This article is a part of the Special Issue on Papers from XIIIth ICBF
Ontogeny of the cortisol stress response and glucocorticoid receptor expression during early development in channel catfish, Ictalurus punctatus

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Highlights

  • Ontogeny of the cortisol and glucocorticoid receptor stress response in channel catfish and several early life stages.

  • Cortisol biosynthesis, integration and maturation of the HPI axis can be observed in channel catfish at hatching.

  • Upregulation of GR-1 and -2 mRNA supports roles for both transcripts in integrating the catfish stress response.

Abstract

Cortisol is a glucocorticoid hormone which is an endocrine signaling molecule in all vertebrates and acts through intracellular glucocorticoid receptors (GRs). Cortisol affects many biological functions including immunity, stress, growth, and reproduction. The objective of this study was to investigate the ontogeny of the cortisol and GR stress response in channel catfish (Ictalurus punctatus) at several early life stages. To accomplish this, resting and stress-induced levels of tissue cortisol and the two catfish GRs (GR-1 and GR-2) expression were measured. Resting cortisol levels in newly fertilized eggs averaged 2.4 ± 0.2 ng/egg and decreased to 0.4 ± 0.01 ng/egg by day 5. Cortisol levels in newly fertilized eggs subjected to an acute stress (lowered dissolved oxygen from 6.5 mg/L to 1.8 mg/L) averaged 2.3 ± 0.1 ng/egg and decreased to 0.3 ± 0.03 ng/egg by day 5. At hatching, resting cortisol levels were 24 ± 1.0 ng/0.1 g tissue while levels increased to 83 ± 2.0 ng/0.1 g tissue in fry subjected to an acute stress (P < .05). Four days post-hatch, resting cortisol levels were 83 ± 1.0 ng/0.1 g tissue while levels increased to 149 ± 4.0 ng/0.1 g tissue in fry subjected to an acute stress (P < .01). There was no significant difference between GR-1 and GR-2 mRNA in stressed and unstressed newly hatched fry. Four days post-hatch, GR-1 mRNA increased 3-fold while GR-2 mRNA increased 2-fold in fry that were subjected to low dissolved oxygen conditions (P < .05). These results indicate that cortisol biosynthesis, integration and maturation of the hypothalamic-pituitary-interrenal (HPI) axis can be observed in channel catfish at hatching. The upregulation of GR-1 and -2 mRNA in stressed fry supports roles for both transcripts in integrating the channel catfish stress response.

Introduction

Stress has been defined as a physiological cascade of events that occurs when an individual attempts to re-establish homeostatic norms in the face of a perceived threat (Schreck et al., 2001). In response to a stressor, cortisol is the primary corticosteroid secreted by the interrenal cells of teleosts and plays a key role in regulating homeostatic and metabolic functions (Schreck et al., 1997; Szisch et al., 2005). One method of determining the stress response in fish is to measure changes in circulating levels of cortisol (Strange, 1980; Donaldson, 1981; Tomasso et al., 1981; Wise et al., 1993). The action of cortisol is at the cellular level by binding to intracellular glucocorticoid receptors and mineralocorticoid receptors (Prunet et al., 2006). Glucocorticoid receptors are expressed in most cells in the body and function through direct activation of gene expression or through non genomic mechanisms (Kumar and Thompson, 2005).

In fishes, the development of interrenal function varies among species (Hwang et al., 1992; Barry et al., 1995a). De novo cortisol synthesis begins as early as one week after fertilization in rainbow trout (Oncorhynchus mykiss, Pillai et al., 1974) but as late as two weeks after hatch in Japanese flounder (Paralichthys olivaceus) (De Jesus et al., 1991). In many fish species including milkfish (Chanos chanos) (Hwang et al., 1992), tilapia (Orechromis mossambicus), (Hwang et al., 1992), Japanese sea bass (Lateolabrax japonicas), (Pérez et al., 1999), Asian sea bass (Lates calcarifer) (Sampath-Kumar et al., 1995), and yellow perch (Perca flavescens) (Jentoft et al., 2002), endogenous cortisol production begins soon after hatching. However, the development of a mature hypothalamic–pituitary–interrenal (HPI) axis able to produce cortisol in response to an external stressor typically occurs much later in development. For example, cortisol production as a response to stress was observed 2 weeks after hatching in turbot and rainbow trout (Stephens et al., 1997; Barry et al., 1995a, Barry et al., 1995b; Pottinger and Mosuwe, 1994), whereas it was observed only one week after hatching in yellow perch (Jentoft et al., 2002). If it is known when cortisol synthesis begins in channel catfish, it will help in understanding the physiological stress response during early development and could have implications in transporting and culturing eggs and fry.

Excluding zebrafish (Danio rerio), all teleosts studied to date have two GR genes that encode different receptor protein isoforms (Ducouret et al., 1995; Takeo et al., 1996; Burry et al., 2003; Greenwood et al., 2003; Terova et al., 2005; Stolte et al., 2006; Vizzini et al., 2007; Alsop and Vijayan, 2008; Schaaf et al., 2009; Stolte et al., 2009; Li and Leatherland, 2012). Recently, two GR genes were cloned and partially characterized in juvenile (120–250 g) channel catfish (Small and Quiniou, 2017). Their results suggest channel catfish GR-1, but not GR-2 expression is significantly increased following a low-water stressor. Together, these GR expression studies show the unique regulation of the corticosteroid axis among teleosts.

Previous research with catfish fry has shown that fry (<1.0 g) could elicit a cortisol response after lowering the water level and chasing the fish with a net (Peterson and Booth, 2010). However, we do not know how soon after fertilization this type of response begins. Furthermore, it is not known what roles GRs play in response to a stressor during early embryo development. This has practical applications as catfish eggs are subjected to a number of potential stressful events such as collection from spawning cans, transportation to the hatchery in trailers with low oxygen, and poor water quality during and after hatching. In addition, it has been shown in rainbow trout that early exposure to stressors can affect the stress response later in life (Auperin and Geslin, 2008). The objectives of the present study were to examine the effects of stress on cortisol and GRs during early development of channel catfish.

Section snippets

Catfish care and use

All animal procedures followed accepted standards of animal care, approved by the Institutional Animal Care and Use Committee (IACUC) according to United States Department of Agriculture, Agricultural Research Service policies and procedures.

Channel catfish development and low DO stress protocol

Gravid channel catfish raised at the National Warmwater Aquaculture Research Unit, Stoneville, MS were selected and were injected with 100 μg luteinizing hormone releasing hormone analog/Kg body weight (BW) following the procedures outlined by Chatakondi et

Cortisol validation for eggs

Sensitivity, accuracy, precision, reproducibility, and parallelism were determined for extracted egg samples. The standard curve for the cortisol RIA covered the range of 10.0–500.0 ng/ml. Cortisol concentrations of serially diluted lipid extracts appeared parallel to the standard curve (data not shown). Sensitivity of the assay was 5.1 ng/egg. After spiking samples with exogenous cortisol, accuracy averaged 100.8% (Table 1). Precision (intra-assay CV) and reproducibility (inter-assay CV) for

Discussion

Common to catfish fry production, high stocking densities as well as intensive handling during transport can result in low dissolved oxygen levels (Abdalla and Romaire, 1996; Torrans et al., 2003), which can result in fish stress. It is not known at what stage of development a catfish egg is vulnerable to stress and how long after fertilization the hypothalamic-pituitary-interrenal (HPI) axis is functional. Understanding the roles that cortisol and GRs play in the stress response of developing

Acknowledgements

We thank Monica Wood and Candace Woodard for help with fish care, sample collection, and running assays. Mention of trade names, proprietary products, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may be suitable.

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