The effect of dissolved oxygen and salinity on oxygen consumption, ammonia excretion and osmotic...

LJournal of Experimental Marine Biology and Ecology,234 (1999) 41–57

The effect of dissolved oxygen and salinity on oxygenconsumption, ammonia excretion and osmotic pressure of

Penaeus setiferus (Linnaeus) juvenilesa , b a c*Carlos Rosas , Evenor Martinez , Gabriela Gaxiola , Roberto Brito ,

a d´Adolfo Sanchez , Luis A. Sotoa ´ ´Laboratorio de Biologıa Marina Experimental, Depto. de Biologıa, Fac. de Ciencias, UNAM, Apdo. Post.

69, Cd. del Carmen, Campeche, Mexicob ´ ´Centro de Investigacion del Camaron, Universidad Centroamericana, Managua, Nicaragua

cCentro de Investigaciones Marinas, Universidad de la Habana, Havana, Cubad ´ ´Laboratorio de Ecologıa del Bentos, Instituto de Ciencia del Mar y Limnologıa UNAM, Mexico 04510

D.F., Mexico

Received 15 October 1997; received in revised form 25 June 1998; accepted 6 August 1998

Abstract

The white shrimp Penaeus setiferus (Linnaeus) is an abundant species in the coastal lagoonsand estuaries of the Gulf of Mexico. This species is well adapted to environments of low salinityand can tolerate low levels of dissolved oxygen. This study was designed to measure the effects ofprolonged hypoxia and salinity level on: (a) the oxygen consumption and ammonia excretion, (b)the metabolic substrate (O:N) on fasting (24 h) and feeding animals, (c) the osmotic pressure ofthe hemolymph and, (d) the body water content of P. setiferus juveniles. The shrimp were exposed

21to different levels of dissolved oxygen (DO of 2, 3, 4 and 5.8 mg l ) and two salinities (15 and35‰) for a period of 60 days. Results indicate that these animals are oxyregulators between 5.8

21 21and 4 mg l DO and oxygen conformers between 3 and 2 mg l DO in both salinities. Therewas not a significant effect of salinity on the oxygen consumption in either nutritional condition.Ammonia excretion was significantly greater in 15‰ than in 35‰. In 15‰ the ammonia excretiondiminished as a function of the DO. In unfed animals the ammonia excretion diminished in adirect proportion to the decrease of the DO, while in fed animals they were ammonia-regulators

21between 5.8 and 4 mg l DO. In 35‰ the ammonia excretion increased in the fed animals21exposed to 2 and 3 mg l DO. In low salinity the animals fundamentally maintained proteins as

their energy substrate at all levels of DO, while in the case of 35‰ of salinity the shrimp changed21 21the metabolic substrate from lipids-proteins (5.8 and 4 mg l DO) to proteins (3 and 2 mg l

DO). These results show that P. setiferus juveniles are capable of changing their energy substratein response to salinity and DO changes. This fact may be related to a possible strategy that allowsthem to obtain energy from proteins. The stability of osmotic pressure between 35 and 5‰ and the

*Corresponding author.

0022-0981/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 98 )00139-7

3184

42 C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 41 –57

changes which have been observed in the total content of water give rise to the supposition thatthe pool of free amino acids, whether of muscular or nutritional origin, are the key to this strategy. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Dissolved oxygen; Oxygen consumption; Ammonia excretion; Osmotic pressure;Salinity; Shrimp; Penaeus setiferus juveniles

1. Introduction

The physiological responses of decapod crustaceans to the decrease of dissolvedoxygen (DO) have been well documented especially in relation to DO effects on therespiratory response. McMahon (1988) carried out an examination of the processesinvolved in the regulation of the ventilating and cardiac mechanisms that contribute tothe maintenance of the oxygen supply to tissues.

In contrast, changes in the nitrogen metabolism which are associated with hypoxia andanoxia have been studied to a lesser degree. Diverse studies have shown that anoxia andhypoxia produce a decrease in the ammonia excretion in several species of decapodcrustaceans, such as Nephrops norvegicus (Hagerman et al., 1990), Saduria entomon(Hagerman and Szaniawska, 1994), Carcinus maenas (Regnault and Aldrich, 1988) andCancer pagurus (Regnault, 1993), among others. These studies suggest that theammonia excretion is diminished as a consequence of a metabolic depression whenoxygen is the limiting factor.

According to Herreid (1980), the mechanisms involved in response to the hypoxiadepend on the environment and the physiological state of the animals. Temperature,salinity, pH, pollutants, activity and size, among others, are the factors that determinechanges in the metabolism of animals exposed to hypoxia. Allan et al. (1990) reportedthat the toxicity of ammonium increased 69% in P. monodon juveniles maintained inhypoxic conditions. Allan and Maguire (1991) reported that a decrease in the pHincreased the sensitivity to low concentrations of oxygen in P. monodon. Martinez et al.(1998) reported that the tolerance to hypoxic conditions of P. setiferus postlarvaediminished in pH 6, as compared with that obtained in pH 8. Rosas et al. (1997) havealso reported that the oxyregulatory capacity (range of dissolved oxygen where therespiratory metabolism is independent upon the oxygen concentration) of P. setiferus andP. schmitti increased when the animals were kept in optimum conditions of salinity of 15and 25‰, respectively.

Salinity is one of the important environmental factors for penaeid shrimp, since itdetermines the distribution of the organisms in estuarine systems (Day et al., 1982).McFarland and Lee (1963) showed the great osmoregulatory capacity of P. setiferus, aspecies that is capable of penetrating the diluted environment of the mouths of rivers.Among the physiological mechanisms that permit shrimp to withstand diluted environ-ments are an increase in the permeability to water, the active uptake of ions and theliberation of osmotic effectors (amino acids and peptides) to the hemolymph. In accordwith different authors, the pool of amino acids which play an osmotic role is deaminated

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 41 –57 43

with a subsequent formation of ammonium (Claybrook, 1983; Ikeda and Dixon, 1984;Hewitt, 1992). Depending on the nutritional balance of the animals, these amino acidsmay or may not be part of the production-of-energy routes in the Krebs cycle (Mayzaudand Conover, 1988).

Penaeid shrimp, unlike other crustaceans, are particularly sensitive to low con-centrations of DO. Egusa (1961) found critical concentrations of oxygen for P. japonicus

21between 4.5 and 5 mg l and Liao and Chien (1994) reported critical levels between 421 21and 4.3 mg l for P. monodon. Other studies indicate that below 2 mg l the growth

rate of P. vannamei and P. monodon were significantly reduced (Seidman and Lawrence,211985). Rosas et al. (1997) have reported critical levels between 4.5 and 5 mg l for P.

setiferus and P. schmitti postlarvae which were exposed to different salinity levels. Areduction between 3.9 and 26% of respiratory metabolism was observed when DOdiminished lower than the critical concentration in both species. In addition it has beenpointed out that the P. aztecus and P. setiferus juveniles are capable of detecting andavoiding hypoxic water (Renaud, 1986). Some authors have proposed that the sensitivityof the decapods at low concentrations of oxygen is due to the limited capacity of theiranaerobic metabolism (Taylor and Spicer, 1987).

It is known that penaeid shrimps use proteins as their major source of energy(Regnault, 1979, 1981). Rosas et al. (1995a), in this respect, have shown that themetabolic substrate functions in both the nutritional state and the protein requirement ofdifferent species of penaeid shrimp which have been studied (P. setiferus, P. schmitti, P.duorarum and P. notialis). The ratio of oxygen consumption to ammonia excretion(O:N) has been amply used to obtain the nature of the metabolic substrate that isoxidized in crustaceans (Regnault, 1979, 1981; Dall and Smith, 1986; Rosas et al.,1995a). Theoretical values of O:N have been the indicators in the use of the differentsubstrates. Values of 3 to 16 have been suggested for proteins, while equal quantities oflipids and proteins correspond to values of O:N between 50 and 60. Greatest valuescorrespond to a substrate that is mixed with proteins and lipids (Mayzaud and Conover,1988). Taking into consideration the fact that DO and salinity can provoke increases inthe pool of free amino acids in the hemolymph (Claybrook, 1983; Hagerman andSzaniawska, 1994), a change in the metabolic substrate (O:N) might be expected as anadaptive strategy associated with the tolerance for hypoxic diluted environments.

The white shrimp of the Gulf of Mexico, Penaeus setiferus (Linnaeus) is an abundantspecies which is exposed to natural fluctuation in the oxygen levels and salinity,characteristic of coastal systems (Renaud, 1986). At the same time, this species ispotentially important for the development of shrimp culture in the Atlantic coastal zoneof America (Hopkins et al., 1993). Taking into account the fact that these shrimps aresensitive to low concentrations of oxygen and have a wide toleration to changes inenvironmental salinity (McFarland and Lee, 1963) it is important to know thephysiological mechanisms which are involved in the adjustments of respiratory andnitrogen metabolism when these two important environmental factors are combined. Thepresent study was designed with the purpose of measuring the effects of prolongedhypoxia on; (a) oxygen consumption and ammonia excretion, and (b) the metabolicsubstrate (O:N) in fasting and feeding P. setiferus juveniles exposed to different salinitylevels. Moreover, in light of the osmoregulatory capacity reported by McFarland and


Lee (1963), the effect of prolonged hypoxia on the osmoregulatory capacity and the totalcontent of water of the juveniles of this species were also studied.

2. Materials and methods

2.1. Origin of organisms and acclimation conditions

P. setiferus juveniles used in the present study were obtained from larvae cultivated inlaboratory conditions. In all cases the larvae were cultivated in natural sea water at

2128628C with a salinity of 3562‰ and an oxygen concentration higher than 5 mg l .Once the animals arrived at the state of PL15, (15 days after the last metamorphicchange), they were randomly distributed into two large groups, one of which wasacclimated at 15‰ and the other was kept at 35‰ of salinity. The salinity of 15‰ wasobtained by diluting sea water with industrialized distilled water. The acclimation of thefirst group was carried out under a gradual change of 5‰ per day (McFarland and Lee,

21963). Once acclimated, the animals were placed in plastic tanks (0.28 m ) at the rate of11 shrimp per tank (12 tanks /salinity). The tanks were connected to a closed system

21with a continual flow of sea water (280 ml min ). The animals were kept there for aperiod of 60 days at distinct levels of DO.

2.2. Experimental design

21The shrimp were exposed to four different levels of DO: 2 mg l DO (27.7% of air21 21saturation 5 % AS), 3 mg l DO (41.7% AS), 4 mg l DO (55.5% AS) and 5.8 mg

21 21 21l DO (control; 80.5% AS) at 15‰ and 2 mg l DO (31.1% AS), 3 mg l DO21 21(46.5% AS), 4 mg l DO (62.1% AS) and 5.8 mg l DO (control; 90% AS) at 35‰.

Each DO level had three repetitions.To achieve adequate control of the oxygen levels, a system similar to that described

by Seidman and Lawrence (1985) and Allan and Maguire (1991) was used. Each DOlevel had five tanks of the same dimensions but located at a distinct height. The lowesttank was used as a discharge tank, water exchange and pumping; the highest tank wasused for re-oxygenation and distribution of water toward the gas-exchange columnwhich, in each system (PVC of a 6-inch diameter and 2 m in height), was connected to anitrogen tank which provided gas from the base of the column, just above the water exittoward the tanks which contained the animals. The experimental tanks were coveredwith a plastic plate to avoid partial water re-oxygenation.

Twice a day the concentration of oxygen and the salinity at both the entrance and theexit of the tanks were determined by means of a digital oxymeter (YSI 50B), which wasconnected to a polarographic electrode and with a hand refractometer, respectively. The

21 21DO in the experimental tanks was 2.0460.01 mg l (nominal 2 mg l ), 3.1560.0321 21 21(nominal 3 mg l ), 4.1260.03 (nominal 4 mg l ) and 5.8360.16 mg l (control).

The salinity was maintained at 1561‰ (nominal 15‰) and at 3561‰ (nominal 35‰).During the experimental period, the temperature of the experimental tanks was2761.58C and pH was 8.260.3.


The animals were kept under these conditions for 60 days during which they were fedwith meal pellets of 50% protein when postlarvae (PL15 to PL35) and 40% of proteinwhen juveniles (Gaxiola, 1994; Rosas et al., 1995a).

2.3. Oxygen consumption

The oxygen consumption by the P. setiferus juveniles (0.52360.070 g ww;0.13160.016 g dw) was measured individually in a flow-respirometer (Martinez-Otero

´and Dıaz-Iglesia, 1975; Rosas et al., 1995b), which was connected to a gas-exchange21column to control the DO levels. The flow in the respirometer was 33 ml min . We

used 20 plastic respirometric chambers of 250 ml, in which animals were placed 24 hbefore any measurements were taken. During this time the animals were given no food.The respirometric chambers were covered to eliminate possible interference by activityin the laboratory. The rate of oxygen consumed was calculated from the differencebetween the concentration of oxygen at the entrance of the chamber and that at the exitand multiplied by the rate of flow. The concentration of oxygen was measured with a

21digital oxymeter (YSI 50B digital) with a polarographic sensor (60.01 mg l ), whichwas previously calibrated with oxygen-saturated sea water at room temperature. Themetabolic rate was measured at time 0 (unfed animals for 24 h). After, a pellet of 0.051g, was placed in each chamber and the oxygen consumption was again measured everyhour for 8 h (between 08:00 and 17:00 h). Two chambers without animals were used asa control so as to detect if the presence of food altered the concentration of oxygen in therespirometric chamber. The oxygen consumption in the control chamber was in-significant.

Once the experiment was concluded the animals were weighed sacrificed and dried at608C to constant weight. The values of oxygen uptake of unfed and fed animals wererelated with the dry weight (dw) of the shrimp.

At the same time oxygen uptake was determined, samples of water were also obtained21whose concentration of N-NH mg l was measured. The ammonia excretion was3

determined from the differences between the ammonia concentration at the entrance andthe exit of each chamber and multiplying that by the rate of water flow. Theconcentration of ammonia was measured with a specific electrode connected to an ionmultianalyzer (ORION 720 A), following the method recommended by Dall and Smith(1986). Each sample of 25 ml of sea water from the respirometer was added with 10 ml

21of a solution with 1 mg l of NH Cl, producing an ammonia concentration in the4

sample where the response of the electrode is linear. This technique was used to measurethe total ammonia concentration in the presence of a large excess of a complexing agent

1 21like K or Mg in sea water (see the Orion ammonia electrode instruction manualModel 95-12). The ammonia excretion of unfed and fed (post-prandial) was related withthe dw of the shrimp.

2.4. The O:N ratio

The atomic ratio of oxygen uptake to ammonia excretion was calculated for both theunfed animals and the animals at the peak of metabolic activity after being fed. The


Mayzaud and Conover (1988) criterion was used to define the catabolism of proteins(3–16) and protein-lipids (50–60).

2.5. Osmotic pressure and total water content

The first experiment was designed to corroborate the osmoregulatory capacity of P.setiferus juveniles, following the same procedure that was described by McFarland andLee (1963). A batch of 320 juveniles were exposed to changes in salinity between 35‰and 1‰ with intervals of 5‰. Animals (20 per salinity 5 160 animals) were exposed tobrusque changes and 160 animals (20 per salinity) were exposed to gradual changes. Inthe first case the shrimp were exposed to experimental salinity for 2 h before the osmoticpressure was measured and in the second case the shrimp were acclimated at the rate of5‰ a day. To avoid interference in the results due to a prolonged period of not beingfed, the animals of both groups were fed with the same balanced food used in growthexperiments.

Subsequently the osmotic pressure of the shrimp that came from different DO andsalinity levels used in the oxygen uptake and ammonia excretion experiments wasanalyzed. In all cases the hemolymph was extracted by heart puncture by means of a1-ml syringe without an anticoagulant. The osmotic pressure of the hemolymph wasanalyzed individually in a micro-osmometer (20 ml / sample) (3 MO-PLUS; AdvancedInstruments Inc.).

The percentage of water of the shrimp of both experimental groups was obtained withthe difference between the wet weight (before the extraction of the hemolymph) and thedry weight. Wet weight was obtained individually in live shrimp. Dry weight wasobtained at 608C until constant weight was obtained. Both the live weight and the dryweight were determined on an analytical balance (Ohaus60.00005 g).

2.6. Statistical analysis

The combined effect of DO and salinity on the oxygen uptake and the ammoniaexcretion were analyzed by means of a two-way ANOVA. The square-root transforma-tion of the sine-arc before analyzing the values given in percentages was used (Zar,1974).

3. Results

3.1. Ammonia excretion

The ammonia excretion from the fed and unfed animals maintained at 15‰ of salinitydiminished in relation to the decrease of DO (Fig. 1a). In the case of the unfed animals,the ammonia excretion diminished proportionately with the decrease of DO with greater

21 21 21 21values in 5.8 mg l DO (0.65 mg g dw h ) and lower values in 2 mg l DO (0.1521 21mg g dw h ) (P , 0.05). In the fed animals the ammonia excretion remained

21 21 21constant between 4 and 5.8 mg l O (average value of 0.88 mg N-NH g dw h ).2 3


21 21Fig. 1. Penaeus setiferus. Nitrogen excretion (mg g h ) of fasting and feeding juvenile shrimp kept at 15‰(A) and 35‰ (B) of salinity and exposed to different levels of DO. Mean6S.E.


A decrease in the ammonia excretion in proportion to the decrease of DO was observed21(Fig. 1a). The ammonia excretion obtained from the fed animals, kept at 2 mg l DO

21 21(0.43 mg g dw h ) was significantly lower than that obtained from the animals kept21between 4 and 5.8 mg l DO (Fig. 1a).

The ammonia excretion of the fed and unfed animals kept at 35‰ of salinity showedirregular behavior (Fig. 1b). The ammonia excretion from the shrimp unfed and exposed

21 21 21to 4 mg l DO (0.11 mg g dw h ) was significantly less than that obtained in 2, 3,21 21 21and 5.8 mg l DO (average value of 0.19 mg g dw h ) (P , 0.05). The ammonia

21 21 21excretion of fed animals and exposed to 4 mg l DO (0.21 mg g dw h ) was21significantly less than that observed in the shrimp kept at 2 and 3 mg l DO (average

21 21 21 21value of 0.4 mg g dw h ) (P , 0.05). An intermediate value of 0.29 mg g dw h21was obtained from the animals kept at 5.8 mg l DO (P , 0.05) (Fig. 1b).

The nitrogen excretion was significantly greater in the animals kept at 15‰ ascompared to those kept at 35‰. The multifactorial ANOVA indicated that the salinity,the DO and the interaction of both factors affected significantly the nitrogen excretion(P , 0.05).

3.2. The oxygen uptake

In the unfed shrimp kept at 15‰ the oxygen uptake was kept constant between 4 and21 21 215.8 mg l DO (average value of 5.67 mg g dw h ), which was significantly greater

21 21 21 21than that obtained at 3 mg l DO (2.34 mg g dw h ) and 2 mg l DO (1.11 mg21 21g dw h ) (P , 0.05) (Fig. 2a). The behavior of oxygen uptake of the fed animals was

21 21very similar with greater values at 5.8 and 4 mg l DO (average value of 7.8 mg g21 21 21 21dw h ) and lesser values in the animals kept at 2 mg l DO (3.88 mg g dw h )

(P , 0.05) (Fig. 2a).In the fed and unfed shrimp kept at 35‰ of salinity the oxygen uptake decreased in

relation to DO decrease (Fig. 2b). The oxygen uptake in the unfed animals kept at 5.821 21 21and 4 mg l DO (average value of 6.1 mg g dw h ) was considerably greater than

21 21 21 21that obtained in those kept at 3 mg l DO (3.22 mg g dw h ) and at 2 mg l (1.7221 21mg g dw h ) (P , 0.05). In the fed animals the oxygen uptake of the animals kept at

21 21 215.8 and 4 mg l DO (average value of 8.23 mg g dw h ) was considerably greater21 21 21than that obtained at 3 and 2 mg l DO (average value of 5.31 mg g dw h ).

The multifactorial ANOVA showed that the oxygen uptake was affected by the DOand nutritional condition and not salinity (P . 0.05).

3.3. The O:N ratio

In both unfed and fed animals, the O:N of the shrimp kept at 15‰ of salinity and atall levels of DO were between 7.7 and 15.33, indicating that proteins were used as anenergy substrate (Fig. 3a).

In the animals kept at 35‰ (Fig. 3b), the values of O:N of those kept at 5.8, 4 and 321mg l DO indicated that different proportions of proteins and lipids were used as an

21energy substrate, with values that were greater in 4 mg l DO (fed and unfed;


21 21Fig. 2. Penaeus setiferus. Oxygen uptake (mg g h ) of fasting and feeding juvenile shrimp kept at 15‰ (A)and 35‰ (B) of salinity and exposed to different levels of DO. Mean6S.E.


Fig. 3. Penaeus setiferus. O:N ratio of fasting and feeding juveniles of white shrimp kept at 15‰ (A) and 35‰(B) and exposed to different levels of DO.


2157.2 5 50% proteins–50% lipids) and smaller in 3 mg l DO (unfed; 18.9 5 84%21 21proteins–16% lipids) (P , 0.05). The fed animals kept at 2 mg l DO and 3 mg l DO

showed lower values of O:N (average of 14.0), indicating a protein catabolism. Unfed21animals kept at 2 mg l also showed O:N values indicating a protein catabolism

21(O:N 5 8.66). In the animals kept at 5.8 and 4 mg l DO, the ratio O:N (33.4 5 73%proteins–27% lipids; 42.4 5 61% proteins–39% lipids, respectively) were significantly

21greater than those obtained from the fed animals kept at 3 and 2 mg l DO (averagevalues of O:N 5 14:proteins), showing a change in the metabolic substrate, of thesubstrates that were mixed into the high DO levels to proteins on low DO levels (Fig.3b).

3.4. The proportion of water and osmotic pressure

An increase in the proportion of water was observed in relation to a decrease insalinity in shrimps exposed to gradual changes, to brusque changes, and those thatformed part of the DO experiments (Fig. 4a). The body water content was considerablylower in animals exposed to 35‰ (66%) than those exposed to salinity between 10 and20‰ (73%) (P , 0.05). The osmotic pressure of shrimps kept between 35 and 5‰

21remained within a relatively narrow interval between 629 to 803 mOsm kg (Fig. 4b).The osmotic pressure of animals exposed to 5 and 1‰ were lower than that obtained inanimals maintained in the isosmotic point (25‰). The animals from DO experiments in

21both salinities fall into this interval with values of 751 mOsm kg in the shrimps kept21at 35‰ and 700 mOsm kg in those that were kept at 15‰. The isosmotic point of the

21P. setiferus juveniles, which was located at 717 mOsm kg , corresponded to a salinityof 24‰ (Fig. 4b).

4. Discussion

The present study corroborates the wide osmoregulatory capacity of the P. setiferusjuveniles, which had been previously reported by McFarland and Lee (1963) (isosmoticpoint of 25‰). The isosmotic point in the present study was obtained at 24‰ of salinity,above which the shrimp were hypo-osmotic and below which they were hyperosmotic.

In both salinities the oxygen uptake of the unfed shrimps were constant between 4 mg21 21l DO (55–62.1% AS) and 5.8 mg l DO (80.5–90% AS), later decreasing as the DO

levels decreased. According with this, the P. setiferus juveniles could be consideredlimited oxyregulators (Herreid, 1980). The point of inflection of the curve between theoxygen uptake and the oxygen concentration has been called the critical oxygen level(COL) (Fry, 1947). Below this point the oxygen uptake became dependent on the DOlevel. When the oxygen uptake is reduced, the physiologically useful energy is alsoreduced (EFU), provoking a general metabolic depression (Guppy et al., 1994; Rosas etal., 1997). In P. setiferus juveniles a reduction in oxygen uptake under the COL, such as

21that observed in this study could mean a loss of between 47.6 and 41.6 J g dw (58.721and 47.6% EFU) in the unfed animals and between 38.9 and 45.8 J g dw (35 and 39%

EFU) in the fed animals in 15 and 35‰, respectively. Recent studies in our laboratory


Fig. 4. Penaeus setiferus. Effect of salinity in body water (A) and osmotic pressure (B) of juvenile whiteshrimp kept at different salinity, including animals from DO experimental design. Mean6S.E.


21have shown that below COL (4.5 mg l DO) the EFU in P. schmitti and P. setiferuspostlarvae may be reduced between 17 and 26%, depending on salinity (Rosas et al.,1997). These results show that P. setiferus and P. schmitti like other species, areespecially sensitive to low concentrations of oxygen (Egusa, 1961; Liao and Chien,1994). This sensitivity has been associated with the limited capacity of the anaerobicmetabolism in shrimp (Taylor and Spicer, 1987). These and other authors (Cochran andBurnett, 1996) stated that the low concentration of stored glycogen deposits in thetissues of Palaemon elegans and P. serratus appears to be the main reason for their lowtolerance of anoxic conditions due to an anaerobic limited metabolism via lactate-glycogen pathways. In P. setiferus this limitation has been justified with the high

´requirements of oxygen which are necessary to maintain high muscular activity (Sanchezet al., 1991). While crabs and sedentary isopods are capable of tolerating severe anoxiafor several hours or even days (Bradford and Taylor, 1982; Regnault, 1993; Hagermanand Szaniawska, 1994), lethal levels (LC 50 24 h to 96 h) have been reported between

210.2 and 2.2 mg l DO for different species of penaeid shrimps (Egusa, 1961; Tournier,1972; MacKay, 1974; Liao and Murai, 1975; Allan and Maguire, 1991; Martinez et al.,1998).

It was observed in this study that fasting condition (24 h) provoked a decrease in themetabolism of the P. setiferus juveniles. In other studies it was similarly observed that inthe postlarvae of P. setiferus, P. schmitti, P. notialis and P. duorarum, both the oxygenuptake and the nitrogen excretion diminished up to 60% when shrimp were exposed atshort periods without food (Rosas et al., 1995a, 1996). The high sensitivity to fooddeprivation may be related to the limited capacity of shrimp to store reserve substances(Dall and Smith, 1986). Regarding the DO, it was observed that the oxygen uptake andthe nitrogen excretion of P. setiferus followed similar behavior patterns in both fed andunfed animals, showing that with this unfed period it is possible to observe the samemechanisms of adaptation as those animals exposed to prolonged hypoxia.

The results obtained in the present study show that P. setiferus juveniles can changetheir metabolic substrate as a function of salinity and DO. This change could be relatedto a possible strategy of the organisms to take full advantage of the capacity of obtainingenergy from proteins. That ammonia excretion increases when organisms are transferredto diluted environments has been amply documented. These changes have been shownfor Carcinus maenas (Haberfield et al., 1975), P. chinensis (Chen and Lin, 1992), P.monodon (Lei et al., 1989) and P. japonicus (Chen and Kou, 1992). In the present studyfed and unfed P. setiferus ammonia excretion was significantly greater in 15‰ than in35‰. While the increase of ammonia excretion in low salinity might be due in part to an

1 1increase in the ionic exchange of NH by Na (Robinson, 1982; Schmitt and Uglow,1997), it can also reflect an accelerated catabolism of amino acids and other nitrogenouscompounds (Claybrook, 1983). The values of O:N obtained in this study show that in15‰, animals used proteins as an energy substrate, reflecting an increase in thedowngrade of the ammonia in the hemolymph. Moreover the changes in the proportionof water for the animals and the stability of the osmotic pressure also show that theamino acids can be more important than the ions for the maintenance of osmoticpressure of P. setiferus, such as was observed by McFarland and Lee (1963) for thesame species.


In general, the ammonia excretion of the fed and unfed P. setiferus kept at 15‰ ofsalinity decreased in relation to the decrease of DO, this showing that in diluted mediaand in the absence of food, the P. setiferus juveniles are incapable of regulating theirnitrogen metabolism. On the other hand, the ammonia excretion of the fed shrimp

21remained constant between 4 and 5.8 mg l DO, indicating a capacity of regulating21their nitrogen metabolism within that interval of DO. Below 4 mg l DO the excretion

of ammonium of the animals became dependent on the DO. The pool of amino acidscould act as a buffer between the downgrade of body proteins and the requirements ofenergy of intermediate metabolism and the osmotic pressure (Claybrook, 1983; Mayzaudand Conover, 1988). According to Mayzaud and Conover (1988), amino acids mighthave an alimentary or muscular origin, depending on nutritional conditions. Rosas et al.(1995a) found that ammonia excretion of fed shrimps can increase as a reflection of anincrease of the amino acids of alimentary origin, which in the case of the fed animalsand those kept in low salinity could have the double function of helping to maintainosmotic pressure and providing metabolic energy to the organism. In this way, in thecase of P. setiferus juveniles fed and kept in low salinity, the regulating capacity of theammonia excretion depends on the proteins of digested food, which was independent of

21DO between 4 and 5.8 mg l DO. In unfed shrimp exposed to low salinity, thisregulatory mechanism could have been limited due to the absence of food since theammonia excretion decreased directly with the DO level. In these circumstances the poolof free amino acids would depend directly on the catabolism of tissue proteins, whichmay depend directly on the DO (Herreid, 1980).

In the animals kept at 35‰ the ammonia excretion diminished significantly in unfedshrimps and, independently of the DO level. The effects of severe hypoxia on theammonia excretion in Cancer pagurus and Carcinus maenas proved to be moredependent on a prolonged unfed period than the DO level (Regnault and Aldrich, 1988;Regnault, 1993). In P. setiferus a similar mechanism may be involved in which theregulation of nitrogen metabolism in high salinity could depend more on the contributionof proteins from food than DO.

21When the DO levels decreased from 5.8 to 2 mg l DO, the ammonia excretion of P.setiferus juveniles kept at 15‰ diminished between 77 and 50% in unfed and fedshrimp, respectively. Regnault (1993) reported a reduction between 50 and 60% of theammonia excretion of C. pagurus after a reduction of DO from 8.12 to 2.0 and 0.8 mg

21l DO, respectively. Hagerman et al. (1990) also reported a 90% reduction in theammonia excretion in the isopod Saduria entomon after 96 h of anoxia. Regnault andAldrich (1988) reported a 40% reduction in the ammonia excretion in the crab Carcinus

21maenas exposed brusquely at 2.8 mg l DO.The ammonia excretion of unfed P. setiferus juveniles kept at 35‰ proved to be

independent of DO and increased in relation to a decrease of DO when the animals werefed. These results suggest a change in the strategy of the organism to tolerate low DO

21levels in hypo-osmotic environments. Under normoxic conditions (4 to 5 mg l DO)the proteins and lipids were used as a source of energy and to supply amino acids forgrowth (Claybrook, 1983). In P. setiferus juveniles these conditions produced O:N ratiosbetween 16 and 60, which revealed a catabolism directed to the breakdown of mixedsubstrates composed of proteins and lipids in which the latter would be utilized as a


preferred source of energy and the proteins as a source of muscle structure (Mayzaud21and Conover, 1988). In hypoxia (2 and 3 mg l DO) P. setiferus juveniles showed a

preference for proteins as a metabolic substrate (O:N between 8 and 18), changing thesource of energy used under normoxic conditions. This change in the energy substrate ispossibly due to an increase of free amino acids in the hemolymph, product of thecatabolism of the alimentary and muscular proteins as a consequence of some anaerobicmechanism, just as has been described in other species of crustaceans (Taylor andSpicer, 1987; Hagerman and Szaniawska, 1994). P. setiferus juveniles are particularlysensitive to a diminution in DO. Presumably the mechanisms involved in the tolerancefor DO are controlled by salinity and by the nutritional condition through the pool offree amino acids, used as a source of energy.

Acknowledgements

´The present study was partially financed by the Direccion General de Asuntos del´Personal Academico of the National Autonomous University of Mexico in the names of

Dr. L.A. Soto and Dr C. Rosas (Project IN200994). We thank the Instituto Nacional dela Pesca (SEMARNAP) for its support in the use of the Centro Regional deInvestigaciones Pesqueras de Lerma, Campeche Mexico, which has a collaborationagreement with the Facultad de Ciencias of the UNAM. We also wish to thank theForeign Office of Mexico for the Scholarship awarded to E. Martinez to carry out hisPh.D. studies. We express our gratitude to the MUTIS foundation for the scholarshipawarded to R. Brito to carry out his Ph.D. studies at the UNAM. CONACYT also

´deserves mention for its support to Dr. E. Dıaz. This is the No. 1 Publication of the´Laboratorio de Biologıa Marina Experimental, Fac. de Ciencias UNAM.

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