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Journal of Experimental Marine Biology and Ecology253 (2000) 193–209

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Effects of temperature and salinity on nitrogenous excretionby Litopenaeus vannamei juveniles

a b a*Dong-Huo Jiang , Addison L. Lawrence , William H. Neill , Hui GongaShrimp Mariculture Project, Texas Agricultural Experiment Station, Texas A&M University System,

1300 Port Street, Port Aransas, TX 78373, USAbDepartment of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA

Received 15 February 2000; received in revised form 27 June 2000; accepted 11 July 2000

Abstract

Excretion rates of ammonia-N, nitrite-N, nitrate-N, and dissolved organic nitrogen (DON) forjuvenile Litopenaeus vannamei (3.8560.83 g) were quantified in response to nine differentcombinations of temperature (24, 28, and 328C) and salinity (10, 25, and 40 ppt) under laboratoryconditions. Results indicated that L. vannamei is ammonotelic, with ammonia-N accounting for61.9–84.3% of total nitrogen (TN) excretion. There were significant effects of temperature andsalinity, but no significant interaction between them, on ammonia-N excretion rate (R ). RAN AN

increased with increasing temperature, over the interval 24–328C. R was lower at 25 ppt than atAN

10 and 40 ppt, at all temperatures. DON excretion rate (R ) was not significantly influenced byDON21 21either temperature or salinity; the overall mean R was about 5.24 mg-N g h . However,DON

the percentages of DON in TN (P ) varied from 15.4 to 36.4% under the various temperature–DON

salinity combinations. P at 28 and 328C was significantly lower than at 248C, and P at 10DON DON

ppt was significantly lower than at 25 and 40 ppt. Only very small amounts of nitrogen wereexcreted by L. vannamei as nitrite-N and nitrate-N. 2000 Elsevier Science B.V. All rightsreserved.

Keywords: Litopenaeus vannamei; Nitrogenous excretion; Salinity; Temperature

1. Introduction

The white shrimp, Litopenaeus vannamei Boone, is a tropical species with its natural

*Corresponding author. Present address: Arizona Mariculture Associates, 50621 Agua Caliente Road,Dateland, AZ 85333, USA. Tel.: 11-520-454-2364; fax: 11-512-454-2364.

E-mail address: [email protected] (D.-H. Jiang).

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 00 )00259-8

194 D.-H. Jiang et al. / J. Exp. Mar. Biol. Ecol. 253 (2000) 193 –209

distribution in the Eastern Pacific Ocean, off Northern Mexico to Northern Peru,between the 208C isotherms (Holthuis, 1980). L. vannamei is the dominant penaeidspecies currently cultivated in the Western Hemisphere. Temperature and salinity are twovery important environmental factors in the culture of this and other shrimp species. Theoptimal temperature for the growth of L. vannamei has been reported to be size-specific,around 28–308C for postlarvae (Ponce-Palafox et al., 1997), greater than 308C for smalljuveniles ( , 5 g) and about 278C for subadults (Wyban et al., 1995). It is known that L.vannamei can tolerate a wide salinity range from brackish water of 1–2 ppt tohypersaline water of 50 ppt (Pante, 1990; Stern et al., 1990). Boyd (1989) consideredsalinity of 15–25 ppt to be ideal for L. vannamei culture. But, in view of inconsistenciesin published information regarding salinity effects on shrimp survival and growth, theoptimum salinity for L. vannamei is still not conclusive. Significant effects oftemperature and salinity have been reported on survival (Ogle et al., 1992), moltingfrequency (Pante, 1990), oxygen consumption (Villarreal et al., 1994; Martinez-Pakacioset al., 1996), and growth of L. vannamei (Huang, 1983; Wyban et al., 1995).

In decapod crustaceans, nitrogen is mainly excreted as ammonia (60–70%), withrelatively small amounts of amino acids, urea, and uric acid (Regnault, 1987). Becauseammonotelism is so dominant in aquatic gill-breathers, ammonia excretion rate typicallyhas been used to evaluate the effects of various factors on total nitrogen excretion bycrustaceans. But the importance of non-ammonia nitrogen excretion (amino acids, urea,etc.) has rarely been scrutinized. Adequate knowledge about nitrogen excretion byshrimp is required for successful design and operation of intensive production systems.Underestimation of total nitrogen excretion is potentially a serious problem, especiallyfor closed recirculating water systems (Wickins, 1985). Quantification of the proportionsof excretory products is also of importance in understanding effects of environmentalfactors on nitrogen metabolism of shrimp.

Nitrogen excretion rates of crustaceans vary with environmental conditions (Regnault,1987). Significant effects of temperature and salinity on ammonia excretion have beendocumented for several commonly cultured species – Macrobrachium rosenbergii(Nelson et al., 1977; Stern et al., 1984), Fenneropenaeus indicus (Gerhardt, 1980), F.chinensis (Chen and Lin, 1992, 1995), Marsupenaeus japonicus (Chen and Lai, 1993),and Penaeus monodon (Chen et al., 1994). However, no comparable data have beenreported for any species of the genus Litopenaeus, which includes the open-thelycumwhite shrimp Litopenaeus vannamei.

The purpose of this study was to determine nitrogenous excretory products and ratesfor L. vannamei juveniles exposed to various combinations of temperature and salinityunder controlled experimental conditions. Temperature and salinity were within thenormal ranges encountered in the culture of L. vannamei.

2. Materials and methods

2.1. Shrimp and acclimation

Specific-pathogen-free L. vannamei were acquired as postlarvae from Harlingen

D.-H. Jiang et al. / J. Exp. Mar. Biol. Ecol. 253 (2000) 193 –209 195

Shrimp Farms, Ltd. (Los Fresnos, TX, USA) and reared in the Nutrition Laboratory ofthe Shrimp Mariculture Project, Texas A&M University System, at 2561 ppt and30618C. Shrimp used for the experiment were acclimated to nine combinations of threetemperatures (24, 28, and 328C) and three salinities (10, 25, and 40 ppt). Watertemperature was adjusted by decreasing or increasing values by 2–48C per day, andsalinity was adjusted by decreasing or increasing values by 2–3 ppt per day until thedesired levels were reached. Before the initiation of the excretion measurements, shrimpwere kept at a constant temperature and salinity for 2–3 weeks to ensure completeacclimation. During the acclimation period, shrimp were fed three times daily. The feedused was prepared in the laboratory with 34% protein and 7.6% total lipid (Table 1).Photoperiod of 12-h light–12-h dark was maintained.

Table 1Ingredients and proximate analysis of the experimental diet

Component Diet (%)aWheat starch 36.2aWheat gluten 18.0

bCasein (vitamin free) 7.4aSoybean protein isolate 2.8

aGelatin 1.6cKrill meal 5.0

dMenhaden fish oil 2.7Soybean oil 1.9

eAcid-washed diatomaceous earth 7.0dMenhaden fish meal 5.0

fLecithin 1.5aCholesterol 0.5

gFish solubles 2.0aCarboxymethyl cellulose (CMC) 4.0

hVitamin mixture 0.5iStable vitamin C 0.3

aSodium chloride 0.4jMineral mixture AIN 76 3.0

kProximate analysisMoisture 6.9Protein 34.0Crude fat 7.6Crude fiber 0.7

a ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA.b United States Biochenicals, Cleveland, OH, USA.c Inual, Santiago, Chile.d Zapata Haynei Corp., Reedville, VA, USA.e Sigma Chemical Company, Cleveland, OH, USA.f Riceland Foods Inc., Stuttgart, AR, USA.g Zapata Protein, Inc., Hammond, LA, USA.h Dawes Laboratories, Arlington Heights, IL, USA.i Roche Vitamins and Fine Chemicals, Pendergrass, GA, USA.j ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA.k Woodson-Tenent Laboratories, Inc., Des Moines, ID, USA.


2.2. Experimental protocol

Filtered (5 mm) and UV-sterilized seawater was used in the experiment. Salinity wasadjusted by adding PHG evaporated salt (Gargill, Inc., MN, USA) or diluting withde-ionized water. Water temperatures were maintained by using water-bath tables

2equipped with thermostat-controlled heaters. Polyethylene tanks (0.09 m bottom area),which had been thoroughly cleaned and contained 8 l of test seawater, were positionedin the water baths. Each individual tank was covered with a plastic lid to prevent shrimpescape and to reduce water evaporation. Water in each tank was gently aerated via a

21single air-stone to keep DO above 6 mg l . In a preliminary experiment, an average21biomass about 1–1.5 g l was established as sufficient to produce an appropriate

amount of nitrogen for accurate analyses, but not so great as to compromise shrimpperformance during a 24-h interval.

Prior to each experimental trial, shrimp were starved for 12 h to ensure maximum gutevacuation. Shrimp of similar size (in the range of 3–5 g) were selected from holdingtanks. To avoid metabolic fluctuation due to molt cycle (Stern and Cohen, 1982), onlyshrimp in the intermolt stage were selected for use. The molt stage was determinedaccording to the methods of Smith and Dall (1985). Sexes were not separated since noprevious study had ever indicated a significant difference in nitrogen excretion betweenmale and female shrimp within the size range used in this study. A group of four or fiveshrimp was transferred into each test tank. Water samples were taken initially and after24 h. Nitrogen analyses were conducted immediately after water samples were collected.After the trial, shrimp were gently dried on paper towels and weighed to the nearest 0.01g by using a digital balance. Each shrimp was used only once in the experiment. Theaverage weight and standard deviation for all tested shrimp was 3.8560.83 g, with nosignificant difference between treatments. There were two or three replicates of eachtreatment each time, and the excretion trial was repeated three times under the sameconditions. In addition, to limit any other potential effects, such as bacterial activity, onetank without shrimp served as a control for each temperature–salinity combination.Water was sampled from the control chamber in the same manner as that from theexperimental chambers; these data then were used as a basis for correction. To determineany ammonia loss into the atmosphere via aerial diffusion during the 24-h interval,

21chambers initially containing water with 0.5 mg-N l ammonia sulfate were processedwith the same procedures as previously described. No significant loss was detected.

2.3. Measurements

2 2Total ammonia-N (TAN), nitrite-N (NO ), and nitrate-N (NO ) were measured by2 3

methods adapted from Solorzano (1969) and Spotte (1979), from Strickland and Parsons(1972) and Spotte (1979), and from Mullin and Riley (1955) and Spotte (1979),respectively. Total dissolved nitrogen (TDN) was measured by the wet-oxidationmethod (Solorzano and Sharp, 1980; Parsons et al., 1984). Dissolved organic nitrogen(DON) was calculated based on the difference between TDN and ammonia-N 1 nitrite-

21 21N 1 nitrate-N. Excretion rates (R, mg-N g h ) of ammonia-N, nitrite-N, nitrate-N,


and DON were calculated by measuring the change in concentrations over theexperimental period using the following formula:

R 5 [(C 2 C ) 3V ] /(W 3 t)f i

21where C is the corrected final concentration of sample (mg-N l ), C is the initialf i21concentration of sample (mg-N l ), V is the water volume of the tank (l), W is the live

weight of shrimp (g), and t is the time interval between initial and final water sampling(h).

The responses of metabolic rate of shrimp to temperature can be expressed as thethermal quotient (Q ). Q values for nitrogen excretion were calculated for each10 10

temperature interval at each salinity using the following formula:

10 / (T 2T )2 1Q 5 (R /R )10 2 1

where, R and R are metabolic rates of shrimp (in terms of ammonia excretion rate,2 1

R ) at temperatures T and T , respectively.TAN 2 1

2.4. Statistical analyses

Repeated measurements of nitrogen excretion resulted in both inter-treatment vari-ability and time-to-time variation. All data, after log transformation, were statisticallyanalyzed by three-way analysis of variance (ANOVA), blocking by trial, to determine theeffects of temperature and salinity on nitrogenous excretion rates. If significantdifferences were indicated at a 50.05, then Duncan’s multiple range test was used toresolve significant differences among treatments (Duncan, 1955). Further, regressionanalysis was used to model the relationships among nitrogen excretion rate, temperature,and salinity (SAS Institute, Inc., 1989–1996).

3. Results

ANOVA indicated that there were significant effects of temperature and salinity onammonia excretion (P,0.05), but the interaction between temperature and salinity wasnot significant (P.0.05). Ammonia excretion rate (R ) significantly increased withTAN

temperature. R at 10 ppt was significantly higher than that at 25 or 40 ppt, with theTAN

difference between rates at 25 and 40 ppt not statistically significant (Fig. 1). Therelationship between R and temperature (T ) and salinity (S) could be described asTAN

0.062T 2 2follows: R 5 4.54e 2 1.13S 1 0.0205S (R 5 0.91, N 5 9). The salinity forTAN

minimum R was estimated to be 27.5 ppt. Relatively large variation was observed inTAN

the excretion rate of dissolved organic nitrogen (R ), both among treatments andDON

within the same treatment. Statistically significant effects of temperature and salinity onR could not be detected (Fig. 1). The overall mean (6standard deviation) of RDON DON

21 21was 5.24 (62.33) mg-N g h .There was no significant interaction between temperature and salinity on either nitrite

excretion rate (R ) or nitrate excretion rate (R ) of shrimp (Fig. 2). R ranged2 2 2NO NO NO2 3 2


21 21Fig. 1. Excretion rates (mean6S.E.M. in mg-N g h ) of ammonia-N and dissolved organic nitrogen (DON)for L. vannamei juveniles exposed to combinations of temperature (8C) and salinity (ppt).


21 21Fig. 2. Excretion rates (mean6S.E.M. in mg-N g h ) of nitrite-N and nitrate-N for L. vannamei juvenilesexposed to combinations of temperature (8C) and salinity (ppt).


21 21from 0.019 to 0.043 mg-N g h ; it increased significantly with salinity and wassignificantly higher at 288C and 328C than at 248C. R ranged from 0.056 to 0.2262NO3

21 21mg-N g h ; it was not influenced significantly by temperature, but increasedsignificantly with salinity.

The excretion rate of total nitrogen (R ) and the percentages of R comprised byTN TN

ammonia-N, nitrite-N, nitrate-N, and DON are presented in Table 2. R was lowestTN21 21 21 21(10.03 mg-N g h ) at 248C and 25 ppt, and highest (30.24 mg-N g h ) at 328C

and 40 ppt. A multiple regression analysis showed that R was significantly affectedTN

both by temperature and salinity, but not by temperature–salinity interaction. RTN

increased with temperature, with R at 28 and 328C significantly higher than at 248C.TN

R at 10 ppt was significantly higher than at 25 ppt, but the difference between 25 andTN

40 ppt was not significant. The relationship between R and temperature (T ) andTN0.051T 2salinity (S) could be described by the equation: R 5 8.10e 2 1.21S 1 0.0228STN

2(R 5 0.91, N 5 9). The salinity for minimum R was thus estimated to be 26.6 ppt.TN

The results indicated that L. vannamei is strongly ammonotelic, with ammonia-Naccounting for 61.9–84.3% of the total nitrogen excreted under the experimentalconditions. Percentage ammonia-N in total nitrogen excreta (P ) was significantlyTAN

influenced both by temperature and salinity. P increased with temperature, with PTAN TAN

at 28 and 328C significantly higher than at 248C. P at 10 ppt was significantly higherTAN

Table 221 21Excretion rate for total nitrogen (R in mg-N g h ) and percentages (%) of R comprised by ammonia-N,TN TN

anitrite-N, nitrate-N and DON for L. vannamei juveniles at various temperatures (T ) and salinities (S)

T S R Percentage of total nitrogen excretion (%)TN

21 21(8C) (ppt) (mg-N g h ) Ammonia-N Nitrite-N Nitrate-N DON

24 10 14.96 66.82 0.06 0.47 32.65(2.32) (4.27) (0.013) (0.15) (4.40)

25 10.03 63.90 0.12 1.44 34.53(1.06) (1.83) (0.016) (0.56) (1.69)

40 14.30 61.93 0.16 1.57 36.35(2.30) (1.88) (0.021) (0.47) (1.63)

28 10 26.20 84.34 0.04 0.24 15.38(2.17) (3.03) (0.011) (0.09) (2.97)

25 18.17 64.17 0.11 0.67 35.06(3.01) (4.10) (0.010) (0.27) (4.38)

40 18.29 73.30 0.17 1.23 25.31(3.66) (2.79) (0.026) (0.37) (2.94)

32 10 28.39 83.74 0.11 0.21 15.94(4.78) (3.16) (0.032) (0.04) (3.21)

25 22.59 77.78 0.10 0.68 21.44(3.34) (4.60) (0.018) (0.25) (4.66)

40 30.24 75.86 0.13 0.80 23.21(2.66) (1.28) (0.016) (0.20) (1.36)

a Values are means. S.E.M.s in parentheses.


Table 3Q values for ammonia-N excretion rate (R ) of L. vannamei juveniles over two temperature ranges at each10 TAN

salinity

Salinity Q calculated by R10 TAN

(ppt) 24–288C 28–328C

10 8.0 1.225 4.7 2.640 3.0 3.8

than that at 25 ppt, but the difference between 25 and 40 ppt was not significant (Table2). The relationship between P , temperature (T ), and salinity (S) could be describedTAN

2 2by the equation: P 5 38.91 1 1.86T 2 1.53S 1 0.0254S (R 5 0.83, N 5 9).TAN

Unlike R , the percentage DON in total nitrogen excretion (P ), which rangedDON DON

from 15.4 to 36.4%, was significantly affected by temperature and salinity. PDON

decreased with increased temperature; P at 28 and 328C were significantly lowerDON

than at 248C (P,0.05). P at 10 ppt was significantly lower than at 25 and 40 ppt, butDON

the difference between 25 and 40 ppt was not significant. The relationship betweenP , temperature (T ), and salinity (S) could be described by the equation: P 5DON DON

2 259.26 2 1.79T 1 1.46S 2 0.0246S (R 5 0.81, N 5 9).There was only a small amount of total nitrogen excreted by L. vannamei as nitrite-N

and nitrate-N. Percentages of nitrite (P ) and nitrate (P ) in total nitrogen excreta2 2NO NO2 3

ranged from 0.04 to 0.16% and from 0.24 to 1.56%, respectively.The Q values for L. vannamei were determined at each salinity for two temperature10

ranges 24–288C and 28–328C (Table 3). In general, larger Q values were observed in10

the temperature range of 24–288C than in range of 28–328C, with an exception atsalinity 40 ppt.

4. Discussion

The results of the present study confirmed that L. vannamei juvenile are primarilyammonotelic, as is the case for other marine crustaceans. Wajsbrot et al. (1989) reportedthat ammonia-N (61–83%) was the dominant form of nitrogen excreted by the greentiger shrimp, P. semisulcatus, with urea-N and other unknown dissolved organic nitrogencompounds comprising the rest (17–39%). Chen et al. (1994) found that ammoniaaccounted for 56.9–78.1%, and urea and other DON accounted for 3.3–25.8% of thetotal nitrogen for P. monodon at salinities of 10–30 ppt. Ammonia accounted for61.9–84.3% of the total nitrogen excreted by L. vannamei juveniles under theseexperimental conditions. Dissolved organic nitrogen (DON), which is considered to bemainly free amino acids and urea (Regnault, 1987), was the second most importantnitrogenous waste, accounting for 15.4–36.4% of the nitrogen excreted by L. vannamei.

In previous studies, ammonia excretion rate (R ) has usually been the basis forTAN


measuring the effect of various factors on nitrogen excretion (Regnault, 1987). Thereexists great variability in the published data for R , even within the same speciesTAN

(Table 4). Part of variance results from variability in experimental methods andenvironmental conditions. In the present study, shrimp were fully adapted to the testmedia prior to testing. The data represent the physiological status of the shrimp at steady

21 21state with ambient environment. R was highest (23.26 mg-N g h ) at 328C and 10TAN21 21ppt and lowest (6.38 mg-N g h ) at 248C and 25 ppt. Since endogenous nitrogen

excretion rate varies with shrimp species, size, molt stage, and environmental conditions(i.e., temperature and salinity), a direct comparison of absolute values between theresults in the present study and those in the literature is difficult. However, the trend andrelative magnitude of R can be compared. Results obtained seem in generalTAN

agreement with those reported by Gerhardt (1980); Wickins (1985); Chen et al. (1993);Koshio et al. (1993); Chen and Chen (1997).

The R of crustaceans is affected both by intrinsic factors such as species, size,TAN

molt stage, and starvation and by external factors such as temperature, salinity, pH, DO,and light intensity. Effects of temperature and salinity on nitrogen excretion bycrustaceans have been reviewed by Regnault (1987). Generally, the R of shrimpTAN

increases with temperature due to a higher metabolic rate at elevated temperatures(Spaargaren et al., 1982; Chen and Lai, 1993; the present study).

The effect of salinity upon nitrogen excretion of crustaceans appears species-specific(Regnault, 1987). For penaeids, an increase in ammonia excretion frequently has beenobserved as salinity decreases. For M. japonicus, R has been reported to increaseTAN

with the decline of salinity in the range of 21–37 ppt (Spaargaren et al., 1982) and in therange of 15–30 ppt (Chen and Lai, 1993); for P. monodon, in the range of 15–35 ppt(Lei et al., 1989) and in the range of 10–30 ppt (Chen et al., 1994); and for F. chinensis,in the range of 10–30 ppt (Chen and Lin, 1995). In this study, R increased for L.TAN

vannamei as salinity decreased from 25 to 10 ppt. The reasons for that could be: (1) adecrease in external osmotic concentration results in an increased metabolic rate ofshrimp (Subramanian and Krishnamurthy, 1986; Chen and Lai, 1993); (2) at lowsalinity, shrimp are prone to use protein, not lipid, as their primary energy source (Chen,1998); (3) at low salinity, the osmotic water inflow to shrimp increases, which iscompensated by increased urine production to maintain water balance. To prevent losses

1 1 1of the alkali ions (Na and K ), increased NH partially replaces alkali ions in the4

formation of urine (Spaargaren et al., 1982); and (4) a decreased concentration of freeamino acids in the tissue (Lange, 1972; Dalla Via, 1986), and an increased catabolism ofamino acids result in evaluated nitrogen excretion which is mainly ammonia (Chen andKou, 1996). Also, it is noteworthy that R of L. vannamei was lower at 25 ppt than atTAN

40 ppt for all temperature levels, even though the difference was not statisticallysignificant. Under hyper-osmotic conditions, more energy is required for osmoregulationwhen the ion gradient between body fluids and external medium is increased (Spaar-garen, 1975, 1976). Total nitrogen excretion of L. vannamei was lowest at 26.6 ppt

21salinity, which is close to its isosmotic point, 718 mOs kg or 24.7 ppt (Castille andLawrence, 1981). The observation that R of L. vannamei was lower at 25 ppt than atTAN

10 ppt and 40 ppt is consistent with those of Kutty et al. (1971), Spaargaren (1975;


Table 4Comparison of the observed rates of ammonia excretion (R ) for some penaeid speciesTAN

Species Weight Conditions R Ref.TAN

21 21(g) T (8C) S (ppt) (mg-N g h )

F. indicus 5.0 28 33 37.5 Gerhardt, 1980

P. semisulcatus 1.3 24 40.5 60.67 Wajsbrot et al., 198910.4 24 40.5 28

aP. esculentus 0.1–0.3 30 35 13.71 Hewitt and Irving, 1990F. chinensis 26.91 25 30 19 Chen et al., 1993P. monodon 1.6 28 20–34 38.75 Wickins, 1985

27.0 28 20–34 12.5

P. monodon 22.1 25.5 30 5.5 Chen et al., 199420 8.810 12.5

M. japonicus 5.0–7.0 10–14 22.1 31.5 Spaargaren et al., 198210–14 36.7 119.28

25 21.9 718.225 35.8 333.2

M. japonicus 11.7 25 30 1.559 Chen and Cheng, 1993aM. japonicus 16.4 25 30 2.310 Chen and Cheng, 1993bM. japonicus 0.4 25 32 10 Koshio et al., 1993

M. japonicus 0.2 15 15 13 Chen and Lai, 199320 825 830 5

25 15 4620 2425 2430 17

35 15 6520 5625 5030 42

M. japonicus 0.3 27 18 33.25 Chen and Chen, 199726 24.1734 19.75

L. vannamei 3.0–5.0 24 10 9.35 Present study25 6.3840 8.67

28 10 21.5025 11.8340 13.39

32 10 23.2625 17.4440 22.85

a Units5mg-N/g dry weight /h.


1976) and Subramanian and Krishnamurthy (1986), all of whom suggested thatmetabolic rates increased as salinity deviated from the isosmotic point because ofincreasing energy-cost due to osmoregulation.

Haberfield et al. (1975) observed that ammonia excretion of Carcinus maenas wasaffected by a salinity decrease while amino acid excretion rate did not change.Spaargaren et al. (1982) documented relative constancy in non-ammonia nitrogenexcretion of M. japonicus at various temperature–salinity treatments. Similarly,Quarmby (1985) found that the organic nitrogen excretion of Pandalus platyceros wasnot affected by either temperature or salinity. In the present study, large variation wasfound in R , but a significant effect of temperature and salinity on R was notDON DON

shown. However, percentage of DON in total nitrogen excretion was significantlyaffected by temperature and salinity. DON is mainly free amino acids and urea(Regnault, 1987). The release of amino acids which are metabolically useful substancesis probably inadvertent and should be considered more as leakage than excretion (Dagg,1976). In the present study, lower P was found at 28 and 328C than at 248C, and atDON

10 ppt than at 25 and 40 ppt. This suggests that the protein metabolism of L. vannameiis more efficient at high temperatures and low salinity.

Spaargaren (1985) reported that nitrate-N excretion of Carcinus maenas was higher atintermediate salinity (22.7–24.7 ppt) than at high salinity (38.5–42.5 ppt) or low salinity(4.7–15.1 ppt). Also, he suggested that nitrate formation may serve in the detoxificationof ammonia and maintenance of hemolymph electroneutrality. Chen et al. (1994)reported both nitrite-N and nitrate-N excretion of P. monodon was higher at 20 ppt thanat 10 or 30 ppt. Nitrite-N excretion increased when shrimp were exposed to ambientammonia (Chen and Cheng, 1993b; Chen et al., 1994), apparently because the ammoniaentering the hemolymph may be converted to nitrite under ammonia stress (Chen andCheng, 1993b). The results of this study showed that, even though the excretion of bothnitrite-N and nitrate-N was affected by temperature and salinity, only very smallamounts of nitrogen were excreted by L. vannamei as nitrite and nitrate. For theconditions of the present study, nitrite-N and nitrate-N make up 0.04–0.16% and0.24–1.56% of nitrogen excretion of L. vannamei, respectively. In contrast, nitrite-N andnitrate-N accounted for about 9.59–14.49% and 6.41–10.31%, respectively, of nitrogenexcretion by P. monodon (Chen et al., 1994).

An experimental period of 24 h has often been used to determine excretion rates ofaquatic animals. Such long periods can minimize the impact of high initial activity dueto transferring shrimp into new tanks and the effect of light cycle on nitrogen excretion.However, bacterial activity during 24 h might introduce an experimental artifact inexcretion studies (Armstrong et al., 1981). But, assuming that autotrophic bacteria takeup ammonia-N while heterotrophic bacteria assimilate organic nitrogen, the apparentexcretion rate of total nitrogen may not be affected much. Moreover, sterilized seawaterand controls (without shrimp) were used in this study; thus, the effect of bacteria onresults was not a large source of error.

Dietary factors also influence nitrogen excretion of shrimp. Regnault (1983) foundthat high-protein diets did not influence growth or body nitrogen storage of shrimpCrangon crangon, but such diets markedly increased the post-digestive ammonia


excretion rate. Koshio et al. (1993) observed that post-feeding ammonia excretion of M.japonicus increased with dietary protein content. They also observed that ammoniaexcretion after 24-h starvation was constant, indicating that a 24-h starvation periodeliminated differences in ammonia excretion rates due to variation in dietary proteincontent. In this study, the shrimp were fully acclimated to temperature and salinity, andhad been starved over 12 h. The test tanks allowed shrimp to move freely but notactively swim, resulting in an uncontrolled but minimum motor activity. According toBrett and Groves (1979), who defined routine metabolism as the metabolic rate of fishduring normal ‘‘spontaneous activity’’, the mean values of nitrogen excretion rate withina 24-h interval should represent the routine level of nitrogen metabolism of shrimp. Inaddition, it is known that in crustaceans protein and lipids appear to be the major sourceof energy (Barclay et al., 1983), while carbohydrates are not used as energy substratesduring starvation (Regnault, 1981). Using P. esculentus, Hewitt and Irving (1990) foundthat carbohydrates and lipids were important energy substrates during the first 8–14 hpost-feeding and that protein catabolism became increasingly significant in the pro-duction of energy thereafter. It is clear that nitrogen excreted by starving shrimporiginates from the endogenous amino acids. Therefore, the rate of nitrogen loss fromthe endogenous pool in fasting L. vannamei due to excretion was estimated to be

210.24–0.73 mg-N g shrimp per day.With intensification of shrimp farming, there is a significant increase in stocking

density and harvest biomass. Accumulation of ammonia is a primary factor constrainingshrimp biomass and stocking density in intensive systems because of its toxicity toshrimp. Using ammonia excretion rate together with a 96-h LC50 value, Wajsbrot et al.(1989) tried to estimate the appropriate biomass density of P. semisulcatus in shrimpponds. But, effects of temperature and salinity should be taken into account because bothammonia toxicity and excretion rate of shrimp are influenced by temperature andsalinity. The ‘‘safe level’’ of ammonia for L. vannamei has been reported to be 2.6 mg-N

21l (Jiang et al., 1999). No attempt is made to calculate the maximum density herebecause it was not an objective of this research. However, the quantitative data obtainedcould be used for designing adequate biofiltration capacities in recirculating culturesystems. Also, by measuring total nitrogen excretion instead of ammonia-N only,nitrogen loading from shrimp could be better estimated for more accurate nitrogenbudgets and dynamic models.

Juvenile shrimp are adapted to estuarine salinities and coastal temperature regimes(Lester and Pante, 1992). Changes in ammonia excretion rate under the experimentalconditions could reflect the responses of the shrimp metabolic rate to temperature andsalinity. Thus, the thermal quotients (Q ) were calculated based on R . However, it10 TAN

is recognized that this calculation may be inappropriate because the response of R toTAN

the temperature is unlikely monotonic. Larger Q values in the temperature range of10

24–288C suggests that L. vannamei juveniles were more sensitive to temperature than at28–328C, especially at low salinity (10 ppt). Coincidentally, in laboratory salinity-preference experiments at 26–328C, Mair (1980) demonstrated L. vannamei has arelatively low salinity preference (1–8 ppt). In nature, the abundance of postlarval L.vannamei is associated with low salinity and high temperature.


5. Conclusions

1. Ammonia is the major end-product of protein catabolism for L. vannamei, accountingfor 61.9–84.3% of total nitrogen excretion.

2. Ammonia-N excretion rate (R ) was significantly affected by temperature andTAN

salinity. R increased with temperature and was significantly higher at 10 ppt thanTAN

at 25 and 40 ppt.3. DON is the second major end-product, accounting for 15.4–36.4% of total nitrogen

excretion.4. The excretion rate of DON (R ) was not significantly influenced by eitherDON

21 21temperature or salinity, with an overall mean of about 5.24 mg-N g h .5. Only very small amounts of nitrogen were excreted by L. vannamei as nitrite-N and

nitrate-N.6. The excretion rate of total nitrogen instead of ammonia is recommended for use in

estimating the nitrogen load from shrimp in nitrogen budgets of production systems.

Acknowledgements

This research was funded in part by Project H-8158 of the Texas AgriculturalExperiment Station and United States Department of Commerce Marine Shrimp FarmingProgram CSREES Grants No. 95-38808-1424 and No. 92-38808-6920. The publicationis also a contribution of the Living Marine Resources Panel under the United States,Peoples’ Republic of China Marine and Fisheries Science and Technology AgreementProgram. [SS]

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