Acute Stress in Lesser-Spotted Catshark (Scyliorhinus canicula Linnaeus, 1758) Promotes Amino Acid Catabolism and Osmoregulatory Imbalances.

Acute-stress situations in vertebrates induce a series of physiological responses to cope with the event. While common secondary stress responses include increased catabolism and osmoregulatory imbalances, specific processes depend on the taxa. In this sense, these processes are still largely unknown in ancient vertebrates such as marine elasmobranchs. Thus, we challenged the lesser spotted catshark (Scyliorhinus canicula) to 18 min of air exposure, and monitored their recovery after 0, 5, and 24 h. This study describes amino acid turnover in the liver, white muscle, gills, and rectal gland, and plasma parameters related to energy metabolism and osmoregulatory imbalances. Catsharks rely on white muscle amino acid catabolism to face the energy demand imposed by the stressor, producing NH4+. While some plasma ions (K+, Cl- and Ca2+) increased in concentration after 18 min of air exposure, returning to basal values after 5 h of recovery, Na+ increased after just 5 h of recovery, coinciding with a decrease in plasma NH4+. These changes were accompanied by increased activity of a branchial amiloride-sensitive ATPase. Therefore, we hypothesize that this enzyme may be a Na+/H+ exchanger (NHE) related to NH4+ excretion. The action of an omeprazole-sensitive ATPase, putatively associated to a H+/K+-ATPase (HKA), is also affected by these allostatic processes. Some complementary experiments were carried out to delve a little deeper into the possible branchial enzymes sensitive to amiloride, including in vivo and ex vivo approaches, and partial sequencing of a nhe1 in the gills. This study describes the possible presence of an HKA enzyme in the rectal gland, as well as a NHE in the gills, highlighting the importance of understanding the relationship between acute stress and osmoregulation in elasmobranchs.

Physiology: An Important Tool to Assess the Welfare of Aquatic Animals.

The assessment of welfare in aquatic animals is currently under debate, especially concerning those kept by humans. The classic concept of animal welfare includes three elements: The emotional state of the organism (including the absence of negative experiences), the possibility of expressing normal behaviors, and the proper functioning of the organism. While methods for evaluating their emotions (such as fear, pain, and anguish) are currently being developed for aquatic species and understanding the natural behavior of all aquatic taxa that interact with humans is a task that requires more time, the evaluation of internal responses in the organisms can be carried out using analytical tools. This review aims to show the potential of the physiology of crustaceans, cephalopods, elasmobranchs, teleosts, and dipnoans to serve as indicators of their wellbeing. Since the classical methods of assessing welfare are laborious and time-consuming by evaluation of fear, pain, and anguish, the assessment may be complemented by physiological approaches. This involves the study of stress responses, including the release of hormones and their effects. Therefore, physiology may be of help in improving animal welfare.

Quercetin attenuates endocrine and metabolic responses to oxytetracycline in silver catfish (Rhamdia quelen).

This study aimed to verify whether dietary quercetin protects against the detrimental effects induced by oxytetracycline (OTC) administration in silver catfish (Rhamdia quelen). Fish were divided into different experimental groups that received OTC and/or quercetin, either during 14 or 21 days. To determine the endocrine system stress response, we have measured the brain mRNA expression levels of corticotropin-releasing hormone (crh), proopiomelanocortins (pomca and pomcb) and some of the pituitary hormones (growth hormone [gh], somatolactin [sl], and prolactin [prl]). We have also quantified the levels of cortisol as well as some metabolites (glucose, glycogen, lactate, and triglycerides) in the plasma. Moreover, the enzymatic activity of hexokinase, phosphorylase (active GPase), fructose-biphosphatase (FBP), glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate dehydrogenase (GDH) and gill Na+/K+-ATPase were measured. The results demonstrated that OTC activates the silver catfish stress response by increasing the plasma cortisol and decreasing the glucose levels at 14 and 21 days. Additionally, OTC also altered the fish hepatic metabolic status as demonstrated by an increase in triglycerides levels and the enzymatic activity of both FBP and GDH after 14 days. OTC also stimulated Na+/K+-ATPase activity in the gill after 14 days and altered the hypophyseal expression of gh (at 14 and 21 days) and prl (at 14 days). The co-treatment with 1.5 g of quercetin could prevent most of the alterations caused by OTC, strongly suggesting quercetin as a beneficial compound when added to the fish diet.

Plasma 1α-Hydroxycorticosterone as Biomarker for Acute Stress in Catsharks (Scyliorhinus canicula).

Glucocorticoids are pleiotropic steroid hormones mediating redistribution of energy. They induce breakdown of glycogen stores and consequent plasma hyperglycaemia after stressful situations. Glucocorticoid actions in most vertebrate species are exerted by cortisol and corticosterone. However, 1α-hydroxycorticosterone is the dominant corticosteroid hormone in elasmobranchs, though its effects as a glucocorticoid are unknown. Here we demonstrate, by using ultra-performance liquid chromatography coupled to tandem mass spectrometry for the quantification of 1α-hydroxycorticosterone in plasma of the elasmobranch Scyliorhinus canicula, the response of this hormone to an acute-stress situation and for the first time its glucocorticoid action in elasmobranchs. After an acute air-exposure challenge, S. canicula increased plasma levels of 1α-hydroxycorticosterone altogether with enhanced glycolysis and gluconeogenesis pathways to fuel energy demanding tissues, such as white muscle, during the first hours after the stress situation. We foresee our study as a starting point to evaluate stress responses in elasmobranchs, as well as for future applications in the management of these key ecosystem species.

Transport and Recovery of Gilthead Seabream (Sparus aurata L.) Sedated With Clove Oil and MS-222: Effects on Stress Axis Regulation and Intermediary Metabolism.

Transport processes between aquaculture facilities activate the stress response in fish. To deal with these situations, the hypothalamic-pituitary-interrenal (HPI) axis releases cortisol, leading to an increase in circulating energy resources to restore homeostasis. However, if the allostatic load generated exceeds fish tolerance limits, stress-related responses will compromise health and welfare of the animals. In this context, anesthetics have arisen as potential agents aiming to reduce negative effects of stress response. Here we assessed the effects of a sedative dose of clove oil (CO) and MS-222 on hallmarks involved in HPI axis regulation and energy management after simulated transport, and further recovery, in gilthead seabream (Sparus aurata L.) juveniles. Fish were placed in a mobile setup of water tanks where transport conditions were simulated for 6 h. Sedation doses of either CO (2.5 mg L-1) or MS-222 (5 mg L-1) were added in the water tanks. A control group without anesthetics was also included in the setup. Half of the animals (n = 12 per group) were sampled immediately after transport, while remaining animals were allowed to recover for 18 h in clean water tanks and then sampled. Our results showed that the HPI axis response was modified at peripheral level, with differences depending on the anesthetic employed. Head kidney gene-expressions related to cortisol production (star and cyp11b1) matched concomitantly with increased plasma cortisol levels immediately after transport in CO-sedated fish, but these levels remained constant in MS-222-sedated fish. Differential changes in the energy management of carbohydrates, lipids and amino acids, depending on the anesthetic employed, were also observed. The use of CO stimulated amino acids catabolism, while MS-222-sedated fish tended to consume liver glycogen and mobilize triglycerides. Further studies, including alternative doses of both anestethics, as well as the assessment of time-course HPI activation and longer recovery periods, are necessary to better understand if the use of clove oil and MS-222 is beneficial for S. aurata under these circumstances.

Transport and Recovery of Gilthead Sea Bream (Sparus aurata L.) Sedated With Clove Oil and MS222: Effects on Oxidative Stress Status.

The use of anesthesia is a common practice in aquaculture to sedate fish and mitigate handling stress. Although the employ of anesthesia is considered beneficial for fish, as it reduces stress and improves welfare, at the same time it may induce hazardous side-effects. The aim of the present study was to investigate the effects of clove oil (CO) and tricaine methanesulfonate (MS222), two of the most used anesthetics, on several oxidative stress related parameters in gilthead sea bream (Sparus aurata), as these types of effects of anesthetics have been seldom investigated. To assess these effects, S. aurata juveniles were placed in a setup of mobile water tanks and were transported during 6 h with either 2.5 mg/L CO or 5 mg/L MS222. After transport, half of the fish were sampled, whereas the remaining fish were transferred to tanks without anesthetics where they were allowed to recover for 18 h before sampling. Changes in the expression levels of several target genes related with the antioxidant response and cell-tissue repair were evaluated in the gills, liver and brain. Those transcripts included glutathione peroxidase 1 (gpx1), catalase (cat), glutathione S-transferase 3 (gst3), glutathione reductase (gr), superoxide dismutase [Zn] (sod2), heat shock protein-70 (hsp70), and metallothionein (mt). Antioxidant enzymatic activities glutathione S-transferase, GST; catalase, CAT; and glutathione reductase, GR, levels of non-enzymatic antioxidants (non-protein thiols - NPT), and pro-oxidative damage, assessed as lipid peroxidation (LPO), were determined in gills, liver and brain. Acetylcholinesterase activity (AChE) was determined in plasma, gills, brain, muscle and heart as an indicator of neuro-muscular alterations. In plasma, the total antioxidant capacity (TAC) and total oxidative status (TOS) were also measured. Results showed that the use of both anesthetic agents, CO and MS222, interferes with fish antioxidant status. All tested biological matrices displayed alterations in antioxidant endpoints, confirming that these substances, although minimizing the effects of transport stress, may have long term effects on fish defenses. This result is of high relevance to aquaculture considering that the oxidative stress, may increase the susceptibility to different environmental or biotic stress and different types of pathologies.

Myrcia sylvatica essential oil mitigates molecular, biochemical and physiological alterations in Rhamdia quelen under different stress events associated to transport.

The effects of pre-transport handling and addition of essential oil of Myrcia sylvatica (EOMS) during transport on stress pathways activation in Rhamdia quelen were investigated. Fish (n=400, 25.2±2.9g) were captured in production ponds and transferred to 100-L tank (density 100g L-1). After 24h, 10 fish were sampled (before transport group). The remaining fish were placed in plastic bags (n=30 or 32 fish per bag, density 150g L-1) containing 5L of water (control), ethanol (315µLL-1, vehicle) or EOMS (25 or 35µLL-1), in triplicate, transported for 6h and sampled (n=10 animals per group). Indicators of stress and metabolism, as well as mRNA expression of brain hormones were evaluated. Previously, full-length cDNAs, encoding specific corticotropin-releasing hormone (crh) and proopiomelanocortins (pomca and pomcb), were cloned from whole brain of R. quelen. Crh expression increased after 24h of capture and handling, whereas cortisol and glucose plasmatics enhanced their values in the control group. Transport with EOMS reduced plasma cortisol and lactate levels, while ethanol and EOMS groups increased Na+/K+-ATPase gill activity compared to control. Gene expression of crh, pomcb, prolactin and somatolactin mRNAs were lower after transport with EOMS compared to control. EOMS was able to mitigate the stress pathways activation caused by transport, maintaining a balance in body homeostasis. Thus, EOMS is recommended as sedative in procedures as transport and the pre-transport handling requires greater attention and use of tranquilizers.