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Metabolic programming of zebrafish Danio rerio uncovered. Physiological performance as explained by dynamic energy budget theory and life-cycle consequence of uranium induced perturbations

Starrlight Augustine has defended her thesis on 23rd April 2012 in Amsterdam.

Document type > *Mémoire/HDR/Thesis

Keywords >

Research Unit > IRSN/DEI/SECRE/LRE

Authors > AUGUSTINE Starrlight

Publication Date > 23/04/2012

Summary

The aim of this dissertation is to characterize the toxicity of uranium on the metabolism of zebrafish, Danio rerio. Because effects of uranium manifest themselves as deviations from the non-stressed situation, the first question this raises is: What do we know about zebrafish metabolism under non-stressed conditions? And the answer is that very little is known, despite the large amount of work on developmental aspects of the zebrafish. This is why the first three chapters are dedicated to characterizing the blank metabolism of zebrafish. I used the Dynamic Energy Budget (DEB) theory for this characterisation; it is presently the only theory that covers the full life cycle of the organism and quantifies feeding, assimilation, growth, reproduction, maturation, maintenance and aging. Any metabolic effect of uranium should appear as effects on one or more of these fundamental processes. Since the life span of zebrafish is some four and a half years, and larger individuals respond slower to chemical stress, the focus was on the early life stages (embryo, juvenile, and reproductive behaviour of the adult) for several reasons. This focus is of importance not only for practical reasons, but the early life stages also seem to be more sensitive and show particular effects (such as on growth) more clearly.

 

Considerable breakthroughs in the quantification of zebrafish development, growth and reproduction have been made. It turned out the zebrafish accelerates its metabolism after birth (when feeding starts) till metamorphosis, when acceleration ceases. This process is seen in some, but not all, species of fish. Another striking conclusion was that somatic maintenance was much higher than is typical for fish. We don't yet have an explanation for this finding. Further it turned out that the details of reproduction matter: allocation to reproduction (in adults) accumulates in a reproduction buffer and this buffer is used to prepare batches of eggs. We needed to detail this preparation process to understand how zebrafish can eliminate uranium via eggs.

 
DEB theory specifies that a particular developmental stage (birth, metamorphosis, puberty) is reached at specified levels of maturity. For different temperatures and food levels, that can occur at different ages and body sizes. We extended this idea to include all the described morphologically defined developmental stages of the zebrafish in the literature; the observed variations in ages and body sizes can now be explained by DEB theory.


To test if DEB theory can also explain perturbations of maturation, we studied developmental patterns in two types of taxonomically related frog species of similar body size. One type shows a typical developmental pattern as embryo, feeding tadpole and juvenile frog. The other type shows, after hatching, but before birth (= start of feeding) a significant acceleration of maturation, which is visible as an increased respiration and a retarded growth, which big effects on the size at the various developmental stages. This acceleration is reduced after metamorphosis (when the tiny froglets leave their drying pool), but, compared to the standard type of frog, it takes considerable time to catch up in growth. All these changes could be captured accurately with DEB theory, by a temporary change in a single parameter: the fraction of mobilised reserve that is allocated to somatic maintenance plus growth, as opposed to maturity maintenance plus maturation. The conclusion is that the observed perturbations of maturation and the age and size variations at various developmental stages provide strong support for how DEB theory incorporates maturation.


We not only required detail on maturation, but also on starvation, especially in the early juvenile stages. The problem is that maintenance is paid, in DEB theory, from mobilised reserve, but when food is scarce or absent, reserve becomes depleted and maintenance can no longer be paid from mobilised reserve. We included more detail on what happens exactly under such conditions. More specifically we modelled the processes of rejuvenation and shrinking (of structure) and their consequences for the hazard rate. We managed to capture observed size and survival trajectories of fish fry under controlled starving conditions. These processes are not only important to capture effects of uranium on feeding, but have a much wider ecological significance in field situations. Many species of fish lay over a million eggs per spawn, yet, in stable populations, each individual fish just replaces itself. The survival process of the early life stages is still the most difficult problem in fish population dynamics.


As a result of my work, there is now a formal basis for understanding (and predicting) how the physiological performance of zebrafish relates to food intake. The model was used to detect uranium induced eco-physiological deviations from the blank. For this purpose we developed a dynamic model for the accumulation-elimination behaviour of uranium in a feeding, growing and reproducing fish. We expected that uranium might affect the immune system and other defence systems. In DEB theory, resource allocation to maturation comprises a (fixed) fraction of mobilised reserve, minus what is required for maturity maintenance. The idea was that uranium might increase the maturity maintenance cost, because defence is paid from this flux, and so delay maturation. I, therefore, paid due attention to maturation rates.


Uranium was shown to alter the histology of the gut wall (major player in nutrient assimilation) and may even modify homeostasis of host-microbe interaction (major players in assimilation and innate immunity). We further found that uranium most likely increases cost for structure, decreases assimilation and, possibly, increases somatic maintenance costs. Surprisingly, we could not detect obvious effects on maturation at very low concentrations, as we expected. Since maturation interacts with growth, reproduction and maintenance, I don't see our work on maturation as lost effort; moreover the topic is of interest in its own right. The toxicity of uranium is such that effects on the costs of structure and somatic maintenance start close to 0 nM uranium in the water.


An important result of my research was that the conditions of the fish (structure, maturity level, reserve, reproduction buffer, stage of batch preparation) at the start of the experiment is very much specific for the individual and determines the response of that individual to toxic stress during the experiment. The problem is severe for adults where the contribution of reproduction buffer to total mass can differ considerably between individuals. This not only affects weight trajectories, but also the concentration of toxicant inside the body, since reproduction represents an important elimination route for uranium. The amount of total reserve material (reserve + reproduction buffer) determines the severity of the toxic effect and contributes in an important way to the scatter in the data. By accounting for differences in initial conditions, I was able to explain the seemingly contradictory results that have been reported in the literature and explain my own results for effects of uranium. The take-home message is: observations on individuals should not be averaged for groups of individuals.


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