Bastien Sadoul

Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts

Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing.

A multivariate analysis using physiology and behavior to characterize robustness in two isogenic lines of rainbow trout exposed to a confinement stress

Robustness is a complex trait difficult to characterize and phenotype. In the present study, two features of robustness in rainbow trout were investigated: sensitivity and resilience to an acute stressor. For that purpose, oxygen consumption, cortisol release, group dispersion and group activity of two isogenic lines of juvenile rainbow trout were followed before and after an environmental challenge. The effect of a 4h confinement protocol (~140kg/m(3)), which is generally considered as a highly stressful challenge, was investigated. Temporal patterns produced by this experiment were analyzed using multivariate statistics on curve characteristics to describe physiological and behavioral adaptive systems for each isogenic line. The two isogenic lines were found to be highly divergent in their corticosteroid reactivity. However, no correlation between physiological and behavioral sensitivity or resilience was observed. Furthermore, the multivariate analysis results indicated two separate and independent fish group coping strategies, i.e. by favoring either behavioral or physiological responses. In addition, considerable intra-line variabilities were observed, suggesting the importance of micro-environment effects on perturbation sensitivities. In this context, cortisol release rate variability was found to be related to the pre-stress social environment, with a strong correlation between pre-stress aggressiveness and cortisol release rate amplitude. Overall, this approach allowed us to extract important characteristics from dynamic data in physiology and behavior to describe components of robustness in two isogenic lines of rainbow trout.

Macronutrient Composition of the Diet Affects the Feeding-Mediated Down Regulation of Autophagy in Muscle of Rainbow Trout (O. mykiss)

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes through a lysosome dependent degradative pathway. Although the induction of autophagy by starvation has been extensively studied, we still know very little about how autophagy is regulated under normal nutritional conditions. The purpose of the present study was to characterize both in vivo and in vitro the response of the autophagy-lysosomal degradative pathway to nutrient (amino acids and carbohydrates) availability in the muscle of the carnivorous rainbow trout. We report that meal feeding is accompanied by a rapid activation of Akt, FoxO1 and the Target of Rapamycin (TOR) signaling pathways and a concomitant decrease of autophagosome formation. We also show that this effect occurs only when the proportion of dietary proteins increases at the expense of carbohydrates. Concurrently, our in vitro study on primary culture of trout muscle cells demonstrates an opposite effect of amino acids and glucose on the regulation of autophagy-lysosomal pathways. More specifically, the addition of amino acids in cell culture medium inhibited the formation of autophagosomes, whereas the addition of glucose had an opposite effect. The effect of amino acids was accompanied by an activation of TOR, considered as an important regulator of autophagosomal formation. However, the mechanisms involved in the effect of glucose were independent of Akt, TOR and AMPK and remain to be determined. Together, these results demonstrated the specific role of macronutrients as well as that of their interactions in the regulation of autophagy and highlight the interest to consider the macronutrient composition of the diets in the control of this degradative pathway.

On the Use of a Simple Physical System Analogy to Study Robustness Features in Animal Sciences

Environmental perturbations can affect the health, welfare, and fitness of animals. Being able to characterize and phenotype adaptive capacity is therefore of growing scientific concern in animal ecology and in animal production sciences. Terms borrowed from physics are commonly used to describe adaptive responses of animals facing an environmental perturbation, but no quantitative characterization of these responses has been made. Modeling the dynamic responses to an acute challenge was used in this study to facilitate the characterization of adaptive capacity and therefore robustness. A simple model based on a spring and damper was developed to simulate the dynamic responses of animals facing an acute challenge. The parameters characterizing the spring and the damper can be interpreted in terms of stiffness and resistance to the change of the system. The model was tested on physiological and behavioral responses of rainbow trout facing an acute confinement challenge. The model has proven to properly fit the different responses measured in this study and to quantitatively describe the different temporal patterns for each statistical individual in the study. It provides therefore a new way to explicitly describe, analyze and compare responses of individuals facing an acute perturbation. This study suggests that such physical models may be usefully applied to characterize robustness in many other biological systems.

Physiological and behavioral flexibility to an acute CO2 challenge, within and between genotypes in rainbow trout

Adaptive capacities, governing the ability of animals to cope with an environmental stressor, have been demonstrated to be strongly dependent upon genetic factors. Two isogenic lines of rainbow trout, previously described for their sensitivity and resilience to an acute confinement challenge, were used in the present study to investigate whether adaptive capacities remain consistent when fish are exposed to a different type of challenge. For this purpose, the effects of a 4-hour hypercapnia (CO2 increase) challenge at concentrations relevant in aquaculture conditions are described for the two isogenic lines. Oxygen consumption, cortisol release, group dispersion and group swimming activity were measured before, during and after the challenge. Sensitivity and resilience for each measure were extracted from temporal responses and analyzed using multivariate statistics. The two fish lines displayed significant differences in their cortisol response, translating differences in the stress axis sensitivity to the stressor. On the contrary, both lines showed, for other measures, similar temporal patterns across the study. Notable within line variability in the stress response was observed, despite identical genome between fish. The results are discussed in the context of animal robustness.

Acute stress response of fathead minnows caged downstream of municipal wastewater treatment plants in the Bow River, Calgary

We examined whether exposure to municipal wastewater effluent (MWWE) compromised the stress performance of laboratory-reared fathead minnows (Pimephales promelas) in a field setting. Adult minnows were caged at two sites upstream and three sites downstream of wastewater treatments plants (WWTPs) discharging MWWE into the Bow River, Calgary, Alberta, Canada. At each site one group of fish was sampled after a 26 day exposure to MWWE, while another group was subjected to 1-min air exposure followed by 60-min confinement and then sampled. Fish morphometrics and proximate composition were measured, and whole-body cortisol, glucose and lactate levels assessed as markers of the stress response. The whole-body protein, glycogen and lipid content were higher at the site closest to a WWTP outfall relative to the other downstream and upstream sites. There were no significant differences in whole-body cortisol levels in minnows sampled at sites either upstream or downstream of WWTPs. Acute stressor exposure significantly elevated whole-body cortisol levels in all groups, and this response was not modified by the location of the sampling sites. The whole-body metabolite profile, including glucose and lactate levels, were significantly higher in fish caged immediately downstream from WWTP inputs relative to upstream sites. There was an acute-stressor-mediated increase in whole-body lactate, but not glucose, levels and this response was independent of sampling site. The results reveal that the capacity to evoke an acute stress response was not compromised in fathead minnows caged for 26 days downstream of WWTPs in the Bow River. However, there were changes in the whole-body proximate composition and metabolite levels immediately downstream from the WWTP outfall suggesting greater accumulation of energy stores in these fish. Taken together, our results suggest that environmental factors in addition to contaminants, including higher water temperature and nutrient availability, influence the impact of MWWEs on fish stress performance.