Swimming ability and physiological response to swimming fatigue in whiteleg shrimp, Litopenaeus vannamei

Some penaeids are active swimmers, undertaking migrations of hundreds of nautical miles. At present, however, very little is known of swimming ability in penaeid shrimps. The aim of the present study is to investigate swimming endurance of whiteleg shrimp, Litopenaeus vannamei, against one of five flow velocities (5.41, 6.78, 8.21, 10.11, and 11.47 cm s(-1)) for up to 9000 s at 20 degrees C in a swimming channel. Body mass, hemolymph total protein concentration, and hemolymph glucose level were measured before swimming and immediately following swimming to evaluate physiological effect of swimming in L. vannamei. No shrimp swam the full 9000 s at any of the velocities tested. The swimming endurance decreased as swimming speed was increased. The relationship between swimming endurance (t, in s) and swimming speed (v, in cm s(-1)) can be described by the Curve Estimation: v.t0.38 = 159.64 (R2 = 0.94). The swimming ability index (SAI), defined as SAI = integral 0-9000 vdt x 10(-4) (cm) was found to be 7.28 cm for the shrimp tested. Swimming to fatigue leads to severe loss of body mass, hemolymph total protein concentration, and hemolymph glucose level in L. vannamei (P < 0.05). Furthermore, these decreases and swimming speed showed significantly polynomial relationships (P < 0.05). The results suggest that the power model fits well to the observed endurance estimates and the SAI is a good index to quantitatively describe the overall swimming ability of L. vannamei. Furthermore, hemolymph total protein concentration may be used as a rapid and reliable indicator to assess the penaeid shrimps' swimming speed and hence swimming ability.     

Effect of salinity on transmission of necrotizing hepatopancreatitis bacterium (NHPB) to Kona stock Litopenaeus vannamei

Elevated salinity and temperature have been observed prior to devastating necrotizing hepatopancreatitis (NHP) outbreaks in several geographically isolated shrimp ponds. These observations have led to the hypothesis that the NHP-bacterium (NHPB) is hindered by reduced salinity, even though the mechanism is not understood. The objective of this research was to examine the effect of salinity on transmission of NHPB. The transmission rate of NHPB was estimated through laboratory experiments whereby individuals of Kona stock Litopenaeus vannamei were orally exposed to a dead NHPB-infected shrimp. For each replicate, 12 susceptible shrimp were placed with a dead NHPB-infected shrimp in a 1 m2 bottom area cylindrical tank maintained at 30 degrees C for a period of 24 h. Four salinities of 10, 20, 30, and 40 per thousand were replicated 2 times in 2 trials, giving a total of 192 shrimp exposed per os to infective material. In each trial, a negative control group was included at each salinity, giving a total of 96 shrimp exposed orally to uninfected material. After the 24 h exposure period, susceptible shrimp were individually isolated at the same physical conditions for up to 60 d to determine NHPB transmission. The NHPB was transmissible regardless of salinity: nearly a quarter of susceptible shrimp exposed to NHPB at the lowest (10 per thousand) and highest (40 per thousand) salinity examined acquired NHPB. Transmission rates were highest at the intermediate salinities of 20 and 30 per thousand, suggesting that those salinities are optimal for NHPB transmission. The observed association between high salinity and NHP outbreak in a shrimp pond is not explained by these results because reduced transmission occurred at very low and very high salinities.     

Osmoregulatory capacity of the shrimp Litopenaeus vannamei at different temperatures and salinities, and optimal culture environment

Osmoregulation in Litopenaeus vannamei was studied in a factorial experiment at four temperatures (20, 24, 28 and 32 degrees C) and six salinities (10, 16, 22, 28, 34 and 40 per thousand). The isosmotic related points for 20, 24, 28, and 32 degrees C were 754, 711, 822, and 763 mmol/kg, respectively. This species hyperregulates between at salinities of 10 and 20 per thousand and hyporegulates between 20 and 40 per thousand. The isosmotic point in L. vannamei exposed to constant salinities changed in relation to temperature from 717 to 823 mmol/kg. For these experimental conditions, the T-S combination of 32 degrees C and 28 per thousand produced the best growth.     

Physiological and behavioral responses of the mud snails Hydrobia glyca and Hydrobia ulvae to extreme water temperatures and salinities: implications for their spatial distribution within a system of temperate lagoons

Physiological responses (oxygen consumption) and behavioral responses (feeding and activity) of the mud snails Hydrobia ulvae and Hydrobia glyca at different salinities (20 per thousand-80 per thousand) and temperatures (20 degrees and 30 degrees C) were studied. After 24 h under experimental conditions, both Hydrobia species already showed maximal activities (>90%) for a wide salinity range (30 per thousand-70 per thousand), with significant differences in activity between species only outside the usual salinity range of the studied lagoon. In contrast, egestion rates of H. glyca were significantly higher at the lowest salinities tested (30 per thousand and 40 per thousand) irrespective of water temperature, whereas egestion rates of H. ulvae were always significantly higher (57% on average) at 20 degrees C than at 30 degrees C and at the usual salinities found in the field (40 per thousand and 50 per thousand). Both species showed an oxyregulatory response to dissolved oxygen concentrations ranging from saturation to 1.5 mg O(2) L(-1), although specific oxygen consumption rates were significantly higher at 30 degrees C than at 20 degrees C (Q(10)=1.47+/-0.08 for H. ulvae and Q(10)=12.1+/-0.06 for H. glyca) and at the lowest salinities (30 per thousand-50 per thousand for H. ulvae and 30 per thousand-40 per thousand for H. glyca). On average, specific rates were higher for the smaller-sized H. glyca (1.64+/-0.03 microg O(2) mg(-1) ash-free dry weight [AFDW]) than for H. ulvae (1.35+/-0.03 microg O(2) mg(-1) AFDW). Despite the overlapping of their tolerances to high temperatures and salinities, the observed interspecies differences could play a certain role in the distribution of H. ulvae and H. glyca in the studied habitat. In particular, the decreasing feeding activity but increasing respiration of H. ulvae at 30 degrees C for salinities that usually occur in the studied lagoon could represent disadvantages to H. glyca during the warm period.     

Metabolism and growth of juveniles of Litopenaeus vannamei: effect of salinity and dietary carbohydrate levels

The present study was designed to understand how carbohydrate (CBH) and protein metabolism are related in the penaeid shrimp Litopenaeus vannamei. With this information, we obtained a comprehensive schedule of the protein-carbohydrate metabolism including enzymatic, energetic, and functional aspects. We used salinity to determine its role as a modulator of the protein-carbohydrate metabolism in shrimp. Two experiments were designed. The first experiment evaluated the effect of CBH-salinity combinations in growth and survival, and hemolymph glucose, protein, and ammonia levels, digestive gland glycogen, osmotic pressure, and glutamate dehydrogenase (GDH) of L. vannamei juveniles acclimated during 18 days at a salinity of 15 per thousand and 40 per thousand. The second experiment was done to evaluate the effect of dietary CBH level on pre- and postprandial oxygen consumption, ammonia excretion, and the oxygen-nitrogen ratio (O/N) of juvenile L. vannamei in shrimps acclimated at 40 per thousand salinity. We also evaluated the ability of shrimp to carbohydrate adaptation. We made phosphoenolpyruvate carboxykinase (PECPK) and hexokinase activity measurements after a change in dietary carbohydrate levels at different times during 10 days. The growth rate depended on the combination salinity-dietary CBH-protein level. The maximum growth rate was obtained in shrimps maintained at 15 per thousand salinity and with a diet containing low CBH and high protein. The protein in hemolymph is related to the dietary protein levels; high dietary protein levels produced a high protein concentration in hemolymph. This suggests hemolymph is able to store proteins after a salinity acclimation. Depending on the salinity, the hemolymph proteins could be used as a source of osmotic effectors or as metabolic energy. The O/N values obtained show that shrimp used proteins as a source of energy, mainly when shrimps were fed with low CBH. The role played by postprandial nitrogen excretion (PPNE) in apparent heat increase (AHI) (PPNE/AHI ratio) is lower in shrimps fed diets containing high CBH in comparison with shrimps fed diets containing low CBH levels. These results confirm that the metabolism of L. vannamei juveniles is controlled by dietary protein levels, affecting the processes involved in the mechanical and biochemical transformations of ingested food. A growth depression effect was observed in shrimps fed with low-CBH protein diets and maintained in 40 per thousand salinity. In these shrimps, the hemolymph ammonia concentration (HAC) was significantly higher than that observed in shrimps fed with low CBH and maintained in 15 per thousand salinity. That high HAC level coincided with lower growth rate, which suggests that this level might be toxic for juveniles of L. vannamei. Results obtained for GDH activity showed this enzyme regulated both HAC and hemolymph protein levels, with high values in shrimps fed with low CBH levels and maintained in 40 per thousand salinity, and lower in shrimps fed with high CBH and maintained in 15 per thousand salinity. These differences mean that shrimp with a high-gill GDH activity might waste more energy in oxidation of the excess proteins and amino acids, reducing the energy for growth. It was evident that L. vannamei can convert protein to glycogen by a gluconeogenic pathway, which permitted shrimp to maintain a minimum circulating glucose of 0.34 mg/ml in hemolymph. A high PECPK activity was observed in shrimps fed a diet containing low CBH level indicating that the gluconeogenic pathway is activated, as in vertebrates by low dietary CBH levels. After a change in diet, we observed a change in PEPCK; however, it was lower and seems to depend on the way of adaptation, because it occurred after 6 days when adapting to a high-CBH diet and with little change for the low-CBH diet.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels

White shrimp Litopenaeus vannamei held in 25 per thousand seawater were injected with TSB-grown Vibrio alginolyticus (1 x 10(4) cfu shrimp(-1)), and then transferred to 5, 15, 25 (control) and 35 per thousand. Over 24-96 h, the mortality of V. alginolyticus-injected shrimp held in 5 per thousand and 15 per thousand was significantly higher than that of shrimp held in 25 per thousand and 35 per thousand, and the mortality of V. alginolyticus-injected shrimp held in 5 per thousand was the highest. Shrimp held in 25 per thousand and then transferred to 5, 15, 25 (control) and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus after 12-72 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 and 15 per thousand after 12 h. It is concluded that the shrimp transferred from 25 per thousand to low salinity levels (5 and 15 per thousand) had reduced immune ability and decreased resistance against V. alginolyticus infection.     

Effects of reduced salinity on survival, growth, reproductive success, and energetics of the euryhaline polychaete Capitella sp. I

Physiological adjustment to water of reduced salinity requires energy expenditure. In this study we sought to determine the fitness costs associated with such adjustment in the euryhaline polychaete Capitella sp. I, and the extent to which such costs could be explained by increased rates of energy expenditure. In a series of experiments conducted at 20 degrees C, salinity was reduced from 30 per thousand to either 25, 20, 15, 12, or 10 per thousand within 72 h after the larvae had been induced to metamorphose. Juveniles were reared on fine, organic-rich sediment. Over the next 15-30 days, we determined survival, growth, fecundity, and rates of respiration and feeding (via fecal pellet production). Larval salinity tolerance was also determined. Juvenile survival at salinities as low as 12-15 per thousand was comparable to that at 30 per thousand. The lower limit of salinity tolerance was 10-12 per thousand at 20 degrees C for both larvae and juveniles. Juveniles grew significantly more slowly at 12-15 per thousand in six of the seven experiments. Fecundity, however, was generally highest at intermediate salinities of 20-25 per thousand, and comparable at 30 and 15 per thousand. No individuals released embryos at 12 per thousand over the approximately 30-day observation periods in any of the three experiments in which the worms were reared at this low salinity. Reduced growth rates were not explained by differences in rates of respiration at different salinities: at reduced salinity, respiration rates were either statistically equivalent to (P>0.10) or significantly below (P<0.05) those recorded for animals maintained at 30 per thousand. Lower growth rates at lower salinities were best explained by reduced feeding rates. Further studies are required to determine whether digestive efficiency, growth hormone concentrations, or reproductive hormone concentrations are also altered by low salinity in this species.     

Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels

Litopenaeus vannamei juveniles (total length 22+/-2.4 mm) were exposed to different concentrations of ammonia-N (un-ionized plus ionized ammonia as nitrogen), using the static renewal method at different salinity levels of 15 per thousand, 25 per thousand and 35 per thousand at pH 8.05 and 23 degrees C. The 24, 48, 72, 96 h LC50 values of ammonia-N on L. vannamei juveniles were 59.72, 40.58, 32.15, 24.39 mg l(-1) at 15 per thousand; 66.38, 48.83, 43.17, 35.4 mg l(-1) at 25 per thousand; 68.75, 53.84, 44.93, 39.54 mg l(-1) at 35 per thousand, respectively. The 24, 48, 72, 96 h LC50 values of NH(3)-N (un-ionized ammonia as nitrogen) were 2.95, 2.00, 1.59, 1.20 mg l(-1) at 15 per thousand; 2.93, 2.16, 1.91, 1.57 mg l(-1) at 25 per thousand; 2.78, 2.18, 1.82, 1.60 mg l(-1) at 35 per thousand, respectively. As the salinity decreased from 35 per thousand to 15 per thousand, susceptibility of ammonia-N increased by 115%, 132%, 140% and 162% after 24, 48, 72 and 96 h exposure, respectively. The "safety level" for rearing L. vannamei juveniles was estimated to be 2.44, 3.55, 3.95 mg l(-1) for ammonia-N and 0.12, 0.16, 0.16 mg l(-1) for NH(3)-N in 15 per thousand, 25 per thousand and 35 per thousand, respectively.     

Effect of chronic Taura syndrome virus infection on salinity tolerance of Litopenaeus vannamei

Taura syndrome virus (TSV) is one of the most important shrimp viruses affecting farmed shrimp worldwide. After an acute phase during which the likelihood of mortality is elevated, infected shrimp enter a chronic phase during which shrimp appear to resume normal behavior and display no gross signs of infection. This study was designed to determine if chronically TSV-infected shrimp Litopenaeus vannamei are compromised by the infection. Specifically we investigated whether chronically infected shrimp could tolerate a drop in salinity as strongly as uninfected shrimp. The study consisted of 3 trials that compared survival of uninfected and chronically TSV-infected L. vannamei after drops in salinity from 24 ppt to salinities varying from 18 to 0 ppt. Logistic regression detected a significant effect of TSV infection on survival of chronically infected shrimp (p < 0.05). Salinity drops from 24 ppt to 3 and 6 ppt resulted in statistically different survivals (p < 0.05). Survival rates were similar among groups for salinity drops to greater than 6 ppt or less than 3 ppt. Salinities at which 50% of the shrimp died (LC50) were 3.06 ppt for the uninfected and 6.65 ppt for the chronically infected groups. Moreover, histopathological analysis of chronically infected shrimp that were moribund or recently dead showed no signs of having reverted to the acute stage of the disease. These results suggest that chronically infected shrimp are not able to tolerate a salinity drop as strongly as uninfected shrimp.     

Oxygen consumption and osmoregulatory capacity in Neomysis integer reduce competition for resources among mysid shrimp in a temperate estuary

Results of field surveys and laboratory measurements of oxygen consumption and body fluid osmolality at different salinities in the mysids Neomysis integer, Mesopodopsis slabberi, and Rhopalophthalmus mediterraneus from the Guadalquivir estuary (southwest Spain) were used to test the hypothesis that osmotic stress (oxygen consumption vs. isosmotic points) was lowest at salinities that field distributions suggest are optimal. The three species showed overlapping spatial distributions within the estuary but clear segregation along the salinity gradient: N. integer, M. slabberi, and R. mediterraneus displayed maximal densities at lower, intermediate, and higher salinities, respectively. Adults of N. integer were extremely efficient hyperregulators (isosmotic point 30 per thousand) over the full salinity range tested (3 per thousand-32 per thousand), and their oxygen consumption rates were independent of salinity; adults of M. slabberi were strong hyper- and hyporegulators at salinities between 7 per thousand and 29 per thousand (isosmotic point, 21 per thousand) and showed higher oxygen consumptions at the lowest salinity (6 per thousand); adults of R. mediterraneus hyperregulated at salinities between 19 per thousand and seawater (isosmotic point, 36 per thousand), with the lowest oxygen consumption at salinity around their isosmotic point (35 per thousand). Thus, the osmoregulation capabilities of M. slabberi and R. mediterraneus seem to determine the salinity ranges in which most of their adults live, but this is not so for adults of N. integer. Moreover, maximal field densities of M. slabberi (males and females) and R. mediterraneus (males) occur at the same salinities as the lowest oxygen consumption. In contrast, field distribution of N. integer was clearly biased toward the lower end of the salinity ranges within which it osmoregulated. We hypothesize that the greater euryhalinity of N. integer makes it possible for this species to avoid competition with R. mediterraneus by inhabiting the more stressful oligohaline zone.     

盐度对凡纳滨对虾体组织蛋白质积累、氨基酸组成和转氨酶活性的影响

本文研究了低、中和高三个盐度水平(分别为3‰、17‰和32‰)对凡纳滨对虾(Litopenaeus vannamei)各组织蛋白质的积累、肌肉谷草转氨酶和谷丙转氨酶活力、肌肉总氨基酸和游离氨基酸组成和含量的影响。结果显示,经过50d不同盐度水平的试验,低盐度组对虾的肝胰腺和血淋巴中可溶性蛋白质含量显著高于中、高盐度组(p<0.05),而肌肉中可溶性蛋白质含量在各处理组间无显著性差异;低、高盐度均导致肌肉中谷丙转氨酶和谷草转氨酶活力升高,但是各处理间的差异不显著;低、高盐度组凡纳滨对虾肌肉总氨基酸和总必需氨基酸含量均显著高于中盐度组(p<0.05),中、低盐度处理组非必需氨基酸含量差异不显著,而低盐度组对虾肌肉中蛋氨酸、丝氨酸、半胱氨酸和脯氨酸含量均显著低于中盐度组(p<0.05),其中脯氨酸为常见的5种主要渗透调节氨基酸之一;低、高盐度组对虾肌肉总游离氨基酸含量显著高于中盐度组(p<0.05),而盐度对机体绝大部分肌肉游离氨基酸含量的影响不显著(p>0.05)。结果显示,当环境盐度偏离凡纳滨对虾最适生长盐度时,其可通过在肝胰腺和血淋巴蛋白质积累及提高自身转氨酶活力,来获得机体在渗透调节供能时所需的氨基酸,而这些氨基酸以脯氨酸为主。     

Salinity effects on viability, metabolic activity and proliferation of three Perkinsus species

Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35 per thousand), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15 per thousand), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25 per thousand) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35 per thousand). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11 per thousand), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11 per thousand) than P. olseni, but exhibited reduced viability at 7 per thousand, even after acclimation.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Characterization and experimental infection of Flexibacter maritimus (Wakabayashi et al. 1986) in hatcheries of post-larvae of Litopenaeus vannamei Boone, 1931.

A preliminary study to characterize filamentous bacteria, whose presence is related to high mortality of Litopenaeus vannamei larvae cultured in Santa Catarina State, Brazil, is reported. The extract of infected larvae was diluted in different concentrations, cultured in marine agar (Difco, Marine Agar 2216) and incubated at 30 degrees C for 48 hours. The biochemical characterization included hydrolytic reactions of starch, gelatin and tyrosine, growth in TCBS agar, growth in 0 and 37 per thousand salinity, pigment production in tyrosine agar, production of H2S, nitrate reduction, congo red reaction, oxidase and catalase. The isolated bacteria belong to the species Flexibacter maritimus, Gram-negative bacilli of 0.4-0.5 microm width and 15 microm length. Experiments were carried out on pathogenicity of F. maritimus in post-larvae of L. vannamei. Survival and symptoms in L. vannamei post-larvae 24 hours after inoculation with F. maritimus and its growth in marine agar were evaluated. Mortality was detected around 92,5% as well as symptoms like melanized lesions in several parts of body, discolouration of gills, bad formation of appendages and of the last abdominal segment, low motility and feeding reduction. The experimental infection results suggested that isolated bacteria of the genus Flexibacter are pathogenic to the shrimp Litopenaeus vannamei post-larvae.     

Osmoregulation, immunolocalization of Na+/K+-ATPase, and ultrastructure of branchial epithelia in the developing brown shrimp, Crangon crangon (Decapoda, Caridea)

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+-ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0 per thousand to 32.2 per thousand, salinity tolerance was generally high (survival rates: 70%-100%) in all developmental stages, but it decreased in media <10.2 per thousand. Zoeal stages and decapodids slightly hyperregulated at 17.0 per thousand and osmoconformed in media > or =25.5 per thousand. At 10.2 per thousand, these stages showed high mortality, and only juveniles survived at 5.3 per thousand. Juveniles hyperregulated at 10.2 per thousand and 17.0 per thousand, osmoconformed at 25.5 per thousand, and hyporegulated in media > or =32.2 per thousand. Large juveniles hyperregulated also at 5.3 per thousand. Expression of the Na+/K+-ATPase and ion-transporting cells was located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion-transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+-ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyperosmoconforming decapodid to an effectively hyper-/hyporegulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion-transporting cells and the expression of Na+/K+-ATPase.     

Influence of salinity and temperature on growth and survival of the planktonic larvae of Marenzelleria viridis (Polychaeta, Spionidae)

In a series of experiments, the planktonic larvae of Marenzelleriaviridis (Verrill, 1873) were exposed to various combinationsof salinity (S = 0.6, 2.5, 5.0, 10 and 20) and temperature (T= 5, 10 and 20C) from the 1-setiger stage to the onset of metamorphosis(16- to 17-setiger stage). One-setiger larvae were unable tocomplete their development to metamorphosis at salinities below5. Metamorphosis was successful at salinities of 10 and 20,when the animals adopted a benthic life mode. Larval developmentwas more rapid at 10 than at 20, and was positively affectedby higher temperatures. Larvae exposed to a salinity of 3.5at the 4- to 5-setiger stage developed and completed metamorphosisto benthic juveniles despite the low salinity. These larvaedeveloped most rapidly at a temperature of 10C. The salinitytolerances (LC₅₀) of M. viridis larvae (t = 48 h), juvenilesand adults (t = 72 h in each case) were determined at 10C.The results showed that all development stages can toleratesalmities <1 The importance of constraints on developmentand tolerance to low salinities for the successful colonizationof oligohaline regions is shown and discussed in connectionwith other brackish-water organisms.     

Bacterial diversity of a cyanobacterial mat degrading petroleum compounds at elevated salinities and temperatures

Cyanobacterial mats of the Arabian Gulf coast of Saudi Arabia experience extreme conditions of temperature and salinity. Because they are exposed to continuous oil pollution, they form ideal models for biodegradation under extreme conditions. We investigated the bacterial diversity of these mats using denaturing gradient gel electrophoresis and 16S rRNA cloning, and tested their potential to degrade petroleum compounds at various salinities (fresh water to 16%) and temperatures (5 to 50 degrees C). Cloning revealed that c. 15% of the obtained sequences were related to unknown, possibly novel bacteria. Bacteria belonging to Beta-, Gamma- and Deltaproteobacteria, Cytophaga-Flavobacterium-Bacteroides group and Spirochetes, were detected. The biodegradation of petroleum compounds at different salinities by mat microorganisms showed that pristine and n-octadecane were optimally degraded at salinities between 5 and 12% (weight per volume NaCl) whereas the optimum degradation of phenanthrene and dibenzothiophene was at 3.5% salinity. The latter compounds were also degradable at 8% salinity. The same compounds were degraded at temperatures between 15 and 40 degrees C but not at 5 and 50 degrees C. The optimum temperature of degradation was 28-40 degrees C for both aliphatics and aromatics. We conclude that the studied microbial mats from Saudi Arabia are rich in novel halotolerant and thermotolerant microorganisms with the potential to degrade petroleum compounds at elevated salinities and temperatures.     

Effect of salinity on the immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae

Addition of NaCl at 2.5% to tryptic soy broth (TSB) significantly increased the growth of Photobacterium damselae subsp. damselae. Tiger shrimp Penaeus monodon held in 25 per thousand seawater were injected with P. damsela subsp. damselae grown in TSB containing NaCl at 0.5%, 1.5%, 2.5% and 3.5% at a dose of 8.48 x 10(4)colony-forming units (cfu)shrimp(-1). Over 24-96 h, the cumulative mortality was significantly higher for the shrimp challenged with P. damselae subsp. damselae grown in 2.5% NaCl than those grown in 0.5%, 1.5% and 3.5% NaCl. In another experiment, P. monodon held in 25 per thousand were injected with TSB-grown P. damselae subsp. damselae (8.48 x 10(4)cfushrimp(-1)), and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand. After 96 h, the mortality was highest for the P. damselae subsp. damselae-injected shrimp held in 5 per thousand, and the lowest for the P. damselae subsp. damselae-injected shrimp held in 25 per thousand. In a separate experiment, P. monodon held in 25 per thousand and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand were examined for immune parameters, and phagocytic activity and clearance efficiency of P. damselae subsp. damselae after 12-96 h. The THC, hyaline cell, phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 per thousand, 15 per thousand and 35 per thousand after 12h. It is concluded that tiger shrimp P. monodon transferred from 25 per thousand to low salinity levels (5 per thousand and 15 per thousand) and high salinity (35 per thousand) had reduced immune ability and decreased resistance against P. damselae subsp. damselae infection.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

The Na+, K+, 2Cl- cotransporter of estuarine pufferfishes (Sphoeroides testudineus and S. greeleyi) in hypo- and hyper-regulation of plasma osmolality

The pufferfishes Sphoeroides testudineus and Sphoeroides greeleyi are estuarine species that osmoregulate efficiently, but S. testudineus tolerates seawater dilution to a much higher degree than S. greeleyi. This study aimed at testing whether NKCC is involved with their differential tolerance of seawater dilution, through the analysis of in vivo furosemide (NKCC inhibitor) injection both on hypo-regulation (in 35 per thousand salinity) and hyper-regulation (in 5 per thousand salinity). After exposure for 6 h or 5 days to both salinities, blood samples were obtained for determination of plasma osmolality, chloride, sodium and hematocrit, and muscle samples for determination of water content. Furosemide injection led to increased plasma osmolality and sodium in 35 per thousand and decreased osmolality and chloride in 5 per thousand, when compared to saline-injected controls. Furosemide injection led to hematocrit reduction in both salinities, and muscle water content increase in 5 per thousand and decrease in 35 per thousand in S. testudineus. The results are compatible with NKCC working in branchial NaCl secretion in 35 per thousand, in both species, and a higher role in cell volume regulation in blood and muscle cells of S. testudineus, in both salinities, which could partially explain the stronger capacity of S. testudineus to tolerate seawater dilution during low tide.