Luis O.B. Afonso

Are hsps suitable for indicating stressed states in fish

SUMMARY In response to most stressors, fish will elicit a generalized physiological stress response, which involves the activation of the hypothalamic-pituitary-interrenal axis (HPI). As in other vertebrates, this generalized stress response comprises physiological responses that are common to a wide range of environmental, physical and biological stressors. Recently, several families of heat shock proteins (hsps) have been proposed as indicators of a generalized stress response at the cellular level. Recent findings that hsp levels, in various fish tissues, respond to a wide range of stressors have supported the use of these proteins as indicators of stressed states in fish. However, the cellular stress response can vary, for example, according to tissue, hsp family and type of stressor. This brief overview of these responses in fish asks the question of whether changes in levels and families of hsps can be used as a suitable indicator of stressed states in fish. By casting this question in the context of the well-established generalized physiological stress response in fish, we argue that the use of hsps as indicators of stressed states in fish in general is premature.

The immune and stress responses of Atlantic cod to long-term increases in water temperature.

Sea-caged cod are limited in their movements in the water column, and thus can be exposed to large seasonal ( approximately 0-20 degrees C) temperature fluctuations. To investigate the physiological response of Atlantic cod to summer-like increases in temperature, we exposed 10 degrees C acclimated juvenile cod to a graded thermal challenge (1 degrees C increase every 5 days) and measured: (1) plasma cortisol and glucose levels; (2) the respiratory burst activity of blood leukocytes; and (3) the expression of specific immune-related genes [MHC Class I, Interleukin-1beta (IL-1beta), beta2-microglobulin (beta2-M), Immunoglobulin M (IgM)-light (L) and -heavy (H) chains] in the blood using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). The experiment was stopped at 19.1 degrees C, with 26.7% of the fish surviving to this point. Plasma glucose levels increased slightly at 16 and 18 degrees C (by 1.39- and 1.74-fold, respectively), in contrast, cortisol levels were elevated significantly (by 2.9-fold) at 16 degrees C but returned to control levels thereafter. The effect of increasing temperature on the expression of immune related genes in blood cells (leukocytes) was variable and depended on the gene of interest. The expression of IgM-H remained stable for the duration of the experiment. In contrast, IL-1beta expression was increased significantly (by approximately 25-fold) at 19 degrees C as compared to time-matched control fish, and changes in the expression of beta2-M, MHC Class I and IgM-L followed a pattern similar to that seen for cortisol: increasing at 16 degrees C (by 4.2-, 5.3- and 17-fold, respectively), but returning to pre-stress levels by 19 degrees C. Interestingly, increasing temperatures had no effect on respiratory burst activity. This study is the first to examine the effects of a chronic regimen of increasing temperature on the stress physiology and immunology of a marine teleost, and suggests that immune function is influenced by complex interactions between thermal effects and temperature-induced stress (elevated circulating cortisol levels).

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.

Sex-related differences in the organismal and cellular stress response in juvenile salmon exposed to treated bleached kraft mill effluent

Exposure of fish to stressors can elicit biochemical and organismal changes at multiple levels of biological organization collectively known as stress responses. The organismal (plasma glucose and cortisol levels) and cellular (hepatic hsp70) stress responses in fish have been studied in several species, but little is known about sex-related differences in these responses. In this study, we exposed sexually immature juvenile chinook salmon (Oncorhynchus tshawytscha) to bleached kraft mill effluent (BKME: 0%, 1%, and 10% v/v) for 30 days and then measured components of their organismal and cellular stress responses. Males exposed to 1% BKME had higher levels of plasma glucose than females. Plasma cortisol levels were unaffected in females exposed to BKME, but males exposed to 10% BKME had significantly higher levels of plasma cortisol relative to non-exposed males. While exposure to BKME did not affect hsp70 levels in males, females exposed to 1% BKME had higher levels of hsp70 relative to non-exposed and 10% BKME groups. Within any given treatment, females had higher levels of hsp70 relative to males. This study demonstrates that sex-related differences exist in commonly used indicators of stress in fish, and points out the importance of considering the sex of the fish in stress research.

O-acetylation of sialic acids in N-glycans of Atlantic salmon (Salmo salar) serum is altered by handling stress

O‐acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O‐acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N‐glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. It is well known that stress may decrease immune function in fish; however, there are only few suitable biomarkers available to monitor the physiological responses under the stress conditions. This study is the first report on the effect of stress on the profile of O‐acetylation of sialic acids in fish serum. In order to preserve the relevant structural characteristics as much as possible, native N‐glycans were directly analyzed using CE‐MS. We have characterized the N‐glycans in serum of salmon (Salmo salar) exposed to long‐term handling stress (15 s out of the water, daily for 4 wk) and compared with the results obtained from sera of control fish. The results indicated that major N‐glycans in salmon serum contained mono‐acetylated sialic acids (83%), and that the O‐acetylation pattern of sialic acids could be altered by long‐term stress.

Effects of the aromatase inhibitor Fadrozole on reproductive steroids and spermiation in male coho / salmon Oncorhynchus kisutch during sexual maturation

Abstract In male teleosts, plasma oestrogen levels are undetectable, or when detectable, are present in very low amounts. Several studies have shown that the testes are incapable of secreting oestrogens. In contrast, other studies have demonstrated that oestrogen can be formed in the male brain, and that oestrogens are involved with neuroendocrine and behavioral responses. Therefore, this study evaluated the effects of inhibiting oestrogen production in male coho salmon (Oncorhynchus kisutch) during sexual maturation using the aromatase inhibitor Fadrozole (AI). Groups of fish, which were approximately 1.5 months before natural spawning, received injections of 1.0 or 10.0 mg AI/kg or five weekly injections of 10 mg AI/kg. Plasma sex steroid hormones (17β-oestradiol, testosterone, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 11-ketotestosterone) were determined before and during 32 days after injection. Injection with AI inhibited 17β-oestradiol secretion by the brain. Fish injected with 10 mg AI/kg or 10 mg AI/kg weekly presented significantly higher plasma 17,20β-P levels (12.11±1.75 and 11.98±2.85 ng/ml, respectively) at 6 h after injection than before injection (0.19±0.19 and 1.00±0.42 ng/ml, respectively). At 16 days after injection, the groups injected with 10 mg AI/kg or 10 mg AI/kg weekly were already spermiating at significantly higher rates than the vehicle group. Gonadosomatic index (GSI) and sperm motility were not different among the groups. This study demonstrated that Fadrozole inhibited 17β-oestradiol in the brain, and prematurely increased plasma 17,20β-P levels.

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.

VALIDATION OF THE ACETO-CARMINE TECHNIQUE FOR EVALUATING PHENOTYPIC SEX IN NILE TILAPIA (Oreochromis niloticus) FRY

In this study a method for evaluating phenotypic sex in Nile tilapia was validated. A technique that uses aceto-carmine squash mounts to stain the entire gonadal tissue for microscopic examination (Method 2- squash mounts) was compared with a technique based on traditional histology. Approximately 2600 Nile tilapia fry weighing and measuring, respectively, between 0.25-2.50g and 26-53mm, aged 35 to 60 days after hatch (DAH), were sexed using this methodology. In situ microscopic examination on the gonads was also performed. A reliable sexing using squash mount was possible with fish weighing more than 0.500g, 45-47 DAH. Results from microscopic observation using the aceto-carmine stain coincided 100% with the histological examination. Male gonadal tissue was characterized by the presence of cysts containing spermatogonia and spermatocytes, while females were easily identified by the presence of oocytes at the perinucleolar stage. The technique proved to be efficient not only in terms of evaluating sex proportion in fish but also because it allows immediate evaluation of gonadal sex and demands less time and labour.

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...