George K. Iwama

Effects of clove oil on the stress response of matrinxã (Brycon cephalus) subjected to transport

Fish transport is one of the most stressful procedures in aquaculture facilities. The present work evaluated the stress response of matrinxa to transportation procedures, and the use of clove oil as an alternative to reduce the stress response to transport in matrinxa (Brycon cephalus). Clove oil solutions were tested in concentrations of 0, 1, 5 and 10 mg/L during matrinxa transportation in plastic bags, supplied with water and oxygen as the usual field procedures in Brazil. Clove oil reduced some of the physiological stress responses (plasma cortisol, glucose and ions) that we measured. The high energetic cost to matrinxa cope with the transport stress was clear by the decrease of liver glycogen after transport. Our results suggest that clove oil (5 mg/l) can mitigate the stress response in matrinxa subjected to transport.

Acute physiological stress down-regulates mRNA expressions of growth-related genes in coho salmon.

Growth and development in fish are regulated to a major extent by growth-related factors, such as liver-derived insulin-like growth factor (IGF) -1 in response to pituitary-secreted growth hormone (GH) binding to the GH receptor (GHR). Here, we report on the changes in the expressions of gh, ghr, and igf1 genes and the circulating levels of GH and IGF-1 proteins in juvenile coho salmon (Oncorhynchus kisutch) in response to handling as an acute physiological stressor. Plasma GH levels were not significantly different between stressed fish and prestressed control. Plasma IGF-1 concentrations in stressed fish 1.5 h post-stress were the same as in control fish, but levels in stressed fish decreased significantly 16 h post-stress. Real-time quantitative PCR (qPCR) analysis showed that ghr mRNA levels in pituitary, liver, and muscle decreased gradually in response to the stressor. After exposure to stress, hepatic igf1 expression transiently increased, whereas levels decreased 16 h post-stress. On the other hand, the pituitary gh mRNA level did not change in response to the stressor. These observations indicate that expression of gh, ghr, and igf1 responded differently to stress. Our results show that acute physiological stress can mainly down-regulate the expressions of growth-related genes in coho salmon in vivo. This study also suggests that a relationship between the neuroendocrine stress response and growth-related factors exists in fish.

Impairment of the stress response in matrinxã juveniles (Brycon amazonicus) exposed to low concentrations of phenol

In this study we measured plasma cortisol, plasma glucose, plasma sodium and potassium, and liver and gill hsp70 levels in juvenile matrinxã (Brycon amazonicus) subjected to a 96 h exposure to phenol (0, 0.2, and 2.0 ppm), and the effect of this exposure on their ability to respond to a subsequent handling stress. Fish were sampled prior to initiation of exposure and 96 h, and at 1, 6, 12, and 24 h post-handling stress. During the 96 h exposure, plasma cortisol and glucose levels remained unchanged in all treatments. While plasma sodium levels were significantly reduced in all groups, plasma potassium levels only decreased in fish exposed to 0 and 0.2 ppm of phenol. Liver hsp70 levels decreased significantly at 96 h in fish exposed to 2.0 ppm of phenol. All groups, except fish exposed to 0.2 ppm of phenol, were able to increase plasma cortisol and glucose levels after handling stress. Fish exposed to 2.0 ppm of phenol showed decreased gill and liver hsp70 levels after the handling stress. Our data suggest that exposure to phenol may compromise the ability of matrinxã to elicit physiological responses to a subsequent stressor.

Supraphysiological Cortisol Elevation Alters the Response of Wild Bluegill Sunfish to Subsequent Stressors

Wild fish are frequently exposed to multiple stressors, but the influence of previous or ongoing stress on an animals subsequent response is poorly understood. Using wild-caught bluegill sunfish (Lepomis macrochirus) as a model, we used exogenous hormone implants to experimentally raise circulating cortisol in a group of fish for ∼10 days. We also maintained sham-treated and control groups of fish. We subjected all animals to a secondary stressor in the form of either a heat challenge or fasting challenge. We compared survival, body condition, and plasma-borne indicators of physiological status among cortisol-treated, sham-treated, and control groups following the secondary stressor. In order to compare short- and long-term effects of cortisol treatment, we initiated the secondary stressor either 4 or 30 days following initial cortisol treatment. Cortisol-treated fish succumbed to the fasting challenge sooner than sham-treated and control fish at both 4 and 30 days. Interestingly, cortisol-treated fish lost equilibrium sooner than sham-treated and control fish during the heat challenge when conducted at 30 days, but not at 4 days. These results demonstrate that multiple simultaneous stressors have cumulative effects on bluegill sunfish. Furthermore, these results demonstrate that supraphysiological cortisol doses alter the long-term responses of bluegill sunfish to additional challenges, even after apparent recovery. Such cumulative and long-term effects may be an important factor in mediating the response of wild animals to natural and anthropogenic stressors, and should be considered in ecological studies.

Triploid and diploid rainbow trout do not differ in their stress response to transportation

Abstract We examined the neuroendocrine and cellular stress responses of diploid and triploid rainbow trout Oncorhynchus mykiss to transportation. Juvenile diploid and triploid rainbow trout (28 and 26 g/fish average weight, respectively) were stocked at 100 g/L in replicate 70-L tanks and subjected to transportation for an 8-h period. Subsequent levels of plasma cortisol and glucose and of cellular hepatic glutathione (GSH) and heat shock protein 70 (Hsp70) were similar between ploidy groups, indicating that triploid fish respond to transportation in much the same way as diploid fish. A stationary treatment was also included that involved confinement of experimental fish in similar tanks without transport to determine to what extent high-density containment contributed to the stress response in the absence of the noise and vibration of transport. Unexpectedly, fish in the stationary treatment had significantly higher plasma cortisol and glucose levels than the transported fish; however, this might be att...

The glutathione antioxidant system is enhanced in growth hormone transgenic coho salmon ( Oncorhynchus kisutch )

Insertion of a growth hormone (GH) transgene in coho salmon results in accelerated growth, and increased feeding and metabolic rates. Whether other physiological systems within the fish are adjusted to this accelerated growth has not been well explored. We examined the effects of a GH transgene and feeding level on the antioxidant glutathione and its associated enzymes in various tissues of coho salmon. When transgenic and control salmon were fed to satiation, transgenic fish had increased tissue glutathione, increased hepatic glutathione reductase activity, decreased hepatic activity of the glutathione synthesis enzyme γ-glutamylcysteine synthetase, and increased intestinal activity of the glutathione catabolic enzyme γ-glutamyltranspeptidase. However, these differences were mostly abolished by ration restriction and fasting, indicating that upregulation of the glutathione antioxidant system was due to accelerated growth, and not to intrinsic effects of the transgene. Increased food intake and ability to digest potential dietary glutathione, and not increased activity of glutathione synthesis enzymes, likely contributed to the higher levels of glutathione in transgenic fish. Components of the glutathione antioxidant system are likely upregulated to combat potentially higher reactive oxygen species production from increased metabolic rates in GH transgenic salmon.

Physiological stress responses in the warm-water fish matrinxã (Brycon amazonicus ) subjected to a sudden cold shock

The present work evaluated several aspects of the generalized stress response [endocrine (cortisol), metabolic (glucose), hematologic (hematocrit and hemoglobin) and cellular (HSP70)] in the Amazonian warm-water fish matrinxa (Brycon amazonicus ) subjected to an acute cold shock. This species farming has been done in South America, and growth and feed conversion rates have been interesting. However, in subtropical areas of Brazil, where the water temperature can rapidly change, high rates of matrinxa mortality have been associated with abrupt decrease in the water temperature. Thus, we subjected matrinxa to a sudden cold shock by transferring the fish directly to tanks in which the water temperature was 10oC below the initial conditions (cold shock from 28oC to 18oC). After 1h the fish were returned to the original tanks (28oC). The handling associated with tank transfer was also imposed on control groups (not exposed to cold shock). While exposure to cold shock did not alter the measured physiological conditions within 1h, fish returned to the ambient condition (water at 28o C) significantly increased plasma cortisol and glucose levels. Exposure to cold shock and return to the warm water did not affect HSP70 levels. The increased plasma cortisol and glucose levels after returning the fish to warm water suggest that matrinxa requires cortisol and glucose for adaptation to increased temperature.

Exogenous glutathione can increase glutathione levels in tissues of rainbow trout (Oncorhynchus mykiss) through extracellular breakdown and intracellular synthesis.

Glutathione (GSH) is an important intracellular antioxidant involved in numerous cellular pathways. However, little is known about the transport of GSH into fish tissues. To determine whether fish tissues took up GSH by extracellular breakdown and intracellular synthesis or by direct cellular transport, we injected rainbow trout (Oncorhynchus mykiss) with exogenous GSH along with blockers of GSH breakdown and synthesis. Exogenous GSH increased GSH levels to the greatest degree in the cells of the posterior kidney, followed by the liver. Exogenous GSH inconsistently increased liver GSH levels independent of GSH synthesis, although this may have been due to disruption of gradient-dependent GSH export, and not necessarily to intact uptake of GSH. The cells of the posterior kidney, liver and gill took up GSH by extracellular breakdown and intracellular synthesis. This indicates that, unlike mammalian tissues, normal cellular GSH levels in fish are not sufficient to inhibit additional GSH synthesis. This may lend flexibility to the GSH system in fish, where levels of GSH may rapidly increase in response to an increased supply of amino acids, or during times of high demand, without increasing synthesis enzymes.