Noninvasive Measurement of Steroid Hormones in Zebrafish Holding-Water

Abstract

Zebrafish (Danio rerio) has recently emerged as a new animal model in neuroendocrinology and behavior (e.g., stress physiology and ecotoxicology studies). In these areas, the concentrations of steroid hormones in the blood are often used to study the endocrinological status of individuals. However, due to the small body size of zebrafish, blood sampling is difficult to perform and the amount of plasma obtained per sample for assaying hormones is very small (ca. 1-5 lL), and therefore most studies have been using whole-body hormone concentrations, which implies sacrificing the individuals and hampers sequential sampling of the same individual. Here a noninvasive method to assay steroid hormones from zebrafish holding-water, based on the fact that steroids are released into the fish holding-water through the gills by passive diffusion, is validated. Cortisol and the androgen 11-ketotestosterone (KT) were measured in water samples and compared to plasma levels in the same individuals. Cortisol released to holding-water correlates positively with plasma concentrations, but there was a lack of correlation between KT water and circulating levels. However, KT levels showed a highly significant sex difference that can be used to noninvasively sex individuals. An ACTH challenge test demonstrated that an induced increase in circulating cortisol concentration can be reliably detected in holding-water levels, hence attesting the responsiveness of holding-water levels to fluctuations in circulating levels.

Disruption of the stress response in wastewater treatment works effluent-exposed three-spined sticklebacks persists after translocation to an unpolluted environment

“…Overall, WC release rates, which provide a surrogate for blood cortisol concentrations (Félix et al 2013;Friesen et al 2012;Gabor and Contreras 2012;Wong et al 2008), were directly proportional to the estimated concentration of WWTW effluent at these sites. Female sticklebacks translocated from the same sites five months previously and subsequently held in pristine aquarium conditions continued to exhibit the same site-related trends in WC release rates as their more recently wild-caught counterparts.…”

“…Instead, the magnitude of the stress response was measured indirectly via the release of cortisol across the gill epithelium to the surrounding water (Scott and Ellis 2007) the rate of which varies in proportion with blood cortisol concentrations (Félix et al 2013;Friesen et al 2012;Gabor and Contreras 2012;Wong et al 2008). During March 2014 five fish were transferred by handnet from a holding tank to a bucket containing 5 litres of water in which they remained for 30 mins before being transferred to individual Nalgene tubs (150 ml, 6.5 cm diameter, with screw-fit lids) containing 100 ml of artificial freshwater (deionised water containing 0.33 g/l aquarium grade sea salt; Klüttgen et al 1994).…”

A comparison of two methods for the assessment of stress axis activity in wild fish in relation to wastewater effluent exposure

“…The rate of release of steroids, from the bloodstream to the surrounding water, across the gill epithelium of fish has been shown to be proportional to the steroid concentration in the blood during the period of collection (Félix et al, 2013;Scott and Ellis, 2007;Sebire et al, 2007) and CRTW estimates may therefore provide a more functionally relevant assessment of HPI activity than WBIC. The cortisol release rates recorded in the present study (100 -3500 pg/g/h) are within the range reported previously for this species (Fürtbauer et al, 2015a;Sebire et al, 2007).…”

“…At the same time, it may be erroneous to assume that the quantity of steroid lost across the gills should be directly proportional to that accumulating in other tissues -diffusional losses across the gills are presumably a function of blood cortisol concentration, and will not necessarily reflect rates of steroid synthesis and metabolism occurring elsewhere in the fish which will, however, affect whole-body concentrations. Few data are available to help interpret these observations since different stress axis endpoints are rarely measured simultaneously in the same individual but a lack of consistent proportionality between WBIC and CRTW in the same individuals has been reported for guppies (Poecilia reticulata) held under laboratory conditions (Fischer et al, 2014) and differences in the relationship between plasma cortisol and CRTW has been reported between the sexes for zebrafish (Danio rerio; Félix et al, 2013) and in the daffodil cichlid (Neolamprologus pulcher; Ligocki et al, 2015). It may also be pertinent that in 18 zebrafish gills cortisol is converted via cortisone to 20β-hydroxycortisone (Tokarz et al, 2012) and in stressed zebrafish the concentrations of sulphated and glucuronidated 20β -hydroxycortisone release across the gills were found to be an order of magnitude greater than the corresponding conjugates of cortisol (Tokarz et al, 2013).…”

“…In fish, circulating steroids, including cortisol, are transferred from the bloodstream to the surrounding water across the gill epithelium (Ellis et al, 2005) and can be quantified after extraction and concentration of water samples from holding tanks or collection vessels. Steroids retained in water samples after transfer across the gill may be a more accurate surrogate of blood steroid levels than the multi-compartment total provided by WBIC: the rate of release of 6 cortisol to water has been shown to be proportional to the concentration of cortisol in the blood in a number of species (Félix et al, 2013;Gabor and Contreras, 2012;Scott and Ellis, 2007;Wong et al, 2008) including three-spined sticklebacks (Sebire et al, 2007). The release of cortisol to water is an endpoint that is ideally suited to the assessment of stress responsiveness in small fish captured from field environments, in circumstances where blood sampling is not possible.…”