Salinity and temperature variations, in addition to other environmental factors in an aquatic environment, trigger adaptive responses that impact various physiological functions, with an impact on the development, growth and survival of organisms, including shrimp (1, 2, 3). Therefore, aquaculture is highly interested in determining the appropriate levels of salinity and temperature for each commercially important shrimp species, especially in larvae or penaeidae, wherein optimum condition studies on these factors have been restricted to only a few species.

Some research studies on penaeidae have found that salinity has a greater influence than temperature on survival during the early larval stages (5,6). Other studies state that salinity and temperature, in addition to various other factors present in water, have an impact on the survival and development of shrimp larvae and post-larvae (7,8). However, some authors state that proper nutrition during the early stages of shrimp larvae favors development, growth and survival (9,10), in addition to resistance to various salinity and temperature conditions during the post-larva stage, when they are moved and planted in shrimp farm tanks.

Conventionally, Litopenaeus vannamei larvae breeding (Boone 1931) has generally been done using saltwater (12,13), given that penaeidae larval stages have little tolerance to changes in high and low salinity (6).

L. vannamei is commonly known as the White Pacific Shrimp, it is a species from the Eastern Pacific Ocean and one of most euryhaline among penaeidae, it is spread out from the coasts of the Gulf of California in Mexico to Peru in South America, it has a high commercial value, and it is the most important species for shrimp breeding in Latin America, commonly bred in Mexico (7,14)

At this point, there is little information on the survival of larvae (nauplius, zoea and mysis) of this species and its tolerance to abrupt changes in high and low salinity in commercial post-larva laboratories in northeast Mexico (6). Furthermore, the effect of different temperatures combined with low and high salinity levels in business breeding tanks has not been properly analyzed during the larval stages of this species (2,6). Therefore, focusing on the variations of these abiotic in large scale breeding (11), in waters with low due to the development of onshore shrimp breeding (12,13), and in waters with high salinity and temperature, where they are vulnerable to disease, have both lead to mortality and difficulties in shrimp production over all sizes and ages bred by different suppliers during the warm or dry season (15,16).

This study assessed survival and development in the different larval stages L. vannamei in response to the combined effects of three temperatures (25, 30 y 35°C) and four levels of salinity (25, 30, 35 y 40ups) under laboratory conditions to establish the most appropriate shrimp larva breeding conditions.

Artemia microalgae and nauplii cultivation. The microalgae cultivated to feed the zoea larvae was Chaetoceros muelleri, taken from the Universidad Autónoma de Sinaloa Faculty of Oceanic Sciences Aquatic Organism Ecophysiology and Support Culture Laboratory (Laboratorio de Ecofisiología de Organismos Acuáticos y Cultivos de Apoyo) culture collection, and hatched Artemia cyst nauplius supplemented with microalgae was used as feed during the mysis stage, cysts were acquired from a commercial company.

Microalgae culture was carried out in transparent PET (Polyethilene Terephthalate or Ethylene Polyterephthalate) carboys with 16 L of seawater filtered at 1 µm and disinfected for 24 h with 1 mL/L sodium hypochlorite at 5%, residual chlorine was neutralized by adding 0.06 g/L sodium thiosulfate at the time of use. Microalgae was cultivated pursuant to (10) in Guillard´s f Medium, with constant aeration, 250-260 µmol/m/s lighting, a temperature of 22 to 25°C, 35ups salinity, pH 8.7-9.6. A 2.04±0.16×106 cells/mL (Loptik Labor) biomass was harvested, which was estimated through hemocytometer counting (Loptik Labor) with a 0.1 mm deep Neubauer camera.

Artemia cysts hatched with the sodium hypochlorite technique using pre-hydrated cysts. Nauplii eclosion was carried out in a conical container with sea water filtered at 1 µm at a temperature of 30°C which was maintained throughout using a heater and a thermal regulator (FINNEX, HMO-50), with bubbling at the bottom to keep the cysts suspended (17). After eclosion (18 h), nauplii were harvested and inactivated in 60°C sea water, they were preserved at -20°C for later use as food within a timeframe no longer than 72 h.

Experimental organism procurement. Shrimp larvae in nauplius stage IV were procured from the “FITMAR” commercial production laboratory located in Sinaloa, Mexico; they were transported in plastic bags contained in an icebox with sea water at oxygen saturation level and at a temperature from 28 to 29°C. Larvae under controlled conditions (30°C and 35ups) were placed in a 400 L plastic tank with sea water and aeration, both filtered at µm.

Experimental design. Once nauplius stage V was reached at 100%, and to establish larva survival and development, a factorial experiment was conducted on two factors, considering as factor A, with four levels (25, 30, 35 y 40ups), and temperature as B, with three levels (25, 30 and 35°C), each treatment (temperature-salinity interaction) was quadrupled, with a total of 48 trial units (aquariums). 12 L plastic containers with water filtered at 1 µm and at the proposed salinity with an initial density of 100 larvae/L or 1200 larvae per aquarium. Trial temperatures were maintained with a heater and a thermal regulator (FINNEX, HMO-50) for every aquarium. To increase salinity to 40 ups, granulated salt (uniodized) was added to the sea water at a ratio of 5 g/L, and fresh water was added to the sea water to decrease salinity, after which the treatment salinity was measured with a digital refractometer (VITALSINE, SR-6), making any necessary adjustments to achieve the desired salinity levels.

Zoea larvae in each aquarium were fed a daily ration of microalgae, which changed according to their development: 100, 120 y 150×10 3 cells/mL for zoea stages I, II and III, respectively. Mysis larvae I, II and III were fed 30, 40 and 50 Artemia/larva nauplii, respectively; in each case, there was also a microalgae supplement at a ratio of 50×103 cells/mL per aquarium. Each day, and before every food ration, feces and the remaining food were removed using syphon protected with a 200 µm nytex mesh, adjusting the volume to 12 L with clean filtered water at the respective temperature and salinity for each aquarium.

Between 25 and 30 larvae were tested in vivo every 24 h to establish their development stages, which were then put back in their corresponding container to minimize sampling mortality. The development index (ID) was calculated as: ID=(∑in i )/N, where i is the absolute value assigned to each larval stage (nauplius V=0; zoea I to III: 1 to 3; mysis I to III: 4 to 6; PL1=7), n is the number of larvae in stage I, and N is the total number of larvae in the sample.

ID data was used to determine the respective food rations, which were modified simultaneously in all the aquariums when larvae started entering the next development stage in at least three replicas of the same treatment.

In addition, survival (S) was assessed, for which the larvae in each trial unit were counted in daily live larva counts in samples with a 0.5 L volume and the number of surviving larvae were calculated in the total volume of their respective aquarium, using the equation: S=(NT/N0)×100, where S is survival expressed as a percentage, NT is the number of remaining larvae at any point during the experiment, and it is NOT the initial larvae amount.

The experiment ended when 50% of the organisms reached stage PL1 in at least one of the temperature-salinity interactions.

Statistical analysis. Normality (Liliefors) and variance equality (Bartlett) tests were conducted on survival data (transformed into arcosene) and larva development index data. Subsequently, they were analyzed using a two-way ANOVA, considering salinity (ups) as factor A and temperature (°C) as factor B as variables to which survival and the development index respond. After the two-way ANOVA proved to be meaningful, Tukey multi-comparison tests were then conducted; an (α) of 0.05 (18) significance level was used in all cases.

In general, regardless of salinity, survival percentages were better at 30°C than at 35 and 25°C. Survival rates above 50% were achieved in treatments of 25 and 30ups in all the temperatures under study. The greatest survival percentage was at 30°C and 30ups (82.2%), followed by 25 ups at 30 and 35°C (71.5 and 71.6%). Organisms kept at 25°C and 40ups had the lowest survival rate throughout the trial period (Figure 1)

Figure 1. Figure 1. Average (%) Litopenaeus vannamei larvae survival at different salinity and temperature levels

Nauplius V metamorphosis until the PL1 stage was successful in some salinity and temperature combinations during the trial (25-35 ups at 30 and 35°C).On the seventh day, the experiment carried out at a temperature of 30° had the highest DI value (6.76) with a salinity level of 30 ups, followed by a DI of 6.74 at a temperature of 35°C and 25 ups salinity). Nevertheless, the lowest DI value was at 25°C and 40 ups, followed by 35, 25 and 30 ups. DI trends during cultivation days at different temperature and salinity levels are shown in Figure 2. At 25 and 35°C, di increase for days three and four was above 30 ups, followed by 25, 35 and 40 ups salinity levels. At 35°C, the DI was similar throughout all the salinities used during the trial for the first two-day period, with a higher value at 25 ups, followed by DI values at 30, 35 and 40 ups salinities until the end of the trial.

Figure 2. Figure 2. Average (%) Litopenaeus vannamei larvae development index at different salinity and temperature levels

The effect of salinity and temperature, in addition to its impact on L. vannamei larvae DI values at the end of the trial, had a significant effect (p<0.05; Table 1).

Table 1. Table 1. Survival and development index (DI) averages (standard deviation in brackets) of Litopanenaeus vannamei larvae at different salinity and temperature combinations at the end of the experiment (day 7). Different letters indicate significant differences (two-way ANOVA).

In particular, average larvae DI values showed significance between 40 ups and 35°C with compared to the remaining salinities used. At 30 and 35°C with salinity between 20 and 40ups, statistical differences were found between DI values, but no statistical differences between DI values were found at 25, 30 and 35ups (the latter at 35°C only) (Table 1).

The development and survival index shows the success or failure of the conditions in which organisms are cultivated (5, 8). Higher survival rates and greater progress in the larval development of shrimp are directly related to appropriate maintenance conditions (7,13); nevertheless, some researchers who studied the combined effect of salinity and temperature in L. vannamei concluded that biomass respiration, excretion and growth were the best survival indicators (19, 20, 21). As in this study, other authors have reported that salinity and temperature have an influence on the larval development index of Penaeus merguiensis and on the survival of Litopenaeus stylirostris (5,22).

The highest survival and larval development index values were found at 30 and 35°C and at 25 and 30 ups. Other salinities analyzed in this trial delayed growth and decreased survival in the larvae of this shrimp type, although the effect was different to the three temperatures of the trial. Results differ from those reported by other authors (9,10), where a salinity of 34 to 35 ups is said to be optimal for the development and survival success of L. vannamei white shrimp larvae. The studies conducted on Penaeus semisulcatus and Penaeus merguiensis (4,5), as well as Penaeus penicillatus, Metapenaeus affinis and Parapenaeopsis stylifera (23), showed that the best temperature and salinity interval for survival and development since nauplius eclosion is between 29-33°C and 30-35 ups.

The results of this study show that salinity had a greater influence on survival and temperature had a greater influence during larval development. The facts show that this shrimp species is more tolerant to low salinity levels, given that survival was considerably low at 40ups, followed by 35ups in all the trial temperatures. Nevertheless, it is known that L. vannamei larval stages (nauplius, zoea, mysis) and PL1 can tolerate salinities between 20 and 45ups (6), due to the fact that it is a euryhaline species under natural conditions (24). Nevertheless, there is evidence to show that penaeidae larvae are less resistant to abrupt salinity and temperature variations (22), mainly due to the fact that its gills and other structures specialized in osmoregulation have not developed completely.

In a trial with P. merguiensis larvae (5), it was found that survival decreases more at low salinities (25 and 30ups) than at 35 and 40 ups with 29 and 33°C temperatures. However, another study (22) reports that L. stylirostis larvae and post-larvae (PL1) survival is lower at 20, 25, 35 and 39ups than at intermediate salinity levels of 27 and 31ups, at a temperature of 29°C. Nevertheless, P. semisulcatus survival starting at the zoea stage I is lower at 34°C than at 26 and 30°C temperatures, with 25, 30 and 35 ups salinity levels (4). The foregoing is explained by the fact that each shrimp species operates in optimum temperatures and salinity levels, out of which, organisms show a lower metabolic efficiency and more energy spending associated to the requirements of metabolical maintenance, which affects development and survival.

Although temperature does not appear to have a drastic impact on survival in this study, its impact on penaeidae growth and on the time to advance through larval stages is known. Another study (4) showed that high temperatures (30 and 34°C) can significantly increase growth rate and metamorphosis during the zoea and mysis stages when compared to a temperature of 26°C.

Similarly, the development index that represents metamorphosis at each larval stage was extended, survival was reduced to 40ups and the metamorphosis process during larval development was faster at 30 and 35°C. These events indicate that L. vannamei is less tolerant to low temperatures during larval development (5,25). Therefore, it is necessary to study the following in future studies: respiration, excretion, longitudinal growth and biomass, as indicators that could lead to a greater understanding of the effects of different temperature and salinity conditions in L. vannamei larvae.

In conclusion, it was found that larvae development and survival were best at temperatures of 30 and 35°C combined with 25 and 30ups salinity levels, which coincided with the implementation of new cultivation methods in most commercial laboratories producing L. vannamei in northeastern Mexico (14).

Projects UAS-CA-162, PROFAPI-159-2014 for the resources provided, CONACYT for the grant awarded to the first author (CVU 206165), the staff at the FITMAR Post-larvae Production Lab and Juan Manuel Flores Alarcón for data collection.