Salinity stress test and its relation to future performance and different physiological responses in shrimp postlarvae

Aquaculture 268 (2007) 123 – 135 www.elsevier.com/locate/aqua-online

Salinity stress test and its relation to future performance and different physiological responses in shrimp postlarvae Elena Palacios ⁎, Ilie S. Racotta Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico

Abstract This review evaluates the use of the salinity stress test (SST) as an index of postlarvae (PL) quality, either at the end of experimental treatments or to predict performance during stocking and growout. The SST is easy to apply and does not require specialized equipment, hence its popularity. However, predictive value of the test seems to be limited to short-term performance during future culture. To determine to what extent SST depends on osmoregulation, the mechanisms in crustaceans are briefly reviewed, with particular emphasis on studies related to SST in PL penaeid shrimp. Two main physiological features of osmoregulation, active ion transport through the Na+/K+-ATPase pump and fatty acid composition of membranes affecting permeability to water and ions, cannot fully explain differential survival to an SST. This observation is derived from different experimental models, which include nutritional conditions or normal (genetic) variation. Other traits of the overall physiological condition appear to be involved and are not related to physiological regulation but rather are a result of a possible increased tolerance capacity. © 2007 Elsevier B.V. All rights reserved. Keywords: Crustacean; HUFA; Membrane permeability; Osmoregulation; Postlarvae quality

1. Introduction The salinity stress test (SST) is widely used to estimate the quality of postlarvae (PL), on the assumption that it will predict further performance during growout, but very few studies have analyzed the certainty of such a predictive relationship. In contrast, a large number of reports have used SST as a final criterion of PL quality during different experimental conditions. Penaeid PL are considered efficient osmoregulators and it has been proposed that tolerance to changes in salinity depends on osmoregulatory mechanisms (Charmantier et al., 1988). An induced (experimental) or natural (genetic) variation ⁎ Corresponding author. Tel.: +52 612 123 8508 or 123 8484x3347; fax: +52 612 125 3625. E-mail address: [email protected] (E. Palacios). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.034

is the basis for tolerance of PL to the SST. However, it is not clear if these variations result from differences in osmoregulatory mechanisms or to other interactive traits associated with the physiological condition of PL. The focus of the present review is to update studies that use the SST as a PL quality criterion (Bray and Lawrence, 1992; Racotta et al., 2003), while attempting to critically analyze its validity as a predictive criterion and its physiological bases, which implies a discussion on osmoregulation mechanisms in crustaceans. 2. Salinity stress test as a quality criterion of postlarvae The production of PL of optimum quality ensures maximum yields during stocking and further growout in

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Specie

Age

Salinity

Tested variable

Survival to SST

L. vannamei

PL1-2 PL4 PL7 PL2

17 psu × 2 h 10 psu × 2 h 3 psu × 2 h 18 psu × 30 m⁎

None (overall variation)

50% (LC50)

Broodstock condition

PL2-5 PL10 PL15 PL20 PL11

20 psu × 2 h 10 psu × 2 h 5 psu × 2 h 2 psu × 2 h 0 psu × 1 h

None (overall variation)

HUFA

NI (1 mg)

DW × 1 h⁎

PC in diet

51–81% Not related to reproductive exhaustion 5–40% 25–30% 75% 86% 18–36% not influenced by diet N84%

PL15

TW × 30 m⁎

PL20

TW × 30 m⁎

Broodstock condition Starvation

PL20

3 psu × 30 m⁎

HUFA

PL20

TW × 30 m⁎

PL35

80 g/l × 30 m

Individual spawn variation HUFA

PL41

TW CM over 1 h

Vitamin C

NI (61 mg)

1.5 psu × 30 m⁎

None (overall variation)

89% 15-d ablation; 39% 75-d ablation 78% 24-h starved PL; 86% fed PL 55% Low and high HUFA; 69% medium HUFA

Performance (P) and SST

Samocha et al. (1998)

↓P, =SST with reproductive exhaustion

Arcos et al. (2005)

Aquacop et al. (1991)

=P, =SST with different HUFA and DHA/EPA ratio ↑P, =SST with intermediate PC levels ↓P, ↓SST with reproductive exhaustion ↓SST with starvation =P, ↑SST with intermediate HUFA but high DHA/EPA

33–42% 46–65% Not influenced by diet 33–90%, 0–2000 mg/kg Vitamin C 96%

References

Wouters et al. (1997) Coutteau et al. (1996) Palacios et al. (1999) Palacios et al. (2004a) Palacios et al. (2004b)

Racotta et al. (2004) ↑P, =SST higher HUFA; =P, =SST difference DHA/EPA ↑P, ↑SST with increasing levels of vitamin C

Wouters et al. (1997) Kontara et al. (1997a) Gomez-Jimenez et al. (2004)

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Table 1 Comparison of SST applied to shrimp PL as a posteriori quality criterion test

P. monodon

L. setiferus

14, 21 psu × 30, 60 m 7, 4 psu × 30, 60 m 0, 4 psu × 30, 60 m 2 psu CM over 2 h

Live and artificial diets + HUFA PC and cholesterol

0–100% depending on. PL age, SST duration and diet 50% (LT50)

PL10

10 psu CM over 2 h

HUFA

PL15

10 psu × 1 h

DHA/EPA ratio

20–95% 0–400 ppm SELCO 27–93%

PL30 PL40

0 psu × 2 h 0 psu CM over 1 h

Z-PL1 PL5-10 PL20 PL32 PL52

20–22 psu × 24 h 14–18 psu × 24 h 6 psu × 24 h DW CM over 1 h

None (overall variation)

50% (LC50)

PC + HUFA

CM index (abstract units)

NI (5 mg)

DW × 30 m⁎

PC in diet

NI (5–22 mg)

0–20 psu × 2 h

PL1-5

16 psu × 1 h

PL10 PL21

10–40 psu CM

None (overall variation) Food quantity during culture None (overall variation)

77–90% after 17 d; 43–87% after 31d 0–100%

Vitamin C and astaxanthin

5–23% 17–73%

7 to 96% 50% (LC50)

↑P, ↑SST with live food and HUFA during culture ↑P, ↑SST with PC and cholesterol supplementation ↑P, ↑SST with intermediate HUFA ↑P, ↑SST with min. HUFA =P, =SST difference DHA/EPA ↑P, ↑SST with increased vitamin C and astaxanthin

Tackaert et al. (1992)

Paibulkichakul et al., 1998 Rees et al., 1994 Kontara et al., 1995

Merchie et al., 1998

Charmantier et al., 1988 ↑P, ↑SST with PC and HUFA supplementation ↑P, ↑SST with PC (17 d)

Tackaert et al., 1991

↓P, ↓SST with PC (31 d)

Duran-Gomez et al., 1991

↑P, ↑SST with intermediate food quantity

Gallardo et al., 1995

Kontara et al., 1997b

Rosas et al., 1999

P = performance (survival and/or growth during culture); PL#, where # represents days since metamorphosis to PL; Z = zoea; NI = not indicated; DW = deionized water; TW = tap water that can range from ∼0 to 5 psu, depending on location and season; CM = cumulative mortality; ⁎ = with recuperation in sea water; LT = lethal time; LC = lethal concentration; PC = phosphatidylcholine; m = minutes; h = hours; d = days.

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P. japonicus

PL5 PL10 PL15 PL15

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ponds. SST is commonly applied in hatcheries to estimate the quality of PL to be used during growout (Tackaert et al., 1989; Dhert et al., 1992; Rees et al., 1994) and for genetic improvement programs (Ibarra et al., 1998). A testing procedure for the immediate evaluation of larval quality was first formulated by Tackaert et al. (1989), although they did not find a correlation between salinity stress resistance of Penaeus monodon and performance in the ponds (Tackaert et al., 1992). Originally, samples of PL of different ages were exposed to several salinities and monitored after 30 and 60 min. Then SST was reported in terms of cumulative mortalities, but the SST has been simplified to abruptly exposing PL during at least 30 min to a reduced salinity that can be variable according to the species and PL stage (Table 1). PL that have a higher survival to an SST are considered to be healthier or of better quality, assuming that a higher physiological condition allows a higher tolerance (survival) to such stress. The SST is easily and rapidly applied and does not require specialized equipment, hence its popularity. In some cases, a second transfer of PL to seawater or to the original salinity for another 30 min is performed and survival is then estimated. As pointed out by Fegan (1992), PL are considered dead when no movement is detected in response to a mechanical stimulus, but these apparently “dead” PL can recover when returned to normal salinities. Indeed, higher survivals were obtained after this recuperation at the original salinity, compared to previously recorded survival, and in addition, PL that were in a good condition showed a better recuperation (Palacios et al., 2004a). For production purposes, PL are stocked in growout ponds when a survival to an SST of at least 60% is recorded. The SST is the basis for price setting according to quality and helps farmers choose among the various PL that are offered for growout (Dhert et al., 1992), with farmers only buying, or willing to pay 30% more for stress-tested PL that may result in N 90% survival during growout (Briggs, 1992). Survival to an SST has been used to predict performance during growout (Aquacop et al., 1991) or during pond stocking (Fegan, 1992), but until recently, there was no direct evidence for an association between survival to an SST and further traits during postlarvae culture, stocking, or growout in ponds (Table 2). Alvarez et al. (2004) evaluated 40 batches of PL and found that batches that had higher survival to the SST also had higher survival during simulated stocking. However, survival to an SST was not correlated to growth or survival during growout after 2 months in ponds. Thus, survival of PL to an SST might be associated with survival for a short-term stress,

Table 2 Comparison of SST applied to shrimp as a priori quality criterion test Species

SST

Related trait on further performance

References

P. monodon

PL10, 10 psu × 2 h 10 to 100%

Rees et al., 1994

L. vannamei

PL1, 18 psu × 30 m⁎ 5 to 95%

L. vannamei

PL15, 3 psu × 30 m⁎ 2 to 82%

Positive correlation with survival to PL15 Positive correlation with survival PL1–PL20 Positive correlation with survival during stocking, no relation with survival and growth during 2 months

Racotta et al., 2004

Alvarez et al., 2004

See Table 1 for abbreviations.

like during further performance to more advanced PL stages, or during stocking in ponds, but it is not necessarily an adequate predictive criterion for a longer good performance. Although developed as an a priori criterion, the SST has been more extensively used as a posteriori criterion to determine the effect of a particular condition or treatment, i.e., diets, environmental conditions, culture schedules, etc., and has now become a standard procedure to evaluate if a treatment has indeed increased the physiological condition or quality of the PL (for a review, see Racotta et al., 2003). As an a posteriori criterion, it has been extensively reported in scientific literature, in contrast to the scant reports that exist for the SST to predict stocking survival or as an a priori criterion (Table 2). Its justification to be used as an a priori criterion is that batches of shrimp in poor condition will be less able to withstand such acute stresses than batches of healthy shrimp (Fegan, 1992). On the other hand, it is used as a posteriori criterion because it is assumed that different treatments will influence several physiological features, which in turn will determine their resistance to stress tests before the impact on growth and eventually survival may be noticed (Dhert et al., 1992). 3. Salinity stress test and osmoregulation It is generally assumed that the SST evaluates indirectly the physiological capacity of osmoregulation. After metamorphosis to PL, osmoregulatory tissues appear in gills and epipodites (Bouaricha et al., 1994). Based on different experimental approaches, it was

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concluded that there is a strong correlation between the development of osmoregulatory epithelia, the ability to osmoregulate, and salinity tolerance (Charmantier et al., 1988; Bouaricha et al., 1991, 1994). Osmoregulatory capacity develops during the migration of shrimp to estuaries, where they are exposed to a wide variation of salinities (Dall, 1981; Charmantier, 1987, 1988, 1998), and can be modified in relation to temperature and season (Péqueux, 1995). The osmoregulatory capacity generally increases with PL age, but depending on the species, adults can be better or worse hyper-regulators (Charmantier et al., 1988; Charmantier-Daures et al., 1988; Bouaricha et al., 1994). The osmoregulation capacity, defined as the difference between osmotic pressure of hemolymph and the external medium (Charmantier et al., 1994), has been proposed as an indicator of the physiological condition and stress indicator for crustaceans and tested in shrimp under several conditions (Charmantier-Daures et al., 1988; Charmantier et al., 1994; Lignot et al., 2000). This test has the advantage of test organisms manifesting differences in osmoregulatory capacity under mild stress conditions that are insufficiently strong or require more time to produce death as the SST. However, the determination of osmoregulatory capacity requires specialized equipment and the hemolymph sample is hard to obtain in PL (Charmantier et al., 1988), in contrast to the simplicity of the SST. Alternatively, osmolarity can be measured in the whole PL (Loeza et al., 2005), as previously standardized for fish larvae (Tandler et al., 2004), although in this way, an average of intracellular (mainly muscle) and extracellular osmolarity is estimated. Thus, in theory, the SST helps decide when the shrimp, that up to that moment have been grown in controlled environments that resemble seawater conditions, are ready to tolerate changes in salinity similar to those found in estuaries, and that could be found during growout in ponds. Because osmoregulatory mechanisms can develop with PL age, batches of PL that have low survival to an SST could be left in controlled conditions for 1 or 2 weeks longer and tested again, and survival should improve, at least up to a certain age (Charmantier-Daures et al., 1988). In this way, the SST would indicate the best age for transference to ponds of a particular batch of PL. However, in the same species and population, genetic variability has been found between batches of the same age, indicating that PL develop their osmoregulation capacities differently under the same conditions and can thus be selected in an genetic improvement program (Ibarra et al., 1998; Laramore et al., 2001).

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4. Active ion transport and osmoregulation Most crustacean species living in seawater are osmoconformers, while those that live in freshwater are hyperosmotic osmoregulators. Hyperosmotic regulators have an osmolarity that is higher than their environment, facing hydration and diffusive loss of ions and requiring energy consumption for osmotic regulation (Péqueux, 1995). Salt movement occurs at the level of permeable surfaces in crustaceans, such as the body wall, gastrointestinal tract, excretory organs, and gills. Gills are among the most permeable external surfaces of crustaceans, and they are considered the primary site for ionic and osmotic regulation (Péqueux and Gilles, 1981; Bouaricha et al., 1994). Gills specialized in osmoregulation have specialized cells for active ion transport or ionocytes, with an apical microvilli and basolateral infoldings that have numerous mitochondria (for review, see Péqueux, 1995; Charmantier, 1998; Towle and Weihrauch, 2001). Permeability can be highly variable at each surface, with the carapace 500–5000 and gill cuticle 50–300 times less permeable in osmoregulating compared to osmoconforming crabs (Péqueux, 1995). The excretory organs, particularly the antennal gland in crustaceans, regulate the volume of body fluids, the concentration of some organic solutes, and divalent ions, and in some crustaceans, the reabsorption of NaCl (Péqueux, 1995). When osmoregulating crustaceans are placed in dilute medium, there is an influx of water and a passive loss of Na+ and Cl− that occurs in permeable epithelia, compensated by an active uptake of Na+ from the water in exchange for H+ or NH4+ at the level of the apical membrane (facing water) of osmoregulatory cells. The Na+ ions are pumped into the hemolymph by the Na + /K + -ATPase located in the basolateral (facing hemolymph) membrane (for a review, see Lucu and Towle, 2003). Intracellular Cl− leaves the cell towards hemolymph space through Cl− channels in the basolateral membrane, possibly driven by the electropositivity of hemolymph (Péqueux, 1995). In addition, evidence for the existence in the basolateral membrane of a channel for K+ that permits recycling of K+ pumped into the cell has been found in crabs (Péqueux, 1995). Posterior gills of osmoregulating crabs have a primary osmoregulatory function compared to anterior gills that function mostly for respiration (Holliday, 1985; Chausson and Regnault, 1995; Genovese et al., 2000), and approximately 75% of the total ATPasespecific activity is found in the posterior gills of crabs (Holliday, 1985). However, Dickson et al. (1991) stated that each gill can contain respiratory and transport filaments. In shrimp, the functional separation of

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anterior and posterior gills into osmoregulating and respiratory functions is not so clear (Lucu and Towle, 2003), but the activity of Na+ /K+ -ATPase in the posterior gills of PL exposed to a salinity challenge can account for up to 85% of the total activity in the gills (Palacios et al., 2004a). The activity of Na+/K+-ATPase is associated with the beginning of osmoregulation capacity during ontogeny in Penaeus japonicus (Bouaricha et al., 1991) and evidence exists for its control by hormones (for reviews, see Péqueux, 1995; Lucu and Towle, 2003). The net movement of Na+ mediated by Na+/K+ATPase is from cell cytosol to hemolymph, but other transporters must mediate movement of Na+ from the environment across the apical membrane to the cytosol (Towle and Weihrauch, 2001), most of which are passive, e.g., they do not require ATP. Among these are the apical membrane Na+/H+ and the Na+-K+-2Cl− exchangers that move Na+ into cytosol, depending on a chemical and electrochemical gradient, respectively (Towle and Weihrauch, 2001). In the apical membrane, the passive exchange mechanism through Na+/NH4+ could also account for a very small part of the total Na+ influx occurring in posterior gills of crabs (Péqueux, 1995). Together with a Na+/H+ apical exchange, and coupled to a Cl−/HCO3− exchange, these mechanisms are able to absorb NaCl in dilute environments (for functional models in crustaceans, see Péqueux, 1995; Towle and Weihrauch, 2001). The H+ exchanged for Na+ at the apical membrane of osmoregulatory cells is released by the enzyme carbonic anhydrase by the formation of HCO3− in the cytoplasm (Henry, 1996). Activity of carbonic anhydrase increases in gills of osmoregulating crabs exposed to low salinities, apparently enhancing the supply of counterions for NaCl uptake (Henry, 1996; Towle and Weihrauch, 2001). The carbonic anhydrase is also active in the respiratory gills to hydrate the CO2 produced by respiration (Henry, 1996). In contrast to Na+/K+-ATPase activity, activity of carbonic anhydrase does not vary throughout development (Bouaricha et al., 1991). No significant differences were found between anterior and posterior gills for carbonic anhydrase, but its activity was only correlated to that of Na+/K+ATPase activity in the posterior, but not in the anterior gills (Palacios et al., 2004a). Activity of these enzymes (Bouaricha et al., 1991) and osmoregulatory structures (Bouaricha et al., 1994) closely match the lethal salinity at different larval and PL stages (Charmantier et al., 1988). An increase in Na+/K+-ATPase activity during an SST (Alvarez et al., 2004) supports the proposition that survival to this test

depends on active ion transport. However, baseline (control salinity of 30 psu) or activated (SST at less than 3 psu) Na+/K+-ATPase activity was not higher in PL batches that had higher survival than in those that had lower survival to an SST (Alvarez et al., 2004), indicating that active ion transport per se is not enough to explain natural (genetic) variability of survival to SST. Another characteristic that is related to active ion transport is the anatomical basis of a larger gill area, which apparently is associated with survival to an SST in several experimental models, particularly to explain individual variation and influence of HUFA. PL fed a rich HUFA diet have larger gill area (Palacios et al., 2004b; Loeza et al., 2005), which agrees with the observation of a more ramified gill structure in HUFA-fed PL (Rees et al., 1994). In addition to the above osmoregulatory mechanisms, a larger gill area has an increased ion transport surface and possibly the number of cells that contain Na+/K+ATPase pumps available to exchange ions. Other possible approaches to establish possible links between Na + /K + -ATPase activity, osmoregulatory mechanisms, and differences in SST survival will be analyzed in further sections. 5. Salinity stress response and diet Survival of PL to a stress test can be influenced by nutrition and culture conditions (Table 1). The SST is widely used in nutritional studies and is generally accepted that a better diet will produce larger PL with a higher rearing survival rate and greater survival during a stress test. However, as shown in Table 1, this relationship is not always the case. Mechanisms that are put into motion during a salinity challenge may not be the same as those used for growth or survival during culturing. Quantity of food, assuming overall energy availability, should affect growth and survival and be directly associated with general performance and survival to an SST. Gallardo et al. (1995) found greater rearing survival, growth, development rates, and survival to an SST in organisms fed more microalgae during larval culture and Artemia during PL culture, but up to a certain limit. Similarly, lower survival to an SST was obtained in 24-h-starved PL compared to fed PL (Palacios et al., 2004a). When comparing osmoregulating mechanisms, the Na+/K+ATPase activity in gills of PL at 30 psu was 50% lower in starved organisms, probably as a consequence of a decrease in the general metabolism due to starving. However, the activity of this enzyme in PL exposed to low salinity (10 psu) was similar between fed and

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starved organisms, indicating that lower survival rate to an SST in starved PL cannot be explained by lower activity of this enzyme alone. The relation between particular biochemical reserves than can be used directly for osmoregulation or to withstand general stress is supported by higher survival to SST for batches of PL that had higher levels of glucose and acylglycerides, compared to same-age batches cultured under the same conditions (Palacios et al., 1999; Alvarez et al., 2004). Chausson and Regnault (1995) found more carbohydrate reserves in posterior than in anterior gills. These reserves can be used as an energy pool for the Na+/K+-ATPase pump. The role of carbohydrate as a source of energy for osmoregulation is illustrated by higher dietary carbohydrate requirements for shrimp exposed to decreasing salinities (Wang et al., 2004). During short-term starvation, carbohydrates are usually the first macronutrients to be depleted, thereafter, lipids and proteins are used. Few studies report on short-time changes in reserve mobilization and substrate oxidation. These changes should be analyzed in an SST to establish the particular contribution of carbohydrate vs. lipid or protein mobilization. Such an analysis was done for proteins, and an increase in NH4+ efflux rate 5 min after exposure to 1.5 psu was found (Gomez-Jimenez et al., 2004). A reversible increase in free amino acids is a well-described mechanism to maintain osmotic balance between intra- and extracellular compartments in sea water (Marangos et al., 1989). But when salinity decreases, the free amino acids might be oxidized with concomitant excretion of NH4+ (Charmantier, 1987) or incorporated into proteins. In turn, the excretion of NH4+ in diluted media can be coupled to Na+ uptake in gills (Holliday, 1985). A mobilization of lipids to gills from the hepatopancreas was suggested in PL submitted to a salinity challenge (Palacios et al., 2004a). Lipids in gills decreased in crabs acclimated to diluted sea water during 72 h (Luvizotto-Santos et al., 2003). Kinsey et al. (2003) concluded that for crabs exposed to hyposmotic conditions for 7 days both gill types oxidized glucose and amino acids, while posterior gills preferentially used fatty acids as metabolic fuels for active ion pumping. A decrease in saturated and monounsaturated fatty acids in neutral lipids of gills after exposure to 10 psu for 3 h can be a result of energy production for osmoregulation by selective fatty acid oxidation (Palacios et al., 2004b). A selective fatty acid oxidation could mean that PL fed a diet rich in saturated fatty acids should have greater survival to the SST because these fatty acids are better energy substrates as they are less oxidized and produce

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more energy per gram than highly unsaturated fatty acids (HUFA). The effect of HUFA enrichment on PL survival to an SST has been extensively studied, compared with other nutrients (Table 1). In general, higher HUFA levels in the diet are considered better. However, results have not always been convincing, because although some investigators found that PL fed enriched HUFA diets had increased performance and survival to an SST, others found no effect on either or an effect on growth, but not SST. Studies conducted with other lipids, such as phosphatidylcholine (PC) or cholesterol have also provided mixed results (Table 1). HUFA and other lipids can effect overall physiological condition as a result of enhanced nutritional status or directly effect osmoregulatory mechanisms. Fatty acid composition of membrane phospholipids in gills can be changed in shrimp fed different levels of HUFA (Palacios et al., 2004b). As will be discussed below, composition of fatty acids of membrane phospholipids can influence water and ions permeability (Morris et al., 1982) and the activity of the Na+/K+-ATPase pump (Tocher et al., 1995). 6. Na+/K+-ATPase activity and lipids An effect of membrane lipids on Na+/K+-ATPase activity was proposed by Towle (1977) when he described how detergents could ‘unmask’ the latent Na + /K + -ATPase pump in fish. He proposed that modulation of membrane lipids may be responsible for the rapid rise (30 min) of Na+/K+-ATPase activity in the gills of fish exposed to changes in salinity, in contrast to changes in gene expression, affecting Na+/K+-ATPase pump numbers or subunit isoform expression on a longterm basis. Membrane bilayers are composed of phospholipids that have an asymmetric arrangement in the membranes, with the outside composed mainly of PC, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) are largely confined to the interior membrane (Chapelle et al., 1976). PE has a higher gel/ fluid transition temperature than, for example, PC of similar fatty acid chain composition, due in part, to hydrogen bonding between the head groups of PE (but not PC), the close packing permitted by the reduced steric bulk of PE, and the greater hydration of PC relative to PE (Hazel and Williams, 1990). PS is acidic and Na+/K+-ATPase selects acidic phospholipids in membranes for interaction (Zwingelstein et al., 1998). The level of phospholipids in the posterior gills differs from that in anterior gills in crabs (Chapelle and Zwingelstein, 1984). Euryhaline crabs increase the synthesis of phospholipids in posterior gills in response

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to changes in salinity (Whitney, 1974; Chapelle et al., 1976; Chapelle and Zwingelstein, 1984), PS and PE particularly act as specific carriers of Na+ (Chapelle et al., 1976). Distribution of different phospholipids in the membranes could be associated with the location of Na+/K+-ATPase pumps (Chapelle et al., 1976) and activity of the Na+/K+-ATPase, which is dependent on the presence of PS in the cellular membrane in the posterior gills of crabs (Chapelle, 1986). In addition to the effect of phospholipids on Na+/K+ATPase, different fatty acids can be selectively incorporated into each phospholipids class of the cellular membrane. Poon et al. (1981) substituted fatty acids from the phospholipids present in the cellular membranes of rat tumor lymphocytes and found that the activity of the Na+/K+-ATPase decreased with saturated fatty acids and increased with HUFA. The lower activation energies observed for crustacean gill Na+/ K+-ATPase may be a consequence of higher concentrations of HUFA in their cell membranes compared with homeotherms (Lucu and Towle, 2003). HUFA, and particularly docosahexanoic (DHA, 22:6n-3) content in membranes can modulate the activity of Na+ /K+ ATPase and other enzymes embedded into the membranes of vertebrates (Turner et al., 2003), an effect that is associated with membrane packing parameters of DHA (Wu et al., 2001; Eldho et al., 2003) and its hundreds of high-probability conformations in a membrane bilayer (Feller et al., 2002). Phospholipids that contain DHA chains seem ideally suited to “solvate” integral membrane proteins, in particular those that undergo rapid structural transitions, and the loss of one double bond in the DHA chain is sufficient to change its properties (Eldho et al., 2003). From this knowledge of other animal models, one may speculate that PL exposed to low salinities have to increase their Na+/K+-ATPase activity to compensate for Na+ loss by increasing the levels of PE and PS, or/ and increasing HUFA proportion in these and possibly other phospholipids. This could be the reason PL fed diets high in HUFA usually have higher survival to an SST. However, the effect of HUFA on survival to an SST and on Na+/K+-ATPase activity is not always clear. For example, Na+/K+-ATPase activity was higher in PL fed diets with intermediate and high levels of HUFA, although survival to an SST was increased only with intermediate levels of HUFA containing a high DHA/ EPA ratio (Palacios et al., 2004b). Crockett (1999) concluded that restructuring of fatty acids and phospholipids in the basolateral membranes of gills does not contribute to the increases in Na+/K+-ATPase activity during seawater acclimation of eels. In Artemia, free

Fig. 1. Relation between Na+/K+-ATPase activity and DHA concentration in the polar fraction of the gills of shrimp (3.5 ± 0.5 g). Circles = acute (15 h) exposure (r2 = 0.87; P b 0.01); triangles = chronic (3 weeks) exposure to changes in salinity (r2 = 0.02; P N 0.05). Adapted from Hurtado et al. (in press).

long-chained fatty acids inhibit the activity of the Na+/ K+-ATPase pump (Morohashi et al., 1991), and a negative correlation has been found between the activity of the Na+/K+-ATPase and DHA levels in gills of shrimp (Fig. 1). It seems that for at least crustaceans, the relationship between survival to an SST and Na+/K+ATPase regulation with HUFA is not simple. Additionally, while Na+/K+-ATPase is the better studied iontransport protein and is particularly sensitive to variation in membrane physical properties (Kimelberg and Papahadjopoulus, 1972), other membrane-bound ion exchangers are involved in osmoregulation, and these proteins can also be affected by lipid membrane composition. In addition, changes in membrane HUFA can also directly affect permeability to water and ions. 7. Membrane lipids and permeability Water transfer across the body surface is primarily through intercellular pathways, the majority through

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pores in the membrane lipid bilayers and approximately 10% directly through the bilayers (Morris et al., 1982; Haines, 1994). Further decrease in permeability or diffusion, once the pores are closed, as might be the case after a salinity challenge, depends upon the permeability of the lipid bilayer, and this observation in turn is affected by the fatty acid composition. Selective changes in fatty acid content in membrane phospholipids in gills can enable organisms to control permeability (Poon et al., 1981; Morris et al., 1982). In freshwater crustaceans, water permeability is lower than in animals living in seawater (Rasmussen and Andersen, 1996) by 50 to 500 times and by a factor of 100 for Na+ (Péqueux, 1995). However, permeability does not depend upon thickness of the cuticle but upon the integrity of the external lipoproteic epicuticule in crustaceans (Péqueux, 1995). Haines (1994) proposed a model for water transport across bilayers packed with straight-chain fatty acids, tested by Paula et al. (1996), who prepared liposomes with chain lengths ranging from 14 to 24 carbon atoms; they found that permeability of water was moderately dependent on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In relation to ions, H+ and Na+ leakage is affected by the number of double bonds in a membrane (Haines, 1994; Porter et al., 1996) and by the length of the fatty acids (Paula et al., 1996). PL exposed to low salinities must decrease their permeability to water and ions, which theoretically could be achieved by increasing the proportion of saturated fatty acids and the chain length of fatty acids in the membrane phospholipids. In crustaceans, an increase in the proportion of saturated fatty acids in cellular membranes, with a concomitant decrease in HUFA, can decrease permeability to water (Whitney, 1974; Morris et al., 1982), and this decrease in amphipods can occur almost instantaneously on transference to dilute water (Morris et al., 1982). However, long-chain saturated fatty acids are not easily incorporated into membrane phospholipids, because they do not have the capacity to fold as fatty acids with double bonds do (Feller et al., 2002). To overcome this physicochemical constrain, long fatty acids with double bonds have to be incorporated into phospholipids, but this compromises membrane permeability. A compromise between fatty acid length and unsaturation has to be reached, and for this reason, feeding HUFA-enriched diets to PL does not necessarily increases PL tolerance to low salinities as discussed in a previous section. In addition to fatty acids, membrane permeability can be regulated by other lipids, such as cholesterol and

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phospholipids (Whitney, 1974; Hazel and Williams, 1990). Although cholesterol in low quantities is essential for shrimp (Teshima, 1982), it is seldom considered as a modulator of osmoregulation in crustaceans. However, cholesterol inhibits water and Na+ permeability through phospholipid bilayers, both by restraining the motion of lipid chains and increasing the phospholipids head group spinning motion, thus saving ATP and energy (Haines, 1994). Accordingly, increased salinity tolerance has been observed in P. monodon PL fed the highest proportion of cholesterol (1%) in comparison to diets with lower cholesterol levels (Paibulkichakul et al., 1998).Uncontrolled variation in cholesterol levels in experimental diets could partially explain different survival to SST. 8. Is the survival to SST a direct index of osmoregulation mechanisms? Two main physiological features of osmoregulation, namely active ion transport through the Na+/K+-ATPase pump and fatty acid composition of membranes, which can affect permeability to water and ions, cannot fully explain the variation in survival to an SST under diverse experimental models. Other osmoregulatory mechanisms must be more thoroughly studied to evaluate their role in PL survival to an SST or quality. In addition to osmoregulatory mechanisms, other variables which perhaps are not related to physiological condition might be involved but could result in an increased survival to an SST. For example, to decrease salinity when performing an SST, some works report using tap water, while others used deionized water. Salinity is often determined as the sum of all ions, particularly Na+ and Cl− in the water, but each ion can be regulated differently (Charmantier, 1987). Ionic composition in tap water might vary substantially from one location to another, and this can affect which transport proteins are used by PL. On the other hand, PL exposed to deionized water might not be able to osmoregulate adequately because of lack of external ions, and thus survival in these conditions might result from a generally lower permeability. In addition to age differences of PL, survival to an SST can also be affected by the stage of molt in PL of the same age and batch. Osmoregulation capacity decreases in late premolt and early postmolt (Charmantier et al., 1994), while water permeability increases in crabs during molting (Rasmussen and Andersen, 1996). The activity of the Na+/K+-ATPase increases in gills and hypodermis of crabs during late premolt and early postmolt stages, suggesting that this enzyme plays an

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important role in driving Na+/Ca++ exchange during mineralization of the cuticle (Lucu and Towle, 2003). If the SST is applied when a higher proportion of PL are in ecdysis, the “quality” of the batch may be underestimated. Molt stage assessment can be complicated in PL, especially on a commercial level at hatcheries, so another SST could be applied the next day to confirm or discard the first results. The time-course of the SST is particularly important because the biochemical reserves and osmoregulatory mechanisms are used differently over time, but Table 1 reveals abundant differences in the time used to evaluate survival of PL after exposure to diluted seawater. For example, there is an active absorption of Na+ in crab gills minutes after a salinity change (Onken, 1996). Cell swelling in crabs after a hyposmotic challenge can be regulated by amino acid leaking out of the cell in minutes (Gilles and Péqueux, 1981). Decreased water permeability in gills of crabs transferred to dilute media after 1 h was reported by Cantelmo (1977). The Na+/K+ATPase can be activated by modulation of membrane lipids in 30 min in fish gills (Towle, 1977). Other rapid changes in Na+/K+-ATPase activity could result from various post-transcriptional modifications that affect turnover number and include reversible phosphorylation and changes in the subcellular distribution of the enzyme (Crockett, 1999). The above mechanisms probably contribute to the stabilization of osmotic pressure in shrimp PL that can be achieved 3 to 6 h after a hyposmotic challenge (Charmantier et al., 1988). Slower modifications (over days) of Na+/K+-ATPase activity can involve changes in gene expression, affecting Na+/K+-ATPase pump numbers or subunit isoform expression on a long term (Holliday, 1985; Crockett, 1999; Lucu and Flik, 1999). Phospholipid decarboxylation and methylation are increased in eurohaline osmoregulating crabs exposed to diluted seawater after 12 h (Zwingelstein et al., 1998), and biosynthesis after 24 h, in contrast to osmoconforming crabs that show no differences in lipid composition (Whitney, 1974). Ionocyte differentiation was reported in posterior gills of crabs exposed to diluted sea water after 48-h exposure to salinity changes (Genovese et al., 2000). A correlation, albeit negative, was found between Na+/K+-ATPase activity and DHA concentration in the membrane lipids of gills on a relatively shortterm salinity exposure (15 h), compared to a chronic exposure, where apparently, membrane lipids in gills do not modulate Na+/K+-ATPase activity (Fig. 1). Thus, evaluating survival after 30 min, 2 h, or 1 day is really indicative of different osmoregulatory responses that are determined genetically and might be affected by the

experimental treatment. In any case, when evaluating survival to SST, only mechanisms activated in minutes can affect survival. Slower activated mechanisms are probably involved in other practices such as acclimation to freshwater. A final question is: to what extent does survival to an SST really reflect the overall physiological condition? Assuming that performance in culture (growth and survival) is determined by general physiological condition, we can analyze the relationship between performance and SST reported in Table 1 for different models of experimental conditions. In eleven cases, an increase (or decrease) in performance was accompanied by a concomitant change in survival to SST, whereas in three cases no changes were observed for any factor. This is equivalent to 80%, where performance and survival to an SST demonstrated an equivalent tendency. In contrast, a positive or negative effect on performance without any changes in the SST occurred in three models (15%), and the contrary, where no changes in performance but SST was affected occurred only once. Even when comparing just one experimental treatment, i.e., HUFA level in diet and survival of PL after 30–60 min, this relation (80%) remains similar. Thus, in most cases the SST does reflect the physiological condition of the PL. However, an SST does not necessarily appear to be more sensitive than an evaluation of general performance during rearing. 9. Conclusions The SST is gaining importance as a PL quality criterion to evaluate the response to a treatment. However, the SST should be standardized, as it can reflect different adaptive mechanism when measured at different times or conditions. SST is not only associated with osmoregulating capacity but also reflects a more general physiological condition of the organism to tolerate adverse environmental conditions (low salinity or others). The general performance during PL culture (final weight and survival) can be more sensitive than SST to larval quality. On the other hand, the use of SST as a predictive tool during growout has probably been overestimated and still needs to be validated. In any case, it should not be the only criterion used to base a decision about the quality of a PL batch. At least the “history” of a particular batch, together with simple biochemical analyses, should be evaluated. On a practical level, the SST could be repeated on the following day to control for molting, and care should be applied when using “tap water” of different origins or seasons.

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