Aquaculture 342–343 (2012) 13–17

Contents lists available at SciVerse ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effects of water temperature and Na +:K + ratio on physiological and production

parameters of Litopenaeus vannamei reared in low salinity water
Martin Perez-Velazquez a,⁎, D. Allen Davis b, Luke A. Roy b, Mayra L. González-Félix a
a
Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Edificio 7-G, Blvd. Luis Donaldo Colosio s/n, e/Sahuaripa y Reforma, Col. Centro, C.P. 83000,
Hermosillo, Sonora, Mexico
b
Department of Fisheries and Allied Aquacultures, Auburn University, 203 Swingle Hall, Auburn, Alabama, 36849–5419, USA

a r t i c l e i n f o a b s t r a c t

Article history: Two experiments were conducted to examine the simultaneous effects of water temperature and Na +:K +
Received 14 December 2011 ratio on growth, survival, and hemolymph osmolality of Litopenaeus vannamei reared in 4‰ salinity well-
Received in revised form 2 February 2012 water. In one study, a factorial experiment was conducted with levels of water temperature of 26 and
Accepted 7 February 2012
30 °C, and Na+:K + ratios of 40:1, 80:1, and 120:1. Growth of shrimp was significantly higher at the Na +:
Available online 22 February 2012
K+ ratio of 40:1 than at 120:1, but not higher than at 80:1. Also, growth of shrimp was statistically greater
Keywords:
at 30 °C than at 26 °C. Survival rate of shrimp was neither affected by Na+:K + ratio nor by temperature. In
Na+: K+ ratio another factorial experiment, a lower range of experimental temperatures (20 and 24 °C) and Na +:K + ratios
Temperature of 40:1 and 120:1 were tested. Both growth and survival of L. vannamei reared at the Na+:K + ratio of 40:1
Litopenaeus vannamei were significantly higher than at the ratio of 120:1. In turn, survival rate of shrimp, but not growth, was sig-
Salinity nificantly higher at 24 °C than at 20 °C. No differences among treatments were observed for shrimp hemo-
lymph osmolality measurements in both experiments. Results of the present study agree with previous
reports of negative effects of high aqueous Na+:K + ratio (or K+ deficient levels) on shrimp performance.
At the same time, they represent the first report of a strong sensitivity of L. vannamei survival to the aqueous
Na +:K + ratio at low temperature.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction
All over the world, farming of penaeid shrimp is a profitable eco-
nomic activity (FAO, Food and Agriculture Organization of the United
Nations, 2009). Although they naturally inhabit the marine environ-
ment, some penaeids, such as the Pacific white shrimp, Litopenaeus
vannamei, are well known for being able to not only tolerate exposure
to low salinity, but also to grow and survive well in this type of environ-
ment (Roy et al., 2010). In fact, a significant part of all farmed penaeids
is raised in low salinity water (Flaherty et al., 2000). Low salinity or
inland shrimp farming is sometimes based upon the use of low salinity
well-waters (Roy et al., 2007a; Saoud et al., 2003). Scientific and eco-
nomic interest in shrimp culture in low salinity stimulated numerous
studies and, to date, considerable progress has been made in understand-
ing the implications of rearing marine shrimp in this rather unusual envi-
ronment. For example, it was found that the ionic composition of well-
waters may be a more important growth- and survival-determining
factor than the salinity itself (Saoud et al., 2003). Also, deficiencies in spe-
cific ions such as potassium (K+) and magnesium (Mg2+) have been
shown to negatively impact shrimp growth and survival (Davis et al.,
⁎ Corresponding author. Tel.: + 52 662 259 2169; fax: + 52 662 259 2197.
E-mail address: mperezv@dictus.uson.mx (M. Perez-Velazquez).
2005; Saoud et al., 2003). Such deficiencies have been amended, with
different degrees of success, by the addition of chemical reagents in
laboratory studies (Roy et al., 2007b) and of K- and Mg-containing fer-
tilizers in field trials (McGraw and Scarpa, 2003; McNevin et al., 2004);
and with more limited effectiveness, by the dietary supplementation
of minerals to shrimp feeds (Roy et al., 2007a, 2009). Evidence also sug-
gests that the sodium (Na+) and K + ratio of low salinity well-waters
may be a critical factor for successful growth and survival of shrimp
(Roy et al., 2007b). It is believed that water temperature interacts with
salinity and ionic ratios to determine growth and survival of shrimp
reared in low salinity water. More knowledge of these phenomena is
needed in order to successfully and consistently rear penaeids in low
salinity well-waters. Consequently, the present study was designed to
evaluate the effects of water temperature and Na +:K + ratio on culture
parameters of L. vannamei reared in low salinity well-water.
2. Materials and methods
2.1. Experimental shrimp
L. vannamei postlarvae, with mean body weight of 0.005 g, were
obtained from the commercial shrimp hatchery Shrimp Improvement
Systems, Islamorada, FL, USA. Shrimp were held at salinities between
25 and 30‰ in a 220-l plastic nursery tank, connected to a biological

0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.02.008
14 M. Perez-Velazquez et al. / Aquaculture 342–343 (2012) 13–17

filter, and were fed a combination of Artemia spp. nauplii and a 40% Table 1
protein commercial feed (PL Redi-Reserve, Ziegler Bros. Gardner, Concentration (mg l− 1) of Na+, K+, and Na+:K+ ratio of experimental waters used to
culture L. vannamei at different temperatures and Na+:K+ ratios in two experiments.
Pennsylvania, USA). At an overall mean body weight of 0.02 ± 0.002 g,
shrimp were acclimated to a salinity of 4‰, decreasing salinity at a Temperature (°C)/Na+:K+ ratio Na+ K+ Na+:K+ ratio
rate not greater than 1‰ h− 1, by gradually adding well water to the Experiment 1
nursery tank until the target salinity was reached. Then, shrimp were 26/40 2999.7 78.6 38
transferred into culture tanks for initiation of two experiments con- 26/80 3143.6 42.6 74
26/120 2982.9 24.4 122
ducted simultaneously.
26/sea salt 4‰ 2807.0 105.4 27
30/40 2770.4 62.8 44
2.2. Experimental culture systems 30/80 3029.9 43.4 70
30/120 2935.4 23.4 125
The study was conducted at the E. W. Shell Fisheries Research Sta- 30/Sea salt 4‰ 2868.8 111.7 26

tion, Auburn University, Auburn, Alabama, USA. Experiment 1 was

conducted in seven independent, identical culture systems. Each
culture system consisted of four 60-L glass culture tanks (each with
bottom area of 0.26 m2) and a sump tank (60-L) with a trickling biolog-
ical filter. Water was recirculated using a submersible pump (Lifegard
Aquatics, Model Quiet One 1200, Cerritos, CA, USA), while temperature
was adjusted with thermostat-controlled submersible aquarium
heaters (Marineland, Model Visi-Therm 300 W, Cincinnati, OH, USA).
Each aquarium was provided with a submerged air stone for water
aeration.
Experiment 2 was carried out in four independent, identical recir-
culating culture systems, each composed of four 220-L polyethylene
tanks (bottom area of 0.36 m2), and each provided with one air stone
for water aeration. Culture tanks were connected to a sump tank that
contained a trickling biological filter, a submersible pump (Danner
Manufacturing, Model MD12, Islandia, NY, USA), a 1/3 HP in-line chiller
(Current-USA, Model 2646, Vista, CA, USA) and thermostat-controlled
submersible aquarium heaters (Marineland, Model Visi-Therm 300 W,
Cincinnati, OH, USA) for temperature control. For both experiments,
fifteen shrimp were stocked into each culture tank (stocking density
of 58 and 42 shrimp per m 2 for experiments 1 and 2, respectively).
2.3. Experimental treatments
The two experiments were conducted at a fixed salinity of 4‰.
Shrimp were fed a 40% protein commercial shrimp feed (Rangen,
Buhl, ID, USA). In experiment 1, feed was administered manually in
slight excess, dividing the daily ration into four equal portions. In ex-
periment 2, feed was administered using automatic feeders that dis-
tributed the ration in four equal portions over 24 h.
2.3.1. Experiment 1
A factorial experiment was conducted with levels of water temper-
ature of 26 and 30 °C, and Na+:K+ ratios of 40:1, 80:1, and 120:1.
Each experimental treatment was assigned to 4 replicate tanks. In
order to obtain the desired Na+:K + ratios, artificial sea salt (Crystal
Sea, Marine Enterprises International, Baltimore MD, USA) was added
to well water until a salinity of 0.5‰ was reached. Then, 0.048, 0.019,
and 0.009 g l− 1 of potassium chloride (KCl) were added for the Na+:
K + ratios of 40:1, 80:1, and 120:1, respectively. Salinity was then raised
to 4‰ with agricultural grade NaCl according to Roy et al. (2007b). As
a reference, a 4‰ artificial sea salt treatment was included in the trial.
Experimental waters were analyzed for Na + and K+ by inductively
coupled argon plasma spectrophotometry (ICAP) and flame photome-
try (Cole Parmer digital flame photometer, Model 2655–00, Vernon
Hills, Illinois, USA) (Clesceri et al., 1998). The determined values for
the concentration and ratio of these ions are shown in Table 1.
Experiment 2
20/40 2845.6 60.9 47
20/120 2737.1 22.5 122
24/40 2893.5 61.8 47
24/120 2770.2 24.7 112
2.4. Water quality parameters
Temperature, dissolved oxygen, and salinity monitored daily
employing a multi-function oxygen meter (YSI, Model Y85, Yellow
Springs, OH, USA). The concentrations of total ammonia nitrogen
(TAN) and nitrite-nitrogen were measured twice weekly following the
procedures described by Solórzano (1969) and Parsons et al. (1985).
2.5. Shrimp performance and hemolymph osmolality
Shrimp were blotted dry at initiation and upon termination of the
28-day experiments to determine group weight. Weight gain was cal-
culated from the difference between final and initial weights and was
expressed as a percent of initial mean weight. Survival was calculated
from the difference between final and initial numbers of shrimp. Also,
the food conversion ratio (FCR) was calculated from the amount of
feed provided and the weight gained by shrimp.
A vapor pressure osmometer (Wescor Vapro, Logan, UT, USA) was
employed to evaluate experimental waters and shrimp hemolymph
osmolality (reported as mmol kg− 1). Hemolymph was withdrawn
from the shrimp pericardial cavity by inserting a 25-gauge needle and
1-cc syringe beneath the carapace at the cephalothorax–abdominal
junction. Hemolymph samples were withdrawn from all shrimp to col-
lect one composite sample per tank and were stored at −20 C. Prior to
analyses, hemolymph samples were thawed on ice and then sonicated
(Omni Ruptor 250, OMNI International, Marietta, GA, USA). The samples
were then centrifuged at 10,000 g for 60 s to separate the clot from
serum. Total osmolality was then measured using 10 μL of serum by
dew point depression. Each hemolymph or experimental water sample
was analyzed in duplicate.
2.6. Statistical analysis
Initial, final weight, percent weight gain and arcsine-transformed
survival (actual survival data are reported) were analyzed by two-way
analysis of variance (ANOVA) using a significance level of P b 0.05. TAN
and nitrite-nitrogen data were analyzed by repeated measures ANOVA
(RMANOVA). Mean separation procedure was analyzed by Tukey's
Honestly Significant Difference. Statistical analyses were performed
using Statistical Analysis System software (SAS Institute Inc. 1999–2000,
Software Release 8.1, Cary, NC, USA).

2.3.2. Experiment 2 3. Results

A factorial experiment was conducted with levels of water tem-
perature of 20 and 24 °C, and Na +:K + ratios of 40:1 and 120:1. Each
experimental treatment was assigned to 4 replicate tanks. The prepa-
ration of experimental waters and analysis of ions (Table 1) was per-
formed according to Roy et al. (2007b), as described above.
3.1. Water quality parameters
Mean values for water temperature, dissolved oxygen, and salinity
in experiments 1 and 2 are shown in Table 2. For both experiments,
 M. Perez-Velazquez et al. / Aquaculture 342–343 (2012) 13–17 15

Table 2 Table 4
Means (± s.d.) of water temperature, dissolved oxygen, and salinity of experimental Experimental water and hemolymph osmolality (means ± s.d.) (mmol kg− 1) of L.
waters used to culture L. vannamei at different temperatures and Na+:K+ ratios in vannamei cultured at different temperatures and Na+:K+ ratios in two experiments.
two experiments.
Experiment 1 Hemolymph Experimental Experiment 2⁎ Experimental
Temperature(°C)/Na+:K+ Temperature Dissolved oxygen Salinity Temp. (°C)/Na+: water Temp. (°C)/Na+: water
ratio (°C) (mg l− 1) (‰) K+ ratio K+ ratio

Experiment 1 26/40 662.5 ± 41.7 120.5 ± 3.7 20/40 125.5 ± 2.1

26/40 25.7 ± 0.3 7.4 ± 0.4 4.0 ± 0.2 26/80 664.0 ± 42.4 128.0 ± 3.4 20/120 118.0 ± 2.0
26/80 25.9 ± 0.5 7.2 ± 0.3 4.0 ± 0.1 26/120 647.0 ± 29.9 129.5 ± 4.7 24/40 120.5 ± 3.5
26/120 25.8 ± 0.3 7.1 ± 0.5 4.1 ± 0.2 26/Sea salt 4‰⁎⁎ 604.5 ± 51.2 127.5 ± 3.7 24/120 122.0 ± 2.8
26/Sea salt 4‰ 25.9 ± 0.4 7.4 ± 0.2 4.0 ± 0.1 30/40 650.0 ± 39.6 134.0 ± 4.4 -
30/40 29.8 ± 0.2 6.8 ± 0.4 4.1 ± 0.3 30/80 630.5 ± 13.4 130.5 ± 5.5 -
30/80 29.7 ± 0.3 6.7 ± 0.3 4.0 ± 0.2 30/120 690.5 ± 27.8 137.0 ± 4.4 -
30/120 29.9 ± 0.3 6.4 ± 0.5 4.0 ± 0.1 30/Sea salt 4‰⁎⁎ 674.5 ± 43.2 129.5 ± 5.7 -
30/Sea salt 4‰ 29.8 ± 0.2 6.2 ± 0.4 4.1 ± 0.2 ANOVA Pr > F
Experiment 2 Temperature 0.9633 0.3639 0.7913
20/40 19.7 ± 0.4 8.1 ± 0.4 4.0 ± 0.2 Na+:K+ ratio 0.6224 0.2624 0.1649
20/120 19.8 ± 0.5 7.9 ± 0.3 4.0 ± 0.1 Temp. x Na+:K+ 0.2527 0.5722 0.0636
24/40 23.9 ± 0.4 7.7 ± 0.4 4.0 ± 0.3 ratio
24/120 23.9 ± 0.2 7.6 ± 0.5 4.0 ± 0.2
⁎ In experiment 2, hemolymph was not withdrawn from shrimp due their small size
and low survival rates upon termination of the study.
⁎⁎ Treatment added as a reference, not included in statistical analysis.

low concentrations of TAN and nitrite-nitrogen were recorded, with-

out significant effects of temperature, Na +:K + ratio, time of sampling,
or the interaction among these factors being detected by RMANOVA.
The overall mean values of TAN and nitrite-nitrogen in experiment
1 were 0.03 ± 0.03 and 0.03 ± 0.02 mg l − 1, respectively, and in exper-
iment 2 they were 0.06 ± 0.08 and 0.04 ± 0.04 mg l − 1, respectively.
3.2. Shrimp performance and hemolymph osmolality
3.2.1. Experiment 1
No statistical differences in shrimp weight were detected at the
beginning of the experiment (Table 3). Upon termination, not only
temperature, but also the Na +:K + ratio of the culture water signifi-
cantly influenced the performance of shrimp. Shrimp maintained at
30 °C had statistically greater final weight, percent weight gain, and
significantly lower FCR than shrimp held at 26 °C. Also, final weight
and percent weight gain of shrimp held at aqueous Na +:K + ratio of
40:1 was significantly greater than that of shrimp held at a ratio of
120:1, but not greater than at a ratio of 80:1. FCR was not statistically
influenced by the water Na +:K + ratio. Survival of shrimp was 100%
across the board (Table 3). No effects of temperature, Na +:K + ratio,
or their interaction, were observed on osmolality of shrimp hemo-
lymph or experimental waters (Table 4).
3.2.2. Experiment 2
No significant differences were observed in shrimp weight at initi-
ation of the study (Table 5). Differences in shrimp growth were not
observed at the temperatures tested (20 °C and 24 °C). However, sta-
tistically higher final weight and weight gain was observed in shrimp
reared at aqueous Na +:K + ratio of 40:1, in comparison with shrimp
reared at a ratio of 120:1. FCR was significantly influenced by temper-
ature (greater at 20 °C than at 24 °C), but not by the water Na +:K +
ratio. Survival rate of shrimp was significantly lower at a temperature
of 20 °C vs. 24 °C. Also, water Na +:K + ratio of 40:1 resulted in signif-
icantly higher survival rate, as compared to the survival rate observed
at the Na +:K + ratio of 120:1. Interactive effects between temperature
and aqueous Na +:K + ratio were not observed for any of these evalu-
ations (Table 5). No effects of temperature, Na +:K + ratio, or their

Table 3
Performance (means ± s.d.) of L. vannamei cultured for 24 days at different temperatures and aqueous Na+:K+ ratios in experiment 1.

Temperature(°C)/Na+:K+ ratio Initial weight (g) Final weight (g) Weight gain (%) FCR Survival (%)

26/40 0.02 ± 0.00 1.0 ± 0.1 4,032.3 ± 487.1 1.5 ± 0.1 100 ± 0
26/80 0.02 ± 0.00 0.8 ± 0.1 3,449.9 ± 508.9 1.9 ± 0.2 100 ± 0
26/120 0.02 ± 0.00 0.9 ± 0.1 3,795.2 ± 844.5 1.7 ± 0.2 100 ± 0
26/Sea salt 4‰⁎ 0.03 ± 0.00 1.1 ± 0.2 4,258.4 ± 942.0 1.3 ± 0.2 100 ± 0
30/40 0.02 ± 0.00 1.2 ± 0.1 5,127.6 ± 201.3 1.2 ± 0.1 100 ± 0
30/80 0.03 ± 0.00 1.2 ± 0.2 4,525.2 ± 927.2 1.3 ± 0.2 100 ± 0
30/120 0.03 ± 0.00 1.0 ± 0.1 4,003.2 ± 431.0 1.5 ± 0.3 100 ± 0
30/Sea salt 4‰⁎ 0.03 ± 0.00 1.5 ± 0.1 5,776.2 ± 553.3 1.0 ± 0.1 100 ± 0

Main effects means

Temperature
26 0.02 ± 0.00 0.9 ± 0.1b 3759.1 ± 626.3b 1.7 ± 0.2a 100 ± 0
30 0.02 ± 0.01 1.1 ± 0.2a 4559.1 ± 725.6a 1.3 ± 0.2b 100 ± 0
Na+:K+ ratio
40 0.02 ± 0.00 1.1 ± 0.1a 4579.9 ± 679.6a 1.4 ± 0.2a 100 ± 0
80 0.02 ± 0.01 1.0 ± 0.2ab 3987.5 ± 899.9ab 1.6 ± 0.4a 100 ± 0
120 0.02 ± 0.01 0.9 ± 0.1b 3899.2 ± 630.6b 1.6 ± 0.2a 100 ± 0
ANOVA Pr > F
Temperature 0.1667 0.0002 0.0057 0.0003 ⁎⁎
Na+:K+ ratio 0.4153 0.0345 0.0346 0.0690 ⁎⁎
Temp. x Na+:K+ ratio 0.0894 0.0698 0.2863 0.0615 ⁎⁎

Values are means of four replicates. Means within columns with the same letter are not significantly different (P b 0.05).
⁎ Treatment added as a reference, not included in statistical analysis.
⁎⁎ 100% survival was recorded for all experimental units. Hence, there was no variability of data.
16 M. Perez-Velazquez et al. / Aquaculture 342–343 (2012) 13–17

Table 5
Performance (means ± s.d.) of L. vannamei cultured for 24 days at different temperatures and aqueous Na+:K+ ratios in experiment 2.

Temperature(°C)/Na+:K+ ratio Initial weight (g) Final weight (g) Weight gain (%) FCR Survival (%)

20/40 0.02 ± 0.00 – – – 0±0

20/120 0.02 ± 0.00 0.1 ± 0.0 303.8 ± 136.7 10.4 ± 4.9 18.3 ± 6.4
24/40 0.02 ± 0.00 0.3 ± 0.0 1,212.8 ± 215.1 2.9 ± 0.2 95.0 ± 3.3
24/120 0.02 ± 0.00 0.1 ± 0.0 514.0 ± 161.1 5.9 ± 1.6 10.0 ± 4.7

Main effects means

Temperature
20 0.02 ± 0.00 0.1 ± 0.0a 303.8 ± 136.7a 10.4 ± 4.9a 18.3 ± 6.4b
24 0.02 ± 0.01 0.2 ± 0.1a 979.9 ± 404.0a 3.9 ± 1.7b 66.7 ± 44.0a
Na+:K+ ratio
40 0.02 ± 0.00 0.3 ± 0.0a 1,212.8 ± 215.1a 2.9 ± 0.2a 95.0 ± 3.3a
120 0.02 ± 0.01 0.1 ± 0.0b 373.8 ± 167.9b 8.9 ± 4.5a 15.6 ± 6.9b
ANOVA Pr > F
Temperature 0.1036 0.1184 0.2141 0.0404 0.0478
Na+:K+ ratio 0.1036 0.0001 0.0027 0.3304 b 0.0001
Temp. x Na+:K+ ratio 0.1930 0.0798 0.1063 0.1585 0.0932

Values are means of four replicates. Means within columns with the same letter are not significantly different (P b 0.05).

interaction, were observed on osmolality of shrimp hemolymph or
experimental waters (Table 4).
4. Discussion
There are numerous commercial shrimp farms producing shrimp
in low salinity water with an odd ionic profile. One of the problems
that have been noted is an inconsistent survival of PL in the spring,
when temperatures may not be optimal. This has brought up the
question of is there an interaction of ionic and temperature stress
that leads to reduced survival. The present study provided, for the
first time, insights into the simultaneous effects of water temperature
and Na +:K + ratio on growth and survival of L. vannamei. It was evi-
dent that, when reared at combinations of water temperatures of
26 °C or 30 °C, and Na +:K + ratios of 40:1, 80:1, or 120:1 in experi-
ment 1, the survival rate of shrimp was not compromised. However,
growth of shrimp was clearly affected by both factors. Significantly
greater final weight and weight gain of shrimp was observed at
30 °C, as compared to 26 °C, and at Na +:K + ratio of 40:1, as compared
to 120:1. Growth and survival of shrimp held at Na +:K + ratio of 40:1
were comparable to those of shrimp of the reference treatment of 4‰
artificial sea salt held at 26 or 30 °C. No differences in growth were
observed between shrimp held at Na +:K + ratios of 40:1 and 80:1.
These results are in agreement with previous observations of in-
creased growth of shrimp with temperature (Ponce-Palafox et al.,
1997; Wyban et al., 1995) and with the results of Zhu et al. (2004),
who calculated that a Na +:K + ratio between 40:1 and 43:1 was nec-
essary for optimal growth of juvenile L. vannamei reared at salinity
and temperature of 30‰ and 25 °C, respectively. Deviations from
this Na +:K + range in either direction, e.g., low (25.6:1) or high
(119.3:1, 187.4:1) Na +:K + ratios, negatively affected the perfor-
mance of L. vannamei. Likewise, Zhu et al. (2006) observed better
growth of L. vannamei within the Na +:K + range of 34.1:1 and
47.3:1, as compared to growth recorded at Na +:K + ratios of 25.6:1,
102.2:1, and 153.3:1 at a salinity of 30‰, but no effects of the Na +:
K + ratio were observed when shrimp were reared at 15‰. Our results
also agree with those of Roy et al. (2007b), who progressively added,
as in the present study, KCl to well water and obtained Na +:K + ratios
of 119:1, 69:1, 48:1, and 29:1 at a fixed temperature of 27.0 ± 0.5 °C.
They observed better growth performance of L. vannamei juveniles as
the aqueous Na +:K + ratio decreased from 119:1 to 29:1. Sowers et al.
(2006) showed that high Na +:K + ratios led to reduced hemolymph
K + levels in L. vannamei. Similarly, other studies have shown that
high Na +:K + ratios elicited decreased growth, survival, gill Na +/K +
ATPase activity, hemolymph osmolality, or increased ammonia toxicity
to penaeids and other crustaceans (Pan et al., 2006; Prangnell and
Fotedar, 2006; Romano and Zeng, 2007a, b, 2011; Tantulo and Fotedar,
2006).
Concurring with observations of depressed growth of shrimp at
low temperature (Ponce-Palafox et al., 1997; Wyban et al., 1995),
poor growth of shrimp was observed in experiment 2, which was
conducted at 20 and 24 °C. At these levels, temperature did not
cause differences in the growth performance of shrimp. In contrast,
significantly higher final weight and weight gain were recorded for
shrimp held at Na +:K + ratio of 40:1, as compared to 120:1. Further-
more, highly significant differences were observed in the survival
rate, greater also at Na +:K + ratio 40:1 than at 120:1, while no effects
of the Na +:K + ratio were observed on the survival rate at high tem-
perature in experiment 1. This is, to our knowledge, the first report
of a strong sensitivity of shrimp survival to the aqueous Na +:K +
ratio at low temperature. These results have implications for main-
taining adequate ionic culture conditions for shrimp that may be ex-
posed to descending temperature and reared in low salinity water.
Measurements of shrimp hemolymph osmolality, with means rang-
ing from 604.5 to 690.5 mmol kg − 1 (Table 4), were within the range of
values previously reported for L. vannamei (609.8–737.3 mmol kg− 1)
reared in low salinity well water (2.5–5.0‰) (Roy et al., 2007b, 2009)
and also are consistent with values obtained from this species when ex-
posed to salinities varying from 0.5 to 40‰ (Bückle et al., 2006; Sowers
et al., 2006). Taking into account that the K + deficient levels (or high
Na+:K + ratios) imposed in the present experiments affected growth
or survival of shrimp, but not the measurements of hemolymph osmo-
lality, the latter was not a sensitive indicator of disrupted osmoregula-
tion when working at a constant salinity. A similar phenomenon was
reported by Roy et al. (2007b) who did not detect differences in hemo-
lymph osmolality, but observed significant differences in growth and
survival of the same species after being exposed to different concentra-
tions of K+ (5 to 40 mg l− 1) and Mg2+ (10 to 160 mg l− 1) at a fixed sa-
linity of 4‰. Interestingly, Sowers et al. (2006) found no differences in
hemolymph osmolality, but showed statistically lower hemolymph K+
concentration in L. vannamei exposed for 7 days to a 2‰ mixed-ion solu-
tion, as compared to 2‰ artificial seawater. This and other measure-
ments could be used in future studies to ascertain effects of the ionic
composition, salinity, and other factors on osmoregulation of shrimp.
No significant effects of the Na +:K + ratio were detected on the
FCR at the levels tested. However, FCR was significantly affected by
temperature, with statistically lower values observed at 30 °C, as
compared to 26 °C in experiment 1, and also lower at 24 °C, as com-
pared to 20 °C in experiment 2. These results can be explained in
terms of greater feed consumption and assimilation by this poikilo-
thermic species at comparatively higher temperatures. At the same
time, because feed was offered to shrimp in moderate excess, they
 M. Perez-Velazquez et al. / Aquaculture 342–343 (2012) 13–17
may reflect greater amounts of uneaten feed at comparatively lower
temperatures.
Adequate culture conditions were provided for execution of both
experiments. The determined values for the Na +:K + ratio, tempera-
ture, and salinity of experimental waters were close to targeted
values. The mean concentration of dissolved oxygen in the water
was always high (≥6.2 mg l− 1); while the mean concentration of
TAN and nitrite-nitrogen, never exceeding values of 0.08 and
0.05 mg l− 1, respectively, were maintained below critical toxic levels
reported for L. vannamei in seawater (2.60–3.95 mg TAN l− 1 and
25.7 mg NO2–N l− 1) (Jiang et al., 1999; Lin and Chen, 2001, 2003) and
below 48-h lethal concentrations (LC50) reported for this species in
low salinity water of 10‰ (39.72 mg TAN l− 1 and 153.75 mg NO2–
N l− 1) (Schuler et al., 2010).
In summary, growth, but not survival of L. vannamei, was nega-
tively affected by high aqueous Na +:K + ratio (120:1, as compared
to 40:1) when reared at a salinity of 4‰ and temperatures of 26 °C
and 30 °C. Also, greater growth response of shrimp was observed
at 30 °C than at 26 °C. In the second trial, growth and survival of L.
vannamei were negatively affected by high Na +:K + ratio (120:1, as
compared to 40:1) when shrimp were reared at temperatures of
20 °C and 24 °C. Survival rate of shrimp was significantly higher at
24 °C than at 20 °C, but no differences in growth were observed.
These results demonstrate a strong sensitivity of L. vannamei survival
to the aqueous Na +:K + ratio at low temperature.
Acknowledgements
This research was supported by the National Sea Grant College
Program of the U.S. Department of Commerce's National Oceanic and
Atmospheric Administration under NOAA Grants NA06OAR4170078,
the Mississippi-Alabama Sea Grant Consortium, project number R/SP-20
and Auburn University. The mention of trademarks or proprietary
products does not constitute an endorsement of the product by Au-
burn University and does not imply its approval to the exclusion of
other products that may also be suitable. The authors would like to ex-
tend their gratitude to the Alabama Agricultural Experiment Station
and the National Oceanic and Atmospheric Administration, Grant
NOAA NAO60AR4170191. Dr. Martin Perez-Velazquez and Dr. Mayra
L. González-Félix were sponsored by the National Council for Research
and Technology (CONACYT), Mexico, Grants 93931 and 93934,
respectively.
References
Bückle, L.F., Barón, B., Hernández, M., 2006. Osmoregulatory capacity of the shrimp
Litopenaeus vannamei at different temperatures and salinities, and optimal culture
environment. Rev. Biol. Trop. 54, 745–753.
Clesceri, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination
of Water and Wastewater, 20th edition. American Public Health Association,
Washington, DC.
Davis, D.A., Saoud, I.P., Boyd, C.E., Rouse, D.B., 2005. Effects of potassium, magnesium,
and age on growth and survival of Litopenaeus vannamei post-larvae reared in in-
land low salinity well waters in west Alabama. Journal of the World Aquaculture
Society 36, 403–406.
FAO (Food and Agriculture Organization of the United Nations), 2009. The State of
World Fisheries and Aquaculture 2008. FAO Fisheries and Aquaculture Depart-
ment, FAO, Rome, Italy. 176 pp.
Flaherty, M., Szuster, B., Miller, P., 2000. Low salinity inland shrimp farming in Thailand.
Ambio 29 (3), 174–179.
17
Jiang, D.H., Lawrence, A.L., Neill, W.H., Grant, W.H., Gong, H., 1999. Lethal effect of
ammonia to postlarval Penaeus vannamei at two temperatures, 25 and 30 °C. Book
of Abstracts, American Aquaculture Society Annual Conference, Tampa, Florida, USA,
January 27–30, p. 77.
Lin, Y.C., Chen, J.C., 2001. Acute toxicity of ammonia on Litopenaeus vannamei Boone
juveniles at different salinity levels. Journal of Experimental Marine Biology and
Ecology 259, 109–119.
Lin, Y.C., Chen, J.C., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone)
juveniles at different salinity levels. Aquaculture 224, 193–201.
McGraw, J.W., Scarpa, J., 2003. Minimum environmental potassium for survival of
Pacific white shrimp Litopenaeus vannamei (Boone) in freshwater. Journal of Shell-
fish Research 22, 263–267.
McNevin, A.A., Boyd, C.E., Silapajarn, O., Silapajarn, K., 2004. Ionic supplementation of
pond waters for inland culture of marine shrimp. Journal of the World Aquaculture
Society 35, 460–467.
Pan, L.Q., Luan, Z.H., Jin, C.X., 2006. Effects of Na+/K+ and Mg2+/Ca2+ ratios in saline
groundwaters on Na+-K+-ATPase activity, survival and growth of Marsupenaeus
japonicus postlarvae. Aquaculture 261, 1396–1402.
Parsons, T.R., Maita, Y., Lalli, C.M., 1985. A Manual of Chemical and Biological Methods
for Seawater Analysis. Pergamon Press, New York, New York, USA.
Ponce-Palafox, J., Martinez-Palacios, C.A., Ross, L.G., 1997. The effects of salinity and
temperature on the growth and survival rates of juvenile white shrimp Penaeus
vannamei, Boone, 1931. Aquaculture 157, 107–115.
Prangnell, D.I., Fotedar, R., 2006. The growth and survival of western king prawns,
Penaeus latisulcatus Kishinouye, in potassium-fortified inland saline water. Aqua-
culture 259, 234–242.
Romano, N., Zeng, C., 2007a. Effects of potassium on nitrate mediated changes to osmo-
regulation in marine crabs. Aquatic Toxicology 85, 202–208.
Romano, N., Zeng, C., 2007b. Ontogenetic changes in tolerance to acute ammonia expo-
sure and associated histological gill alterations during early juvenile development
of the blue swimmer crab, Portunus pelagicus. Aquaculture 266, 246–254.
Romano, N., Zeng, C., 2011. Importance of balanced Na(+)/K(+) ratios for blue swim-
mer crabs, Portunus pelagicus, to cope with elevated ammonia-N and differences
between in vitro and in vivo gill Na(+)/K(+)-ATPase responses. Aquaculture
318, 154–161.
Roy, L.A., Davis, D.A., Saoud, I.P., Henry, R.P., 2007a. Supplementation of potassium,
magnesium, and sodium chloride in practical diets for the Pacific white shrimp,
Litopenaeus vannamei, reared in low salinity waters. Aquaculture Nutrition 13,
104–113.
Roy, L.A., Davis, D.A., Saoud, I.P., Henry, R.P., 2007b. Effects of varying levels of aqueous
potassium and magnesium on survival, growth, and respiration of the Pacific white
shrimp, Litopenaeus vannamei, reared in low salinity waters. Aquaculture 262,
461–469.
Roy, L.A., Davis, D.A., Nguyen, T.N., Saoud, I.P., 2009. Supplementation of chelated mag-
nesium to diets for Litopenaeus vannamei reared in low salinity waters of west
Alabama. Journal of the World Aquaculture Society 40, 248–254.
Roy, L.A., Davis, D.A., Saoud, I.P., Boyd, C.A., Harvey, J.P., Boyd, C.E., 2010. Shrimp culture
in inland low salinity waters. Reviews in Aquaculture 2, 191–208.
Saoud, I.P., Davis, D.A., Rouse, D.B., 2003. Suitability studies of inland well waters for
Litopenaeus vannamei culture. Aquaculture 217, 373–383.
Schuler, D.J., Boardman, G.D., Kuhn, D.D., 2010. Acute toxicity of ammonia and nitrite to
Pacific white shrimp, Litopenaeus vannamei, at low salinities. Journal of the World
Aquaculture Society 41, 438–446.
Solórzano, L., 1969. Determination of ammonia in natural waters by the phenolhypo-
chlorite method. Limnology and Oceanography 14, 799–801.
Sowers, A.D., Young, S.P., Grosell, M., Browdy, C.L., Tomasso, J.R., 2006. Hemolymph
osmolality and cation concentrations in Litopenaeus vannamei during exposure to
artificial sea salt or a mixed-ion solution: Relationship to potassium flux. Compar-
ative Biochemistry and Physiology, Part A 145, 176–180.
Tantulo, U., Fotedar, R., 2006. Comparison of growth, osmoregulatory capacity, ionic
regulation and organosomatic indices of black tiger prawn (Penaeus monodon, Fab-
ricius, 1798) juveniles reared in potassium fortified inland saline water and ocean
water at different salinities. Aquaculture 258, 594–605.
Wyban, J., Walsh, A.W., Godin, D.M., 1995. Temperature effects on growth, feeding rate
and feed conversion of the Pacific white shrimp (Penaeus vannamei). Aquaculture
138, 267–279.
Zhu, C., Dong, S., Wang, F., Huang, G., 2004. Effects of Na/K ratio in seawater on growth
and energy budget of juvenile Litopenaeus vannamei. Aquaculture 234, 485–496.
Zhu, C., Dong, S., Wang, F., 2006. The interaction of salinity and Na/K ratio in seawater on
growth, nutrient retention and food conversion of juvenile Litopenaeus vannamei.
Journal of Shellfish Research 25, 107–112.