Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming

Since 1998, Haliotis tuberculata mass mortalities have been occurring regularly in wild abalone populations in France during their reproductive period and in conjunction with seawater summer temperature maxima and Vibrio harveyi presence. To confirm the importance of bacterial exposure, temperature and reproductive status on abalone susceptibility, experimental infections via bath exposure were performed using abalone ranging from immature to reproductively mature. Ripe abalone were more susceptible to the bacterium than immature specimens ( P <0.001), and a difference of only 1 °C in temperature had a highly significant impact on the mortalities ( P <0.001). The natural mortalities that were surveyed during summer 2007 confirmed that recent epidemic losses of European abalone appeared in conjunction with host reproductive stress, elevated temperatures and presence of the pathogen V. harveyi . In view of the elevation of the mean summer temperatures observed in Brittany and Normandy over the last 25 years, this temperature-dependent vibriosis represents a new case of emerging disease associated with global warming.     

Health management during handling and live transport of crustaceans: a review

Best practice approaches used in the live transport of commercial crustacean species groups are reviewed and the physiological responses to handling practices are described. Codes of practice aimed at providing technological guidelines in handling and transportation of live prawns, lobsters, crabs and freshwater crayfish are examined. While some handling and transport practices are common across species groups, for example purging and chilling, recommended practices vary with species group. The influence of stress responses on health and survival during live transport is discussed and research investigations on the effect of stressors, in particular air exposure, handling and physical disturbances and temperature fluctuations on physiological processes are reviewed for the six species groups, crabs, freshwater crayfish, clawed and spiny lobsters, freshwater prawns and marine prawns. Investigations on the assessment of immune responses to live transport stressors using haemograms, clotting times, phenoloxidase activity, phagocytic activity, bacteremia and antibacterial activity and haemolymph protein concentration are described. A combination of physiological parameters is desirable in the assessment of stress response or health status in crustacean species transported live to seafood markets.     

Hormonal status and phagocytic activity in sea bream infected with vibriosis

Serum taken from female, sexually mature, silver sea bream (Sparus sarba) displaying either no symptoms of vibriosis, mild infection, severe infection or moribundity were assayed for a number of key hormones. Serum cortisol levels were not significantly different among symptomless, mildly- and severely-infected groups, whereas moribund fish displayed hypercortisolemia with a 14-fold increase in serum cortisol in comparison to symptomless fish. Serum estradiol levels were significantly reduced 19-fold in mildly-infected fish and remained at a low level as infection progressed, whereas serum testosterone increased gradually during vibriosis with a 1.8-fold increase in moribund groups in comparison to symptomless groups. Both serum thyroxine (T(4)) and triiodothyronine (T(3)) gradually decreased during vibriosis being 26- and 2.8-fold lower, respectively, in moribund fish in comparison to symptomless fish. The non-specific immune response, as determined by phagocytic activity, was also assessed using macrophages isolated from the pronephros and spleen of infected fish. Phagocytic indices significantly increased in mildly- and severely-infected fish and then decreased from these stimulated levels in moribund fish.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.     

Effects of the dietary administration of sodium alginate on the immune responses and disease resistance of Taiwan abalone,Haliotis diversicolor supertexta

Sodium alginate extracted from brown algae was reported to enhance the immune response and resistance of fish and shrimp. In this study, survival rates of the abalone, Haliotis diversicolor supertexta, against Vibrio parahaemolyticus, and its non-specific immune parameters such as the total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency to V. parahaemolyticus by H. diversicolor supertexta were determined when abalone (4.5 ± 0.4 g) were fed diets containing sodium alginate at 0, 1.0, 2.0, and 3.0 g kg^?1. Abalone fed a diet containing sodium alginate at 2.0 and 3.0 g kg^?1 for 14 days and at 1.0 g kg^?1 for 21 days had significantly higher survival rates than those fed the control diet after challenge with V. parahaemolyticus. The relative survival percentages of abalone fed the 1.0, 2.0, and 3.0 g kg^?1 sodium alginate-containing diets for 14 and 21 days were 16.1%, 40.0%, and 48.0%, and 63.6%, 27.3% and 22.6%, respectively. The PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus of abalone fed the sodium alginate-containing diets at 1.0, 2.0, and 3.0 g kg^?1 were significantly higher than those of abalone fed the control diet for 14 days. After 21 days, the PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus by abalone fed the sodium alginate-containing diet at 1.0 g kg^?1 were significantly higher than those of abalone fed the other diets. It was concluded that sodium alginate can be used as an immunostimulant for abalone through dietary administration to enhance immune responses of abalone and resistance against V. parahaemolyticus, which were related to the dose and timing of administration.     

In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata

Triclosan (2,4,4'-trichloro-2'-hydroxy-diphenyl ether; TCS) is an antibacterial agent incorporated in a wide variety of household and personal care products. Because of its partial elimination in sewage treatment plants, TCS is commonly detected in natural waters and sediments. Moreover, due to its high hydrophobicity, TCS accumulates in fatty tissues in various aquatic organisms. TCS can be converted into methyl-triclosan (2,4,4'-trichloro-2'-methoxydiphenyl ether; MTCS) after biological methylation. In this study, the acute cytotoxicity of TCS and MTCS in short-term in vitro experiments was assessed on cell cultures from the European abalone Haliotis tuberculata. The results showed that morphology and density of hemocyte are affected from a concentration of 8 μM TCS. Using the XTT reduction assay, TCS has been demonstrated to decrease hemocyte metabolism activity in a dose- and time-dependent exposure. The IC(50) was evaluated at 6 μM for both hemocyte and gill cells after a 24 h-incubation with TCS. A significant cytotoxicity of MTCS was also observed from 4 μM in 24 h-old hemocyte culture. Our results reveal a toxic effect of TCS and MTCS on immune (hemocytes) and/or respiratory cells (gill cells) of the abalone, species living in coastal waters areas and exposed to anthropogenic pollution.     

Electroantennogram response and attraction of Anastrepha suspensa to volatiles of various sugar sources and aged sugar solutions

With the aim of finding new, sugar‐based volatile attractants for economically important tephritid fruit fly species, we used electroantennography (EAG) to quantitate olfactory responses of female Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae), to volatiles of six sugar sources (refined white and brown cane sugar, coconut sugar, date sugar, date jaggery, and cane panela). Laboratory‐strain and wild flies, both sexually immature and mature, were tested for EAG responses to the volatiles of dry crystallized sugar sources and 10% (wt/vol) aqueous solutions that had aged in the laboratory for 0–7 days. In general, wild flies exhibited higher EAG responses than laboratory flies, and immature females responded more strongly than mature females. With the exception of date jaggery and cane panela, volatiles of dry sugar sources and 0‐ and 1‐day‐old solutions elicited lower EAG responses than any of the aged solutions. Most solution volatiles elicited the strongest EAG response after 2 days of aging. Of the treatments evaluated, volatiles of the 5‐day‐old date jaggery solution elicited the highest‐amplitude EAG responses (39%) in A. suspensa females. On the basis of the latter, we tested the attraction response of mature and immature females to date‐jaggery solutions aged over 2 and 4 days in two‐choice flight tunnel bioassays. With both mature and immature females, the 2‐day‐old solution was more attractive than the 4‐day‐old jaggery solution, but significantly more mature females (70% of captures) were attracted to 2‐day‐old jaggery solution. We discuss our results with respect to the improvement of fruit fly lures and attractants by incorporating elements from aged date‐jaggery sugar.     

Reproductive biology of the southern thorny skate Amblyraja doellojuradoi (Chondrichthyes,Rajidae)

The total lengths (L_T) of 193 males (209–556 mm) and 130 females (275–515 mm) of Amblyraja doellojuradoi, a commercial by‐catch species on the Argentinean continental shelf, which are increasingly retained, were analysed. No sexual dimorphism was observed in the L_T at which 50% of individuals were sexually mature; males matured at 448 mm and females at 411 mm, c. 80 and 82% of maximum L_T. The hepato‐somatic index was similar among sexes, but significantly different between maturity stages, being lower in mature than immature specimens. Males had no seasonal difference in the hepato‐somatic index and females had the lowest index in autumn. The gonado‐somatic index was lower in males than in females and significantly higher in mature than immature specimens of both sexes. Males had the highest index in autumn and females had no seasonal difference. Collectively, these results would indicate that A. doellojuradoi breeds in autumn.     

Effect of nitrite on immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalones Haliotis diversicolor supertexta held in 30% per thousand seawater and 26 degrees C were injected with tryptic soy broth (TSB)-grown Vibrioparahaemolyticus (1.6 x 10(5) CFU [colony-forming units] abalone(-1)), and then placed in water containing different concentrations of nitrite-N (nitrite as nitrogen): 0.01 mg l(-1) (control), 1.05, 3.04, 5.10 and 10.06 mg l(-1). Mortality of the abalones increased in direct parallel to ambient nitrite-N concentration. Over 12 to 48 h, the mortality of V. parahaemolyticus-injected abalones held in 3.04 mg l(-1) nitrite-N was significantly higher than that of abalones in the control solution. Abalones that had been exposed to control, 0.96, 2.95, 5.03 and 10.16 mg l(-1) nitrite-N for 24, 72 and 120 h were examined for THC (total hemocyte count), phenoloxidase activity, respiratory bursts (release of superoxide anion), phagocytic activity, and clearance efficiency of V. parahaemolyticus. The THC increased in abalone after 72 h exposure to 0.96 and 2.95 mg l(-1) nitrite-N, but decreased in abalones after 24 h exposure to 5.03 and 10.16 mg l(-1) nitrite-N. Phenoloxidase activity and respiratory bursts increased, while phagocytic activity and clearance efficiency decreased in abalones exposed to > or = 0.96 mg l(-1) nitrite-N for 24 h. It is concluded that nitrite-N in water at concentrations as low as 0.96 mg l(-1) weakens the immune response and increases mortality of H. diversicolor supertexta infected with V. parahaemolyticus.     

Effects of holding temperature and handling stress on the upper thermal tolerance of threadfin shad Dorosoma petenense

The critical thermal maxima (T_MAX) of threadfin shad Dorosoma petenense exposed to standardized stress (30 s handling in a dip‐net), simulating stressors endured during fish loading before transport, were measured over a range of holding temperatures (15, 20 and 25° C). Dorosoma petenense T_MAX showed a significant thermal effect, displaying mean ±s.d . critical thermal maxima of 26·5 ± 1·6, 30·9 ± 1·2 and 33·3 ± 1·4° C, when tested at temperatures of 15, 20 and 25° C, respectively. Dorosoma petenense T_MAX levels were also affected by stress, with handled fish showing significantly lower values than control fish exposed to 15 (mean ±s.d . T_MAX = 25·6 ± 2·0° C), 20 (27·6 ± 2·8° C) and 25° C (32·0 ± 2·6° C). In addition to providing basic information on D. petenense thermal tolerance, experimental results suggest that fishery managers should consider the whole suite of potential stressors, such as air exposure during handling and fish loading, when developing management criteria.     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

Similar metabolic rate-temperature relationships after acclimation at constant and fluctuating temperatures in caterpillars of a sub-Antarctic moth

Temperature compensation in whole-animal metabolic rate is one of the responses thought, controversially, to characterize insects from low temperature environments. Temperature compensation may either involve a change in absolute values of metabolic rates or a change in the slope of the metabolic rate – temperature relationship. Moreover, assessments of compensation may be complicated by animal responses to fluctuating temperatures. Here we examined whole animal metabolic rates, at 0 °C, 5 °C, 10 °C and 15 °C, in caterpillars of the sub-Antarctic moth, Pringleophaga marioni Viette (Tineidae), following one week acclimations to 5 °C, 10 °C and 15 °C, and fluctuating temperatures of 0–10 °C, 5–15 °C, and 10–20 °C. Over the short term, temperature compensation was found following acclimation to 5 °C, but the effect size was small (3–14%). By comparison with caterpillars of 13 other lepidopteran species, no effect of temperature compensation was present, with the relationship between metabolic rate and temperature having a Q₁₀ of 2 among species, and no effect of latitude on temperature-corrected metabolic rate. Fluctuating temperature acclimations for the most part had little effect compared with constant temperatures of the same mean value. Nonetheless, fluctuating temperatures of 5–15 °C resulted in lower metabolic rates at all test temperatures compared with constant 10 °C acclimation, in keeping with expectations from the literature. Absence of significant responses, or those of large effect, in metabolic rates in response to acclimation, may be a consequence of the unpredictable temperature variation over the short-term on sub-Antarctic Marion Island, to which P. marioni is endemic.     

Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei,Callichthyidae)

The present study aimed to investigate in Hoplosternum littorale (Hancock, 1828) the effects of different water temperatures (10 °C, 25 °C-control group- and 33 °C) on physiologic and metabolic traits following acute (1 day) and chronic (21 days) exposures. We analyzed several biomarker responses in order to achieve a comprehensive survey of fish physiology and metabolism under the effect of this natural stressor. We measured morphological indices, biochemical and hematological parameters as well as oxidative stress markers. To evaluate energy consumption, muscle and hepatic total lipid, protein and glycogen concentrations were also quantified. Extreme temperatures exposures clearly resulted in metabolic adjustments, being liver energy reserves and plasma metabolites the most sensitive parameters detecting those changes. We observed reduced hepatosomatic index after acute and chronic exposure to 33 °C while glycogen levels decreased at both temperatures and time of exposure tested. Additionally, acute and chronic exposures to 10 °C increased liver lipid content and plasma triglycerides. Total protein concentration was higher in liver and lower in plasma after chronic exposures to 10 °C and 33 °C. Acute exposition at both temperatures caused significant changes in antioxidant enzymes tested in the different tissues without oxidative damage to lipids. Antioxidant defenses in fish failed to protect them when they were exposed for 21 days to 10 °C, promoting higher lipid peroxidation in liver, kidney and gills. According to multivariate analysis, oxidative stress and metabolic biomarkers clearly differentiated fish exposed chronically to 10 °C. Taken together, these results demonstrated that cold exposure was more stressful for H. littorale than heat stress. However, this species could cope with variations in temperature, allowing physiological processes and biochemical reactions to proceed efficiently at different temperatures and times of exposure. Our study showed the ability of H. littorale to resist a wide range of environmental temperatures and contributes for the understanding of how this species is adapted to environments with highly variable physicochemical conditions.     

Effects of temperature change on the innate cellular and humoral immune responses of orange-spotted grouper Epinephelus coioides and its susceptibility to Vibrio alginolyticus

Orange-spotted grouper Epinephelus coioides held at 27 °C were then further cultured at 19, 27 (control), and 35 °C, and were examined for innate cellular and humoral responses after 3–96 h. The total leucocyte count, respiratory burst, and phagocytic activity significantly decreased 3, 48, and 96 h after fish were transferred to 19 and 35 °C. Both the alternative complement pathway (ACH₅₀) and the lysozyme activity significantly decreased at 3–96 h after fish were transferred to 19 and 35 °C. In another experiment, groupers reared at 27 °C at 34‰ salinity were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at a dose of 2.3 × 10⁹ colony-forming units (cfu) fish^?1, and then further reared in water temperatures of 19, 27 (control), and 35 °C. The cumulative mortalities of V. alginolyticus-injected fish held in 19 and 35 °C were significantly higher than that of injected fish held in 27 °C. Resistance had decreased after 12 h for the challenged grouper held at 35 °C. All injected fish held in 19 °C had died after 72 h. It was concluded that at 12 h after transfer of grouper from 27 to 19 and 35 °C, immunity was suppressed and resistance against V. alginolyticus had decreased.     

Ca and Mg control of cytostatic factors from Rana pipiens oocytes which cause metaphase and cleavage arrest.

A Ca-sensitive cytoplasmic factor is extractable from mature oocytes of Rana pipiens, but not from immature oocytes or fertilized embryos, and is capable of inducing cleavage arrest at metaphase when microinjected into one blastomere of a cleaving embryo. This factor, designated “primary cytostatic factor” (1°CSF), is active in the presence of Mg or EGTA, but is rapidly inactivated by Ca at concentrations as low as 10 μM or by EDTA. 1°CSF is also inactivated in vivo by injection into the cytoplasm of a zygote during the first 45 min following insemination. Another factor, the “secondary cytostatic factor” (2°CSF), develops in vitro during the aging of extracts prepared from immature oocytes, mature oocytes, and fertilized eggs, and is also capable of arresting cleavage at metaphase. 2°CSF is insensitive to exogenous Ca or EDTA, and also remains active when injected into a zygote at any time following insemination. Since the responses of the fertilized egg to 1°CSF and to mature oocyte cytoplasm are quite similar, it appears that 1°CSF is equivalent to the factor which maintains metaphase II arrest in the mature oocyte. The possibility of 1°CSF inactivation by endogenous free Ca at the time of fertilization is discussed in the context of changes in the free Ca concentration.     

Tissue-Specific Induction of Hsp90 mRNA and Plasma Cortisol Response in Chinook Salmon following Heat Shock, Seawater Challenge, and Handling Challenge

In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6°C; + 10.6°C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold. Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight. Received October 1, 1999; accepted January 21, 2000