A multi-targeted investigation of Deepwater Horizon crude oil exposure impacts on the marine teleost stress axis

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).

The stress response in teleost fish shows many similarities to that of the terrestrial vertebrates. These concern the principal messengers of the brain-sympathetic-chromaffin cell axis (equivalent of the brain-sympathetic-adrenal medulla axis) and the brain-pituitary-interrenal axis (equivalent of the brain-pituitary-adrenal axis), as well as their functions, involving stimulation of oxygen uptake and transfer, mobilization of energy substrates, reallocation of energy away from growth and reproduction, and mainly suppressive effects on immune functions. There is also growing evidence for intensive interaction between the neuroendocrine system and the immune system in fish. Conspicuous differences, however, are present, and these are primarily related to the aquatic environment of fishes. For example, stressors increase the permeability of the surface epithelia, including the gills, to water and ions, and thus induce systemic hydromineral disturbances. High circulating catecholamine levels as well as structural damage to the gills and perhaps the skin are prime causal factors. This is associated with increased cellular turnover in these organs. In fish, cortisol combines glucocorticoid and mineralocorticoid actions, with the latter being essential for the restoration of hydromineral homeostasis, in concert with hormones such as prolactin (in freshwater) and growth hormone (in seawater). Toxic stressors are part of the stress literature in fish more so than in mammals. This is mainly related to the fact that fish are exposed to aquatic pollutants via the extensive and delicate respiratory surface of the gills and, in seawater, also via drinking. The high bioavailability of many chemicals in water is an additional factor. Together with the variety of highly sensitive perceptive mechanisms in the integument, this may explain why so many pollutants evoke an integrated stress response in fish in addition to their toxic effects at the cell and tissue levels. Exposure to chemicals may also directly compromise the stress response by interfering with specific neuroendocrine control mechanisms. Because hydromineral disturbance is inherent to stress in fish, external factors such as water pH, mineral composition, and ionic calcium levels have a significant impact on stressor intensity. Although the species studied comprise a small and nonrepresentative sample of the almost 20,000 known teleost species, there are many indications that the stress response is variable and flexible in fish, in line with the great diversity of adaptations that enable these animals to live in a large variety of aquatic habitats.