Effects of rearing temperature on growth,metabolism and thermal tolerance of juvenile sea cucumber,Apostichopus japonicus Selenka: critical thermal maximum (CTmax) and hsps gene expression

Effects of different rearing temperatures (16, 21 and 26°C) on growth, metabolic performance and thermal tolerance of juvenile sea cucumber Apostichopus japonicus (initial body weight 7.72 ± 0.96 g, mean ±SD) were investigated in this study. During the 40‐day experiment, growth, metabolic performance, food intake and energy budget at different reared temperatures were determined. Sea cucumbers rearing at 16°C obtained better growth (final body weight 11.96 ± 0.35 g) than those reared at 21 (10.33 ± 0.41 g) and 26°C (8.31 ± 0.19 g) (P < 0.05), and more energy was allocated for growth at 16°C (162.73 ±11.85 J g^?1 d^?1) than those at 21(79.61 ± 6.76 J g^?1 d^?1) and 26°C (27.07 ± 4.30 J g^?1 d^?1) (P < 0.05). Critical thermal maxima (CTmax) values of juvenile sea cucumbers reared at 16, 21 and 26°C were 33.1, 34.1 and 36.6°C, respectively, and the upregulation of hsps in sea cucumbers reared at 26°C was higher than those acclimated at lower temperatures (16 and 21°C), indicating that temperature acclimation could change the thermal tolerance of the sea cucumber, and CTmax and hsps were sensitive indicators of the sea cucumber's thermal tolerance.     

Effects of temperature and dietary protein on gene expression of Hsp70 and Wap65 and immunity of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed diets containing 303.4, 321.7, 341.2, 361.0 and 379.1 g kg^?1 proteins, respectively, and reared at different water temperatures (18, 23 and 28°C) for 60 days. Gene expression of heat shock protein gene (Hsp70) and the warm temperature acclimation‐related 65 kDa protein gene (Wap65), immunity and antioxidant status in the carp were investigated. Results indicated that the contents of serum complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM), as well as activities of liver superoxide dismutase (SOD) and lysozyme (LSZ) were significantly enhanced with increasing dietary protein (P < 0.05), while content of malondiadehyde (MDA) decreased. Gene expression level of Wap65 in the liver significantly increased with dietary protein, while gene expression of Hsp70 decreased. The contents of C3, C4 and IgM, the activities of SOD and LSZ and gene expression level of Wap65 in the liver significantly increased with temperature. These results suggest that: Serum immune parameter, antioxidant enzymes and Hsp70 and Wap65 expression interact in fish to improve ability to adapt to the environment; and the optimal conditions for the immunity of carp are 348.1?354.5 g kg^?1 protein at 18°C, 352.3?364.9 g kg^?1 at 23°C and 360.2?364.3 g kg^?1 at 28°C, and the optimum temperature for carp is 23°C.     

Acclimation of <Emphasis Type="Italic">Anabas testudineus</Emphasis> (Bloch) to three test temperatures influences thermal tolerance and oxygen consumption

Teleost fish have developed their own specific adaptive mechanism, both behavioral and physiological, to maintain homeostasis in response to unfavorable temperatures. Therefore, this study was aimed at assessing the critical thermal maxima (CT_Max), critical thermal minima (CT_Min), and oxygen consumption rate of Anabas testudineus (17.03 ± 1.2 g) after acclimating to three preset temperatures (25, 30, and 35°C) for 30 days. The CT_Max and CT_Min were 40.15, 41.40, 41.88°C and 12.43, 13.06, 13.94°C, respectively, and were significantly different (P < 0.05). The thermal tolerance polygon for the specified temperatures was 278.30°C². The oxygen consumption rate (117.03, 125.70, 198.48 mg O₂ kg⁻¹ h⁻¹, respectively) increased significantly (P < 0.05) with increasing acclimation temperatures. The overall results indicate that the thermal tolerance and oxygen consumption of A. testudineus are dependent on acclimation.     

Effect of thermal stress on Hsp70 gene expression and female reproductive performance of giant freshwater prawn,Macrobrachium rosenbergii

Using Hsp70 as a biomarker, thermal stress impinges on reproductive organs, ovary and hepatopancreas were being analyzed by determining the expression of Hsp70 mRNA inside the organs after the adult inter‐molt females were subjected to thermal treatment at 35, 30 and 28°C (Control). Results showed the expression of Hsp70 mRNA under thermal treatment of 35°C after 2 hr recovery in ovary were upregulated at 2, 4, 6, 12, 24 hr and 30 days compared to control whereas in hepatopancreas under similar treatment, the expression of Hsp70 mRNA were significantly higher than control at 6, 24 hr and 30 days. Frequency of reproductive molt at 35°C showed the ovary of females were failed to develop and only entered common molt along three consecutive molt cycles. For 30°C thermal treatment, the expression of Hsp70 mRNA was significantly higher than control after 2 hr recovery but returned to normal afterwards until 30 days’ thermal treatment. Maternal heat shock for 2 hr at 35°C were found to give significantly lower frequency of reproductive molt and longer duration of ovarian development and incubation period whereas maternal heat shock for 2 hr at 30°C gave lower frequency of reproductive molt, slower development of embryo and lower hatching success compared to untreated control. This study suggests that short and long‐term thermal stress at 30 and 35°C were found to affect the induction of Hsp70 mRNA in reproductive organs of Macrobrachium rosenbergii and also influence their reproductive performance.     

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h^?1) from 10 °C to the fish's critical thermal maximum (CT_Max). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U_crit) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT_Max and U_crit tests (i.e. a reduction of 30–40%), it appears that the Atlantic cod can largely compensate for these Loma‐induced cardiorespiratory limitations. For example, (i) CT_Max (21.0 ± 0.3 °C) and U_crit (~1.75 BL s^?1) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.     

Thermal tolerance, oxygen consumption and haemato-biochemical variables of Tor putitora juveniles acclimated to five temperatures

A 30-day acclimation trial was conducted using Tor putitora to elucidate its thermal tolerance, oxygen consumption, haemato-biochemical variables and selected enzymatic activities at five acclimation temperatures (AT). Juveniles of T. putitora were randomly distributed among five treatment groups (20, 23, 26, 29 and 32 ± 0.5 °C). There was a significant curvilinear increase in critical thermal maxima (CT_max) (y = ?0.0693x ² + 1.7927x + 34.628, R ² = 0.996) and lethal thermal maxima (LT_max) (y = ?0.1493x ² + 2.3407x + 35.092, R ² = 0.991) with increasing AT. The oxygen consumption rate increased significantly with increasing AT. The Q ₁₀ values were 1.16 between 20 and 23 °C, 3.09 between 23 and 26 °C, 1.31 between 26 and 29 °^C and 1.76 between 29 and 32 °C of AT. The acclimation response ratios were ranged between 0.37 and 0.59. Catalase, superoxide dismutase and ATPase activities were increased linearly in liver, gill and kidney, while brain acetylcholine esterase activity decreased linearly with increasing AT. Blood glucose remained unchanged up to AT of 26 °C and increased significantly at AT of 29 and 32 °C. Haemoglobin content was increased linearly with increasing AT. The highest WBC count was observed at 20 °C, and no significant changes found till AT of 26 °C and significantly decreased at 32 °C. Total serum protein and globulin were significantly decreased with increasing AT. Highest values were observed at 20 °C and remained consistent till 26 °C, then decreased significantly. There was no significant change in A/G ratio through the AT 20–29 °C and increased significantly at 32 °C. The increase in CT_max, LT_max and oxygen consumption rate with increasing AT may suggest that the thermal tolerance of T. putitora is dependent on its prior thermal exposure history, and it could adapt to higher AT by altering its haemato-biochemical variables.     

Heat-shock protein70 gene expression in four hatchery Pacific Abalone Haliotis discus hannai Ino populations using for marker-assisted selection

Heat shock proteins (Hsps) are molecular chaperones that help organisms cope with stressful conditions. Here, we report on the growth rates and Hsp70 expressions in inbred and hybrid populations of abalone Haliotis discus hannai Ino. In abalone, inbred populations expressed more Hsp70 than hybrid populations at all temperatures, except at very high temperatures close to the physiological limit. At benign temperatures, there was a clear trend towards higher Hsp70 expression in inbred than hybrid populations, whereas at higher temperatures, a trend in the opposite direction was observed. The temperature of maximal Hsp70 expression (T_peak) varied with the population type. The T_peak of inbred populations (26°C) was lower than that of the hybrid populations (28°C). The maximal inducible Hsp70 of inbred populations was higher than that of hybrid populations. The results showed a trend towards higher expression in inbred population at a lower temperature. These results provide direct experimental evidence that hybrids can cope with the intrinsic stress even at non‐stressful temperatures. The constitutive Hsp70 may therefore be used for marker‐assisted selection in a breeding programme.     

Coping with climate change: Phenotypic plasticity in an imperilled freshwater fish in response to elevated water temperature

1. Climate change has emerged as an increasingly important threat to freshwater systems. To cope with rapidly changing thermal regimes, freshwater fishes must either relocate or adjust through genetic adaptation and/or phenotypic plasticity. Short-term responses to elevated water temperature have been well studied in freshwater fishes; however, far less is understood about change induced by long-term exposure. Furthermore, few studies have investigated the effects of temperature on already imperilled species, which may be more sensitive to environmental change.
2. This study investigated the effects of rearing temperature on critical thermal maximum (CT_max), agitation temperature (T_ag, temperature at which fish show behavioural signs of thermal stress) and gill size in pugnose shiner, Notropis anogenus, a threatened species in Canada. Juvenile pugnose shiner were reared for 4 months across five different ecologically relevant temperatures. CT_max and T_ag were measured under normoxia and acute exposure to hypoxia to test for oxygen sensitivity of the upper thermal limits in this species.
3. CT_max increased with elevated water temperature. T_ag also increased with rearing temperature and occurred, on average, 4.3°C above acclimation temperatures. The CT_max and T_ag were lower when fish were exposed acutely to hypoxia. Interestingly, gill size (e.g. total gill filament length) increased with rearing temperature, which may increase oxygen uptake capacity and support increased metabolic demands of warmer waters.
4. Overall, pugnose shiner show plasticity in several traits in response to long-term exposure to elevated water temperature that may facilitate persistence in warmer waters. However, acute hypoxia exposure reduced thermal tolerance, stressing the importance of evaluating interactive effects of multiple stressors.
5. Identifying source populations of pugnose shiner with greater thermal tolerance or implementing captive breeding under higher temperature regimes may improve the success of re-introduction efforts in the face of climate change, but the consequences to fitness of increased thermal tolerance should be examined.

     

The induction of Hsp70 synthesis by non‐lethal heat shock confers thermotolerance and resistance to lethal ammonia stress in the common carp,Cyprinus carpio (Linn)

Exposure to heat‐shock protein (Hsp) stimulating factors induces Hsp accumulation and confers tolerance to lethal ammonia stress on the common carp Cyprinus carpio. This study investigated whether a non‐lethal heat shock bestowed similar protective effects against ammonia and induced thermotolerance, both thought to be rendered by increased amounts of Hsps. The 30‐min lethal temperature (30 min LHT) and 1‐h lethal ammonia concentration (1 h LCT) for this species occurred at 41°C and 14.2 mg/L NH₃ respectively. Heating juvenile carp (5 cm) from 28°C to 32, 34 and 38°C, with a subsequent 8‐h recovery period augmented tolerance to lethal heat and ammonia perturbation by two to threefold as compared with animals held at 28°C. Protection occurred in conjunction with Hsp70 accumulation in gills, substantiating the role of this Hsp in enhancing the stress tolerance of common carp.     

Effects of temperature on growth of juvenile blackfoot abalone, Haliotis iris Gmelin

The effects of temperature on growth and survival of juvenile blackfoot abalone, Haliotis iris, were investigated. Animals of 10, 30 or 60 mm initial shell length were exposed to ambient (6–10°C), 14, 18, 22 and 26°C for 112 days in a flow‐through culture system. Maximum growth occurred at 22°C for the 10 and 30 mm size classes and at 18°C for the 60 mm size class. Regression analysis identified the optimal temperature for growth (T_optG) at around 21°C for the 10 and 30 mm size classes and at 17–18°C for the largest size class. In a second experiment, the critical thermal maximum of H. iris was determined as a measure of thermal tolerance. Abalone were subjected to increasing water temperatures at a rate of 2°C h^?1 until they detached from the substrate. Abalone of 10 mm displayed greater thermal tolerance than abalone of 30 and 60 mm in length. CT₅₀ temperatures were 28.8, 27.7 and 27.8°C, yielding deduced T_optG values of 19.7, 18.3 and 18.4°C for the 10, 30 and 60 mm size classes respectively. The size‐dependent nature of the relationship between growth and temperature could be capitalized upon in recirculating aquaculture systems.     

Temperature preference and oxygen consumption of the largemouth bass Micropterus salmoides (Lacépède) acclimated to different temperatures

The behavioural and metabolic responses of the largemouth bass Micropterus salmoides (Lacépède) to temperature were determined to define optimal thermal conditions. The final preferendum of largemouth bass juveniles determined with acute and gravitation methods was independent of the method (29.0–28.1 °C). The displacement velocity in the horizontal thermal gradient of bass juveniles was 22.4 cm h⁻¹ in the light phase and 22.6 cm h⁻¹ in the dark phase. Oxygen consumption rates in the largemouth bass increased significantly (P<0.05) from 48.8 to 69.4 mg O₂ Kg⁻¹ h⁻¹ with an increase in the acclimation temperature from 20 to 33 °C. The temperature quotient (Q₁₀) in the juveniles was 1.37–2.00 in the range of acclimation temperatures of 26–29 and 29–32 °C. The optimum temperature range for growth calculated using Jobling's equation was 28.1–28.6 °C and for Q₁₀ values 26–29 °C. The results are discussed in relation to the use of this information in aquaculture.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.     

Physiological and biochemical responses of Zhikong scallop,Chlamys farreri,to different thermal stressors

Water temperature is a significant environmental stressor that affects physiology and biochemical activities of bivalves. Here, temporal variations in physiological and biochemical parameters of Zhikong scallop, Chlamys farreri, under three water temperature treatments were investigated. For the first treatment, scallops were transferred from rearing temperature (15°C, control temperature) to 5, 10, 20 and 25°C, respectively. The second group of scallops was gradually acclimated to above experimental temperatures at a rate of 1–2°C day^?1. The third group was transferred directly between 15 and 7°C every 12 h and for six times. Results showed that significantly higher oxygen consumption and ammonia‐N excretion together with a significant lower ingestion rate was observed for the acute temperature change treatment compared to those in the equivalent gradual temperature variation treatment (P < 0.05). In acute temperature change treatment, hepatopancreas antioxidant enzyme activities, that is, superoxide dismutase (SOD) and catalase (CAT), immune enzyme activities, that is, acid phosphatase (ACP) and lysozyme (LSZ), and heat‐shock protein 70 gene (Hsp70) expression levels of scallops increased substantially within 48 h. Significant increases in SOD, CAT, ACP and LSZ activities, and malondialdehyde content occurred under exposure to fluctuating temperatures (P < 0.01). Gill and hepatopancreatic Hsp70 expression levels also increased significantly in response to water temperature fluctuations (P < 0.05). The study provides basic knowledge about thermal stress in Zhikong scallop and may contribute to the management of scallop mortalities.     

Temperature preference and acclimation in Poecilia sphenops (Pisces, Poeciliidae)

The preferred temperature (PT) of Poecilia sphenops (Pisces, Poeciliidae) (n = 240; 1.13 ± 0.06 g wet weight; 3.26 ± 0.88 cm standard length) from Presa Piedra Azul, Oaxaca, Mexico, was measured at acclimation temperature of 20 °C, 23 °C, 26 °C, 29 °C, 32 °C and 35 °C. The thermal preference of males was 25.5 °C in summer and 29.6 °C in winter; whereas females preferred 29 °C in both seasons. Fish held at 20 °C and 23 °C preferred temperatures 36% higher than their acclimation temperature (AT), and those held at 35 °C and 32 °C preferred 30% and 20% lower temperatures than their AT. In intermediate temperatures, no significant differences between PT and AT were found. The interval of thermal selection based on the higher (HAT) and lower (LAT) avoidance temperatures was of 10–14 °C and not different between sexes in both seasons. Knowledge of the thermal biology of P. sphenops based on the preferred and avoidance temperatures was very important in order to understand the capacity of the species to adapt to the thermal changes occurring in its habitat. Based on the results, we can recommend the use of P. sphenops in aquaculture owing to its thermal plasticity.     

Thermal tolerance of paralarvae of Patagonian red octopus Enteroctopus megalocyathus

Patagonian red octopus, Enteroctopus megalocyathus, is a merobenthic octopus whose paralarvae have been successfully cultured up to juvenile octopuses. At present, high mortality during the paralarval period prevents the scaling from experimental rearing to commercial aquaculture. The aim of the study was to determine upper (CTMax) and lower (CTMin) thermal tolerance, acclimation response ratio (ARR) and thermal tolerance polygon of paralarvae from different culture conditions and subjected to seven acclimation temperatures (6, 8, 10, 12, 14, 16, 18°C) during the first 5 days of paralarval life. Culture conditions were two types of egg incubation (maternal care and artificial incubators) and two feeding regimes (fed or starved). Fed paralarvae showed thermal preferendum, while unfed paralarvae preferred much higher temperatures than those of acclimation. CTMin and CTMax increased along with the acclimation temperature. Lower values of ARR were obtained in paralarvae from artificial incubation, with this type of paralarva showing the least adaptability to thermal changes. Starved paralarvae showed the lowest values for thermal tolerance range (TTR) and smaller areas of thermal polygon than fed paralarvae. Rearing temperatures above 16°C may be considered suboptimal to paralarvae and affected by the conditions during the embryonic incubation. Rearing temperatures below 8°C may be considered suboptimal for all hatched paralarvae. Therefore, the other temperatures within this range could be used in the context of improving the culture management of paralarvae.     

Thermal tolerance and compatibility zones as a tool to establish the optimum culture condition of the halibut Paralichthys californicus (Ayres, 1859)

California halibut, Paralichthys californicus (Ayres, 1859) juveniles were studied to ascertain the thermal tolerance and the compatibility zone where these species can be cultivated. Juvenile halibut acclimated at 15, 18, 21 and 24 °C preferred temperatures of 15.1, 18.2, 18.5 and 24.7 °C respectively. The final preferendum (FP) was 18.4 °C, equivalent to the temperature where the physiological processes are more efficient and the optimum growth temperature of 18.02 °C was calculated using the Jobling (1981) equation. The maximum average weekly temperature that must not be exceeded in a juvenile cultivation system is 22.6 °C. Juveniles avoided temperatures of 10.8 and 29.1 °C if they were acclimated between 15 and 24 °C. The thermal tolerance range of the juvenile halibut, having low and high lethal temperatures of 5.0 and 31 °C, characterizes it as a eurythermic organism. The tolerance of the halibut did not increase with the acclimation temperature corresponding to the ultimate upper incipient lethal temperature of 31 °C that differed by only 0.83 °C to the value calculated using the Jobling (1981) equation. The thermal tolerance and compatibility zone for the California halibut were 242.8 and 121.5 (°C)², respectively; they characterize the thermal niche that includes the FP supporting an optimal growth of juveniles.     

The effect of temperature on the development of yolk‐sac larvae of European hake (Merluccius merluccius L.) under laboratory conditions

The effect of temperature (10, 13, 16, 19 and 22°C) on hatching, development and survival of yolk‐sac larvae of European hake, Merluccius merluccius, was studied. At 22°C the experiment was suspended because all eggs died a few hours after incubation. Five morphological indicators of larval development (standard length – SL, yolk‐sac volume – YSV, oil globule diameter – OD, body height – BH and body wet weight – BW) were analysed. SL, YSV, OD and BW of newly hatched larvae were biggest in size and weight at low temperatures (P < 0.05). Throughout the experiments, the rates of changes in SL, YSV and OD varied with temperature (P < 0.05). By contrast, BH and BW remained constant and did not show significant relationship with the effect of temperature (P > 0.05). The potential model showed that the SL growth rate varied from 0.05 to 0.08 mm day^?1 from 10.5 to 19.5°C respectively. However, at all temperatures two growth phases were identified: a rapid growth phase followed by a slow one. Rapid growth rate in length and depletion in yolk‐sac and oil globule occurred at high temperatures. After total YSV and OD consumption (death by starvation), larvae died between 8 and 14 days at 19.5 and 10.5°C respectively, reaching a maximum length of 4.3 mm at 10.5°C. Metabolic indices such as: lowest threshold temperature (T₀); effective larval development (); cumulative thermal units (TC) to complete larval development; the temperature coefficient on growth rate in length (Q₁₀) and the condition factor (K) were also studied.     

Acute toxicity of ammonia in Meagre (Argyrosomus regius Asso, 1801) at different temperatures

Argyrosomus regius (3.0 ± 0.9 g) were exposed to different concentrations of ammonia in a series of acute toxicity tests by the static renewal method at three temperature levels (18, 22 and 26°C) at a pH of 8.2. Low temperature clearly increased the tolerance of the fish to total ammonia nitrogen (TAN) and unionized ammonia (NH₃) (P < 0.05). While the 96‐h LC₅₀ values of TAN were 19.79, 10.39 and 5.06 mg L^?1, the 96‐h LC₅₀ of NH₃ were 1.00, 0.70 and 0.44 mg L^?1 at 18, 22 and 26°C respectively. The safe levels of NH₃ for A. regius was estimated to be 0.10, 0.07 and 0.04 mg L^?1 at 18, 22 and 26°C respectively (P < 0.05). This study clearly indicates that A. regius is more sensitive to ammonia than other marine fish species cultured on the Mediterranean and Eastern Atlantic coasts.     

Salinity and temperature tolerance of brown-marbled grouper Epinephelus fuscoguttatus

Grouper have to face varied environmental stressors as a result of drastic changes to water conditions during the storm season. We aimed to test the response of brown-marbled grouper to drastic and gradual changes in temperature and salinity to understand the grouper’s basic stress response. The results can improve the culture of grouper. Brown-marbled grouper, Epinephelus fuscoguttatus (6.2 ± 0.8 g) were examined for temperature and salinity tolerances at nine different environmental regimes (10, 20, and 33 ‰ combined with 20, 26 and 32 °C), in which the fish were subjected to both gradual and sudden changes in temperature and salinity. The critical thermal maximum (50 % CT_MAX) and the upper incipient lethal temperature (UILT) were in the ranges of 35.9–38.3 and 32.7–36.5 °C, respectively. The critical thermal minimum (50 % CT_MIN) and the lower incipient lethal temperature (LILT) were in the ranges of 9.8–12.2 and 14.9–22.3 °C, respectively. The critical salinity maximum (50 % CS_MAX) and the upper incipient lethal salinity (UILS) were in the ranges of 67.0–75.5 and 54.2–64.8 ‰, respectively. Fish at temperature of 20 °C and a salinity of 33 ‰ tolerated temperatures as low as 10 °C when the temperature was gradually decreased. Fish acclimated at salinities of 10–33 ‰ and a temperature of 32 °C tolerated salinities of as high as 75–79 ‰. All fish survived from accumulating salinity after acute transfer to 20, 10, 5, and 3 ‰. But all fish died while transferred to 0 ‰. Relationships among the UILT, LILT, 50 % CT_MAX, 50 % CT_MIN, UILS, 50 % CS_MAX, salinity, and temperature were examined. The grouper’s temperature and salinity tolerance elevated by increasing acclimation temperature and salinity. On the contrary, the grouper’s temperature and salinity tolerance degraded by decreasing acclimation temperature and salinity. The tolerance of temperature and salinity on grouper in gradual changes were higher than in drastic changes.     

Effects of air exposure on the lysosomal membrane stability of haemocytes in blacklip abalone, Haliotis rubra (Leach)

The neutral red retention (NRR) assay was used to evaluate the effects of air exposure on lysosomal membrane integrity in the haemolymph of blacklip abalone, Haliotis rubra, and its subsequent recovery in water. After acclimation in 16°C water for 7 days, abalone were exposed to an air temperature of 7, 16 or 23°C for 12 h in the air exposure experiment or to these three air temperatures, e.g., for 12, 24 or 36 h, followed by re‐immersion in 16°C water in the lysosomal membrane stability recovery experiment. Statistical analyses of the air exposure experiment showed that when abalone were exposed to different air temperatures (7, 16 or 23°C), the lysosomal membrane stability was significantly affected by the air temperature, the exposure duration and their interaction. Air temperature similar to the acclimation temperature had a significantly lower impact on the lysosomal membrane stability within the initial 4.5 h in comparison with the other two temperatures in the same period. The lysosomal membrane stability recovery experiment showed that after air exposure durations of 12, 24 or 36 h, the re‐stabilization of the lysosomal membrane was faster in the animals exposed to lower temperatures than those exposed to higher temperatures. The recovery of the lysosomal membrane stability in abalone exposed to lower 7°C air temperature was not significantly affected (F_{2, 66}=0.251, P=0.779) by the exposure durations (12, 24 and 36 h) used in this study. Alternatively, the lysosomal membrane stability in abalone exposed to higher air temperatures of 16 or 23°C recovered at a faster rate when subjected to shorter durations of air exposure (F_{2, 66}=3.663, P=0.031 and F_{1, 44}=17.057, P<0.001 for 16 and 23°C respectively).