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CARBOHYDRATE-RICH DIETS MEET ENERGY DEMANDS OF GAMETOGENESIS IN HATCHERY-CONDITIONED MUSSELS (MODIOLUS CAPAX) AT INCREASING TEMPERATURES.

INTRODUCTION

In marine bivalves, gonad conditioning is controlled by many biotic and abiotic factors, particularly temperature and diet (Utting & Millican 1997. Fabioux et al. 2005. Helm et al. 2006). Temperature acts as a key regulator of reproductive events, such as sexual differentiation, growth, maturation, and release of gametes, as well as nutrient recycling when environmental conditions are adverse (Barber & Blake 2006, Thompson & MacDonald 2006). Studies have reported that many tropical, subtropical, and temperate bivalves breed in summer in response to increasing temperature regimes that trigger spawning (Chavez-Villalba et al. 2002. Delgado & Perez-Camacho 2007. Rodriguez-Jaramillo et al. 2008. Saucedo & Southgate 2008, Angel-Dapa et al. 2010, Castillo-Duran et al. 201.3). In contrast, other species reproduce in winter when water temperature decreases (Velez & Epifanio 1981, Fearman & Moltschaniwskyj 2010). Each species has a unique temperature threshold that strongly influences gametogenesis. particularly oocyte growth and proliferation (Chavez-Villalba et al. 2002. Rodriguez-Jaramillo et al. 2008. Castillo-Duran ct al. 2013) and the formation of yolk components (Fabioux et al. 2005. Fearman & Moltschaniwskyj 2010). Changes in water temperature also allow activating, accelerating, and stopping gametogenesis at the laboratory depending on nutritional conditions.

Historically, bivalve hatcheries have relied on the culture of microalgae as the main source of food (Helm et al. 2006, Gonzales-Araya et al. 2012). Despite the advantages of using more than one species of microalgae to irnprove nutrition, their production is expensive and may account for up to 50% of hatchery operating costs (Coutteau & Sorgeloos 1992, Knauer & Southgate 1999). Consequently, there has been a growing interest in diets that substitute the proportion of live microalgae to reduce operation costs at the hatchery. Among a wide variety of products investigated, carbohydrate-rich cereal flours are particularly important, given their low cost, ease of preparation, ability to be metabolized, and promising results in covering tnost nutritional requirements of conditioned broodstock (Mazon-Suastegui 1988, Knauer & Southgate 1999) and grown-out juveniles (Perez-Camacho et al. 1998, Fernandez-Reiriz et al. 1999. Knauer & Southgate 1999, Albentosa et al. 2002. Mazon-Suastegui et al. 2008. Mazon-Suastegui et al. 2009). Carbohydrates provide an immediate energy source to fuel and sustain gametogenesis (Darriba et al. 2005. Barber & Blake 2006. Mladineo et al. 2007. Gonzales-Araya et al. 2012); they can also be converted into neutral lipids to be either used for catabolic processes or stored as energy reserves in somatic tissues (Gabbott 1975, Saucedo et al. 2001, Martinez-Pita et al. 2012). Previous reports indicated an increase in spawning frequency of the bivalves Modiolus capax (Conrad, 1837) and Pteria sterna (Gould, 1851), conditioned with cornstarch and rice flour as supplements to live microalgae (Mazon-Suastegui 1988).

Bahia de La Paz, in Mexico, is a transition zone between temperate and tropical latitudes, which makes farming and production of some commercial bivalve species difficult (Sicard ct al. 2006). The mussel Modiolus capax offers the potential to develop a sustainable aquaculture industry based on the use of edible native resources. This species is distributed from Santa Cruz, CA to Paita. Peru, but it is more abundant in the Gulf of California where it inhabits areas of abundant particulate organic matter between the intertidal and subtidal zones (Farfan et al. 2007). At Bahia de La Paz, reproduction of wild M. cupax starts at 21[degrees]C and peaks at 24[degrees]C-26[degrees]C (Garcia-Corona 2014). Given the lack of sufficient scientific knowledge relative to the reproductive physiology of the species for aqua-culture practices, this study expands on the criteria for conditioning M. capax broodstock with different diet--temperature combinations at the hatchery. The goal was not only to define strategies for ensuring maturation of the species outside of the main breeding season but also to understand the mechanisms of using energy reserves by interactions between diet--temperature in mussels fed carbohydrate-rich diets during conditioning periods larger than the 30 days usually recommended for tropical bivalves.

MATERIAL AND METHODS

Origin of Mussels and Experimental Design

A total of 1,080 wild Modiolus capax were collected at Bahia de La Paz, Gulf of California, Mexico (24[degrees]13'38.9" N, 110[degrees]18'44.9" W), during February 2013. All mussels were transported to the laboratory, cleaned of fouling, and acclimated for 2 wk at 18.5[degrees]C in a 2,500-1 container providing a 1:1 blend of Tisochrysis lutea (Bendif et al. 2013) and Chaetoceros calcitrans (Takano, 1968) at 150 x [10.sup.3] cells/ml using a continuous flow-through system.

Once the acclimation period finished, all mussels were conditioned in 18 maturation units with 80-1 capacity. Each unit was used in duplicate and held 60 adult mussels. Maturation units were adapted with an open flow-through system that provided 1-[micro]m filtered and UV-sterilized seawater with food. They were set at 37-38 g/l salinity.

Mussels were conditioned with three natural and artificially enriched diets with different proximal composition, particularly in relation to carbohydrate content (Table 1); (1) Micro 100 diet was composed of a 1:1 blend of the microalgae Tisochrysis lutea and Chaetoceros cakitrans given daily at 4% dry weight of soft tissues; (2) MicroW diet was composed of 4% dry weight of live microalgae (Micro 100) and 3% dry weight of wheat flour (Espuma de Chapala. Zapopan, Mexico); and (3) MicroC diet was composed of 4% dry weight of live microalgae (Micro100) and 3% dry weight of cornstarch (Maizena Tutitlan, Mexico). Each diet was given daily and evaluated at three conditioning temperatures (22, 24, and 26[degrees]C), yielding nine different diet--temperature treatments. The conditioning trial lasted 4 months.

Three samplings were conducted during the conditioning period, at the start (day 0), halfway through (day 60), and at the end (day 120). At each sampling, 14 mussels were randomly sampled from each duplicated maturation unit to extract the gonad and somatic tissues (digestive gland, muscle, and mantle). One part of the gonad was preserved in Davidson solution for 48 h for histological and histochemical analyses; the other part of the gonad and remaining tissues were maintained at -80[degrees]C for biochemical analyses. Tissues (digestive gland, muscle, and mantle) from all 28 mussels per treatment were analyzed separately.

Qualitatire Histological Analysis

Preserved gonad samples from each treatment and sampling were dehydrated, embedded in Paraplast XT (SPI Supplies, West Chester, PA), cut at 4-[micro]m sections, and stained with hematoxylin-eosin (Kim et al. 2006).

Male and female sexual developmental stages were classified as inactive, early- or late-developed, ripe, partially spawned, and spent according to Rodriguez-Jaramillo et al. (2008).

Quantitative Histological and Histochemical Analyses

The protocol of Rodriguez-Jaramillo et al. (2008) was used to quantify the gonad coverage area (GCA) of samples collected at days 0, 60, and 120 for each treatment. Three randomly selected digital images taken at 4x were processed with Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD). The information was used to determine the GCA with the formula

GCA % = [[area occupied by the gonad]/[total area ot image]] x 100

Female gonad samples were also stained with Sudan Black B to identify oocyte neutral lipid content (Sheehan & Hrapchak 1973). An oocyte lipid index (LIo) was determined per treatment and sampling by taking three randomly digital images at 20x and processing them with Image Pro Plus. Thereafter, 30 late-developing oocytes were randomly selected to quantify the area occupied by black hues (lipids) inside the oocytes (Rodriguez-Jaramillo et al. 2008, Angel-Dapa et al. 2010). With these data, the LIo was determined with the formula

LIo % = [[area occupied by lipid droplets inside oocyte]/[total area of oocyte]] X 100

Biochemical Analysis

Preserved tissue samples collected from each treatment and sampling were weighed to 0.001 g, lyophilized, rehydrated in 1-ml cold saline solution, and homogenized to obtain crude extracts, which were then used to determine (1) total proteins according to Smith et al, (1985) using a BCA reagent (#B9643; Sigma-Aldrich) and bovine serum albumin (#9048-46-8; Sigma-Aldrich) as standard; absorbance was read at 562 nm; (2) total carbohydrates according to Roe et al. (1961), using a reagent blank and dextrose solution as standard (#3803; Vedco); absorbance was read at 630 nm; and (3) total lipids, following a modified version of Barnes and Blackstock (1973) using a microplate with 20 [micro]l supernatant extract previously digested with sulfuric acid and 200 [micro]l lipid reactive solution; absorbance was recorded at 540 nm.

Statistical Analysis

For each duplicated treatment, group normality was initially analyzed with the Kolmogorov--Smirnov test and then confirmed with the Levene test for homogeneity of variances. Two-way analysis of variance (n = 2 duplicated tanks, each with 14 mussels) was run to assess significant differences in the histological and biochemical indicators of tissues as a function of diet and temperature. The analyses were run separately halfway through (day 60) and at the end (day 120) of the trials. As needed, post hoc multiple range mean comparisons with Tukey's test (HSD) were included. The level of significance was set at P < 0.05 for all analyses.

RESULTS

Histological Analysis

At the beginning, female gonad development was minimal in all treatments with high frequency of inactive (50%) and spent (25%) mussels (Fig. 1). At day 60. most females were partially spawned, particularly those fed MicroC and MicroW, and kept at 26[degrees]C (73%), compared with Micro 100 at 22[degrees]C (21 %). At day 120, most gonads experienced a rematuration process in all treatments, but Micro 100 at 26[degrees]C had the highest frequency of partially spawned gonads (78%), compared with Micro100 at 22[degrees]C (10%).

In males, higher numbers of spawned gonads occurred at day 60 in mussels fed MicroC and MicroW at 26[degrees]C (86%) and lower numbers with Micro 100 (33%) and MicroW (9%) at 22[degrees]C (Fig. 1). Males also rematured at day 120 and increased the frequency of late-developing gonads in all treatments, particularly with MicroW at 26[degrees]C (82%). The lowest frequency of developing gonads occurred in males fed Micro 100 at 26[degrees]C (56%), 24[degrees]C (50%), and 22[degrees]C (50%).

Quantitative Histological and Histochemical Analyses

In all treatments, mean GCA increased halfway through the trial (females 26%-38%, males 22%-50%), compared with the initial values (5% in females, 13% in males) (Fig. 2). In females, diet significantly affected the GCA (P = 0.001) al day 60; values were higher in mussels fed MicroW at 22[degrees] (33%)) than MicroC at 26[degrees]C (26%). At day 120, temperature (P = 0.001) and the combination of both factors (P = 0.007) significantly increased the values of GCA; particularly in females fed Micro 100 at 24[degrees]C (44%). compared with almost all treatments with the exception of MicroC (33%), MicroW (38%), and Micro 100 (33%) at 22[degrees]C.

In males, differences were only significant at day 60, where temperature (P = 0.006) and diet (P = 0.0002) exerted the greatest impact on the GCA; particularly in males fed MicroW at 22[degrees]C (50%) and Micro 100 at 22[degrees]C (44%), 24[degrees]C (46%), and 26[degrees]C (44%), compared with MicroC at 26[degrees]C (22%) (Fig. 2).

In females, the LIo was significantly affected by temperature (P = 0.0), diet (P = 0.02), and the interaction of both factors (P = 0.001); higher LIo occurred in mussels fed at day 60 with MicroC at 26[degrees]C (22%), compared with almost all treatments (16%-18%) with the exception of Micro 100 at 26[degrees] C (19%). At the end, differences in the LIo were not significant (Fig. 3).

Biochemical Analysis

Total Carbohydrates

In the gonad, temperature (P = 0.001), diet (P = 0.005), and the combination of factors (P = 0.03) significantly affected the carbohydrate content of mussels at day 60. Higher values occurred in mussels fed MicroC at 24[degrees]C (128 mg/g) compared with Micro 100 (54 mg/g), MicroW (66 mg/g), MicroC at 26[degrees]C (59 mg/g), and Micro 100 at 24[degrees]C (47 mg/g) (Fig. 4). At day 120, temperature (P = 0.000001), diet (P = 0.001), and again the combination of factors (P = 0.02) significantly increased the carbohydrate content of mussels fed MicroC (181 mg/g) at 22[degrees]C, compared with all other treatments (50-97 mg/g) with the exception of MicroW at 22[degrees]C (122 mg/g).

In the digestive gland, temperature (P = 0.003), diet (P = 0.003), and the combination of factors (P = 0.01) significantly increased the carbohydrate content of mussels at day 60, particularly those fed MicroC at 22[degrees]C (57 mg/g), compared with MicroC at 26[degrees]C (34 mg/g). Micro 100 at all temperatures (36-40 mg/g), and MicroW at 26[degrees]C (40 mg/g) (Fig. 4). At day 120, temperature (P = 0.00009) and diet (P = 0.01) significantly increased the carbohydrate content of mussels conditioned at 22[degrees]C with all diets (109-126 mg/g); lowest carbohydrate values occurred with the MicroC treatment at 26[degrees]C (68 mg/g).

The carbohydrate content in mantle tissue at day 60 was significantly affected by temperature (P = 0.009) and temperature/diet (P = 0.01). Values were higher in mussels from the MicroW treatment at 24[degrees]C (50 mg/g) and lower with MicroW at 22[degrees]C (30 mg/g) (Fig. 4). At day 120, temperature (P = 0.005), diet (P = 0.04), and both factors combined (P = 0.002) significantly affected carbohydrate content; higher values occurred in the MicroC treatment at 22[degrees]C (107 mg/g) and lower values in most of the treatments (46-61 mg/g) (Fig. 4).

In muscle, the carbohydrate content of mussels was significantly influenced at day 60 by temperature (P = 0.000001), diet (P = 0.0), and the combination of factors (P = 0.000004). Higher values occurred with MicroC at 22[degrees]C (52 mg/g) compared with all treatments (12-36 mg/g) with the exception of MicroW at 22[degrees]C (38 mg/g) (Fig. 4). At the end, temperature (P = 0.0), diet (P = 0.00002), and both factors combined (P = 0.0) had a significant effect in carbohydrate content with higher values with MicroC and MicroW at 22[degrees]C (113 mg/g, 86 mg/g) and lower values with all other treatments (27-52 mg/g).

Total Lipids

At day 60, the gonad lipid content was significantly affected by temperature (P = 0.02). Higher values occurred with the MicroC treatment at 26[degrees]C (130 mg/g), compared with Micro 100 at 24[degrees]C (72 mg/g). At the end, no significant differences were found in gonad lipid content (Fig. 5).

The lipid content of the digestive gland at day 60 was significantly affected by temperature (P = 0.02), diet (P = 0.002), and the combination of factors (P = 0.02); higher values occurred in mussels from the MicroW diet at 22[degrees]C (56 mg/g) in contrast to the Micro 100 at 26[degrees]C (35 mg/g) and MicroC diet at 24[degrees]C (31 mg/g) (Fig. 5). At day 120, temperature (P = 0.0), diet (P = 0.0), and both factors combined (P = 0.008) significantly increased the lipid content of mussels fed all diets at 22[degrees]C (35-47 mg/g) and Micro 100 at 24[degrees]C (35 mg/g) compared with MicroC diet at 26[degrees]C(12mg/g).

In mantle tissue, significant differences in the lipid content occurred only halfway through the trial by effect of temperature (P = 0.003) and diet (P = 0.002); mussels fed MicroC at 26[degrees]C (47 mg/g) had higher values compared with those fed Micro 100 at 22[degrees]C (26 mg/g) (Fig. 5).

Lipid reserves in the muscle significantly increased at day 60 by effect of temperature diet (P = 0.0) when using the MicroC diet at 24[degrees]C (15 mg/g), 22[degrees]C (13 mg/g), and 26[degrees]C (14 mg/g) in contrast to the Micro 100 diet at 22[degrees]C (6 mg/g). At day 120, significant differences in the lipid content only occurred by effect of temperature/diet (P = 0.002); Micro 100 at 26[degrees]C had higher values (28 mg/g) compared with MicroW at 22[degrees]C (19 mg/g) (Fig. 5).

Total Proteins

At day 60, the protein composition of the gonad was significantly affected by temperature (P = 0.02) and temperature/diet (P = 0.000006); higher values occurred in mussels fed MicroC diet at 24[degrees]C (425 mg/g) compared with the MicroC diet at 22[degrees]C (211 mg/g). At the end, protein composition of the gonad was not significantly affected (Fig. 6).

In the digestive gland, diet (P = 0.008) and temperature/diet (P = 0.04) significantly increased the protein content of mussels fed at day 60 with MicroW at 26[degrees]C (363 mg/g) and Micro 100 at 22[degrees]C (356 mg/g) and 26[degrees]C (350 mg/g); in contrast, lower values occurred with MicroC at 26[degrees]C (259 mg/g). At day 120, the protein content was significantly influenced by temperature (P = 0.005) and temperature/diet (P = 0.02); higher values occurred with the MicroC diet (468 mg/g) at 22[degrees]C and MicroW (464 mg/g), whereas lower values occurred with MicroC at 24[degrees]C (380 mg/g) and 26[degrees]C (391 mg/g) (Fig. 6). In mantle tissue, no significant differences in the protein content were found.

In muscle, protein values significantly increased in response to temperature (P = 0.0002), particularly with MicroW (409 mg/g) at 22[degrees]C and decreased with MicroW (256 mg/g) and Micro 100 (260 mg/g) at 24[degrees]C. Significantly higher protein values occurred at day 120 by effect of temperature (P = 0.00007) and temperature/diet (P = 0.003) with the MicroC diet at 22[degrees]C (615 mg/g); lower values occurred with the MicroC diet at 26[degrees]C (459 mg/g).

DISCUSSION

Compared with the natural diet exclusively prepared with live microalgae, the two artificially enriched diets given at 7% total dry weight (4% dry weight of live microalgae and 3% cereal flours) covered most nutritional needs of hatchery-conditioned Modiolus capax, particularly at higher temperatures (26[degrees]C). Several authors have reported that cereal flours can be used as partial substitutes to live microalgae to improve growth and survival of juvenile Ruditapes decussatus (Linnaeus, 1758), Ruditapes philippinarum (Adams & Reeve, 1850), Nodipecten subnodosus (Sowerby, 1835), and Crassostrea corteziensis (Hertlein, 1951) (Perez-Camacho et al. 1998, Fernandez-Reiriz et al. 1999, Albentosa et al. 2002, Mazon-Suastegui et al. 2008, 2009). Cereal flours have also been successfully used to enhance gonad conditioning in the bivalves M. capax, Pinctada mazadanica (Hanley, 1856), and Pteria sterna (Mazon-Suastegui 1988).

Marine bivalves display different strategies to regulate growth, reproduction, and overall metabolism (Gonzales-Araya et al. 2012, Martinez-Pita et al. 2012, Irisarri et al. 2015), which are closely related to variations in temperature and food availability. The Manila clam Ruditapes philipinarum broodstock require greater demands of the microalgae Isochrysis galbana when conditioned at 22[degrees]C rather than at 14[degrees]C (Delgado & Perez-Camacho 2007); this represents greater energy consumption at increasing temperatures as gametogenesis proceeds. In the black-lip pearl oyster Pinctada margaritifera (Linnaeus. 1758), greater maturation occurs in adults fed higher food ration (40 x [10.sup.3] cells/ml) and higher temperature (28[degrees]C) compared with: (1) low food ration (10 x [10.sup.3] cells/ml) and low temperature (24[degrees]C); (2) low food ration (10 x [10.sup.3] cells/ml) and a high temperature (28[degrees]C); and (3) high food ration (40 x [10.sup.3] cells/ml) and low temperature (24[degrees]C) (Teaniniuraitemoana et al. 2016). Findings in this study agree with these authors and confirm that energy-rich diets are needed when energy demands increase because of gamete development and increments in metabolic rates as a result of rising temperatures (MacDonald et al. 2006). If food availability is limiting or energy reserves are low, some species are not able to efficiently sustain gametogenesis.

Results from this study also suggest that the thermal threshold of Modiolus capax lays 26[degrees]C and occurred around day 60. Mazon-Suastegui (1987) reported 100% maturation in M. capax and spawning success after 50-day conditioning using temperatures from 23[degrees]C to 24[degrees]C.

It has been observed that most male and female gonads experienced a rematuration process at day 60 and further at day 120, indicating that overall conditions at the hatchery were still suitable for reproduction, particularly with the MicroC diet at 26[degrees]C. Warmer temperatures (25[degrees]C-29[degrees]C) have proven to increase gametogenesis in many tropical and subtropical bivalves, such as the mother-of-pearl Pinctada mazatlanica (Saucedo & Monteforte 1997), Cortez oyster Crassostrea corteziensis (Rodriguez-Jaramillo et al. 2008), and smooth clam Chioe fluctifraga (Sowerby, 1853) (Castillo-Duran et al. 2013). In contrast, warmer temperatures reduce gametogenesis in other subtropical and temperate bivalves, such as the winged pearl oyster Pteria sterna (Saucedo & Monteforte 1997), king scallop Pecten maximus (Linnaeus, 1758) (Saout et al. 1999), blue mussel Mytilus galloprovincialis (Lamarck, 1819) (Fearman & Moltschaniwskyj 2010), and pen shell Atrina maura (Sowerby, 1835) (Angel-Dapa et al. 2010). In this study, low temperatures (22[degrees]C) favored the storage of energy reserves in somatic tissues, particularly carbohydrates. This trend gives temperature, and increasing temperatures in particular, greater importance for promoting metabolic reserve intake (carbohydrates) needed to fuel reproduction in this subtropical bivalve species. In the gonad, a direct relationship between high lipid and low carbohydrate contents was determined, which is mainly attributed to lipid storage in unreleased mature oocytes, particularly with the MicroC diet at 26[degrees]C that was also consistent with the highest LIo values.

In bivalve molluscs, particularly females, an increase in neutral lipid content in the gonad has been related to the occurrence of late-developing and ripe oocytes (Angel-Dapa et al. 2010, Gonzales-Araya et al. 2012). The protein content attributed to yolk formation can also increase during oocyte growth (Arcos-Ortega et al. 2015), as opposed to protein increase in males that appears to play a structural role (Darriba et al. 2005). In bivalves, low GCA values are also attributed to spawning events in females where lipid and protein reserves in the gonad are depleted. In the Cortez oyster Crassostreu corteziensis, for example, a decrease in the lipid content is consistent with a low GCA because of spawning events (Rodriguez-Jaramillo et al. 2008). In the blue mussel Mytilus edulis (Linnaeus, 1758), the main spawning period is associated with decreasing gonad protein contents (Fernandez et al. 2015). In our study, higher lipid and protein concentrations in female gonads were mostly related to peak frequencies of partially spawned mussels and low numbers of unreleased ripe oocytes. Thus, it is likely that lipid or protein concentrations in the gonad are closely related to the timing and intensity of spawning events. This result supports the occurrence of a low-intensity partial spawning, given the lack of significant differences in the GCA between most of the treatments. In the gonad, high GCA values have not only been associated with the occurrence of late-developing and ripe oocytes but also with partially spawned gonads because of unreleased ripe oocytes (Rodriguez-Jaramillo et al. 2008).

The peaks in the lipid composition of mantle and muscle tissues occurred in mussels fed only the MicroC diet. Consistently, a greater gain of lipid reserves in juveniles of the Manila clam Ruditapes philippinarum was observed when the micro-algal-based diet was supplemented with wheat germ flour (Albentosa et al. 2002). This result confirms again the role of carbohydrate-rich diets as synthesis facilitators of lipid reserves within body tissues, and supports previous findings that carbohydrates can be metabolized, via lipogenesis, into neutral lipid components that are essential for gonad (yolk) development (Gabbott 1975, Racotta et al. 2003, Pogoda et al. 2013). Carbohydrates have been reported to promote lipids accumulation in body tissues as gametogenesis proceeds (Darriba et al. 2005, Barber & Blake 2006).

Protein content in mantle and muscle tissues did not show any relationship with gonad development and ripening in Modiolus capax. Similar scenarios showing lack of correlation between protein content in the gonad and gametogenesis have been reported in the flat Japanese oysters Ostrea edulis (Linnaeus, 1758) and Crassostreu gigas (Thunberg, 1793) (Pogoda et al. 2013). In M. capax, it is likely that mantle and muscle proteins were used as structural components rather than as energy substrates for gametogenesis. At the end of the trials, the digestive gland had a different role, as the greatest depletion of lipid reserves coincided with the highest frequencies of late-development and ripening stages. Similarly, other bivalve species use protein and lipid reserves from the digestive gland during the ripening peaks, such as the mother-of-pearl Pinctada mazatlauica (Sauccdo et al. 2001). bay scallop Argopecten irradians (Lamarck, 1819) (Barber & Blake 2006), and Mediterranean mussel Mytilus galloprovincialis (Irisarri et al. 2015). This pattern confirms the role of the digestive gland as a short-term repository of lipid reserves used during the main breeding season (Saucedo & Southgate 2008). In contrast, protein content in the digestive gland appears to be mostly associated with the protein composition of the diets, particularly MicroW and Micro 100.

In summary, the energy demand for gonad development in conditioned Modiolus capax appears to be more dependent on temperature than on diet. One effective way of providing this energy and covering most nutritional needs of gametogenesis was through low-cost, carbohydrate-rich products such as cereal flours, especially cornstarch. The combination of high temperature and a diet prepared from cornstarch favored gonad development and reduced conditioning timing, particularly outside of the natural breeding season. Both factors jointly also allowed a rematuration process of broodstock by day 60 and further by day 120, which has important implications for activities of the species spat production. If needed, though, mussels able to spawn for hatchery production of spat may be conditioned in shorter periods of time (e.g., 30 days). Transcriptomic analyses are recommended to broaden the understanding of metabolic and reproductive patterns when carbohydrate-rich diets are used for hatchery conditioning of this and other bivalve species of commercial value.

ACKNOWLEDGMENTS

This study was funded by SEP-CONACYT Basic Science grant No. 258282 "Experimental evaluation of homeopathy and new probiotics in the cultivation of moUusks, crustaceans, and fish of commercial interest" under the academic responsibility of JMMS. The authors are grateful to Acuacultura Robles and technical staff at CIBNOR: Delfino Barajas, Pablo Ormart, Julian Garzon, Eulalia Meza, Roberto Herrera, and Diana Fischer for editorial services in English. JALC and JMMS designed the study and carried out zootechnical work; JALC and CRJ ran the histological analyses, JALC, JMMS, and PES wrote the manuscript and made suggestions to improve it. JALC and JLGC are recipients of a master's fellowship from CONACYT (#301921 and #590925). All authors approved the final version of this manuscript.

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JESUS ANTONIO LOPEZ-CARVALLO, PEDRO ENRIQUE SAUCEDO, CARMEN RODRIGUEZ-JARAMILLO, ANGEL ISIDRO CAMPA-CORDOVA, JOSE LUIS GARCIA-CORONA AND JOSE MANUEL MAZON-SUASTEGUI (*)

Centro de Investigaciones Biologicas del Noroeste S.C. (CIBNOR), Calk I.P.N. 195, Col. Playa Palo de Santa Rita Sur. La Paz, B.C.S. 23096, Mexico

(*) Corresponding author. E-mail: jmazon04@cibnor.mx

DOI: 10.2983/035.036.0314
TABLE 1.
Biochemical composition expressed as percentage dry weight biomass of
the different diet constituents for conditioning Modiolus capax
broodstock at different temperature--diet treatments.

                          Carbohydrates    Proteins    Lipids
                          (%)              (%)         (%)

Tisochrysis lutea         12.3             32.1        22.6
Chaetoceros calcitrans     6.9             25.6        34.3
Cornstarch (Maizena)      94                0           0
Wheat flour               65-70             8-13        0.8-1.5
(Espuma dc Chapala)

                          Reference

Tisochrysis lutea         Mazon-Suastegui et al. (2008)
Chaetoceros calcitrans    Mazon-Suastegui et al. (2008)
Cornstarch (Maizena)      Product label
Wheat flour               Product label
(Espuma dc Chapala)
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Author:Lopez-Carvallo, Jesus Antonio; Saucedo, Pedro Enrique; Rodriguez-Jaramillo, Carmen; Campa-Cordova, A
Publication:Journal of Shellfish Research
Article Type:Report
Date:Dec 1, 2017
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