Effects of temperature and salinity fluctuation on the oxygen consumption, ammonium excretion and osmoregulation of the blue shrimp Litopenaeus stylirostris (stimpson).

ABSTRACT This study presents data on the effect of temperature and fluctuations in salinity on the oxygen consumption, nitrogen excretion, and osmoregulation of Litopenaeus stylirostris juveniles to determine the combinations, so these can be used to optimize the culture conditions. The oxygen consumption rate of juveniles acclimated to 23[degrees]C, 28[degrees]C, and 33[degrees]C was measured. Fluctuating salinity levels were applied to these organisms in a sequence of 40[per thousand], 32[per thousand], 25[per thousand], 16[per thousand], 25[per thousand], 32[per thousand], and back to 40[per thousand]. The results indicate that metabolic rate of blue shrimp exposed to salinity fluctuation was not affected significantly. When the salinity was reduced from 40[per thousand] to 25[per thousand] the ammonium excretion of the shrimp was increased, in an 16[per thousand] salinity in all temperatures the ammonium production increased in average of 220%, when the salinity was increased from 16[per thousand] to 40[per thousand] the ammonium excretion of the shrimp was reduced. Hemolymph osmolarity of the organisms was hyposmotic as the salinity was reduced form 40[per thousand] to 33[per thousand], hyperosmotic during the 25[per thousand] to 16[per thousand] interval and hyposmotic as the salinity increased from 25[per thousand] to 40[per thousand]. The isosmotic points had an interval of 676.8-700.7 mmol [Kg.sup.-1] (23.7[per thousand] to 24.6[per thousand]). The excretion rate of ammonium of the shrimp acclimated at temperatures of 23[degrees]C, 28[degrees]C, and 33[degrees]C was related with the pattern of osmoregulation, when the shrimp were hyper-regulators, ammonium increased and diminished when they were hypo-regulators. To optimize the culture of L. stylirostris in controlled conditions, we propose that they be cultivated in salinities of 25[per thousand] and temperatures of 28[degrees]C because these are considered the optimal conditions, also this environment is free of stress, and for this reason the growth of shrimp is increased.

KEY WORDS: shrimp, temperature, salinity fluctuation, oxygen consumption, ammonium excretion, osmoregulation, litopenaeus stylirostris

INTRODUCTION

Temperature and salinity of the water are among the most important environmental factors that affect the life of the penaeids shrimp (Chen & Nan 1995). The adaptative capacity of the penaeids is specific and determined by a number of factors, that have caused shrimp species to be distributed differently in the marine-estuarine gradient (Claybrook 1983). In recent years the studies on the physiology of the penaeids have increased, due to interest in the aquacultural practice with these organisms; through these investigations optimal conditions for their cultivation can be delimited (Brito et al. 2000).

One of the physiologic responses that can be correlated with the changes of the environmental parameters is the oxygen consumption rate, because it is related with the metabolic work and energy flow that the organisms must destine for the mechanisms of homeostatic control (Salvato et al. 2001). Therefore the measurement of the oxygen consumption in the aquatic animals is a valid method to evaluate the effect of environmental factors such as temperature, salinity, exposition to pollutants, light intensity, and the dissolved oxygen, because it allows the determination of the energetic cost associated with the physiologic stress that these combinations impose on the organisms (Kinne 1970, Villarreal & Rivera 1993, Brown & Terwilliger 1999, Lemos et al, 2001, Altinok & Grizzle 2003).

The ammonium excretion rate has been used to evaluate the effect of various environmental factors on the physiology of the crustaceans (Jiang et al. 2000). The ammonium represents 40% to 90% of the total nitrogen excreted by the crustaceans and is continuously released through the branchial epithelium (Hartenstein 1970, Kinne 1977, Regnault 1987). A decrease of the environmental osmotic concentration gives a result of a decrease on the amino acids in the tissues and an increase in the ammonium excretion on the organisms (Lange 1972). Significant effects of temperature and salinity on ammonium excretion have been quantified in some species of crustaceans of commercial importance as in Macrobrachium rosenbergii, De Man (Nelson et al. 1977, Stern et al. 1984, Diaz Herrera & Buckle Ramirez 1993); Fenneropenaeus indicus, Milne-Edwards (Gerhardt 1980); F. chinensis, Osbeck (Chen& Lin 1992, 1995); Marsupenaeusjaponicus, Bate (Chen & Lai 1993); Penaeus monodon, Fabricius (Chen et al. 1994); Farfantepenaeus aztecus, Ives (Hernandez & Diaz 1995); and Litopenaeus vannamei, Boone (Jiang et al. 2000, Diaz et al. 2001).

The environmental variation of the salinity disorganizes the osmotic balance in the penaeids, provoking the readjustment of the osmotic concentration of the hemolymph on these organisms. The ability for osmoregulation in decapods was evaluated by Charmantier et al. (1988, 1989) through the osmoregulatory capacity (OC), defined as the difference between the osmolality of the hemolymph and the external medium osmolality for a given salinity. The OC is a useful tool to evaluate the physiologic condition of the shrimps as well as to detect the sublethal effects of the stress in the culture systems (Lignot et al. 2000).

During the last 4 decades, the osmoregulatory physiology of different species of penaeids has been studied to evaluate the optimal conditions for their culture as in: Farfantepenaeus aztecus (Bishop et al 1980, Castille & Lawrence 1981, Howe et al. 1982, Hernandez & Diaz 1995); in Litopenaeus setiferus, Linnaeus L. stylirostris Stimpson and L. vannamei (Williams 1960, Castille & Lawrence 1981, Rodriguez 1981); in P. esculentus, Haswell; Fenneropenaeus neropenaeus merguiensis, De Man; and Metapenaeus bennettae Racek and Dall (Dall 1981); in F. indicus (Parado-Estepa et al. 1987); in P. monodon (Cawthorne et al. 1983, Ferraris et al. 1986); in Marsupenaeus japonicus (Charmantier-Daures et al. 1989); in Fenneropenaeus chinensis (Chen & Lin 1994, Chen et al. 1995, Chen & Lin 1998); and P. semisulcatus, De Harm (Clark 1992). However, the osmoregulation processes in these organisms were studied in regimes of constant salinity, whereas in the lagoon-estuarine systems in which these crustaceans normally inhabit, the factors are unstable and present marked variations in temperature, salinity, and dissolved oxygen throughout the year (Salvato et al. 2001).

The blue shrimp Litopenaeus stylirostris is distributed from Punta Abreojos, Baja California down to Tumbes, Peru. This species is commercially cultivated in Ecuador and Mexico, inhabits lagoons, estuaries and bays, places with a varying hydrography through the year, affecting organisms that inhabit in these aquatic systems, due to well defined periods of rain and drought (low water) (Rodriguez 1981, Aragon 2000). The aquacultural potential that the blue shrimp L. stylirostris has in Mexico makes it necessary to know the effect of the variations of temperature and salinity on the oxygen consumption, nitrogen excretion and the osmoregulatory capacity, to determine the adequate environmental conditions to optimize its cultivation.

MATERIALS AND METHODS

A number of Litopenaeus stylirostris juveniles (n = 1500) was acquired in the production farm "Camaron Dorado S.A." located in Huatabampo, Sonora, Mexico. The organisms were placed on three reservoirs of 2000 1 in the laboratory with continuous flow of seawater and constant aeration. They were maintained at a temperature of 28[degrees]C [+ or -] 1[degrees]C and a salinity of 35[per thousand] during a week. The shrimps (n = 900) with a wet weight of 2.8 to 5.0 g were transferred to three circular reservoirs of 5001 for acclimation during 30 days to the temperatures of 23[degrees]C, 28[degrees]C, and 33[degrees]C + 1 [degrees]C and 35[per thousand] salinity. The temperatures of 23[degrees]C and 33[degrees]C were obtained by decreasing or increasing the temperature of the water of the reservoirs at a rate of 2[degrees]C by day. During this phase the shrimps were fed twice a day with Rangen diet for shrimp with 35% of protein. Once the period of thermal acclimation was over, salinity was increased to 40[per thousand] [+ or -] 1[per thousand] and remained in these conditions for 6 days, sufficient time for the shrimps to stabilize their internal medium (Bishop et al. 1980, Rosas et al. 2001).

Of each experimental condition, 20 organisms in intermoult stage were selected. The stage of the moult cycle was identified according to the procedure described by Huner and Colvin (1979), 12 h before initiating the measurements they were introduced individually in a semi-opened respirometer as described by Diaz et al. (1989) which consists of 21 respirometric chambers of 1 l. These organisms, as well as the remainders (n = 140) that stayed in the reservoirs, were exposed during 4 h in a cycle of 28 h simulating a natural variation similar to an estuarine system, to the effect of the salinity fluctuation as the salinities were changed in sequence to 40[per thousand], 32[per thousand], 25[per thousand], 16[per thousand], 25[per thousand], 32[per thousand], and 40[per thousand], according to the methodology described by Vanegas et al. (1988). To obtain the salinities lower than 35[per thousand] dilutions were carried out using freshwater and the salinity of 40[per thousand] was obtained by the addition of Instant Ocean salts. The shrimps were not fed 24 h before beginning the determination of oxygen consumption, ammonium excretion, and osmoregulatory capacity.

The measurements of oxygen consumption and ammonium excretion were quantified simultaneously in 20 organisms placed in the respirometric chambers, the water flow on the chambers remained open for 3 hours to let the adjustment of the internal medium of the organisms exposed to a change of salinity (Diaz et al. 2001). Before closing the flow, two water samples were taken to measure the initial oxygen and ammonium concentration. Subsequently the chambers remained closed for 1 hour to avoid a reduction of the dissolved oxygen of 25% to 30%, and this constituted a stress factor (Stem et al. 1984). Before the water flow was re-established in the chambers, two water samples were taken to measure the final dissolved oxygen and ammonium production. The dissolved oxygen concentration in the water samples were measured with a dissolved oxygen meter YSI, model 52, provided with a polarographic sensor, which was inside of an acrylic hermetic chamber with a 10-mL capacity with adequate stirring. The rate of oxygen consumption of the shrimps was calculated as the difference between the initial and the final concentrations and it was expressed as mg [O.sub.2] [h.sup.-1] [g.sup.-1] of dry weight (d.w.) calibration in air at 100% relative humidity, to achieve this field probes can be placed in the plastic calibration bottle with a moistened sponge. For the quantification of ammonium excretion we used the phenolhypochlorite method (Rodier 1979). The ammonium production of the shrimps in each experimental condition was calculated as the difference between the final concentration and the initial and is expressed in mg NH[4.sup.+] [h.sup.-1] [g.sup.-1] d.w. The 21st chamber was used as a control to measure the oxygen consumption and the ammonium production by the microorganisms and to make the necessary corrections.

The determination of the osmotic pressure was carried out in the shrimps that remained in the reservoirs and they were exposed to the same regimen of salinity fluctuation. Before the salinity changed, 20 shrimps of the reservoir were extracted at random. To each organism a single hemolymph sample of 10 [micro]L was extracted with an automatic pipette directly off the thoraco-abdominal membrane (previously dried with absorbent paper). Osmolality of the hemolymph and the external medium were measured with a Wescor 5520 vapor osmometer, calibration was run a 290 mmol/kg standard and 1000 mmol/kg standard to establish baseline calibration for these standards, and the data were expressed in mmol [Kg.sup.-1]. The linear relationship in the decreased or increased salinity and osmolality at different temperatures were tested using linear regression (Sigma-Stat). The isosmotic points (where hemolymph osmolality is equal to medium osmolality) were determined using the formula of Ferraris et al. (1986). The osmoregulatory capacity (OC), in the blue shrimp was calculated according to Lignot et al. (2000) as the difference between the hemolymph osmolality and the medium osmolality.

Immediately after ending the experiments the shrimps were killed by immersion in boiling water, dried overnight at 60[degrees]C, placed on a desiccator for 2 hours and weighed on a balance (OHAUS Explorer) to determine the d.w.

The data of the oxygen consumption and ammonium excretion of the shrimps exposed to the different experimental conditions were plotted in parallel boxes (Tukey 1977). Within the boxes 50% of the data were distributed in the median and the confidence intervals, the other 50% remained distributed in each bar. A 2-way analysis of variance was used as previous determination of the normality and homoscedasticity of the data (Sigma-Stat), to determine the effect of the temperature and salinity fluctuation on the oxygen consumption, nitrogen excretion and the hemolymph osmotic pressure of L. stylirostris.

RESULTS

The oxygen consumption rate in Litopenaeus stylirostris juveniles exposed to fluctuations of salinity and maintained in 23[degrees]C was 1.1-1.51 mg [O.sub.2] [h.sup.-1] [g.sup.-1] d.w. This rate was of 1.70-1.98 mg 02 [h.sup.-1] [g.sup.-1] d.w. in organisms acclimated to 28[degrees]C and it increased 230% in comparison with those exposed to 33[degrees]C (Fig. 1). An analysis of variance indicated that temperature had a significant effect (P < 0.05) on the oxygen consumption of the blue shrimp, but the effect of salinity and the interaction temperature-salinity was not significant (P > 0.05).

[FIGURE 1 OMITTED]

L. stylirostris juveniles exposed to salinity of 40[per thousand] and acclimated to 23[degrees]C and 28[degrees]C the lower ammonium excretion rate of 0.04-0.08 mg NH[4.sup.+] [h.sup.-1] [g.sup.-1] d.w. was obtained. In the shrimps maintained in 33[degrees]C the excretion rate increased significantly up to 0.18 to 0.35 mg NH[4.sup.+] [h.sup.-1] [g.sup.-1] d.w. When the organisms were exposed to descending salinities of 40[per thousand] to 25[per thousand], ammonium excretion increased. In salinity of 16[per thousand] and in the three temperatures in which the shrimps were maintained, the ammonium production increased an average of 220%. When the organisms were exposed to ascending salinities, the excretion of the shrimps was reduced significantly (P < 0.05) (Fig. 2). An analysis of variance indicated that there was a significant effect of temperature and salinity on the ammonium excretion rate of L. stylirostris (P < 0.05), the interaction between temperature and salinity was not significant (P > 0.05).

[FIGURE 2 OMITTED]

Hemolymph osmolarity of L. stylirostris acclimated to 23[degrees]C, 28[degrees]C, and 33[degrees]C and exposure to salinity fluctuation was related linearly; the obtained slopes had an interval of 0.113 to 0.264 (Table 1). When salinity was reduced from 40[per thousand] to 33[per thousand] (1,278-965 mmol [Kg.sup.-1]) the hemolymph concentration was hypo-osmotic with an interval of 873-777 mmol [Kg.sup.-1] in respect of the external medium. In the interval of salinities of 25[per thousand] to 16[per thousand] (752-556 mmol [Kg.sup.-1]) the hemolymph of the shrimps was hyperosmotic with an interval of 677-770 mmol [Kg.sup.-1]. When the salinity was increased from 25[per thousand] to 40[per thousand] the osmolality of the internal medium of the shrimps was hypo-osmotic with an interval of 737-848 mmol [Kg.sup.-1] (Fig. 3). An analysis of variance indicated that the fluctuation of salinity had a significant effect (P < 0.05) on the hemolymph concentration of the shrimps; the temperature and the interaction between temperature and salinity did not have a significant effect (P > 0.05).

The acclimation temperature did not affect significantly (P > 0.01) the values of the isosmotic points of the organisms exposed to salinity fluctuation. The isosmotic points obtained in the shrimps exposed to salinity fluctuation had an interval of 726-758 mmol [Kg.sup.-1] (24.9-25.9[per thousand]), when the increased salinity had an interval of 751-766 mmol [Kg.sup.-1] (25.7-26.2[per thousand]) (Fig. 3).

The osmoregulatory capacity (OC) in juveniles of the blue shrimp was modified significantly by salinity (P < 0.05) the hemolymph was hypo to hyper-osmotic since in the salinities of (40[per thousand], 32[per thousand], 40[per thousand]) had an interval of-417 to -167, whereas the salinities of (25[per thousand], 16[per thousand], 25[per thousand]) increased from 18[per thousand] to 143[per thousand] (Fig. 4). The temperature did not affect significantly (P > 0.05) the osmoregulatory capacity of the organisms.

DISCUSSION

The respiratory rate in the crustaceans is modified by external factors as salinity, light intensity, dissolved oxygen and temperature (Kutty et al. 1971, Bishop et al. 1980, Dalla-Via 1986, Kurmaly et al. 1989, Martinez-Palacios et al. 1996). In Litopenaeus stylirostris juveniles a direct relationship was obtained between the oxygen consumption rate and temperature. This same response was obtained by Kutty et al. (1971) in Fenneropenaeus indicus, in Penaeus monodon by Kurmaly et al. (1989) and Chen and Lai (1993), in Farfantepenaeus californiensis (Holmes) by Villarreal and Ocampo (1993), and Martinez-Palacios et al. (1996) in Litopenaeus vannamei. It has been emphasized by Venkataramiah et al. (1974) that the respiratory rate cannot be considered as a good index of the salinity tolerance, except in the cases where the experimental temperature correspond to the natural characteristics of the habitat of the organisms. For blue shrimp a temperature of 28[degrees]C corroborates the above-mentioned fact because it corresponds to the preferred temperature determined for this species by Re et al. (2000). Furthermore it is within the average interval experienced in their natural environment.

The oxygen consumption of L. stylirostris exposed to salinity fluctuation was not affected significantly. In other euryhaline decapods salinity was not found to significantly affect the oxygen consumption if the experimental organisms were acclimated to the test salinities and if this were not extreme (Bishop et al. 1980, Gaudy & Sloane 1981, Diaz-Herrera et al. 1992, Villarreal & Rivera 1993, Villarreal et al. 1994, Salvato et al. 2001). In aquatic organisms that have been acclimated to a new salinity, Kinne (1967) described four types of metabolic response. The blue shrimp exposed to salinity fluctuation exhibited the type I response because the oxygen consumption was not modified significantly. According to Newell (1976) physiologic rate independence from salinity obtained for blue shrimp is a characteristic of animals that experience highly variable environmental conditions. For other osmoregulator decapods it has been demonstrated, that salinity did not have a pronounced effect on the oxygen consumption if the experimental organisms were acclimated to salinities and if these are not extreme (Bishop et al. 1980, Gaudy & Sloan 1981, Villarreal & Rivera 1993, Salvato et al. 2001). Acclimation time for the Litopenaeus stylirostris juveniles to each condition of salinity was sufficient to stabilize their internal medium (Diaz et al. 2001). Furthermore, the intervals of the used salinities correspond to the natural variations to which this species is exposed (Aragon 2000).

In the juveniles exposed to salinity fluctuation ammonium excretion rate was increased, when salinity was reduced from 40[per thousand] to 16[per thousand]. This response is related to an increase in the catabolism of the amino acids, because; according to Chen (1998) the shrimps in the lower salinities use proteins as the primary source for requirements of energy. This response has been reported in penaeids such as M. japonicus, F. chinensis, P. monodon, F. aztecus, and L. vannamei (Spaargaren et al. 1982, Chen & Lin 1992, Chen & Lai 1993, Chen et al. 1994, Hernandez & Diaz 1995, Jiang et al. 2000, Diaz et al. 2001).

In the blue shrimp exposed to salinity fluctuations, the ammonium excretion rate was related to the processes of osmoregulation, because excretion increased when the shrimps were hyper-regulators and reduced when they were hypo-regulators. It has been demonstrated that there is an increase in the active incorporation of Na+ followed by the transfer from the organisms to dilute mediums, and this was related to an increase in ammonium excretion because the sodium absorption is required to compensate its lost, caused by the operation of the exchange pump [Na.sup.+]/NH[4.sup.+] (Mangum et al. 1976, Pressley et al. 1981). To balance the osmotic concentration of the hemolymph when exposed to dilute mediums, the shrimps use these mechanisms. The quantification of nitrogen excretion rate in the shrimps exposed to the effect of different environmental factors is an important requirement for the design and operation of the intensive production systems (Jiang et al. 2000).

In the L. stylirostris juveniles, the osmoregulation pattern obtained was typical of many crustaceans that inhabit the lagoon-estuarine systems. They were hypoosmoregulators and hyperosmoregulators above and below the isosmotic point (Vernberg & Vernberg 1972, Mantel & Farmer 1983). The isosmotic points for the juveniles of the blue shrimp acclimated to different temperatures and exposed to salinity fluctuations had an interval of 726-766 mmol [Kg.sup.-1] (24.9[per thousand] to 26.2[per thousand]). These values are within the interval of isosmotic points reported by Brito et al. (2000) and Diaz et al. (2001) in 8 and 11 different species of penaeid respectively. Lemaire et al. (2002) reported an isosmotic point 735 mmol [Kg.sup.-1] for juvenile shrimp L. stylirostris maintained in constant salinities. In the L. vannamei juveniles exposed to salinity fluctuations and acclimated to different temperatures. Diaz et al. (2001) obtained that the isosmotic points had an interval of 712-777 mmol [Kg.sup.-1]. These values are greater than those reported by Castille and Lawrence (1981) and Rodriguez (1981) of 680 (24[per thousand]) and 610 (21[per thousand]) mmol [Kg.sup.-1] respectively. The differences can be attributed to the different experimental conditions used in these studies, because it is known that factors like temperature, dissolved oxygen, molt stage, the size of the organisms, and the nutritional state have an influence on the hemolymph osmolality control of the penaeid shrimp (Williams 1960, Charmantier et al. 1994, Lignot et al. 1999). Our observations were conducted under standardized experimental conditions, and allow us to isolate the effect of temperature salinity fluctuations and repeated the experiments.

To evaluate the osmoregulatory capacity of L. stylirostris the values of the slopes of the relationship between the osmotic concentration of the medium and the hemolymph were compared with those obtained for other penaeids maintained in constant salinities, which had an interval of 0.11-0.73 (Diaz et al. 2001). A deviation of the slope of the isosmotic line reflects the degree of the regulation capacity (slope = 0 osmoregulator; slope = 1 osmocon-former). The values of the slopes of the juveniles acclimated to 23[degrees]C, 28[degrees]C, and 33[degrees]C and exposed to decreasing salinity were 0.26[per thousand], 0.17[per thousand], and 0.11[per thousand] and for ascending salinities 0.26[per thousand], 0.19[per thousand], and 0.18[per thousand], respectively. The organisms with higher slopes as those maintained in 23[degrees]C had a weak regulation and their osmoregulatory capacity was poor. According to Vernberg and Silverthon, (1979) the temperature affect osmoregulatory capacity in crustacean by influencing water movement across cell membrane and in the uptake and loss of ions. The lower values of the slopes for the organisms maintained in 28[degrees]C and 33[degrees]C reflected that in these conditions the shrimps had a hyperosmoregulatory capacity in higher salinities and hyperosmoregulatory in lower salinity. Therefore, they can be characterized as strong regulators, because they adapt rapidly to the new salinities by increasing and decreasing the hemolymph osmotic concentration. For L. stylirostris sub-adults, Lemaire et al. (2002) obtained that reducing the temperature from 26[degrees]C to 15[degrees]C, there was a significant decrease of the osmoregulatory capacity in organisms maintained in constant salinities. This different response can be considered from the adaptive point of view because the lower sensibility in the osmoregulatory capacity shown by the juveniles permits living in the lagoon-estuarine environment in which there are significant variations of temperature.

To optimize the culture of the blue shrimp in controlled conditions it is recommended that temperatures of approximately 28[degrees]C be used. Because the best salinity has not been determined experimentally, we propose that 25[per thousand] be considered as the optima for this species, because it corresponds with the isosmotic point determined for the blue shrimp. According to Pannikar (1968), in this condition the shrimps expend less energy for the maintenance of the osmotic gradient and therefore the growth increases when the organisms are cultivated in a stress-flee environment.

TABLE 1. Relationship between the hemolymph osmolality (y) and medium
osmolality (x) for Litopenaeus stylirostris acclimated different
temperatures and fluctuating salinities.

                                    Relationship

Temperature
([degrees]C)    Salinity Decreased    [R.sup.2]    Salinity Increased

23              y = 536.5 + 0.261x      0.98       y = 552.6 + 0.264x
28              y = 624.1 + 0.173x      0.86       y = 603.1 + 0.197x
33              y = 672.3 + 0.113x      0.79       y = 621.7 + 0.189x

                Relationship
Temperature
([degrees]C)     [R.sup.2]

23                 0.89
28                 0.90
33                 0.96

ACKNOWLEDGMENTS

The authors thank the Laboratory El Camaron Dorado located in Huatabampo, Sonora, for the donation of the specimens used in this investigation. SIMAC-CONACYT gave the economical support to the projects 98011110606 and 200017509. We also appreciate the graphical material; elaborated by Jose M. Dominguez and Francisco J. Ponce from the Drawing Department of CICESE.

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FERNANDO DIAZ, (1), * ANA DENISSE RE, (1) ELIZABETH SIERRA (1) AND EUGENIO DIAZ-IGLESIAS (2)

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

(1) Departamento de Biotecnologia Marina, Centro de Investigacion Cientifica y de Educacion Superior de Ensenada (CICESE), Kin. 107, Carretera Tijuana-Ensenada, Ensenada Baja California, Mexico and (2) Departamento de Acuicultura, Centro de Investigacion Cientifica y de Educacion Superior de Ensenada (CICESE), Km 107, Carretera Tijuana-Ensenada, Ensenada Baja California, Mexico