accumulated mean values from June to September, a pattern can be seen. The site with the least milligram of ACHL- a available per ATD was Bacorehuis, followed by Ceuta and Rancho Bueno. The observed growth (body weight) between culture sites for both ploidies followed a pattern of best growth occurring within the site with the highest ACH L-a: ATD ratio and the least growth occurred at the site with the lowest ACHL-a:ATD ratio.
Our results indicate the following main findings: (1) growth of Crassostrea gigas in coastal areas of Mexico is accelerated, with oysters reaching market size in one growing season; (2) regardless of ploidy, growth was less at the tropical sites than at the temperate site, but triploids grew better than diploids at the tropical sites in spite of higher GV and higher fecundity of female triploids than that found at the temperate site; (3) diploids survival was higher at the temperate site than at the two tropical sites, but survival of triploids was not different between sites; (4) the percentage of triploidy decreased significantly during the culture months only at the temperate site, the only site showing less survival of triploids than diploids; (5) regardless of ploidy, gonad development was continuous at the tropical sites when compared with the temperate site; and. (6) changes in male and female proportions were more predominant at the tropical sites, and were more accentuated for triploids than diploids.
Growth and Survival
Studies have shown that growth and survival of Crassostrea gigas depends strongly on the combined effects of salinity, temperature, and food availability (Brown 1988. Brown & Hartwick 1988a, 1988b, Bougrier et al. 1995, Costil et al. 2005, Degremont et al. 2005). Growth of C. gigas in temperate and tropical areas has been reported to be accelerated when compared with temperate-cold areas, reaching market size in less than 1 y. For example, oysters grown in Laguna Manuela (Pacific Coast of the Baja California Peninsula) were found to grow 1 cm per month, reaching 7.56 cm with a mortality of only 20% in 6 mo when growing from November to April (Isias et al. 1982). Also, Ochoa-Araiza and Fimbres-Pena (1984) evaluated C. gigas growth and survival rates at a tropical area within the Gulf of California in Sonora State (El Soldado) for 10 mo, from June to March, finding that this species grew 0.71 cm per month reaching market size in 10 mo, with an absolute mortality of 30% during that period. In agreement with those authors, C. gigas showed an accelerated growth in our study, reaching market sizes in less than 1 y particularly at the temperate site. Nevertheless, although a higher growth rate was found than that commonly seen at temperate-cold areas, less growth was attained in the two tropical culture sites, and this was associated with higher temperatures and lower productivities, as well as an early onset and sustained gonad maturation. Lower growth was particularly evident at Ceuta and Bacorehuis, the two tropical sites within the Gulf of California evaluated in this work, where monthly average temperatures ranged from 18.8[degrees]C to 31.5[degrees]C during the grow-out period in comparison with the growth attained at Rancho Bueno, a temperate site characterized by high productivity and lower temperatures (17.2-26.5[degrees]C). The effect of environmental conditions was a clear factor that negatively impacted the growth rates of diploid C. gigas (total body weight) at the tropical sites in comparison with the climatic conditions found at the temperate site, finding that growth was less than half that at the temperate site. The estimated average gain at Ceuta of total body weight per month was 5.47 g and shell height was 0.56 cm, whereas at Bacorehuis was 4.64 g or 0.56 cm; this contrasts markedly with the temperate site Rancho Bueno where growth rates reached 11.65 g or 0.88 cm per month. Furthermore, the estimated growth rates for total body weight at the tropical sites were mainly caused by shell growth, as tissue weight at Ceuta did not increase after April and at Bacorehuis it did not after June. Other authors have also found a significant effect of temperature on growth; for example, in controlled laboratory experiments, Shpigel et al. (1992) found no differences in daily growth rate of 10-g diploids grown at 8-15[degrees]C or 30[degrees]C, but dry tissue weight of diploids grown at 30[degrees]C was less than half that at 8-15[degrees]C after 35 days. Flores-Vergara et al. (2004) found that when diploid C. gigas spat was raised at 32[degrees]C for 60 days, poor growth resulted regardless of the diet provided, and survival was less than 50% after 5 wk when maintaining the oysters at that temperature, whereas at all other temperatures (23[degrees]C, 26[degrees]C, and 29[degrees]C) survival was significantly higher (88%). In the present study, month survival differences for diploids between tropical and temperate sites were significant, and became more evident when absolute survival, for example, between Bacorehuis and Rancho Bueno, are compared for the 8-mo period of this study; survival at the tropical site Bacorehuis was only 50% of that at the temperate site Rancho Bueno.
When growth rates of triploid Crassostrea gigas was compared across sites, the differences between sites were of a lesser magnitude than with diploids, but still a larger average gain in live body weight and shell height per month was seen when grown in the temperate site Rancho Bueno (12.37 g, 0.86 cm) than when grown at the tropical sites, Ceuta (9.29 g, 0.78 cm) and Bacorehuis (7.59 g, 0.72 cm). The triploids used in the present study were biological triploids with expected higher heterozygosity than that seen for meiosis II triploids (Wang et al. 2002), and where an advantage over diploids is expected in growth and metabolic efficiency (Hawkins et al. 1994. 2000), a superiority that was seen especially when triploid oysters were grown at the tropical sites characterized by high temperatures and low productivity. The expected higher metabolic efficiency of triploids over diploids was evident when contrasting growth between diploid and triploid C. gigas within culture sites, as triploid oysters performed better in growth rates than diploids especially at the tropical sites. For example, triploids at Ceuta gained 70% more weight per month than diploids, and at Bacorehuis they gained 64% more, but at Rancho Bueno triploids were gaining only 6% more weight monthly and gained less in shell height than diploids. Results presented agree with Shpigel et al. (1992), whom after a short-term (35 days) evaluation, found that triploid C. gigas had a significantly increased growth rate over diploids when grown at 30[degrees]C, but not when the temperature was 8-15[degrees]C. Other authors have also concluded that the superior growth rate of triploid oysters is more evident when culture sites have stressful environmental conditions (Davis 1988, Maguire et al. 1995, Kesarcodi-Watson et al. 2001, Garnier-Gere et al. 2002), although the magnitude of the stressors and growth differences was less than in the present study when comparing temperate and tropical sites.
Decreased Survival Was Site Specific and Also Caused by OsHV-1 Virus
Although growth across sites was largely affected by environmental conditions in the present study, less marked differences were seen for month survival between sites, although significantly higher survivals were seen at the temperate site Rancho Bueno when compared with the two tropical sites. One of the causes for the survival difference was the large spat mortality caused by the OsHV-1 virus at Ceuta early during the evaluations. OsHV-1 is a herpes-like virus that can infect oysters and is associated with high seed mortalities. Its existence has been reported since the 1990s for Pacific oysters grown in France (Renault et al. 1994), New Zealand, and the United States (Cheney et al. 2000). In Sonora Mexico, mortalities of oyster spat and adults are known to be site dependent, they occur when temperatures are increasing in the spring or decreasing after summer, and have been found to be associated with the herpes virus OsHV-1 and with a light infection of a protozoan (SAGARPA 2008), recently identified as Perkinsus marinus (Enriquez-Espinoza et al. 2010). Large mortalities are known to occur in Sonora state (Chavez-Villalba et al. 2010), but until this study, they had not been reported for the state of Sinaloa, where Ceuta is located. This study found that this site has had mortality problems of spat and early juveniles of Crassostrea gigas when planting them from November to March, or during the coldest months in tropical Mexico, when temperatures are around 20[degrees]C (Jorge Guevara, Ostricola Guevara, personal communication). Bacorehuis, which borders the states of Sonora and Sinaloa, however did not present a mortality of spat as that seen in Ceuta, agreeing with the fact that the mortalities caused by the OsHV-1 virus are site dependent. In temperate countries such as France where significant mortalities have been reported in the last decade, mortalities of spat (1 g) occur during the warming months in the summer, but juvenile-adult (25-35 g) mortalities are also observed in spring (Soletchnik et al. 2007). Summer mortalities of spat were found to be caused by the herpes virus (Nicolas et al. 2008). Mortalities associated with OsHV-1 in temperatecold areas occur at similar temperatures than those seen for tropical areas during autumn-winter, although in temperatecold climates they occur during the summer, which is the season when temperatures are between 16[degrees]C and 25[degrees]C, and are therefore known as summer seed mortalities (Cheney et al. 2000, Burge et al. 2006, 2007).
Besides the mortalities caused by the OsHV-1 virus at Ceuta, when comparing Crassostrea gigas diploids and triploids survivals within culture sites, our results were variable and site dependent. Reports on survival between diploids and triploids of C. gigas have varied in their conclusions, indicating either higher survival of triploids than diploids, higher survival of triploids in low productivity sites, equal survival rates, and lower survival rates (Allen & Downing 1986, Maguire et al. 1995, Cheney et al. 2000, Garnier-Gere et al. 2002, Gagnaire et al. 2006). This variation of survival rates can be attributed at least partially because of the different culture and environmental conditions present during each study just as in the present study, where environmental conditions were different between sites. In the present work, significant month survival differences between diploids and triploids were not seen with the exception of the temperate site Rancho Bueno, where less survival of triploids occurred, a result that we will discuss ahead because it might explain the decreased percentage of triploids at this site. At one of the tropical sites, Ceuta, no differences were seen between ploidy groups, with the triploid condition not conferring any resistance to the OsHV-1 virus, as it affected equally diploid (67%) and triploid (66%) juveniles planted at this site. At Bacorehuis there was a tendency for triploid monthly survival to be higher than diploids, and although those differences were not statistically significant, when absolute survival is considered, more triploids (69%) survived to the end of the evaluation than diploids (49%). The variable results observed for triploids survival between tropical sites cannot be explained by just different environmental conditions present at each of the sites. On the other hand, the interaction of environmental conditions with the energetic costs involved with the predominant sex and the stage of gonad development might. This topic will be discussed with reproduction in more detail later.
Low Chi-a and High SST at Tropical Sites Associate with Less Growth and Survival hut High Chl-a at the Temperate Site Associated with Low Triploid Survival
The thermal degree methodology used here has been primarily used for crop growth prediction (Bell & Wright 1998) but has also been used to compare the effects of chronic exposure to high temperatures for fish (Bevelhimer & Bennett 2000). We found by comparing sites by ATD or ACHL-a by themselves that it provided some explanation for the site dependent growth differences, because the estimated ratio that took into account the relationship between those two variables shows a clear pattern that corresponds largely with the growth observed at the different sites. The growth differences between temperate and tropical sites are explained by a continuous thermal stress resulting in increased energetic costs for maintenance of homeostasis, and a low food acquisition capability due to low productivity, especially at Bacorehuis. Physiological characteristics of Crassostrea gigas might have further impacted the negative effects on growth as it has been reported that clearance (filtration) rates of C. gigas decrease above 19[degrees]C and with advanced maturation stages (Soletchnik et al. 1997, Lefebvre et al. 2000). Furthermore, increases in temperatures are known to result in increased oxygen consumption in diploids of C. gigas. In addition, oxygen consumption has been reported as higher for males, as well as for oysters in postspawned condition (Bougrier et al. 1995, Le Moullac et al. 2007, Tran et al. 2008). Interestingly, this could explain both the differences in growth between tropical and temperate sites, and also between the tropical sites, Ceuta and Bacorehuis, because the proportion of mature males was consistently higher (above 60%) from April to July for Bacorehuis (see Fig. 8). Although in triploids, the same effect of temperature on oxygen consumption would be expected according to Shpigel et al. (1992), if triploids are metabolically more efficient, use less energy for basal metabolism, and have higher filtration or clearance rates than diploids (Hawkins et al. 1994, Hawkins et al. 2000), their growth and survival rates would be expected to be greater than for diploids. Indeed, in this study triploids at the tropical sites had higher growth rates than diploids and showed a tendency to have larger monthly survival rates.
In contrast, the temperate site of Rancho Bueno is located within Bahia Magdalena system, which is characterized as a Biological Activity Center because larger than average productivity is present, that is particularly evident from April to July. This site has been shown to have the largest phytoplankton concentration of the three Biological Activity Centers present in the Baja California Peninsula of Mexico (Garate-Lizarraga et al. 2000, Lluch-Belda 2000, MartinezLopez & Verdugo-Diaz 2000). In previous studies at this site it was found that triploids of the scallop Nodipecten subnodosus (Sowerby I, 1835) did not grow better than diploids, which was explained by proposing that either, the reproductive strategy of this scallop at this site was opportunistic as suggested by Racotta et al. (2003), such that the energy stored for maturation in diploids is not used, thus allowing them to grow continuously, or that the high productivity characterizing this site might be negating the growth rate advantage that triploids typically have over diploids (Maldonado-Amparo et al. 2004, Racotta et al. 2008). Furthermore, the high productivity might be affecting their survival because under normal temperature conditions triploid Crassostrea gigas is known to have a higher filtration rate than diploids. But just as low concentrations of microalgae result in higher clearance rates of triploids, high concentrations of microalgae are known to reduce efficiency in the clearance rate of triploid oyster Saccostrea commercialis (Iredale and Roughley, 1933) (Hawkins et al. 2000. KesarcodiWatson et al. 2001). Whether triploid C. gigas grown under high productivity environmental conditions are negatively affected because of their potential high filtration rate, or because there is a reduced efficiency in clearance rates when high concentrations of microalgae are present is unknown. Nevertheless, if it does, that might explain the marginal advantage of triploid oysters at Rancho Bueno, a site that, in a comparative growth/site context, was the best culture site regardless of ploidy, but was also the only site where survival of triploids was less than diploids. In addition, this site was characterized by the largest decrease in the percentage of triploidy that was particularly low in April, and continued to decrease until September. This declining trend cannot be attributed to temperature, because the range was within acceptable levels of 18-25[degrees]C. On the other hand, it may be significant that Chl-a concentrations increased three times in April and remained the highest from April to July (>4 mg/[m.sup.3]) as shown in Figure 10, and during this period survival of the triploid group was the lowest (Fig. 4). Whether the decrease in triploidy was caused by selective mortality of triploids within the group or by reversal of triploids to diploids is not known for sure, but very few mosaic individuals were found during ploidy evaluations by cytometry that could explain a reversal to the diploid condition. Furthermore, all triploids sampled each month (n = 90 per site) were evaluated individually, such that the chance of errors regarding their ploidy is null. Hence, selective mortality of triploids within the triploid group is the most probable cause for the decreased percentage of triploids at this site.
Mean GV and Fecundity Were Higher for Triploids at the Tropical Sites
Partial gonad sterility in triploid Crassostrea gigas is known to cause reduced fecundity; however, reduced fecundity does not imply that no gonad development can occur in triploids, because follicle area has been found to increase in a similar manner in both diploid and triploid oysters (Guo & Allen 1994, Allen & Downing 1986, Normand et al. 2009). In the present work, fecundity was used as a measure of reproductive output to compare both ploidies and culture sites. It was found that when the number of oocytes for female triploid oysters was contrasted with diploids, only the temperate site Rancho Bueno had significant differences, with female triploid oysters having only 18% of the oocytes observed for diploids. On the other hand, the numbers of oocytes in triploids grown at the two tropical sites were not significantly different from diploids, representing 60% of those in diploid oysters. Just as number of oocytes indicated a larger reproductive output for triploids at the tropical sites compared with the temperate one, the mean GV indicated that regardless of gender, maturation at the tropical sites was continuous, with no defined resting stage during the experimental period as was seen for triploids at the temperate site during June.
It is known that depletion of energy reserves of molluscs depends not only on the stage of gonad development, but on environmental effects that impact metabolism, as well as the quantity of food available (Bayne & Newell 1983). The minimum temperature for maturation to proceed in Crassostrea gigas is 16[degrees]C, and differences between culture sites for maturation of same origin Pacific oysters have been principally associated with culture site temperatures, with temperatures of 18[degrees]C or greater inducing gonad ripening and gamete release (Mann 1979, Hickey 1997, cited by Castanos et al. 2009, Chavez-Villalba et al. 2002, Enriquez-Diaz et al. 2009).
In temperate-cold areas, maturation of Crassostrea gigas follows a defined seasonal pattern that is associated with gradual increases in temperature and productivity, with increasing gonad development from spring to summer when productivity is increasing, reaching maturity and spawning during the summer, and reabsorbing the gonad thereafter (Mann 1979, Berthelin et al. 2000, Costil et al. 2005, Royer et al. 2008, Castanos et al. 2009, Enriquez-Diaz et al. 2009, Normand et al. 2009). On the other hand, when comparing growth and maturation at temperate-cold areas with tropical or temperate-warm areas, it is clear that large differences exist. In tropical and temperate-warm areas, C. gigas is known to grow to similar market sizes than those reached after 2-3 y when grown at temperate-cold areas, although in tropical areas market sizes are attained in only 1 y (Islas et al. 1982, Ochoa-Araiza & Fimbres-Pena 1984, Chavez-Villalba et al. 2010), and growth occurs in parallel to maturation and spawning, with no resting period to acquire or store new energetic reserves for continued growth or gonad maturation as it occurs in temperate-cold areas.
When only considering if there were temperature effects on when first gonad maturation occurred, we observed differences between sites in the present study, because it was found that both diploid and triploid oysters at Ceuta began maturation earlier than at the other tropical site, and this was associated with differences in SST at each site when planting. At Ceuta, SST was 21.6[degrees]C in January, whereas at Bacorehuis, it was 18.7[degrees]C. At both tropical sites, temperatures increased thereafter, with a sharp increase to ~26[degrees]C from April to May, whereas at the temperate site Rancho Bueno temperature decreased from January (22.3[degrees]C) until May (17.2[degrees]C), which explains why the percentage of mature diploid and triploid oysters at the temperate site decreased from April to June. Chl-a concentration was low until March at this site, whereas at the tropical sites it was from 2 to 3 times higher during January and February. Further causes for the differences in growth between tropical and temperate sites are the generally earlier maturation of diploid and triploid oysters in the tropical sites in response to high and increasing temperatures (21-31.5[degrees]C) when Chl-a was decreasing, and the lack of a resting period after first maturing, partially spawning, and new postdevelopment occurring. Just as seen in the present work, an accelerated reproductive effort beginning as early as February and March has been reported for diploid Crassostrea gigas grown in other tropical sites within the Gulf of California (Chavez-Villalba et al. 2007). These authors concluded that there was continuous oocyte development to vitellogenesis without generation of new oocytes when temperatures were above 23[degrees]C; however, sex changes were not analyzed. In a previous study, Chavez-Villalba et al. (2002) found that when oysters were conditioned at increasing temperatures from 16[degrees]C to 25[degrees]C oocytes reached maturity 2 wk sooner than at higher temperatures. Based on the male and female changes observed in the present work (Fig. 9), and the lack of a significant interaction between ploidies and culture months indicating that similar proportions of males and females occurred for diploids and triploids during the culture months, we propose that more than one gametogenic cycle occurred at both tropical sites, Bacorehuis and Ceuta. Because gametogenesis in triploids was retarded when compared with diploids, as has been previously reported to occur for C. gigas (Allen & Downing 1990), in the present study this allows for observing gametogenesis of triploid males and females at the tropical sites in "slow motion" (Fig. 11). In Bacorehuis, maturing triploid males in March were replaced by mature females in April, which were then replaced gradually by males and hermaphrodites until July. During this time, no spawned or undifferentiated oysters were found, but the changes in male and female proportions were significant (see results section Quantitative Analyses of Proportion of Males versus Females). In Ceuta, more maturing triploid males were initially seen in February, which by April were replaced by mature females and by May by maturing and mature males, changes that also were significant (also see results section Quantitative Analyses of Proportion of Males versus Females). Then females increased again up to July. Few triploid oysters were found in the spawn stage, and only in July. This contrasts with what was found at the temperate site Rancho Bueno, where no significant differences in genders were seen for triploid or diploid oysters. For diploids at the temperate site Rancho Bueno, males and females partially spawned from May to July, but by August both males and females had again reached maturity, and spawned by September. Detailed histological observations indicated that spawned organisms in May and June were reabsorbing gametes. At the tropical sites, partial spawns in diploids were recorded from April onward for Ceuta or in May onward for Bacorehuis; however, differently from what occurred in the temperate site, histological observations indicated that the partial spawns occurred in parallel in the same cohort with postdevelopment of new gametes, and no resting period or reabsorbed gonads was seen at the tropical sites as was seen for the temperate site.
Not only was the intensity of maturation different between the temperate site and the tropical sites, but also the maturing sexes were different. Sex- or gender-specific disparities have been reported for the Pacific oyster in response to productivity, with more females found when Chl-a concentrations peaked, and more males when temperatures increase (Baghurst & Mitchell 2002, Santerre etal. 2013). Whether the high productivity seen at Ceuta was a cause for the larger frequency of females is unknown, because OsHV-1 virus may have been a second factor playing a role in inducing sex to change. In France, it has been observed that sex is biased toward more females after a summer mortality event, although this does not necessarily implies that males are more susceptible to summer mortalities (Costil et al. 2005), nor that spat and adult mortalities have the same causal pathogenic agent. Regardless of the cause, for diploid Pacific oysters sex and stage of gonad development have been found to differentially impact on the physiological status through oxygen consumption demands, such that male oysters have twice the consumption rate than females, and postspawned oysters of both sexes have higher oxygen demand than prespawned (Soletchnik et al. 1997, Tran et al. 2008). When considering gonad development at Ceuta and Bacorehuis, part of the growth differences between these tropical sites might be as a result of reproduction and the predominant sex during maturation at each site. At Bacorehuis, a higher percentage of diploid oyster matured as males, whereas at Ceuta they did as females. If male oysters have higher energy expenditure due to higher oxygen demand, this might explain the differences in growth rate observed between this site and Ceuta, especially when considering that SST and Chl-a were similar for these two sites after maturation began, although Chl-a was higher at Ceuta at the onset of maturation. As age (month) and size increased at both tropical sites, with SST increasing and Chl-a decreasing, partial spawns together with environmental conditions might have played a role in the effects of maturing sexes on growth.
In conclusion, we have found that Crassostrea gigas growth and survival are less under extreme temperatures as those present in the Gulf of California than at the temperate site at the Pacific Coast of the Baja California Peninsula in Mexico. The use of triploid oysters will improve total production, although not to the same yields that can be obtained at temperate areas. Selective breeding for temperature tolerance might result in better performing oysters at the tropical sites. Furthermore, monitoring of available food will provide with important information to be used in estimating the index between ACT and ACHL-a to define the best culture sites and to prevent mortalities resulting from negative energy balances associated with high temperatures and low productivity. Models that integrate multiple factor for identifying suitable sites for mollusc aquaculture have been proposed (i.e., Radiarta et al. 2008) based on geographic information systems and remote sensing-derived data, and is expected that they will have an important predictive role also for tropical mollusc aquaculture.
Further culture of this species at the tropical site of Ceuta must consider the critical months associated with the presence of OsHV-1 for spat planting to avoid massive mortalities, as well as the monitoring of OsHV-1 using sentinel oysters before actual winter planting. Because adult oysters are known to be carriers of the OsHV-1 virus and the risk of resistant individuals to be carriers is high (Arzul et al. 2002), movements of oysters from sites known to have this virus to free-virus sites should be avoided. Development of resistant oyster strains is important because this virus is known to be present today in multiple oyster aquaculture areas in northwestern Mexico, including the Baja California Peninsula. A selective breeding program carried in a "clean" area that evaluates replicated families at sites known to have the herpes virus, or under laboratory controlled conditions is highly recommended. Such a program is expected to be successful because genetic variability for resistance to the Os-HV-1 has been reported (Burge et al. 2007, Sauvage et al. 2009, Degremont et al. 2015).
This research was supported by SAGARPA-CONACYT grant 2003/030 awarded to AM Ibarra. We are thankful to the commercial producers (Ostricola Guevara-Ceuta, Cultemar-Rancho Bueno, Ostricola Silvia Ramirez-Bacorehuis) that aided with maintenance of experimental groups at their field installations. Satellite-derived dataseis (1-km resolution) were made available by Dr. Mati Kahru, Scripps Institution of Oceanography. We are also grateful to NASA Ocean Color Processing Group and NASA GSFC for their technical assistance. Eulalia Meza Chavez aided with histology. Dr. Miguel Cordoba-Matson, a native English editor, edited the manuscript.
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ANA M. IBARRA, (1) * ROBERTO ASCENCIO-MICHEL, (1) JOSE L. RAMIREZ, (1) MARLENNE MANZANO-SARABIA (2) AND CARMEN RODRIGUEZ-JARAMILLO (1)
(1) Centro ele Investigaciones Biologicas de! Noroeste S.C., Ave. Instituto Politecnico Nacional No. 195, La Paz BCS 23096, Mexico; (2) Facultad de Ciencias del Mar, Universidad Autonoma de Sinaloa, Paseo Claussen s/n, Col. Los Pinos, Mazatlan, Sinaloa 82000, Mexico
* Corresponding author. E-mail: firstname.lastname@example.org
Caption: Figure 1. Location of culture sites evaluated with diploid and triploid Pacific oysters (Crassostrea gigas) in northwestern Mexico. Circles indicate the location of sites and the coastal areas where temperature and chlorophyll-a were estimated by means of satellite data.
Caption: Figure 2. Percentage of triploid Pacific oysters (Crassostrea gigas) per month and site. Each site legend key indicates mean percentage of triploids per site for the experiment, with different capital letters indicating significant differences between sites (P < 0.05).
Caption: Figure 3. Growth of Pacific oysters (Crassostrea gigas) at the three sites in northwest Mexico: triploids dashed lines and black squares; diploids continuous lines and open circles. Comparison of the mean for each trait during the culture for each site are indicated for diploids (2n) and triploids (3n) where different letters indicate significant differences (P < 0.05) in the ploidy groups and between sites. Up-down lines for each mean represent 95% confidence intervals.
Caption: Figure 4. Survival of Pacific oysters (Crassostrea gigas) at each of the experimental sites: (A) monthly surv ival (untransformed) of diploids (continuous line, white circles) and triploids (dashed line, black squares) grown at the three sites in northwestern Mexico; different letters indicate significant differences (P < 0.05); up-down lines at each mean indicate 95% confidence intervals; mean survivals (back transformed to percentages) during the culture time per site are presented for diploids (2n) and triploids (3n), with different letters indicating significant differences between ploidies and sites (P < 0.05); (B) absolute survivals observed during the culture months for diploids (left) and triploids (right) at each site.
Caption: Figure 5. Gonad maturation stages for diploids and triploids Pacifie oysters (Crassostrea gigas) found from February to September 2007 at each culture site. 0 = inactive, I - early gametogenesis, II = growth, IIIA = partial maturation, IIIB = maturation, IV = spawn, V = reabsorption.
Caption: Figure 6. Mean G V estimated from gametogenic stages for diploids and triploids of Pacific oysters (Crassostrea gigas) at each site and during the culture months. Up-down lines at each mean indicate 95% confidence intervals used to establish significant differences during the culture months and between culture sites.
Caption: Figure 7. Number of oocytes per 0.322 [mm.sup.2] for diploid and triploid Pacific oysters (Crassostrea gigas) at each site from February to September 2007 (left). Analyses of number of oocytes estimated per culture site and monthly for diploids (right-top) and triploids (right-bottom). Up-down lines at each mean indicate 95% confidence intervals. Different letters between months and sites indicate significant differences (P < 0.05) between them. For diploids, horizontal lines below or on top of letters indicates that all those belonged to the same group.
Caption: Figure 8. Gender proportions of diploids and triploids of Pacific oysters (Crassostrea gigas) grown at three culture sites in northwestern Mexico during an 8-mo period (February to September 2007). U = undifferentiated, M = males, H = hermaphrodites, F = females.
Caption: Figure 9. Proportion of male:female Pacific oysters (Crassostrea gigas) (with males modeled) found for diploid and triploid oysters at each of the evaluated sites. Up-down lines at each mean indicate 95% confidence intervals used to establish significant differences during the culture months.
Caption: Figure 10. (A) SST and (B) degrees of anomalies and MEI; (C)Chl-a concentrations and (D) percent Chl-a anomalies registered at the culture sites in northwest Mexico evaluated during 9 mo after a prefattening period of 2 mo at Rancho Bueno.
Caption: Figure 11. Maturation stages per sexes for diploids and triploids Pacific oyster Crassostrea gigas from February to September at each culture site. FI-FV = females in maturation stages I-V, HER = hermaphrodite, 10 = undifferentiated, MI-MV - males in maturation stages I-V.
TABLE I. Gametogenic stages for diploid and triploid Crassostrea gigas. Gametogenic stage Diploids Triploids 0, inactive Germinal epithelium Same than diploids not differentiated. none or few follicles I, early Elongated and Same than diploids gametogenesis isolated follicles within connective tissue; large number of spermatogonia or oogonia; few spermatocytes or previtellogenic oocytes 11, growth In females, large In females, abundant interval of gametic oogonias, cells, from oogonia. previtellogenic previtellogenic, and ooyctes, but few vitellogenic oocytes, early vitellogenic with the last one oocytes. In males, dominating. Some abundant postvitellogenic spermatogonia. oocytes. In males several layers of there are primary spermatogonia, large spermatocytes, few numbers of secondary spermatocytes. spermatocytes and spermatids, and some spermatids, and no spermatozoa spermatozoa IIIA, partial Not observed in Large frequency of maturation diploids developing gametes (vitellogenic oocytes and spermatids), and very few mature gametes (postvitellogenic and spermatozoa) dispersed in the follicle. Both, ooogonias and spermatogonias are still present at the follicle wall IIIB, maturation In females the This stage is only follicles are full present in with postvitellogenic hermaphrodite oocytes of similar triploids. with the size; few remaining numbers of previtellogenic and vitellogenic oocytes vitellogenic oocytes. being less than among In males numerous diploids spermatozoa are present in the follicle lumen IV, spawn Distended follicles, Residual mature with some still gametes retaining numerous (postvitellogenic gametes. In some oocytes or sperm) are cases there are new observed in the spermatogonia and gonadal ducts of oogonia apparent at individuals with low the follicle wall. degree of sterility. Gonoducts with The gonads are not postvitellogenic and emptied completely degenerating oocytes between each spawning, with many immature gametes present V, reabsorption Follicles almost Same than diploids empty, with when this stage was phagocytic cells observed in triploids, with many phagocytes TABLE 2. ACHL-a and ATD, and the ratio of chlorophyll-a available per each thermal degrees accumulated during the summer (June to September), at each site where Crassostrea gigas was evaluated. Rancho Bueno Ceuta Bacorehuis ACHL-a 10,044 4,688 3,056 ATD 2,736 3,635 3,640 ACHL-a/ATD (mg/[m.sup.3]/ 3.67 0.77 0.84 [degrees]C) Mean total weight (g) 56.6 40.2 29.0 Diploids 53.5 33.2 22.7 Triploids 59.7 47.3 35.3 Mean total weight for diploids and triploids at each site is shown to follow a direct relationship with ACHL-a/ATD.
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|Author:||Ibarra, Ana M.; Ascencio-Michel, Roberto; Ramirez, Jose L.; Manzano-Sarabia, Marlenne; Rodriguez-Jar|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2017|
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