A multi-season survey for infectious myonecrosis in farmed shrimp, Litopenaeus vannamei, in Pernambuco, Brazil

Infectious myonecrosis (IMN), caused by infectious myonecrosis virus (IMNV), is the disease of greatest impact on shrimp farming in the northeast region of Brazil. The occurrence of IMN remained restricted to northeastern Brazil until 2006, when its presence was also confirmed in Indonesia. To determine the occurrence and evolution of IMN in Litopenaeus vannamei farmed along the coast of the state of Pernambuco, Brazil, histopathological examinations were performed on 60 samples collected from four farms in both predominant seasons in the northeastern region: dry and wet seasons. Samples made up of ten specimens were collected monthly from each pond. Histopathological results were associated to wet-mount exams and rearing performance data. Lesions suggestive of IMN (coagulative necrosis, hemocytic infiltration in the musculature, ectopic spheroids in the lymphoid organ) were identified in all the farms, with a higher occurrence during the dry season. Longer rearing periods and higher stocking densities were the variables with the most significant influence (p < 0.05) in the occurrence of IMN.     

Influence of highly unsaturated fatty acids on the responses of white shrimp (Litopenaeus vannamei) postlarvae to low salinity

Salinity stress tests are commonly applied in shrimp hatcheries to estimate the quality of postlarvae (PL) to be used during growout. Higher larval survival during culture and to a salinity stress test in both fish and crustaceans have been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids (HUFA). However, it is not clear if increased survival is a result of better overall physiological condition resulting from the diet or a specific effect of HUFA on osmoregulatory mechanisms. This study analyzed if HUFA-rich diets could modify the fatty acid composition of membranes in gills, and if this change in composition could affect the activity of the Na⁺/K⁺ ATPase pump and carbonic anhydrase in relation to changes in salinity. One-day-old postlarvae (PL1) pooled from different spawns were fed for 20 days with Artemia sp. nauplii enriched with three levels of HUFA: low, medium and high. At PL20, survivals during culture and to salinity stress test (tap water for 30 min) were evaluated. Also at this stage, Na⁺/K⁺-ATPase and carbonic anhydrase activity, morphometric variables, and fatty acid composition in the hepatopancreas and gills were measured after they were submitted to a salinity challenge in dilute seawater (10 ppt) for 3 h. No significant differences were observed in survival rates during culture, but survival to a salinity stress test was higher and gill area was larger in PL20 fed the Artemia sp. nauplii enriched with medium HUFA levels, probably as a result of an increased 22:6n-3 content and higher 22:6n-3/20:5n-3 ratio in this diet and in the tissues of the organisms fed this diet. Na⁺/K⁺-ATPase specific activity was significantly higher in posterior gills, while the specific activity of the carbonic anhydrase was higher in anterior gills. Enzymatic activities increased significantly in PL20 submitted to a salinity challenge, and HUFA levels in the diet affected both. The proportion of fatty acids in hepatopancreas and gills were significantly affected not only by diet, but also by exposure to dilute media. This effect is discussed in relation to an increase in gill surface and changes in fatty acid composition in the phospholipids present in gill membranes, which can modify the permeability and the activity of the Na⁺/K⁺-ATPase pump. The beneficial effect of HUFA supplementation in the diet on survival to salinity stress test is partially related to modification of fatty acid composition of gills and to a larger gill area, which in turn enhances osmoregulatory mechanisms, namely Na⁺/K⁺-ATPase and carbonic anhydrase activities.     

Thermal preference,tolerance and oxygen consumption of adult white shrimp Litopenaeus vannamei (Boone) exposed to different acclimation temperatures

Final temperature preferendum of white shrimp adults were determined with acute and gravitation methods. The final preferendum was similar, independent of method (26.2–25.6 °C). A direct relationship was determined between the critical thermal maxima values and the acclimation temperatures (P<0.05). The end point of Critical Thermal Maxima (CTMax) for adults was defined as the loss of righting response (LRR). The acclimation response ratio (ARR) for adults of white shrimp had an interval of 0.36–0.76, values that agreed with others obtained for crustaceans from tropical and subtropical climates. The oxygen consumption rates increased significantly (P<0.05) from 39.6 up to 90.0 mg O₂ kg⁻¹ h⁻¹ wet weight (w.w.) as the acclimation temperature increased from 20 to 32 °C. The range of temperature coefficient (Q₁₀) of the white shrimp between 23 and 26 °C was the lower 1.60. The results obtained in this work are discussed in relation to the species importance in the reproductive scope and maintenance of breeders.     

Arginine kinase of Litopenaeus vannamei involved in white spot syndrome virus infection

Virus–host interaction is important for virus infection. White spot syndrome virus VP14 contains transmembrane and signal peptides domain, which is considered to be important for virus infection. Until now, the function of this protein remains undefined. In this study, we explored the interaction of VP14 with host cell. A new shrimp protein (arginine kinase of Litopenaeus vannamei, LvAK) is selected and its localization in shrimp cells is also confirmed.     

Thermal tolerance of Litopenaeus vannamei (Crustacea: Penaeidae) acclimated to four temperatures

Critical thermal minima (CTMin) and maxima (CTMax) values were determined for the Pacific white shrimp Litopenaeus vannamei post-larvae and juveniles at four different acclimation temperatures (15, 20, 25, and 30 °C). The CTMin of shrimp at these acclimation temperatures were 7.82, 8.95, 9.80, and 10.96 °C for post-larvae and 7.50, 8.20, 10.20, and 10.80 °C for juveniles, respectively, at 1 °C h⁻¹ cooling rate. The CTMax values were 35.65, 38.13, 39.91, and 42.00 °C for post-larvae and 35.94, 38.65, 40.30, and 42.20 °C for juveniles at the respective acclimation temperatures. Both acclimation temperature and size of the shrimp affected CTMin values of L. vannamei (P<0.01). Overall, juveniles displayed significantly lower CTMin values than the post-larvae (P<0.0001). However, the CTMax response by post-larvae and juveniles were not significantly different from each other and no interaction was determined between the acclimation temperature and development stage (P>0.01). The area of the thermal tolerance polygon over four acclimation temperatures (15, 20, 25, and 30 °C) for the post-larvae of L. vannamei was calculated to be 434.94 °C². The acclimation response ratio (ARR) values were high ranging from 0.35 to 0.44 for both post-larvae and juveniles. L. vannamei appears to be more sensitive to low temperatures than other penaeid species and its cold tolerance zone ranged from 7.5 to 11 °C. In successful aquaculture temperature must never fall below 12 °C to prevent mortalities. Upper thermal tolerance is less of a problem as in most subtropical regions maximum water temperature rarely exceeds 34 °C, but care should be given if shallow ponds with low water renewal rate are being used.     

Effect of temperature on hemolymph lactate and glucose concentrations in spiny lobster Panulirus interruptus during progressive hypoxia

Some metabolic fuels in hemolymph samples from the sinuses at the base of the third walking leg (pre-branchial blood) and from the cardiac sinus (post-branchial blood) in the red lobster, Panulirus interruptus, were evaluated during normoxia, hypoxia, and at the critical oxygen point (P_cr) at two temperatures of acclimatization. Three-way ANOVA indicated a significant effect of oxygen saturation R(6140)=19.84 in metabolic fuel concentrations. Lactate varied from 0.01 to 0.29 mg/ml at 20 °C, from 0.02 to 0.29 mg/ml at 27 °C, and increased significantly during hypoxia (P<0.05). Glucose varied from 0.10 to 0.27 mg/ml at 20 °C, from 0.13 to 0.33 mg/ml at 27 °C, and increased with temperature. Proteins varied from 80.78 to 119.99 mg/ml at 20 °C and from 82.99 to 130.30 mg/ml at 27 °C.     

Combined effects of temperature and salinity on critical thermal minima of pacific white shrimp Litopenaeus vannamei (Crustacea: Penaeidae)

Critical thermal minima (CTMin) were determined for the Pacific white shrimp Litopenaeus vannamei juveniles from four different acclimation temperatures (15, 20, 25, and 30 °C) and salinities (10‰, 20‰, 30‰, and 40‰). The lowest and highest CTMin of shrimp ranged between 7.2 °C at 15 °C/30‰ and 11.44 °C at 30 °C/20‰ at the cooling rate of 1 °C h⁻¹. Acclimation temperature and salinity, as well as the interaction of both parameters, had significant effects on the CTMin values of L. vannamei (P<0.01). Yet, the results showed a much more profound effect of temperature on low thermal tolerance of juveniles. Only 40‰ salinity had an influence on the CTMin values (P<0.01). As the acclimation temperature was lowered from 30 to 15 °C thermal tolerance of the shrimp significantly increased by 3.25–4.14 °C. The acclimation response ratio (ARR) of the Pacific white shrimp exposed to different combinations of salinity and temperature ranged between 0.25 and 0.27. When this species is farmed in sub-tropical regions, its pond water temperature in the over-wintering facilities (regardless of the water salinity level) must never fall below 12 °C throughout the cold season to prevent mortalities.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28°C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.     

Estimation of genetic variation in Cynodon dactylon accessions using the ISSR technique

Genetic variation in 55 accessions of Cynodon dactylon was estimated using inter-simple sequence repeat (ISSR) markers. The plant materials used in this study originated from 17 countries. A total of 236 ISSR fragments were generated with 14 primers. Fragment sizes ranged from 200 to 3000 bp. All scorable bands were polymorphic in nature and none of the primers used produced monomorphic bands, indicating a high level of genetic variation in this grass. The accessions were found to be clustered into eight major groups through the unweighted pair-group method with arithmetic averages. Genetic similarity coefficients (GSC) among the 55 accessions ranged from 0.52 to 0.95. The results clearly indicate that a high level of variation exists in Cynodon accessions. This study shows that the ISSR technique is a reliable tool for differentiating Cynodon accessions and for determining the genetic relationships among them.     

Combined ventilatory responses to aerial hypoxia and temperature in the South American lungfish Lepidosiren paradoxa

The control of pulmonary ventilation in South American lungfish Lepidosiren paradoxa is poorly understood. Interactions between temperature and hypoxia are particularly relevant due to large seasonal variations of its habitat. Therefore, we tested the hypothesis that the ventilatory responses to aerial hypoxia of Lepidosiren are highly dependent on ambient temperature. We used a pneumotachograph to measure pulmonary ventilation (V_E), tidal volume (V_T) and respiratory frequency (f_R) during normoxic (21% O₂) and hypoxic (12%, 10% and 7% O₂) conditions at two temperatures (25 and 35 °C). Blood gases, arterial PO₂ (PaO₂), arterial PCO₂ (PaCO₂) and arterial pH (pH_a) were also evaluated. At 25 °C, V_E increased significantly at 10% and 7% hypoxic levels when compared to the control value (21% O₂). At 35 °C, all hypoxic levels elicited a significant increase of V_E relative to control values. V_E is augmented mostly by increases of respiratory frequency (f_R), and there were significant interactions (p<0.001) between aerial hypoxia and temperature. PaCO₂ increased from ∼22 mmHg (normoxic value at 25 °C) to ∼32 mmHg (normoxic value at 35 °C). Concomitantly, the pH_a decreased from 7.51 (25 °C) to 7.38 (35 °C). Hypoxia-induced hyperventilation caused a reduction in PaCO₂ and an increase in pH_a, which were more pronounced at 35 °C than at 25 °C, reflecting an increased hyperventilation under the high temperature. In conclusion, the magnitude of ventilatory response is highly temperature-dependent in L. paradoxa, which is important for an animal experiencing large seasonal variations.     

Hormonal and metabolic responses to nesting activities in the green turtle, Chelonia mydas

We examined the association between green turtle nesting activities and plasma profiles of hormones that are widely implicated in aspects of heightened metabolism and energy regulation; epinephrine (EPI), norepinephrine (NE) and corticosterone. In conjunction, we examined plasma profiles of glucose and lactate to infer metabolic processes associated with green turtle nesting behaviour. Finally, because these hormones are also involved in mediating behaviour and physiology associated with stressful situations, we examined the effect of a stressor encountered during nesting, physical disturbance, on hormone levels. Plasma profiles of epinephrine, norepinephrine and corticosterone were not significantly altered across different stages of nesting. Plasma glucose and lactate both exhibited significant increases related to nesting activity; glucose increased dramatically during the emergence stage of nesting before stabilizing, and lactate levels continued to increase throughout the nesting process. There was no significant association between plasma hormones and glucose. For female turtles that abandoned nesting activities due to competition for nest space, there was no significant difference in plasma levels of epinephrine, norepinephrine and corticosterone compared to females that persisted with nesting activities. Overall, while distinct metabolic changes took place in nesting females, there was little association in profiles of hormones typically considered important for regulating heightened metabolism and nesting activity. This disassociation could arise because hormonal action may be altered in breeding female green turtles to facilitate reproductive processes.     

Identification of a type of human IgG-like protein in shrimp Penaeus vannamei by mass spectrometry

Recently, infectious diseases have delayed the growth of shrimp aquaculture. Interest has been focused on immune molecules and defense mechanisms to reduce these diseases in shrimp aquaculture. In invertebrates, various immunoglobulin superfamily (IgSF) molecules have been characterized in body tissues and fluids, which play a significant role in innate defense. In the current study, we found that a protein in shrimp serum, referred as an IgG-like protein, could be reacted with goat anti-human IgG, specifically. The IgG-like protein was purified from the serum of shrimp Penaeus vannamei by affinity chromatography using CNBr-activated sepharose 4B. The purified protein was subsequently analysed using one-dimensional sodium sulphate-polyacrylamide gel electrophoresis (1-DE), two-dimensional sodium sulphate-polyacrylamide gel electrophoresis (2-DE) and immunoblotting. Furthermore, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass fingerprint (PMF) and sequencing of the protein, respectively. Sequence information and PMF queried against the NCBI database confirmed the identity of the protein as hemocyanin. Conserved domain search showed that there was an Ig-like conserved domain of 252 amino acid residues in the C-terminus of arthropoda hemocyanins. In addition, four and one conserved regions were found between hemocyanin and human Ig heavy chain and Ig kappa chain, respectively. These results indicate that in addition to copper-binding domains hemocyanin has an Ig-like conserved domain, which would confer some new functions to multifunctional respiratory pigment of crustaceans.     

Effects of feeding and hypoxia on cardiac performance and gastrointestinal blood flow during critical speed swimming in the sea bass Dicentrarchus labrax

Previous studies have shown that if European sea bass are exercised after feeding, they can achieve a significantly higher maximum metabolic rate (MMR) than when fasted. They can meet combined metabolic demands of digestion (specific dynamic action, SDA) and maximal aerobic exercise, with no decline in swimming performance. If, however, exposed to mild hypoxia (50% saturation), bass no longer achieve higher MMR after feeding but they swim as well fed as fasted, due to an apparent ability to defer the SDA response. This study explored patterns of cardiac output (Q_A) and blood flow to the gastrointestinal tract (Q_GI) associated with the higher MMR after feeding, and with the ability to prioritise swimming in hypoxia. Sea bass (mean mass ~ 325 g, forklength ~ 27 cm) were instrumented with flow probes to measure Q_A and Q_GI during an incremental critical swimming speed (U_crit) protocol in a tunnel respirometer, to compare each animal either fasted or 6 h after a meal of fish fillet equal to 3% body mass. Feeding raised oxygen uptake (M_O2) prior to exercise, an SDA response associated with increased Q_A (+ 30%) and Q_GI (+ 100%) compared to fasted values. As expected, when exercised the fed bass maintained the SDA load throughout the protocol and achieved 14% higher MMR than when fasted, and the same U_crit (~ 100 cm s^-1). Both fed and fasted bass showed pronounced increases in Q_A and decreases in Q_GI during exercise and the higher MMR of fed bass was not associated with higher maximum Q_A relative to when fasted, or to any differences in Q_GI at maximum Q_A. In hypoxia prior to exercise, metabolic and cardiac responses to feeding were similar compared to normoxia. Hypoxia caused an almost 60% reduction to MMR and 30% reduction to U_crit, but neither of these traits differed between fed or fasted bass. Despite hypoxic limitations to MMR and U_crit, maximum Q_A and patterns of Q_GI during exercise in fasted and fed bass were similar to normoxia. Estimating GI oxygen supply from Q_GI indicated that the ability of bass to prioritise aerobic exercise over SDA when metabolically limited by hypoxia was linked to an ability to defer elements of the SDA response occurring outside the GI tract.     

Reproductive fitness,population size and genetic variation in Muscari tenuiflorum (Hyacinthaceae): The role of temporal variation

In plant populations a positive correlation between population size, genetic variation and fitness components is often found, due to increased pollen limitation or reduced genetic variation and inbreeding depression in smaller populations. However, components of fitness also depend on environmental factors which can vary strongly between years. The dry grassland species Muscari tenuiflorum experiences long term habitat isolation and small population sizes. We analyzed seed production of M. tenuiflorum in four years and its dependence on population size and genetic variation. Genetic diversity within populations was high (AFLP: H_e = 0.245; allozymes: H_e = 0.348). An analysis of molecular variance revealed considerable population differentiation (AFLP: 26%; allozyme: 17%). An overall pattern of isolation by distance was found, which, however was not present at distances below 20 km, indicating stronger effects of genetic drift. Genetic diversity was positively correlated to population size. Self pollination reduced seed set by 24%, indicating inbreeding depression. Reproductive fitness was not correlated to genetic diversity and a positive correlation with population size was present in two of four study years. The absence of a general pattern stresses the importance for multi-year studies. Overall, the results show that despite long term habitat isolation M. tenuiflorum maintains seed production in many years independent of population size. The long term persistence of populations is thus expected to depend less on intrinsic genetic or demographic properties affecting seed production but on successful plant establishment and persistence, which latter are based on conservation and protection of suitable habitats.     

Persistence of changes in the genetic covariance matrix after a bottleneck

Abstract.— Genetic variance, phenotypic variance, and the genetic covariance matrix ( G ) can change as a result of genetic drift. These changes will persist over time to some extent and will continue if population size remains relatively small. Nine populations founded by a single pair of Drosophila melanogaster were measured for a series of six morphological characteristics for a large number of parent-offspring families at both the third generation after the bottlenecks and after 20 generations. From these data, the phenotypic variance, additive genetic variance, and G were estimated for each line at each generation. Phenotypic and genetic variances were highly correlated over time, so that the measurements made at the third generation were predictive of the state of the population 17 generations later. Genetic covariances were also somewhat stable over time; however, the G matrices of some lines changed significantly over the intervening generations. This change did not return the populations toward their original state before the population bottlenecks. We conclude that the genetic covariance matrix can change as a result of mild genetic drift over a short span of time.     

Ontogenic changes in tolerance to hypoxia and energy metabolism of larval and juvenile Japanese flounder Paralichthys olivaceus

Changes in tolerances to hypoxia and sodium azide, an indicator of cellular respiration, and activities of various energy metabolism-related chemical components were studied in Japanese flounder Paralichthys olivaceus during its early life stages from 3.5 to 20.5 mm in total length (TL). They showed flexion stage around 10.4 mm TL. Lethal levels of hypoxia increased with growth from 3.5 to 8 mm total TL, and the levels remained high in larvae, until 10.4 mm TL, decreased significantly thereafter. The 50% lethal concentration of sodium azide temporarily increased at 4.5 mm TL, diminished drastically between 4.5 and 10.4 mm TL, and then increased again in post-flexion larvae. Cytochrome c oxidase activity was highest in larvae around flexion, at 10.4 mm TL, and subsequently decreased. In post-flexion larvae at 13.0 mm TL, lactate dehydrogenase (LDH) and creatine kinase activities increased; LDH activity decreased at the juvenile stage. The adenosine triphosphate content and energy charge in fish were consistently higher in the larval stage than in the juvenile stage. These results indicated that, from just before flexion to the post-flexion stage, the energy metabolism of larvae is higher due to activated aerobic and subsequent anaerobic metabolism for metamorphosis; as a consequence, hypoxia tolerance in fish is the lowest during the increase of aerobic metabolism just before and around flexion.