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Growth and Lipid Metabolism of the Pacific White Shrimp Litopenaeus vannameiat Different SalinitiesAuthor(s): Ke Chen, Erchao Li, Lei Gan, Xiaodan Wang, Chang Xu, Heizhao Lin, Jian G. Qin and LiqiaoChenSource: Journal of Shellfish Research, 33(3):825-832. 2014.Published By: National Shellfisheries AssociationDOI: http://dx.doi.org/10.2983/035.033.0317URL: http://www.bioone.org/doi/full/10.2983/035.033.0317

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GROWTH AND LIPID METABOLISM OF THE PACIFIC WHITE SHRIMP

LITOPENAEUS VANNAMEI AT DIFFERENT SALINITIES

KE CHEN,1 ERCHAO LI,1* LEI GAN,1 XIAODAN WANG,1 CHANG XU,1 HEIZHAO LIN,2

JIAN G. QIN3 AND LIQIAO CHEN1*1School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China;2Shenzhen Base of South China Sea Fisheries Research Institute, 83 Dadui Village, Shenzhen 518121,China; 3School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide SA 5001, Australia

ABSTRACT Juvenile white shrimp Litopenaeus vannamei (1.98 ± 0.28 g) were fed a commercial diet for 8 wk in triplicate to

investigate growth and lipidmetabolism at 3 salinities (3, 17, and 30). Shrimpweight gain and survival at 3 were significantly less than

that at 17 and 30. No differences were found in whole-body proximate composition. Linolenic acid (18:3[n-3]) and (n-3) long-chain

unsaturated fatty acid levels in the hepatopancreas, and n-3 long-chain polyunsaturated fatty acid level, especially eicosapentaenoic

acid (EPA; C20:5[n-3]) and docosahexaenoic acid (DHA; C22:6[n-3]) inmuscle at 3 were significantly greater than at other salinities.

Fatty acid synthase, hormone sensitive lipase, lipoprotein lipase, adipose triacylglycerol lipase, acyl-CoA, diacylglycerol

acyltransferase 2, elongase of very long-chain fatty acid 6, and D5 and D6 fatty acid desaturase activity was detected and showed

a negative trend with an increase of salinity, and no significant differences were found among salinity groups (P > 0.05). The results

indicate that the low salinity of 3 decreases the growth of L. vannamei. Although L. vannamei could not synthesize either DHA or

EPA de novo, it possibly has the potential ability to convert linolenic acid to DHA and EPA regardless of salinity. However, the

factors influencing this ability remain unknown and need further study.

KEY WORDS: shrimp, Litopenaeus vannamei, salinity, lipid metabolism, fatty acids, osmoregulation

INTRODUCTION

Salinity is one of the most important factors that influencethe physiological status of aquatic animals. Previous studies

have shown that aquatic euryhaline animals depend on energetic

reorganization to cope with ambient salinity changes (Tseng &

Hwang 2008), showing that the osmoregulation of aquatic

animals is an energy–cost (20%–50% of total metabolic energy)

process to maintain intracellular and extracellular osmotic

equilibrium (Evans et al. 2005, Tseng&Hwang 2008). Therefore,studies on the energy use of aquatic animals under salinity stress

are significant in understanding the mechanism of osmoregula-

tion for aquatic animals.

Of the major energy-yield nutrients, lipids are of the greatestenergy density, and many fatty acids from lipid metabolism are

essential for normal growth and development of aquatic animals.

In addition, phospholipids and glycolipids are indispensable

components of the cell membrane and can affect osmoregulatory

capacity by changing the contents in the cell membrane (Li et al.

2006, Tseng & Hwang 2008). Therefore, lipid metabolism mustfunction correctly when responding to ambient salinity changes

in aquatic animals by altering the permeability of the cell

membrane (Tseng & Hwang 2008).

The Pacific white shrimp Litopenaeus vannamei is 1 of themost important shrimp species cultured worldwide (Hu et al.

2004). During the past decade, the expansion of inland saline

water farming, coupled with the wide range of salinity tolerance

from 0.5–50 (Samocha et al. 2002), makes L. vannamei an

attractive species in aquaculture at low salinity inmany countries

(Saoud et al. 2003, Cheng et al. 2006). Previous studies on theisosmotic point of L. vannamei are controversial. The white

shrimp L. vannamei obtained optimal growth at salinity ranges

of 15–30 (Bray et al. 1994, Ponce-Palafox et al. 1997). Huang

et al. (2004) found that 20 was the best salinity for the growth

performance of L. vannamei. Although greater production can

be obtained from low-salinity (<5) farming of white shrimp,

many problems have been found, such as reduction of growth

and survival (Ponce-Palafox et al. 1997, Rosas et al. 2001,

Palacios et al. 2004a, Li et al. 2007) and low stress resistance (Li

et al. 2007, Li et al. 2008). Therefore, it is necessary to explore

a practical way to improve the performance of white shrimp

farmed at low salinities. The L. vannamei in either a hypo- or

hypersaline condition has greater amylase activity and a lower

number of R cells, which is for nutrient reserve in the

hepatopancreas (the main site for lipogenesis) of shrimp,

compared with shrimp at normal seawater salinity (Li et al.

2008, Al-Mohanna &Nott 1989), indicating that lipid nutrition

may have an important role in improving osmoregulation

capacity. So far, although many studies have been conducted on

various aspects of lipid nutrition in L. vannamei (Gonzalez-Felix

et al. 2002a, Gonzalez-Felix et al. 2002b, Zhu et al. 2010, Niu et al.

2011, Ju et al. 2012), information on lipid metabolism at different

salinities is still limited, and a successful method for nutrient

modulation to improve the physiological status of L. vannamei

at low salinity has not yet been reported because of the poor

understanding of the lipid mechanisms in L. vannamei under

salinity stress.

Therefore, this study aims to understand the salinity-dependentgrowth and lipid metabolism of white shrimp by analyzing

fatty acid composition of different tissues, enzyme activity

related to fatty acid catabolism (hormone sensitive lipase

[HSL], lipoprotein lipase [LPL], adipose triacylglycerol lipase

[ATGL]) and synthesis (elongase of very long chain fatty acids 6

[ELOVL6], fatty acid synthase [FAS] complex, D5 fatty acid

desaturase [D5FAD], and D6 fatty acid desaturase [D6FAD]),

and the contents of metabolic products ofLitopenaeus vannamei

at 3 salinities. It is hoped that the findings from this study

*Corresponding authors. E-mail: ecli@bio.ecnu.edu.cn (E. Li) and

lqchen@bio.ecnu.edu.cn (L. Chen)

DOI: 10.2983/035.033.0317

Journal of Shellfish Research, Vol. 33, No. 3, 825–832, 2014.

825

provide insight to understanding the mechanism of osmoregu-lation in L. vannamei at different salinities, which should be very

instructive, especially from the perspective of lipid metabolism.

MATERIALS AND METHODS

Experimental Animals, Design, and Facilities

Juvenile white shrimpwere obtained from the Shenzhen baseof South China Sea Fisheries Research Institute, Shenzhen,

China, and were stocked in 9 tanks at a density of 40 shrimp pertank (500 L) at a salinity of 17 for 1 wk. Then, shrimp in 2 of 3tanks were acclimated to 3 and 30, respectively, by a daily

change of 2 prior to the start of the 8-wk experiment. During theacclimation and experimental periods, shrimp were fed 3 timesdaily at 0800 HR, 1600 HR, and 2200 HR with a commercial dietcontaining 10% moisture, 40% crude protein, 8% crude lipid,

12% ash, and 30% carbohydrates, and a digestible energy of16.7 kJ/g. The known fatty acid and main ionic compositionsare shown in Table 1. Based on the amount of food remaining

the following day, daily rations were adjusted to a feeding levelslightly more than satiation. The uneaten food was removeddaily with a siphon tube. The photoperiodwas 12 h light and 12 h

dark. Seawater was pumped from the Dayawan Coast inShenzhen and filtered through an activated carbon cartridgefor at least 3 days before entering the culture system. Tap water

was aerated before being added to the tank to adjust the salinitylevel. During the experiment, water was exchanged once dailywith one third of the tank volume. Water-quality parameterswere monitored 2–3 times a week throughout the feeding trial,

and were maintained at a pH of 7.5–7.9, a temperature of 26–28�C, a dissolved oxygen level of 4.8–6.4 mg/L, and a totalammonia nitrogen level of less than 0.02 mg/L during the trial.

Some mineral ion information for all salinity treatments isshown in Table 2.

At the end of the experiment, shrimp were deprived of food

for 24 h before being bulk weighed and accounted. Five shrimpat intermolt stage C in each tank were used for the bodycomposition analysis. Another 10 shrimp at intermolt stage C ineach tank were dissected to obtain muscle, hepatopancreas, and

gill, and were stored at –80�C for biochemical analysis andenzyme essay. Weight gain and survival were calculated to assessthe growth performance of shrimp. Weight gain (percent) was

calculated as follows:

Weight gain ¼ Wt �W0

W03 100;

where W0 is the initial weight and Wt is the final weight.

Survival rate (percent) was calculated as follows:

Survival rate ¼ Final shrimp number

Initial shrimp number3 100:

Whole-Body Proximate Compositions

Each experimental sample consisted of five randomly col-

lected shrimp from each tank, and was analyzed in triplicate forproximate composition following the standard methods (Associ-ation of Official Analytical Chemists 2000). Moisture was de-

termined by oven drying at 105�C to a constant weight. Samplesused for dry matter were digested with nitric acid and incineratedin a muffle furnace at 600�C overnight to determine ash content.

Protein wasmeasured by the combustionmethod using an FP-528nitrogen analyzer (Leco). Lipid content was determined by theether extraction method using the 2055 Soxtec system (Foss,

Sweden).

Fatty Acid Analysis

Total lipids of the gill, muscle, and hepatopancreas wereextracted in triplicate using chloroform:methanol (2:1, v/v)(Folch et al. 1957). The saponifiable lipids were converted to

their methyl esters using the standard boron trifluoride–methanolmethod (Morrison & Smith 1964). Fatty acid methyl esters wereanalyzed using an Agilent 6890 gas chromatograph (AgilentTechnologies) equipped with a flame ionization detector and an

SP-2560 fused silica capillary column (100 m, 0.25 mm i.d., and0.20-mm film thickness). Injector and detector temperatures were

TABLE 1.

Fatty acid composition (percent by weight of total fatty acids)and minerals (percent of diet) of the commercial diet used in

this study.

Feed content

Fatty acid

14:0 1.89

16:0 16.63

18:0 4.36

24:0 0.51P

SFA 24.34

16:1 2.30

18:1(n-9)c 26.21

20:1n-9 1.43

24:1n9 0.23PMUFA 30.60

18:2(n-6)c 28.16

18:3(n-3) 2.72

20:5(n-3) 4.16

22:6(n-3) 7.23P

PUFA 45.07

(n-3) 15.31

(n-6) 29.75

(n-3/n-6) 0.51

Cations

Ca++ 0.74

Na+ 0.44

K+ 0.91

Mg++ 0.28

MUFA, monounsaturated fatty acid; SFA, saturated fatty acid.

TABLE 2.

Contents of some keyminerals in water used in this experiment

(grams per kilogram).

Salinity

3 17 30

Sodium 0.35 4.30 9.28

Magnesium 0.04 0.52 1.12

Calcium 0.01 0.16 0.35

Potassium 0.01 0.16 0.33

Chloride 0.63 7.73 16.70

CHEN ET AL.826

270�C and 280�C, respectively. The column temperature was heldat 120�C for 5min then programmed to increase at 3�C/min up to

240�C, where it was maintained for 20 min. Carrier gas washelium (2 mL/min), and the split ratio was 30:1. Identification offatty acids was carried out by comparing the sample fatty acidmethyl ester peak relative retention times with those obtained for

the Sigma-Aldrich (St. Louis, MO) standards. The concentrationof individual fatty acids was calculated and expressed as the masspercentage of total identified fatty acids.

Enzyme Activity, Lipoprotein, Total Cholesterol, and Triglyceride

Content Determination

Activity assays of all enzymes and lipoproteins tested in thisstudy were determined using an enzyme-linked immunosorbent

assay kit (Xinyu, Shanghai) according to protocols, with specificantibodies corresponding to the parameter. Enzyme activity ofFAS, HSL, LPL, ATGL, acyl-CoA, diacylglycerol acyltransfer-

ase 2 (DGAT2), ELOVL6, D5FAD, and D6FAD are expressedas units per gram protein and were detected in the hepatopan-creas. The protein content of each sample was determined using aUV-Vis spectrophotometer (NanoDrop 2000; Thermo Scientific).

Total triacylglycerol (TAG) and total cholesterol are expressed asnanomoles per liter hemolymph. The contents of high-densitylipoprotein (HDL) and low-density lipoprotein (LDL) are

expressed as micromoles per liter hemolymph. The content ofvery low-density lipoprotein (VLDL) is expressed as microgramsper milliliter hemolymph.

Statistical Analyses

Data are expressed as mean ± SE and were subjected to1-way analysis of variance (SPSS for Windows, version 11.5) to

determine significant differences among treatments. If a signif-icant difference was identified, differences between means werecompared using Duncan�s multiple range test. The level of

significance was set at P < 0.05.

RESULTS

Weight gain of shrimp at 3 was significantly less than that ofshrimp at 17 and 30 (Table 3). There were no differences in shrimpsurvival and weight gain between salinities of 17 and 30. Whole-

body proximate composition (protein, lipid, ash, and moisture)was not affected by salinity (Table 3).

Table 4 shows the fatty acid composition in muscle, gill, andhepatopancreas of the shrimp at different salinities. No differ-

ences were found in hepatopancreas 18:2(n-6) (LA), eicosapen-taenoic acid (EPA), docosahexaenoic acid (DHA),

Psaturated

fatty acid (SFA),P

polyunsaturated fatty acid (PUFA) and

P(n-6). Shrimp hepatopancreas 18:3(n-3) (linolenic acid

[LNA]) at 3 was significantly greater than that of shrimp at

17 and 30. BothP

(n-3) andP

monounsaturated fatty acid(MUFA) of shrimp at 3 were more than those at othersalinities, and

PMUFAof shrimp at 3 was significantly greater

than that of shrimp at 30. ShrimpP

(n-3) at 3 was significantly

more than that of shrimp at 17. With regard to muscle tissue,salinity did not affect LNA,

PMUFA, and

PPUFA contents.

The LA andP

(n-6) of shrimp at 30 were significantly more

than those at the other 2 salinities, and those of shrimp at17 were significantly more than shrimp at 3. However, EPAhad the opposite trend compared with LA and

P(n-6). The

DHA of shrimp at 3 was significantly more than that at 17, andP(n-3) at 3 was more than that at 30. For gill, a significant

difference was found only inP

SFA content, which was lowerat 3 than at 30.

Table 5 shows lipid metabolites of white shrimp at differentsalinities. No differences were found in total cholesterol, LDL,and VLDL contents among all treatments, but values of these

parameters at 3 were the lowest. The HDL content of shrimp at3 was lowest and differed from that of shrimp at 30. Totaltriacylglycerol content of shrimp at 17 was lowest and differed

from that of shrimp at 30, but no significant difference between3 and 30 was found.

No difference was found in activities of FAS, HSL, LPL,

ATGL, DGAT2, D5FAD, and D6FAD (Table 6; (P > 0.05). Atthe 3 salinity, ELOVL6, HSL, D5FAD, and D6FAD werelowest (P > 0.05), and all enzyme activity values tested inshrimp at 3 were less than in shrimp at 30 (P > 0.05).

DISCUSSION

In the current study, Litopenaeus vannamei at the mediumsalinity of 17 (in the range of salinity for the optimal growth ofL. vannamei) had the best growth performance compared withwhite shrimp at either low salinity or high salinity. Similar

reports have demonstrated that the optimal salinity ofL. vannamei ranges from 15–30 for optimal growth performance(Bray et al. 1994, Ponce-Palafox et al. 1997). Similarly, the

optimal salinity for the growth of L. vannamei was found to bearound 20 (Huang et al. 2004), and much higher or lowerambient salinity would affect shrimp growth negatively (Li

et al. 2007). This study further confirms that low salinity candecrease growth performance ofL. vannamei, and there is a needto solve this problem and to improve the growth performance ofL. vannamei at low inland salinities.

When challenged with ambient salinity stress, a high pro-portion of the metabolic energy budget (20%–50%) is neededfor osmoregulation, leading to low energy available for growth

TABLE 3.

Growth, survival, and whole-body proximate composition (percent live weight) of white shrimp at different salinities (percent).

Salinity Survival Weight gain Moisture Protein Lipid Ash

30 93.33 ± 1.67 652.87 ± 24.27a 73.61 ± 1.56 18.04 ± 0.83 1.36 ± 0.045 2.83 ± 0.094

17 88.33 ± 3.00 645.89 ± 15.61a 72.81 ± 1.66 19.31 ± 0.98 1.37 ± 0.054 2.92 ± 0.095

3 65.00 ± 9.61 548.34 ± 21.95b 72.94 ± 0.22 19.38 ± 0.18 1.37 ± 0.069 2.62 ± 0.067

P value 0.07 0.02 0.90 0.42 0.99 0.12

Values within the same column with different letters represent significant difference (P < 0.05).

LIPID METABOLISM OF L. VANNAMEI AT DIFFERENT SALINITIES 827

in many fish, such as Spams sarba (Woo & Kelly 1995), Salmo

salar (Handeland et al. 1998), Anarhichas minor (Foss et al.2001), Scophthalmus maximus (Imsland et al. 2001), Pagrusauratus (Fielder et al. 2005), and Sparus aurata (Laiz-Carrion

et al. 2005). However, previous research in crustacean specieshas showed only that energy expenditure increases whenambient salinity changes, including Penaeus setiferus (Rosas

et al. 1999), Penaeus latisculcatus (Sang & Fotedar 2004),Marsupenaeus japonicas (Setiarto et al. 2004), and Litopenaeusvannamei (Silvia et al. 2004), but a specific discussion of theenergy budget used for osmoregulation is not reported in these

studies. The current study, there was an extra energy expendi-ture during osmoregulation that resulted in slow body weightgain at a salinity of 3 (Rosas et al. 1999, Gomez-Jimenez et al.

2004, Setiarto et al. 2004, Li et al. 2007).The white shrimp Litopenaeus vannamei is a euryhaline

decapod species and is generally considered an osmoconformer

(Dall et al. 1990, Kirschner 1991). To maintain homeostasis byosmoregulation, shrimp require energy from nutrients viaa compensatory process, and lipids play significant roles in thisprocess (Lemos et al. 2001, Luvizotto-Santos et al. 2003, Sang&

Fotedar 2004, Palacios et al. 2004b). Therefore, it is reasonable

TABLE4.

Fattyacidcomposition(percentbyweightoftotalfattyacids)in

muscle,gill,andhepatopancreasofwhiteshrimpgrownatdifferentsalinities(percent).

Hepatopancreas

Muscle

Gill

30

17

3Pvalue

30

17

3Pvalue

30

17

3Pvalue

18:2(n-6)

26.87

±0.38

26.10

±0.66

25.91

±0.14

0.34

14.72

±0.19c

13.68

±0.07b

12.71

±0.31a

0.01

14.33

±0.94

13.88

±0.31

13.69

±0.28

0.25

18:3(n-3)

0.17

±0.02a

0.29

±0.07a

0.64

±0.10b

0.01

0.52

±0.03

0.46

±0.02

0.46

±0.03

0.27

0.71

±0.01

0.71

±0.43

0.71

±0.40

0.99

EPA

2.72

±0.11

2.69

±0.07

2.97

±0.07

0.12

14.52

±0.17a

15.36

±0.23b

16.26

±0.20c

0.01

12.69

±0.25

13.22

±0.58

13.32

±0.36

0.55

DHA

6.90

±0.11

6.63

±0.18

6.84

±0.17

0.48

9.27

±0.08

8.88

±0.16

9.61

±0.09

0.01

12.62

±0.31

13.29

±0.26

13.45

±0.36

0.21

PSFA

23.08

±0.06

22.69

±0.75

22.63

±0.23

0.76

32.24

±0.20a

33.24

±0.36b

33.57

±0.24b

0.03

35.02

±0.12b

34.20

±0.24ab

33.80

±0.32a

0.03

PMUFA

35.85

±0.45

36.55

±0.13

36.90

±0.15

0.10

20.23

±0.15

19.79

±0.19

19.75

±0.21

0.19

19.51

±0.17

19.62

±0.64

19.86

±0.42

0.86

PPUFA

41.08

±0.42

40.76

±0.88

40.47

±0.38

0.79

47.53

±0.29

46.97

±0.49

46.68

±0.22

0.30

45.47

±0.26

46.18

±0.48

46.34

±0.17

0.22

P(n-3)

10.95

±0.17ab

10.74

±0.24a

11.67

±0.33b

0.01

30.47

±0.30

31.11

±0.38

32.05

±0.46

0.07

29.59

±0.42

30.68

±0.76

31.07

±0.52

0.25

P(n-6)

28.40

±0.38

28.17

±0.90

27.24

±0.08

0.37

14.720.19c

13.68

±0.17b

12.71

±0.31a

0.01

14.52

±0.12

14.09

±0.34

13.85

±0.12

0.31

(n-3)/(n-6)

0.4

0.4

0.4

2.1

2.3

2.5

2.0

2.1

2.2

Values

inthesamerow

withdifferentlettersrepresentasignificantdifference

(P<0.05).DHA,docosahexaenoic

acid;EPA,eicosapentaenoic

acid;MUFA,monounsaturatedfattyacid;PUFA,

polyunsaturatedfattyacid;SFA,saturatedfattyacid.

TABLE 5.

Lipid metabolism related to biochemical parameters inhemolymph of white shrimp grown at different salinities.

30 17 3 P value

TAG (nmol/L) 16.13 ± 0.52a 13.61 ± 0.09b 15.44 ± 0.40a 0.01

TC (nmol/L) 1.42 ± 0.05 1.39 ± 0.08 1.25 ± 0.03 0.16

HDL (mmol/L) 8.47 ± 0.22b 6.68 ± 0.25a 6.37 ± 0.06a 0.01

LDL (mmol/L) 8.08 ± 0.20 8.93 ± 0.97 6.78 ± 0.34 0.11

VLDL (mg/mL) 5.79 ± 0.25 5.78 ± 0.31 5.42 ± 0.20 0.56

Values in the same row with different letters represent a significant

difference (P < 0.05). HDL, high-density lipoprotein; LDL, low-density

lipoprotein; TAG, total triacylglycerol; TC, total cholesterol; VLDL,

very low-density lipoprotein.

TABLE 6.

Lipid metabolism related to enzyme activities (in units per

gram protein) in hepatopancreas of white shrimp grown at

different salinities.

30 17 3 P value

ATGL 2.22 ± 0.42 1.55 ± 0.22 1.73 ± 0.15 0.312

HSL 1.17 ± 0.19 0.67 ± 0.10 0.96 ± 0.20 0.184

LPL 0.35 ± 0.06 0.23 ± 0.03 0.30 ± 0.05 0.250

FAS 1.73 ± 0.35 1.15 ± 0.21 1.17 ± 0.10 0.233

DGAT2 2.87 ± 0.50 2.24 ± 0.42 2.06 ± 0.16 0.376

ELOVL6 0.22 ± 0.04 0.16 ± 0.02 0.16 ± 0.01 0.169

D6FAD 0.95 ± 0.12 0.66 ± 0.09 0.64 ± 0.04 0.081

D5FAD 1.07 ± 0.17 0.75 ± 0.10 0.80 ± 0.04 0.194

D5FAD, D5 fatty acid desaturase; D6FAD, D6 fatty acid desaturase;

ATGL, adipose triacylglycerol lipase; DGAT2, diacylglycerol acyltrans-

ferase 2; ELOVL6, elongase of very long chain fatty acids 6; FAS, fatty

acid synthase; HSL, hormone sensitive lipase; LPL, lipoprotein lipase.

CHEN ET AL.828

to assume that sufficient energy provided from lipid metabo-lism can improve osmoregulation efficiency, survival, and

growth of cultured shrimp in a low-salinity condition. In thecurrent study, ATGL, LPL, and HSL activities of shrimp atboth 3 and 30 were more than those at 17, indicating that lipidmobilization has increased, because ATGL (TAG hydrolysis),

LPL, and HSL are crucial enzymes in lipid mobilization byperforming the first step in hydrolyzing TAG to generatediacylglycerol and free fatty acid (Osuga et al. 2000, Jenkins

et al. 2004, Villena et al. 2004, Zimmermann et al. 2004). BothLPL and HSL are factors in lipolysis. Lipoprotein lipase isa ‘‘gatekeeper’’ for fatty acid uptake (Greenwood 1985), and

HSL as the rate-limiting enzyme is thought to break down TAGfunctionally into diacylglycerol and then into monoacylglycerol(Holm et al. 2000, Lampidonis et al. 2011).

In addition, we observed that lipid synthesis of shrimp at

both 3 and 30 was slightly enhanced with increased FASactivity. Total triacylglycerol of shrimp at both 3 and 30 weresignificantly greater than at the optimal salinity of 17 (Li et al.

2007). Fatty acid synthase and DGAT2 are the key enzymesrelated to lipid synthesis. Fatty acid synthase is a complexmultifunctional enzyme consisting of a protein with 7 catalytic

domains and acts on energy homeostasis by catalyzing thesynthesis of myristate, palmitate, stearate, and long-chainSFAs (Chirala & Wakil 2004). Diacylglycerol acyltransferase

2 can catalyze the final step in TAG biosynthesis by convertingacyl-CoA and DAG in TAG (La Russa et al. 2012, Gong et al.2013). Therefore, this finding indicates that both high andlow salinity can enhance lipid synthesis and stimulate lipolysis

capacity.Lipoproteins function in the transportation of lipids, in-

cluding PUFAs, that cannot be synthesized de novo andmust be

obtained from the diet (Teshima & Kanazawa, 1971). In thecurrent study, HDL, LDL, and VLDL shrimp contents at 3were less than those at 17, indicating that more lipids are used

during the process of b-oxidation to supply enough energy forosmoregulation. In addition, among lipids, cholesterol maydecrease gill permeability via increasing membrane stability(Coutteau et al. 1996). In the current study, total cholesterol in

hemolymph at low salinity was less than in the other 2 groups,and it could likely be used in synthesizing cell membrane ofother tissues to improve permeability for regulating osmotic

stress.Previous studies have shown that an (n-3) PUFA-rich diet

can improve resistance to osmotic shock in aquatic animals

because (n-3) HUFAs, especially DHA, are incorporatedprimarily in cell membranes and can increase membranepermeability, and hence their fluidity (Martins et al. 2006, Sui

et al. 2007). Free fatty acids, especially long-chain PUFAs, havethe potential to modulate fatty acid composition on the gillmembrane and thus increase enzymatic efficiency (Palacioset al. 2004b, Hurtado et al. 2007). The modification of fatty

acid composition in the gills with higher levels of (n-3) PUFAscan result in a larger gill area to enhance the osmoregulatorycapacity of shrimp at low salinities, and increase survival

(Palacios et al. 2004a). In the current study, in gill, muscle,and hepatopancreas,

P(n-3) long chain-PUFAs—andDHA, in

particular—at 3 were greater than those in the other 2 groups

(30 and 17), especiallyP

(n-3) long-chain PUFAs in hepato-pancreas andDHA inmuscle, indicating that white shrimp needmore (n-3) long-chain PUFAs at 3. This is in agreement with the

finding that a greater DHA content results in better tolerance tolow salinity in Chinese mitten crab (Eriocheir sinensis) larvae

(Sui et al. 2007).The pathways from LNA to EPA and DHA involve

desaturation at the D6 and D5 positions of the carbon chain,and an intermediate-chain elongation step. However, there is

wide variation among aquatic animals in their ability tosynthesize HUFAs (Sargent et al. 2002). Shrimp have a limitedability to synthesize (n-6) and (n-3) families of fatty acids

de novo, including LA and LNA (Suprayudi et al. 2004).Marine shrimp also have a limited ability to elongate anddesaturate LNA toHUFAs, such as 20:4(n-6), EPA, andDHA

(Kayama et al. 1980). Because of this, long-chain PUFAs suchas fish oil have been added to the diets of marine shrimp. Incontrast to marine species, freshwater species have a lowerrequirement for (n-3) HUFAs and a greater capacity to

elongate and desaturate PUFAs from shorter chain fatty acidssuch as 18:3(n-3) (Sargent et al. 1999). In the current study,ELOVL6, D6FAD, and D5FAD activity was detected in

Litopenaeus vannamei hepatopancreas, and this enzyme activ-ity tended to increase at low salinity. It was also noted that18:3(n-3) was significantly greater in shrimp hepatopancreas

at 3. In shrimp muscle and gill, both DHA andP

(n-3) long-chain PUFAs at 3 were increased compared with the other2 salinities. These findings, including the relevant enzyme

activity and DHA and EPA accumulation (4.5%–6%) inmuscle and hepatopancreas of shrimp fed diets containingextremely low or no DHA and EPA (<0.1%), together with thefindings of the key genes in the process for lc-HUFA synthesis

(K. Chen, E. Li, and L. Chen, unpubl. data) could indicatethat, as a marine species, L. vannamei has an ability tosynthesize both DHA and EPA from LNA, and ambient low

salinity may improve its ability to synthesize more long-chainPUFAs, especially EPA and DHA, to reduce stress at lowsalinities.

In summary, Litopenaeus vannamei showed a series ofphysiological responses to ambient salinity challenges(Fig. 1). Gills responded first to osmotic stress as the pri-mary organ for osmoregulation (Pequeux 1995), and SSFAwas used through b-oxidation to supply enough energy forosmoregulation (Deering et al. 1997), resulting in low SSFAin gills, because osmoregulation needs more energy to main-

tain intracellular and extracellular osmotic equilibrium(Evans et al. 2005, Tseng & Hwang 2008). However, aquaticanimals need to produce sufficient energy to cope with long-

term stress (not stress limited to the scope of 96 h). Totaltriacylglycerol, HSL, and ATGL activity was increased inhepatopancreas, which is the main lipid reserve, lipid metabo-

lism, and fatty acid synthesis site in crustaceans (Boer et al. 2007),and more fatty acids were hydrolyzed from monoglycerol,diacylglycerol, and TAG. Those fatty acids were transferred tothe gill by lipoprotein to meet the energy requirement for

osmoregulation.Last, 18:3(n-3) and (n-3) long-chain PUFA levels increased

in gill, hepatopancreas, and muscle under either hypo- or

hyperosmotic stress to limit the salinity impact on Litopenaeusvannamei. During this long-term stress, ELOVL6, D5FAD, and6FAD activity was inhibited as a result of (n-3) long-chain

PUFA accumulation in the organism (feedback regulation). Inaddition, (n-3) series releasing in all tissues would havepositive effects during ambient osmotic stress, as reported in

LIPID METABOLISM OF L. VANNAMEI AT DIFFERENT SALINITIES 829

other aquatic animals (Palacios et al. 2004a, Martins et al. 2006,Sui et al. 2007). Detained function of (n-3) series in osmoticregulation of aquatic animals needs further investigation.

ACKNOWLEDGMENTS

This research was supported by grants from the NationalNatural Science Foundation of China (nos. 31472291 and31172422), the Special Fund for Agro-scientific Research in

the Public Interest (nos. 201003020 and 201203065), the Na-tional ‘‘Twelfth Five-Year’’ Plan for Science & Technology

Support (no. 2012BAD25B03), the National Basic Research

Program (973Program, no. 2014CB138603), and the Shanghai

University Knowledge Service Platform Shanghai Ocean Uni-

versity Aquatic Animal Breeding Center (no. ZF1206), and in

part by the E-Institute of Shanghai Municipal Education

Commission (no. E03009) and the ECNU innovation fund.

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