Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss).

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin-globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.

Population genetics of the African snakehead fish Parachanna obscura along West Africa's water networks: Implications for sustainable management and conservation.

An essential factor for aquatic conservation is genetic diversity or population divergence, which in natural populations reflects the interplay between geographical isolation with restricted gene flow and local evolution of populations. The long geological history of Africa may induce stronger among-population divergence and lower within-population divergence in fish populations of African watersheds. As an example, we studied population structure of the African snakehead fish Parachanna obscura. Our study aimed: (1) to develop a set of highly polymorphic microsatellite markers suitable for the analysis of genetic diversity among P. obscura and (2) to study the genetic diversity and structure of P. obscura populations from the West Africa region and mostly from Côte d'Ivoire, with respect to the effects of climate region and geographical distance on the genetic differentiation. A total of 259 specimens from 15 locations of P. obscura were collected over the West Africa region reflecting a high variability of pairwise geographical distances and variability of hydrological connectivity of the area. We developed a set of 21 polymorphic microsatellite markers for studying population genetics of the fish. The results showed relatively low intragenetic diversity for all the 15 locations, certainly attributable to confinement of fish in segregated catchments, resulting in limited gene flow. We also found evidence for local adaptation processes, suggested by five out of 21 microsatellite loci being under putative selection, according to BAYESCAN analysis. In turn, there was strong genetic differentiation (F ST > 0.5) among fish from most locations, reflecting the allopatric development in watersheds without hydraulic connectivity. Neighbor-joining dendrogram, Principal Coordinate Analysis, and analysis of ancestral groups by STRUCTURE suggested that the 15 locations can be divided into three clusters, mainly matching the dominant climate zones and the segregation of the watersheds in the region. The distinct genetic structure of the fish from the 15 locations obtained in this study suggests that conservation and sustainable management actions of this fish resource should avoid genetic mixing of potentially locally adapted populations.

A 180 Myr-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes.

Several hypotheses explain the prevalence of undifferentiated sex chromosomes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered conserved heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferentiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

A novel transcriptome-derived SNPs array for tench (Tinca tinca L.).

Tench (Tinca tinca L.) has great economic potential due to its high rate of fecundity and long-life span. Population genetic studies based on allozymes, microsatellites, PCR-RFLP and sequence analysis of genes and DNA fragments have revealed the presence of Eastern and Western phylogroups. However, the lack of genomic resources for this species has complicated the development of genetic markers. In this study, the tench transcriptome and genome were sequenced by high-throughput sequencing. A total of 60,414 putative SNPs were identified in the tench transcriptome using a computational pipeline. A set of 96 SNPs was selected for validation and a total of 92 SNPs was validated, resulting in the highest conversion and validation rate for a non-model species obtained to date (95.83%). The validated SNPs were used to genotype 140 individuals belonging to two tench breeds (Tabor and Hungarian), showing low (FST = 0.0450) but significant (<0.0001) genetic differentiation between the two tench breeds. This implies that set of validated SNPs array can be used to distinguish the tench breeds and that it might be useful for studying a range of associations between DNA sequence and traits of importance. These genomic resources created for the tench will provide insight into population genetics, conservation fish stock management, and aquaculture.

Phylogeographic Identification of Tench Tinca tinca (L., 1758) (Actinopterygii: Cyprinidae) from the Northern Balkans and Adjacent Regions and its Implications for Conservation.

Jelena Lujic, Klaus Kohlmann, Petra Kersten, Zoran Marinovic, Miroslav Cirkovic, and Vladica Simic (2017) The tench, Tinca tinca, is an endangered freshwater fish species in the Balkans. However, there are no management and conservation strategies developed for this species so far. In order to be able to develop such strategies, we first determined the phylogeographic identity of 70 tench individuals from four countries (Serbia, FYRO Macedonia, Hungary and Croatia) by PCR-RFLP analyses of two nuclear markers (Act and RpS7) and one mitochondrial marker (Cytb). All makers enabled the identification of two major geographic clades of tench (Western and Eastern), while nuclear markers additionally enabled the identification of hybrids between the two clades. Based on the mitochondrial marker Cytb, tench populations can be separated into two distinct areas: areas north of the Danube River with the dominant Western origin, and areas south of the Danube River with the dominant Eastern origin. Data obtained for the Act gene demonstrated Eastern origin for most individuals (88.23%) while data obtained for the RpS7 gene demonstrated mixed origin with a high percentage of hybrids. The presence of high numbers of individuals with Western alleles for the RpS7 gene in areas south of the Danube may indicate a natural invasion of this phylogroup. According to these results, areas north and south of the Danube are identified as two main management units. Additionally, we identified the rare western haplotype W2 based on the Cytb marker which clearly indicated human-aided dispersals of tench in the investigated region and since some individuals with W2 origin were cultured, attention must be given to the genetic structure and identity of the introduced individuals, whether during introduction or reintroduction since biological and ecological consequences of the hybridization between the two major clades are still unknown. Finally, we propose and discuss management and conservation strategies for tench of both management areas.

Population differentiation of zander (Sander lucioperca) across native and newly colonized ranges suggests increasing admixture in the course of an invasion.

In addition to ecological factors, evolutionary processes can determine the invasion success of a species. In particular, genetic admixture has the potential to induce rapid evolutionary change, which can result from natural or human-assisted secondary contact between differentiated populations. We studied the recent range expansion of zander in Germany focusing on the interplay between invasion and genetic admixture. Historically, the rivers Elbe and Danube harboured the most north-western source populations from which a north-westward range expansion occurred. This was initiated by introducing zander outside its native range into rivers and lakes, and was fostered by migration through artificial canals and stocking from various sources. We analysed zander populations of the native and invaded ranges using nuclear and mitochondrial genetic markers. Three genetic lineages were identified, which were traced to ancestral ranges. Increased genetic diversity and admixture in the invaded region highlighted asymmetric gene flow towards this area. We suppose that the adaptive potential of the invading populations was promoted by genetic admixture, whereas competitive exclusion in the native areas provided a buffer against introgression by novel genotypes. These explanations would be in line with evidence that hybridization can drive evolutionary change under conditions when new niches can be exploited.

Isolation and characterization of nine microsatellite loci from the pike-perch, Sander lucioperca (Linnaeus, 1758).

In order to facilitate studies on the genetic structure of wild populations as well as to monitor genetic changes in cultured stocks, nine polymorphic microsatellite loci were isolated from pike-perch (Sander lucioperca). Single loci allele numbers varied between two (loci MSL-3 and MSL-7) and six (loci MSL-1 and MSL-2), and observed heterozygosities ranged from 0.36 (locus MSL-2) to 1.00 (locus MSL-9) in a test panel of 25 individuals. Only one pair of loci (MSL-5 and MSL-8) displayed significant linkage disequilibrium after sequential Bonferroni corrections. Hardy-Weinberg tests revealed significant excesses of heterozygotes at three loci (MSL-1, MSL-7, and MSL-9).

The parthenogenetic Marmorkrebs (marbled crayfish) produces genetically uniform offspring.

Genetically identical animals are very much in demand as laboratory objects because they allow conclusions about environmental and epigenetic effects on development, structures, and behavior. Furthermore, questions about the relative fitness of various genotypes can be addressed. However, genetically identical animals are relatively rare, in particular, organisms that combine a high reproduction rate and a complex organization. Based on its exclusively parthenogenetic reproduction mode, it has been suggested that the Marmorkrebs (Crustacea, Decapoda, Astacida), a recently discovered crayfish, is an excellent candidate for research addressing the aforementioned questions. However, until now, a study using molecular markers that clearly proves the genetic uniformity of the offspring has been lacking. Here, with this first molecular study, we show that this crayfish indeed produces genetically uniform clones. We tested this with 19 related individuals of various generations of a Marmorkrebs population by means of six different microsatellite markers. We found that all examined specimens were identical in their allelic composition. Furthermore, half of the analyzed loci were heterozygous. These results and the absence of meioses in previous histological studies of the ovaries lead us to conclude the Marmorkrebs propagates apomictically. Thus, a genetically uniform organism with complex morphology, development, and behavior is now available for various laboratory studies.