Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

Experimental infection of Pacific white shrimp Litopenaeus vannamei with Necrotizing Heptopancreatitis (NHP) bacterium by per os exposure

Necrotizing Hepatopancreatitis Bacterium (NHPB), which causes Necrotizing Hepatopancreatitis, was successfully transmitted in individually isolated Kona stock Litopenaeus vannamei through per os exposure. Animals (140) were individually exposed orally to a 0.05 g piece of an NHPB-infected hepatopancreas and 120 control animals were each exposed to a 0.05 g piece of NHPB-negative hepatopancreas. Shrimp were maintained in Sterilite containers with approximately 41 of artificial seawater at 30 per thousand salinity and 30 degrees C for 60 d. Mortality of infected shrimp was observed from Day 16 to Day 51 post-exposure. Infected animals sustained reduced feeding activity and displayed empty guts. Some infected animals developed a pale hepatopancreas noticeable through the carapace. Survival probabilities fit a Weibull distribution and parametric survival analysis revealed lowered survival due to NHPB infection. Median survival time of NHPB-infected animals was 34.5 d. After correcting for background daily mortality in the controls, mean acute daily mortality of NHPB was estimated at 0.09, a value much lower than that estimated for other diseases in Kona stock L. vannamei such as White Spot Syndrome Virus (0.40) and Taura Syndrome Virus (0.30). A chronic, or carrier, state was not demonstrated in NHPB epizootics because all NHPB-positive animals experienced mortality and no animals surviving to 60 d post-exposure were diagnosed NHPB-positive through PCR or histology.     

Preservation of necrotizing hepatopancreatitis bacterium (NHPB) by freezing tissue collected from experimentally infected Litopenaeus vannamei

Necrotizing hepatopancreatitis (NHP), a severe disease of penaeid shrimp, is caused by bacteria (NHPB) that have previously been demonstrated to reside in tubular epithelial hepatopancreatic (HP) cells of infected shrimp. There has yet to be a successful in vitro culture method to grow the intracellular organism; therefore, it must be propagated in vivo via transmission from NHPB-infected shrimp to healthy individuals. In our studies, NHPB propagation tanks containing infected shrimp were used to maintain a constant supply of organisms for experiments. In order to develop a method for storing infectious NHPB material for future challenge studies, we collected HP tissue containing NHPB by flash freezing whole, fresh HPs at -80 degrees C for up to 80 d and used it to successfully infect specific pathogen-free Litopenaeus vannamei per os in controlled experiments. HP tissue samples were collected from dead shrimp, and PCR was performed to confirm the presence of NHPB. Our results demonstrate that the infectivity of NHPB in tissue is not altered after being frozen at -80 degrees C when compared to NHPB in fresh tissue. Thus, the continual propagation of NHPB in vivo is not required to assure a source of the infectious agent.     

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.     

The effects of temperature and salinity on the swimming ability of whiteleg shrimp, Litopenaeus vannamei

Swimming endurance of whiteleg shrimp, Litopenaeus vannamei exposed to various temperatures (15, 20, and 25 degrees C) and salinities (15, 32, and 40 per thousand) was determined in a swimming channel 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. No shrimp swam the full 9000 s throughout the experiment. The swimming endurance decreased as swimming speed was increased at any of the temperatures and salinities tested and was significantly affected by temperature and salinity (P<0.05). The power model (nu x t(b) = a) showed a better fit to the relationship between swimming endurance (t, in s) and swimming speed (nu, in cm s(-1)) at any of the temperatures and salinities tested. The swimming ability index (SAI), defined as SAI = integral(0)(9000) vdt x 10(-4) (cm), was found to be temperature- and salinity-dependent in L. vannamei. The optimum temperature and salinity and corresponding maximum SAI were Topt = 21.3 degrees C and SAI(max21.3) = 7.37 cm; Sopt = 27.6 per thousand and SAI(max27.6) = 7.47 cm, respectively. The range of temperatures and salinities within which SAI is >90% of the maximum was estimated between 17.6 and 24.9 degrees C and between 18.5 and 36.7 per thousand, respectively. 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, temperature and salinity can limit the swimming performance of L. vannamei.     

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.     

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.     

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.     

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.     

Effect of salinity changes on the bacterial diversity, photosynthesis and oxygen consumption of cyanobacterial mats from an intertidal flat of the Arabian Gulf

The effects of salinity fluctuation on bacterial diversity, rates of gross photosynthesis (GP) and oxygen consumption in the light (OCL) and in the dark (OCD) were investigated in three submerged cyanobacterial mats from a transect on an intertidal flat. The transect ran 1 km inland from the low water mark along an increasingly extreme habitat with respect to salinity. The response of GP, OCL and OCD in each sample to various salinities (65 per thousand, 100 per thousand, 150 per thousand and 200 per thousand) were compared. The obtained sequences and the number of unique operational taxonomic units showed clear differences in the mats' bacterial composition. While cyanobacteria decreased from the lower to the upper tidal mat, other bacterial groups such as Chloroflexus and Cytophaga/Flavobacteria/Bacteriodetes showed an opposite pattern with the highest dominance in the middle and upper tidal mats respectively. Gross photosynthesis and OCL at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP, OCL and OCD rates at salinities > 100 per thousand. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.     

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.     

Effects of osmotic stress on crustacean larval growth and protein and lipid levels are related to life-histories: the genus Armases as a model

In the larval stages of three euryhaline species of the genus Armases, we tested if changes in biomass (dry mass, W; protein; lipid) under hyposmotic stress were related to their salinity tolerance, capabilities of osmoregulation, and migration patterns. As model species, we compared Armases miersii, which breeds in supratidal rock pools, the riverine crab Armases roberti (showing a larval export strategy), and Armases ricordi, whose larvae probably develop in coastal marine waters. At each stage, larvae were exposed to different salinities (selected according to previous information on larval survival; range: 5 per thousand-32 per thousand for A. miersii, 10 per thousand-32 per thousand for A. roberti, and 15 per thousand-32 per thousand for A. ricordi). Biomass was measured in early postmoult and intermoult. The larvae of the strongly osmoregulating species A. miersii, which develop in habitats with highly variable salinity conditions, showed the smallest variations in biomass. The effect on A. roberti varied during its ontogeny: the Zoea I and the Megalopa, which carry out downstream and upstream migrations, respectively, showed lower biomass variations than the intermediate zoeal instars, which develop in coastal waters. The larvae of A. ricordi showed generally the highest variations in biomass, reflecting poor adaptation to salinity variations. In addition, a common pattern was found for these estuarine species: the maximum of biomass shifted during ontogeny from 32 per thousand to 25 per thousand, reflecting changes of the iso-osmotic point. The ontogeny of osmoregulation reflected ontogenetic migration patterns, which allow for avoiding detrimental effects of salinity variations.     

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.     

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.     

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.     

Effects of long-term exposure to different salinities on the location and activity of Na+-K+-ATPase in the gills of juvenile mitten crab, Eriocheir sinensis

The euryhalinity of mitten crab, Eriocheir sinensis, is based on osmoregulation, and thus on the activity of Na(+)-K(+)-ATPase. We studied location and activity of this enzyme in gills of juvenile crabs exposed to 5 per thousand, 25 per thousand, and 40 per thousand salinity. The posterior gills showed always a high number of immunopositive cells (IPC), staining with fluorescent antibody against Na(+)-K(+)-ATPase, covering at 5 per thousand the entire lamellae. At 25 per thousand, they showed fewer IPC which occurred only at the bases of the lamellae. Enzyme activity was consistently higher in posterior than in anterior gills. Low salinity stimulated the activity only in posterior gills. Both histochemical and enzymatic results are consistent with previous ultrastructural observations showing that the epithelial cells of the posterior, but not the anterior gills exhibit typical traits of ionocytes. While an increase in Na(+)-K(+)-ATPase activity at a reduced salinity is consistent with a strong hyper-osmoregulatory capacity in juvenile crabs, a low activity at an enhanced salinity suggests a physiological response, directed towards a reduction of Na(+) uptake. The activity increase of ion-transporting enzymes is directly related to spatial changes in their distribution along the osmoregulatory tissue, i.e. an enhanced number of IPC scattered along the entire lamellae. In juveniles, this allows for successful development and growth at reduced salinities.     

Ontogeny of osmoregulation in the crayfish Astacus leptodactylus

Osmoregulation was studied during the postembryonic development of Astacus leptodactylus Eschscholtz 1823 in juvenile stages 1-8 and in adults. Juveniles hatch and later stages develop in freshwater or in moderately saline waters. The time of acclimation from freshwater to a saline medium increased from early juveniles to adults. At all stages, it was longer than in comparable stages of marine crustaceans, reflecting the high impermeability of the teguments to water and ions. All stages were able to hyperisoosmoregulate. In freshwater, the ability to hyperosmoregulate was established at hatching and increased during development. The hemolymph osmolality increased from 286 mosm kg-1 in stage 1 juveniles to 419 mosm kg-1 in adults. All stages also hyperregulated at low salinities (7 per thousand and 13 per thousand salinity) and were osmoconformers at higher salinities up to 21 per thousand salinity. The lowest isosmotic salinity tended to increase with the developmental stages. The ability to osmoregulate at hatch and throughout postembryonic development is probably a key physiological adaptation in this and other freshwater crayfish.     

Transmission of white spot syndrome virus (WSSV) to Litopenaeus vannamei from infected cephalothorax, abdomen, or whole shrimp cadaver.

Shrimp viruses can remain infectious in frozen shrimp tissue and have been found in frozen commodity shrimp. Therefore, the threat of viral outbreaks in wild and cultured shrimp via frozen commodity shrimp exists. Because frozen shrimp are imported with and without the cephalothorax, more knowledge is needed concerning the infectivity of a cephalothorax relative to that of an abdomen. We compared the mortality rates from shrimp exposed to a WSSV-infected cephalothorax, abdomen, or whole shrimp cadaver. Estimates of transmission coefficients from the exposures to the infected cephalothorax, abdomen, or whole shrimp were also calculated because the transmission coefficients account for differences in the initial doses. In addition, we compared the variability in infectivity of pieces of shrimp by feeding 24 equal-sized pieces of cephalothorax and abdomen to 24 individually isolated shrimp. In Expt 1, susceptible shrimp did not completely consume the infected abdomen, and a significant difference was detected among shrimp exposed to the abdomen (mortality rate = 0.40), cephalothorax (mortality rate = 0.75), and whole shrimp cadaver (mortality rate = 0.67). The calculated transmission coefficients were 0.95 from an infected cephalothorax, 0.59 from an infected abdomen, and 0.69 from an infected whole shrimp cadaver. In Expt 2, susceptible shrimp were starved to ensure complete ingestion of each dose. No significant difference was observed in the estimated mortality rates from an infected cephalothorax (0.58), abdomen (0.63), or whole shrimp (0.67). The calculated transmission coefficients were 0.84 from an infected cephalothorax, 0.83 from an infected abdomen, and 0.60 from an infected whole shrimp cadaver. In Expt 3, no difference was observed in the mortality rates resulting from exposures to pieces of infected cephalothorax (0.57) or abdomen (0.58). Our results suggested that there was no difference in the viral loads of a WSSV-infected cephalothorax or abdomen, but that the cephalothorax was more infectious, probably because it was more palatable. In addition, our results are inconsistent with some assumptions of pathogen transmission used in epidemiological models. Some shrimp may be less aggressive feeders; therefore, susceptible shrimp are differentially contacting the dead infected shrimp in the exposure tanks, violating the random mixing assumption. Moreover, virus is probably not homogeneously distributed throughout an infected shrimp, suggesting that contacts between susceptible and infected shrimp are not equally likely to result in transmission.     

Beta-Alanine and other free amino acids during salinity adaptation of the polychaete Nereis japonica

The free amino acid pool was measured in the body wall muscle and in coelomic cells (eleocytes) of the polychaete Nereis japonica following adaptation to salinities between 6 and 44 per thousand. Beta-Alanine and glycine were the major amino acids comprising 35-60% of the total free amino acid pool in the body wall. In eleocytes, glutamate and lysine in addition to beta-alanine and glycine were the dominant free amino acids. In the body wall, the concentrations of beta-alanine were closely correlated with the ambient salinity between 12 and 35 per thousand. The concentrations of glycine rose initially but remained unchanged at concentrations above 26 per thousand. In both body wall and eleocytes, the mean total primary amine concentrations were correlated with the ambient salinities between 12 and 35 per thousand. The sum of amino acids determined by HPLC showed the same correlation in both tissues, but accounted only for 60-85% of the concentrations of total primary amines. The total protein content of the body wall was slightly higher at 44 per thousand compared to the lower salinities indicating dehydration of the tissues. Eleocytes swell at 6 per thousand and showed irregular amino acid concentrations indicating a loss of metabolic integrity.     

Effects of low salinity on juvenile pink shrimp Farfantepenaeus paulensis (Perez-Farfante 1967, Crustacea)

It is of paramount importance to study salinity tolerance of commercially important crustaceans, such as the pink shrimp Farfantepenaeus paulensis to determine possible mortality causes in the wild and in aquaculture in oligohaline waters. The aim of this study was to determine the lethal salinity concentration (LC50) for juvenile pink shrimp F. paulensis and measure its oxygen consumption and ammonia excretion at different salinity levels. Shrimp of two length classes (49.4?±?4.3 and 78.5?±?5.5?mm) were placed in 10-L containers and exposed to salinity levels of 35, 30, 25, 20, 15, 10 and 5. The experiments were tripled, with seven shrimp in each container. The average lethal concentration (LC50s) for an exposure of 24?h was 13.33 (11.26–15.78) and 10.26 (8.60–12.64), respectively, for the two classes of juveniles. For an exposure of 48?h, LC50s were 12.71 (10.68–15.12) for the larger animals and 9.20 (7.34–11.52) for the smaller ones. There was an inverse relationship between salinity and rates of oxygen consumption and ammonia excretion. The average reduction in specific oxygen consumption in salinities 20, 25 and 30 showed a decrease in metabolic rate of 63, 80 and 82%, respectively, in relation to salinity level 0. The same occurred for the averages of ammonia excretion at salinity levels of 15, 20, 25, 30 and 35, which represented low metabolic rates of 57, 61, 70, 71 and 74% respectably in relation to salinity level 0.