How Can We Advance Integrative Biology Research in Animal Science in 21st Century? Experience at University of Ljubljana from 2002 to 2022.

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health.

Neotype designation for Thymallus aeliani Valenciennes, 1848 from a museum topotype specimen and its affiliation with Adriatic grayling on the basis of mitochondrial DNA.

In 1848, the grayling Thymallus aeliani (Valenciennes) was described from Lake Maggiore, Italy, in the north Adriatic basin. Genetic analyses of the mitochondrial control region showed a unique evolutionary history of grayling inhabiting the rivers of northern Adriatic basin, from the upper reaches of the Po River and its left tributaries in the west to the Soca River in the east, which resulted in the designation of this phylogenetic lineage as Adriatic grayling. Consequently, the name T. aeliani was connected to the Adriatic lineage, re-establishing the validity of this taxon. However, the mitochondrial haplotypes belonging to Adriatic grayling were never compared with the type specimens of T. aeliani, as their whereabouts were unknown. In this study, a neotype for T. aeliani was designated using topotypical specimens stored at the Natural History Museum in Vienna. The neotype (NMW 68027:2 labelled as "Lago Maggiore, Bellotti, 1880") was designated pursuant to the conditions stipulated in Article 75.3 of the International Code of Zoological Nomenclature. Furthermore, the mitochondrial control region of the neotype was compared to haplotypes of the Adriatic lineage and showed high genetic similarity, which therefore connects the species name T. aeliani to the Adriatic grayling. This crucial step in fixing nomenclatural status of this species is very important for its protection and management.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Molecular phylogeny of Salmo of the western Balkans, based upon multiple nuclear loci.

BACKGROUND: Classification of species within the genus Salmo is still a matter of discussion due to their high level of diversity and to the low power of resolution of mitochondrial (mt)DNA-based phylogeny analyses that have been traditionally used in evolutionary studies of the genus. We apply a new marker system based on nuclear (n)DNA loci to present a novel view of the phylogeny of Salmo representatives and we compare it with the mtDNA-based phylogeny. METHODS: Twenty-two nDNA loci were sequenced for 76 individuals of the brown trout complex: Salmo trutta (Danubian, Atlantic, Adriatic, Mediterranean and Duero mtDNA lineages), Salmo marmoratus (marble trout), Salmo obtusirostris (softmouth trout), and Salmo ohridanus (Ohrid belvica or belushka). Sequences were phylogenetically analyzed using maximum-likelihood and Bayesian Inference methods. The divergence time of the major clades was estimated using the program BEAST. RESULTS: The existence of five genetic units i.e. S. salar, S. ohridanus, S. obtusirostris, S. marmoratus and the S. trutta complex, including its major phylogenetic lineages was confirmed. Contrary to previous observations, S. obtusirostris was found to be sister to the S. trutta complex and the S. marmoratus clade rather than to the S. ohridanus clade. Reticulate evolution of S. obtusirostris was confirmed and a time for its pre-glacial origin suggested. S. marmoratus was found to be a separate species as S. trutta and S. obtusirostris. Relationships among lineages within the S. trutta complex were weakly supported and remain largely unresolved. CONCLUSIONS: Nuclear DNA-based results showed a fairly good match with the phylogeny of Salmo inferred from mtDNA analyses. The comparison of nDNA and mtDNA data revealed at least four cases of mitochondrial-nuclear DNA discordance observed that were all confined to the Adriatic basin of the Western Balkans. Together with the well-known extensive morphological and genetic variability of Balkan trouts, this observation highlights an interesting and variegated evolutionary history of Salmo in this area.

Phylogeographic structure and demographic patterns of brown trout in North-West Africa.

The objectives of the study were to determine the phylogeographic structure of brown trout (Salmo trutta) in Morocco, elucidate their colonization patterns in North-West Africa and identify the mtDNA lineages involved in this process. We also aimed to resolve whether certain brown trout entities are also genetically distinct. Sixty-two brown trout from eleven locations across the Mediterranean and the Atlantic drainages in Morocco were surveyed using sequence analysis of the mtDNA control region and nuclear gene LDH, and by genotyping twelve microsatellite loci. Our study confirms that in Morocco both the Atlantic and Mediterranean basins are populated by Atlantic mtDNA lineage brown trout only, demonstrating that the Atlantic lineage (especially its southern clade) invaded initially not only the western part of the Mediterranean basin in Morocco but also expanded deep into the central area. Atlantic haplotypes identified here sort into three distinct groups suggesting Morocco was colonized in at least three successive waves (1.2, 0.4 and 0.2-0.1 MY ago). This notion becomes more pronounced with the finding of a distinct haplotype in the Dades river system, whose origin appears to coalesce with the nascent stage of the basal mtDNA evolutionary lineages of brown trout. According to our results, Salmo akairos, Salmo pellegrini and "green trout" from Lake Isli do not exhibited any character states that distinctively separate them from the other brown trout populations studied. Therefore, their status as distinct species was not confirmed.