New genomics and transcriptomics tools toward improving conservation strategies for sturgeons

Sturgeons (Family Acipenseridae) are one of the largest and most primitive fish families on Earth. Although they have always been typically distributed throughout the Northern Hemisphere, during the last decades wild populations have declined due to anthropogenic factors such as overfishing, poaching, pollution, and habitat loss. The situation is particularly dramatic for Atlantic sturgeon, one of the most ancient species among the family, which is currently extinct in Europe. In order to reintroduce the Atlantic sturgeon in Europe, several Baltic countries have been working together for more than two decades to build an ex-situ broodstock locally with fish derived from Canada, where the populations are not threatened, and releasing juveniles into the rivers flowing to the Baltic Sea.However, rearing fish aimed for restocking in the same manner as aquacultured fish has been shown to impact the post-release survival of juveniles in the long term. For example, in aquaculture conditions, fish are often maintained in high densities, at constant photoperiod and temperature conditions, and feeding on commercial pellets, leaving them cognitively naïve when released into natural environments. Additionally, increased water temperatures due to global warming have a strong influence on the geographic distribution of the species,\nresulting in local extinctions and population shifts. Although the effect of heat and cold\nstress on the juvenile and embryonic development have been assessed for several sturgeon species, most studies on gene expression have looked at a very limited number of genes due to the lack of sequence information and genomic resources. Also, most studies use other sturgeon species such as Siberian, Japanese, and white sturgeons, which are widespread aquacultured species. Exposing fish to temperatures higher than the optimal can trigger phenotypic adaptations leading to increase thermotolerance and potentially improve postrelease survival, however the impact of temperature-training protocols on the response to a subsequent heat shock has not yet been assessed in sturgeons.\nTherefore, the main aim of this thesis was to generate genomic and transcriptomic resources for Atlantic sturgeon, which are essential to improve and promote research in many fields, such as ecology, physiology and evolutionary studies. Moreover, it provides a reference for RNAseq-mediated transcriptome mapping. Additionally, we have used these resources to develop and evaluate the impact of novel rearing techniques toward improving restoration success, focusing on temperature training. First, we have assembled a high-quality de novo transcriptome, made an inventory of all the heat shock protein (HSP) gene family members and exposed a cell line derived from Atlantic sturgeon larvae to a moderate and severe heat shock in order to identify all heat-responsive genes using an RNAseq approach (Paper I).\nWe found 76 HSP genes in the Atlantic sturgeon transcriptome, only 16 of which were responsive to at least one of the applied heat shock protocols, and only 5 of which were consistently upregulated after both moderate and severe heat shock at all the tested timepoints. After building the reference transcriptome and annotating all the HSP genes, we have evaluated the differences in liver transcriptome between temperature-trained and nontrained juveniles upon exposure to a new heat shock (Paper II). After four weeks of treatment, fish exposed to temperature training showed between 2 to 4 fold less dysregulated genes in response to a new heat shock than the non-trained group, indicating their improved ability to maintain transcriptomic homeostasis during a new heat shock. Again, like in the in vitro experiment, very few of the annotated HSP genes were dysregulated in response to heat shock in the liver transcriptome, namely hspa1, hspc1 and dnajb4. Overall, the response to heat shock in the liver transcriptome was milder than the in vitro response, which is likely a consequence of the activation of compensatory mechanisms. These mechanisms include the neuroendocrine system and result in increased tissue protection and thermogenic capacities, especially in the trained fish. We therefore propose that temperature-training protocols like the one tested in this thesis should be included in the set of new rearing techniques for fish used for restocking; however, other protocols should be investigated.\nSince the main bottleneck in the evaluation of the effect of such training is the lack of sequence information and a reference genome for RNAseq experiments, we have additionally assembled a reference genome for Atlantic sturgeon using a combination of short and long-read sequencing technologies (Paper III). The assembled genome provides for the first time clear evidence of a sturgeon-specific whole-genome duplication event (SR), independent from the American paddlefish (Polyodon spathula), which is the main representative of the sister Family (Polyodontidae) within the same Order (Acipensiformes). The presence of duplicated Hox clusters, together with synteny and phylogenetic studies of these developmental genes, and the results of microsatellite loci analysis, suggests that sturgeons have a paleotetraploid origin, and that a rediploidization process is still ongoing.\nIn summary, the results presented in this thesis advance the field of sturgeon research. We hypothesized that temperature training has a positive effect during the exposure to a subsequent heat shock, but its potential to improve post-release survival in the long term should still be assessed. We therefore suggest that future work should be aimed at the optimization of rearing methods for stocking programs and that a reference genome should be used.

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