Thesis (PhD.)--University of Canberra, 2007. / Includes bibliography (p. 152-176)

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

Habitat requirements and habitat use for Pseudophryne australis and Heleioporus australiacus were investigated to aid management of these threatened frogs around Sydney, Australia. Much of the work focussed on roads, commonly encountered features in the habitat of both species. The habitat requirements based on locality records of both frogs in the Sydney Basin were investigated at four spatial scales. Both species are habitat specialists. They showed a strong geological association with Hawkesbury Sandstone and occupy upper topographic areas with ephemeral watercourses of gentle gradients. Both frogs occur predominantly in areas of higher precipitation and milder temperature regimes compared to averages representative of the region. Leaf litter is an important feature of P. australis breeding sites, whereas H australiacus generally associate with crayfish burrows. Both species are dependent on natural vegetation with a complex structure. H australiacus have a relatively long larval period (3 - 12 months) and breed in ephemeral pools, exposing their tadpoles to the risk of dying due to early pond drying. In the laboratory, tadpoles responded to decreasing water levels by shortening their larval periods and metamorphosing earlier than siblings held at constant water level. Despite this plastic response, a number of pools in the field failed to produce metamorphs due to early drying, an observation also made on P. australis. Regular monitoring of breeding sites revealed increased reproductive success away from roads for both species probably because of relatively longer hydroperiods. Spatial distributions and associations with habitat features, and movement patterns of both frogs were further investigated using mark-recapture methods. Both species showed strong site fidelity. P. australis formed small aggregations and predominantly selected leaf litter piles despite their relatively low availability. Leaf litter piles in creeks moved over time and the animals moved with these piles. In contrast, H australiacus individuals formed no aggregations and showed no preference for any available structural vegetation type. Locations of individuals were independent of relative distances to creeks and artificial drains, but males appeared to be more common near culverts. However, individuals were randomly distributed in space and nearest-neighbour distances were high relative to individual movement distances, suggesting minimal overlap between relatively large home ranges. Radio-telemetry demonstrated that some H australiacus individuals burrow in the road environment. There they would be at risk of being dug up and possibly injured during road works. The results are discussed in relation to the spatial requirements of both species and the protection of utilised habitat features. Management options are suggested to mitigate the impacts of road works. Differences in spatial dynamics of both frogs with overlapping habitats highlighted in this study require species-specific management approaches.

The phylogenetic relationships of Pseudophryne and the closely related monotypic Metacrinia are resolved with the use of morphological characters and comparison with representatives species of Crinia, Uperoleia, and Taudactylus as defined out-groups. Characters describing musculature are not sufficient to resolve the relationships, but do provide support when used in combination with osteological and external characteristics. When all data are considered, parsimony and maximum likelihood analyses yield the same hypothesis of relationships within Pseudophryne + Metacrinia. Thus, a monophyletic lineage of Pseudophryne + Metacrinia is supported by four synapomorphies: (1) the absence of toe fringes, (2) wide frontoparietal fontanelle, (3) m. abductor indicis longus arising from the humerus and radioulna, and (4) neopalatine in contact with the maxilla. The columella is shown as a relatively plastic characteristic, present ancestrally but lost in the common ancestor to Pseudophrye+Metacrinia and then reappearing within that taxon as well as one outgroup species (Crinia riparia). Analyses of size data reveal shape trends among the outgroup taxa that differ from the in-group of Pseudophryne and Metacrinia. Interpretations of shape differences are congruent with the placement of Uperoleia as more closely related to Pseudophrye. This study suggests support for re-synonymy of Metacrinia nichollsi with Pseudophryne, but formal change of status depends on access to more complete data.

The decline and extinction of amphibian species over the past three decades is widely acknowledged as one of the greatest biodiversity crises of modem time. Providing convincing data to support hypotheses about these declines has proved difficult, which has greatly restricted the development and implementation of management actions that may prevent further amphibian declines and extinctions from occurring. In this thesis, I present research that was undertaken as part of the recovery programs for the southern corroboree frog (Pseudophryne corroboree), and the Booroolong frog (Litoria booroolongensis); two species that underwent very rapid declines in distribution and abundance during the 1980's. More specifically, I investigated potential causal factors in the declines of both species using experimental and correlative studies, and examined the mechanisms by which one threatening process (chytridiomycosis) may be causing continued decline and extinction in P. corroboree. I also examined the implications of population dynamics for monitoring L. booroolongensis, and suggest a possible monitoring strategy that may reliably facilitate the implementation of recovery objectives for this species. I also tested one possible reintroduction technique aimed at preventing the continued decline and extinction of P. corroboree populations. In Chapters 2 and 3, I present the results from a series of experiments in artificial enclosures designed to examine whether the tadpoles of L. booroolongensis are susceptible to predation by co-occurring introduced predatory fish species; brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), European carp (Cyprinus carpio), redfin perch (Percafluviatilis), and mosquito fish (Gambusia holbrooki). I demonstrated that the tadpoles of L. booroolongensis, and a closely related species Litoria lesueuri, were palatable to non-native trout species, but not to two native predatory fish species, Gadopsis bispinosus and Galaxias olidus. A pond breeding frog species included in this experiment, Limnodynastes tasmaniensis, was palatable to both the native and non-native fish species. In a separate experiment I also demonstrated that the tadpole of L. booroolongensis is palatable to the three other introduced fish species examined in this study; C. carpio, P. fluviatilis, and G. holbrooki. In three of the experiments, the provision of rock within enclosures as a potential refuge habitat did not afford protection to L. booroolongensis tadpoles from predation by any of the five introduced fish species examined. While all the introduced fish species tested here did consume L. booroolongensis tadpoles, the results also suggested that chemical unpalatability might afford some level of protection against some of these fish species. Firstly, the addition of alternative prey items in one of the experiments reduced the proportion of tadpoles consumed, suggesting that L. booroolongensis may not be a preferred prey item. Secondly, the proportion of tadpoles consumed varied greatly among the different fish species examined, suggesting differing levels of palatability. Overall, this study supports previous research in suggesting that chemical unpalatability may be an important strategy for the tadpoles of riverine frog species in south-eastern Australia to avoid predation by native fish species, and that this strategy is less effective against introduced fish species. While L. booroolongensis currently persists in streams inhabited by a number of introduced fish species, this study supports the likelihood that these species are having a negative impact on populations of L. booroolongensis in the wild. In Chapter 4, I present the results of a study aimed at examining potential monitoring techniques for L. booroolongensis. The results of a mark-recapture exercise demonstrated that L. booroolongensis may exhibit large fluctuations in abundance from one year to the next, and through a prospective power analysis approach, I demonstrated that it would be difficult to confidently identify population trends of interest using either indices or estimates of abundance for this species. An assessment of the capacity to identify the presence or absence of L. booroolongensis using nighttime spotlight surveys demonstrated the high detectability of this species using this technique, at both the scale of 300-meter sections of stream and individual breeding areas (typically less than 10-meters of stream). This study suggests that the monitoring objectives of the L. booroolongensis recovery program would be most effectively achieved using presence/absence surveys at different scales. In Chapter 5, I present the results of a field survey aimed at determining the current distribution and habitat requirements of L. booroolongensis in the South West Slopes region of New South Wales. Of the 163 sites I surveyed across 49 streams,I located L. booroolongensis along 77 of these sites from 27 streams. Based on population and habitat connectivity, this study identified 18 populations of L. booroolongensis that are likely to be operating as independent populations. Twelve of these populations are not represented in conservation reserves, but rather occur along streams that flow through the agricultural landscape. A broad scale habitat analysis identified a positive relationship between extent of rock structures along the stream and the occurrence of L. booroolongensis, and a negative relationship between the proportion of canopy cover and this species' occurrence. At the breeding habitat scale, this study identified a positive relationship between the presence of breeding males and; number of rock crevices in the aquatic environment, extent of emergent rocks, and proportion pool. This analysis also detected a negative relationship between occupancy and water depth. These results confirm previous work suggesting the importance of rocky stream habitats to the persistence of L. booroolongensis, but also suggest how disturbance processes, such as increasing sedimentation and weed invasion, may reduce the suitability of rocky structures as breeding sites. In Chapter 6, I investigated current levels of amphibian chytrid fungus (Batrachochytrium dendrobatidis) infection in corroboree frog populations, and used retrospective screening of museum specimens to assess the possibility that this pathogen was implicated in the initial decline of the corroboree frogs. Using histology, I did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline, however using the same technique, moderate levels of infection were detected in post-decline populations of both species. Real-time PCR screening of skin swabs identified much higher overall infection rates in post-decline populations of P. corroboree (between 44% and 59%), while significantly lower rates of infection were observed in P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species. In Chapter 7, I investigated how B. dendrobatidis may be causing the continued decline of P. corroboree through the presence of an abundant reservoir host for this pathogen. I found that populations of adult C. signifera in sub-alpine bogs carry high B. dendrobatidis infection rates (86%), but appear unaffected by this infection. An experiment involving the release of P. corroboree tadpoles into 15 natural pools resulted in metamorphs from seven of these pools testing positive for B. dendrobatidis, with all these individuals dying soon after metamorphosis. These results support the possibility that B. dendrobatidis infection in P. corroboree populations is being facilitated by the presence of large numbers of infected C. signifera in the shared environment. Chapter 8 presents the results of a population augmentation study for P. corroboree. I investigated the extent to which increasing recruitment to metamorphosis may result in population recovery in this species. This was undertaken by harvesting eggs from the field and rearing them through to mid stage tadpoles over the winter period prior to being released back to their natal ponds in spring. While I was able to increase recruitment to metamorphosis by an average of 20 percent, this did not result in a noticeable influence on the subsequent adult population size, as both manipulated and non-manipulated sites declined over the course of this study by an average of 80 percent. I observed a positive relationship between natural recruitment to a late tadpole stage and subsequent adult male population size, however there was considerable variation associated with this relationship. The relationship between recruitment and subsequent population size at the augmentation sites was consistent with the relationship observed at the non-manipulated sites. These results suggest that recruitment to metamorphosis may not be the most important life stage restricting the population recovery of P. corroboree, but that mortality during post-metamorphic stages may be more important in regulating current population size. Hence, further attempts to use captive rearing to increase P. corroboree populations in the wild should focus on the release of post-metamorphic frogs. Overall, this thesis demonstrates the value of quantitative research to the implementation and progress of threatened species recovery programs. While this research will specifically contribute to the recovery programs for L. booroolongensis and P. corroboree, it more broadly contributes to the understanding and capacity to respond to the concerning levels of amphibian extinctions currently occurring throughout the world.

The documented decline of amphibian populations over the past two decades has increased attention towards amphibian conservation. Much of this attention has been focused on testing hypotheses as to the causal factors of these declines, however providing convincing data to support any of these hypotheses has proved difficult. The testing of these hypotheses and the implementation of endangered species recovery programs has been restricted by a lack of knowledge of the ecology and population demography of amphibian species that have suffered dramatic declines. This thesis presents aspects of the research phase of the recovery program for the Southern Corroboree Frog, Pseudophryne corroboree, a species that declined to very low numbers during the early 1980's. In particular, this research aimed to determine the distribution, abundance, population dynamics and demography of this rare species. A complete reassessment of the conservation status of P. corroboree was undertaken and the nature of the persistence of this species across the landscape was analysed. Temporal trends in abundance and its relationship with population size were also investigated. Early life-history survivorship and recruitment to metamorphosis were studied at the scale of individual nest sites and populations, and the adult male population age structure and annual mortality were investigated using skeletochronology. The shout/response survey technique was used to survey and monitor the number of breeding male P. corroboree during this study. This method was found to provide consistent results when the surveys were conducted over a short (two week) period during the peak breeding season in January. Neither time of day, nor the number of males present at a pool, was found to influence the level of responsiveness of male P. corroboree to the shout/response technique. Variation in the number of responding males to the shout/response technique through the breeding season, assessed at a single site over two seasons, was unimodal with the peak responding period occurring during the last two weeks of January during both the 1998 and the 1999 breeding seasons. A systematic survey covering 213 sites across the entire historic distribution of P. corroboree found this species to be persisting at 79 sites. The majority of these sites were in the northwestern portion of the species former range, around the Jagungal Wilderness area, while no extant sites were found in the south-eastern portion of the species former range in the Smiggin Holes and Perisher Blue ski resorts area. The overall abundance of males at persistent sites was extremely low, with 92 percent of sites having fewer than ten responding males. Only one site was found to support greater than fifty responding males. A logistic regression analysis found the persistence of P. corroboree to be associated with increased number of pools within a site, decreased distance to nearest extant population and geographic position (latitude and longitude) in the landscape. While annual variation was observed in the number of breeding males for individual sites, there was no overall trend for an increase or decrease in the number of males, regardless of population size. The average annual extinction rate for local populations was five percent during this study, with those populations becoming extinct having very few breeding males (between one and three) during the previous season. Embryonic and tadpole survivorship was monitored for individual nests at three sites across three years. Recruitment to metamorphosis for P. corroboree was characterised by high variation in survivorship between nest sites, populations and years, while overall recruitment for nest sites was skewed towards lower survivorship. Average nest survivorship to metamorphosis across all sites and years was ten percent but the skewed nature of this survivorship meant that the majority of nest sites attained very low or no survivorship. The low proportion of nest sites that did attain high survivorship provided the greatest contribution to overall recruitment. The levels of embryonic and tadpole mortality observed in this study would be providing a considerable contribution to the regulation of current population sizes. The greatest level of early life-history mortality was observed during the late autumn/winter egg and tadpole stage, with high survivorship during the summer and early autumn egg stage and the post-winter tadpole stage. The estimated sex ratio for seven populations, based on the number of eggs within male nest sites, indicated that for most populations, regardless of population size, there was a greater proportion of females to males. In general, the estimated sex ratio of smaller populations showed greater annual variation and had a lower average number of females to males than the single large population. Tadpole surveys conducted across remnant populations during both 1998 and 1999 found recruitment to metamorphosis to be very low for the majority of populations. A third of all populations during both years attained no recruitment to metamorphosis, with those populations that did attain recruitment typically having fewer than 20 tadpoles. While sites with more frogs generally recruited more tadpoles, there was no strong relationship between population size and the number of tadpoles recruited per male at the scale of either pool or site. There was also no significant difference in recruitment levels between the two years. Tadpole surveys across breeding pools within the single large population also found very low tadpole abundance. There was no strong relationship between the number of male frogs at a pool and the number of tadpoles per male and there was no significant difference in tadpole abundance between the two years. Based on the low density of males at pools and sites (typically less than five), and the skewed nature of nest survivorship identified from monitoring individual nest sites, it seems likely that both deterministic and stochastic factors are influencing recruitment levels in remnant populations of P. corroboree. This study determined that adult male P. corroboree could be accurately aged using the technique of skeletochronology, and this technique was used to determine the adult male population age structure for three populations. The results indicated that adult male P. corroboree can reach sexual maturity from metamorphosis in three years, but the majority of individuals take four years. The oldest individual identified in this study was nine years old from metamorphosis. The adult male age structure at the single large site showed very little annual variation, whereas the two smaller populations showed highly pulsed age structures from one year to the next. The size of adult males was found to be a poor predictor of age. Annual adult male survivorship, calculated by following cohorts from one year to the next, was 55 percent. Based on this calculation of annual adult male survivorship, it seems likely that the initial decline in P. corroboree involved increased levels of adult mortality. The results of this study indicate that the persistence of. corroboree in the wild is precarious in the short-term. For this reason, it is recommended that efforts be undertaken to secure this species ex situ. Attempts to increase population numbers in the wild would greatly benefit from determining the factor(s) that have caused the decline in this species, however, failure to do so should not preclude field experimental management aimed at developing technique to increase the size of remnant populations. This is because it is likely that small population stochasticity is contributing to the current regulation of population size and it is possible that the factors that caused the decline in this species cannot be removed from the environment.

Ce travail de thèse s'est intéressé aux effets de l'uranium appauvri (U) sur le poisson zèbre, Danio rerio. L'hypothèse de travail majeure est que les effets de l'U peuvent se traduire par des modifications du métabolisme. Par conséquent nous avons caractérisé la performance physiologique par le biais de la théorie des bilans d'énergie dynamique (DEB) car c'est la seule théorie qui quantifie simultanément l'ingestion, l'assimilation, la croissance, la reproduction, la maturation, la maintenance et le vieillisse¬ment au cours du cycle de vie entier à des niveaux de nourriture variable. Un modèle DEB a ainsi été construit et a permis de quantifier et de prédire la manière dont la performance physiologique du poisson zèbre dépend de son niveau de nutrition (et de la température). Nous avons montré que le développement s'accélère après la naissance jusqu'à la métamorphose où l'accélération cesse. De plus les coûts de maintenance somatique sont très élevés.Un module spécifiant la toxico-cinétique de l'U, chez un individu qui se nourrit, croit et se reproduit, a été incorporé dans le modèle DEB. Le modèle a été appliqué aux données de toxicité (publiés et acquis pendant la thèse) afin de découvrir quel processus est affecté par l'U. Les résultats montrent qu'à partir de 0 nM, l'U augmenterait les coûts de croissance et diminuerait l'assimilation et/ou augmenterait le coût de la maintenance somatique. Nous n'avons pas pu détecter d'effets notables sur la maturation. Une étude histologique révèle que l'U altère l'intégrité de la paroi intestinale et pourrait perturber l'homéostasie des interactions hôte-bactéries. / The aim of this dissertation is to characterize the toxicity of depleted uranium (U) on the metabolism of zebrafish, Danio rerio. The underlying hypothesis of this work is that effects of U show up as effects on the metabolism of the individual. Consequently, we characterized physiological performance using Dynamic Energy Budget (DEB) theory since it is the only theory which simultaneously specifies ingestion, assimilation, growth, reproduction, maturation, maintenance and ageing over the whole life-cycle at varying food availability. Thus a DEB model was built which quantifies and predicts how the physiological performance of zebrafish relates to food level (and temperature). We showed that development accelerates after birth until metamorphosis after which acceleration ceases. Furthermore, somatic maintenance costs are very high.A module specifying toxico-kinetics of U in a feeding, growing and reproducing individual was incorporated into the DEB model. The model was then applied to toxicity data (from the literature or acquired during this thesis) in order to determine which processes are affected by U. Our results show that, from 0 nM onwards, U increases costs for growth and either increases somatic maintenance or decreases assimilation. We were unable to detect effects on maturation. A histological study showed that U alters histology of the gut wall and may perturb host-microbe homeostasis. By accounting for differences in initial conditions between individuals we were able to explain a number of seemingly contradictory results. The take home message is: observations on individuals should not be averaged for groups of individuals.

L'objectif de ces travaux de thèse était de caractériser la toxicité de l'uranium sur le métabolisme du poisson zèbre, Danio rerio. Puisque les effets de l'uranium se traduisent par des modifications de la performance du métabolisme, la question suivante se pose: que savons-nous du métabolisme du poisson zèbre témoin? Très peu de chose. En effet, nos connaissances à ce sujet sont assez limitées en dépit d'un grand nombre de travaux sur le développement de ce poisson. C'est pourquoi les trois premiers chapitres de ce manuscrit sont dédiés à la caractérisation du métabolisme témoin du Danio. J'ai utilisé la théorie des bilans d'énergie dynamique (DEB) pour procéder à cette caractérisation; à l'heure actuelle c'est la seule théorie qui quantifie l'ingestion, l'assimilation, la croissance, la reproduction, la maturation, la maintenance et le vieillissement pendant le cycle de vie entier d'un organisme. L'effet de l'uranium sur l'organisme implique un effet sur au moins une des processus cités ci-avant. Étant donné que la longévité du poisson zèbre est d'environ quatre ans et demi, et que l'intensité des effets liés à un stress chimique est inversement proportionnel à la taille, nous avons centré nos efforts sur les stades de vie précoces (embryon, juvénile et reproduction adulte). De surcroît, les stades de vies précoces semblent plus sensibles aux effets de l'uranium surtout au niveau des effets sur la croissance. <br /><br /> D'importants progrès ont été réalisés dans le domaine de la quantification du développent, de la croissance et de la reproduction du poisson zèbre. Il s'est avéré que le poisson zèbre accélère son développent après la naissance (c'est-à-dire l'instant où l'individu commence à se nourrir), jusqu'à la métamorphose, où l'accélération cesse. Ce processus a été constaté chez d'autre espèces de poissons, mais pas toutes. Une autre conclusion surprenante était que la maintenance somatique est beaucoup plus élevée que la valeur typique d'un poisson. Nous n'arrivons pas encore à expliquer pourquoi. De plus nous avons découvert que les détails sur la physiologie reproductive sont importants pour caractériser les effets de l'uranium: chez l'adulte les ressources allouées à la reproduction sont stockées dans un compartiment où siègent les processus de préparation de "batch " d'œufs (=buffer de reproduction). Il est donc important de comprendre ce processus pour comprendre comment le poisson zèbre élimine l'uranium via les œufs. <br /><br /> La théorie DEB spécifie que l'individu atteint un stade de développement à un niveau de maturité donné. Selon la température et/ou la nourriture, ce niveau de maturité peut être atteint à des tailles ou des âges différents. Nous avons élargi le concept pour inclure tous les stades de développement (définis sur la base de critères morphologiques) publiés dans les atlas de développement. Ce travail nous a permis d'expliquer par la théorie DEB à présent la variabilité en termes de taille et d'âge. <br/><br /> Dans le but de tester si la théorie DEB peut expliquer des perturbations au niveau de la maturation, nous avons étudié le développement de deux espèces de grenouilles taxonomiquement proches et de taille similaires. Une des espèces possède un développement typique comprenant un stade embryonnaire, un stade têtard qui se nourrit et puis un stade juvénile avec la morphologie typique d'une grenouille. Par contre la deuxième espèce témoigne d'une accélération du développement après l'éclosion mais avant la naissance- qui correspond au stade de développement où l'individu commence à se nourrir. Cette accélération est trahie par une augmentation de la respiration et un retard de la croissance avec au final une diminution de la taille à chaque stade de développement par rapport à la première espèce. Cette accélération s'estompe après la métamorphose (le moment où les jeunes grenouilles quittent l'eau). Toutes les différences entre les deux types de développement ont été expliquées par la théorie DEB en considérant qu'un seul paramètre changeait temporairement de valeur: la fraction de la réserve mobilisée vers la croissance et la maintenance somatique. La conclusion est que les perturbations observées au niveau de la maturation et de la variabilité de l'âge et la taille entre les différents stades de développement soutiennent empiriquement la façon que la théorie DEB incorpore la maturation. <br /><br /> Non seulement notre étude requérait une quantification détaillée de la maturation, mais elle requérait aussi la prise en compte de périodes (prolongées) de jeune, et ce plus particulièrement pour les stades précoces. Selon la théorie DEB la maintenance est alimentée avec l'énergie mobilisée de la réserve. Dès lors que la nourriture devient rare ou disparait cette dernière ne suffit plus pour alimenter la maintenance somatique. Nous avons détaillé ce cas de figure en modélisant le lien entre les processus de rajeunissement et d'amaigrissement extrême et la probabilité de survie. Les prédictions du modèle sont en accord avec les trajectoires de survie de larves obtenues en conditions de laboratoire. Certaines poissons libèrent plus d'un million d'œufs par événement de ponte et pourtant, si la dynamique de la population est stable, à chaque génération chaque poisson n'est remplacé que par un seul individu. Le processus de survie des larves représente une grande énigme irrésolue dans le domaine de la dynamique de populations de poisson. <br /><br /> Par le biais de ces travaux de doctorat, nous disposons à présent d'un outil permettant de comprendre, et de prédire, la manière dont la performance physiologique du poisson zèbre dépend de son niveau de nutrition. Le modèle a été utilisé pour détecter les modifications induites par l'uranium sur la performance physiologique d'un individu exposé par rapport à celle du témoin. A cette fin, nous avons développé un modèle dynamique qui spécifie la manière dont l'uranium s'accumule et s'élimine chez un individu qui se nourrit, grandit et se reproduit. Nous avions imaginé que l'uranium pourrait affecter le système immunitaire ainsi que d'autres mécanismes de défense cellulaire (e.g. système antioxydant). Selon la théorie DEB, l'allocation des ressources à la maturation comprend une fraction fixe de la réserve mobilisée auquel est soustrait le coût de maintenance de la maturité. Notre idée est que les coûts du système immunitaire et de défense cellulaire contribuent à la maintenance de la maturité. Si l'uranium augmentait les coûts de ce dernier alors la maturation ralentira, ainsi j'ai porté une attention soutenu aux taux de maturation. <br /><br /> Nous avons montré que l'uranium altère l'histologie de la paroi intestinale (acteur majeure dans l'assimilation des nutriments) et pourrait potentiellement modifier l'homéostasie des interactions hôte-bactérienne (acteur majeur dans l'assimilation et l'immunité inné). De plus nos travaux suggèrent que l'uranium augmenterait les coûts de synthèse de la structure et diminuerait l'assimilation et/ou augmenterait le coût de la maintenance somatique. Chose étonnante, malgré ce que nous pensions, nous n'avons pas pu détecter d'effets notables sur la maturation à ces faibles concentrations. Puisque la maturation interagit avec la croissance, la reproduction et la maintenance, je considère néanmoins que les travaux que j'ai pu mener sur la maturation sont pertinents. La toxicité de l'uranium est telle que les effets sur le coût de la synthèse de la structure et de la maintenance somatique sont estimés proches de 0 nM d'uranium dans l'eau. <br /><br /> Un résultat très important se dégageant de ces travaux est que la condition des poissons (structure, maturité, réserve, buffer de reproduction, stade de préparation des "batch") au début de l'expérience dépend de l'individu et conditionne la réponse de celui-ci au stress pendant (toute) l'expérience. Ce problème s'aggrave lorsque nous travaillons avec des poissons zèbres adultes car la contribution de la masse du buffer de reproduction par rapport à la masse totale diffère de manière important entre chaque individu. Ceci affecte alors non seulement les trajectoires de masse dans le temps, mais aussi la concentration interne, car la reproduction représente une voie importante d'élimination de l'uranium. La quantité totale de réserve (à savoir : réserve + buffer de reproduction) conditionne la sévérité de l'effet toxique contribuant ainsi à la variabilité dans les données. En prenant en compte les différences entre les conditions initiales de chaque individu, j'ai pu expliquer les résultats contradictoires publiés dans la littérature ainsi qu'expliquer mes propres résultats sur les effets de l'uranium. La leçon à retenir est que des données acquises sur des individus ne devraient pas être moyennées sur des groupes d'individus. <br /><br />