Klaus Kohlmann

Genetic variability and structure of common carp (Cyprinus carpio) populations throughout the distribution range inferred from allozyme, microsatellite and mitochondrial DNA markers

Domesticated/captive stocks and wild/feral populations of common carp from Europe, Central Asia and East/South-East Asia were examined for allozyme (23 populations), microsatellite (11 populations) and mitochondrial DNA (21 populations) variation. Allozyme variability (1.06–1.81 alleles per locus, expected heterozygosity 0.006–0.136 at 16 loci) was much lower than microsatellite variability (2.5–14.0 alleles per locus, expected heterozygosity 0.426–0.887 at four loci). Differences in variability between domesticated/captive stocks and wild-caught ones were more pronounced at microsatellite loci than at allozyme loci, suggesting that microsatellites are better suited to detect population bottlenecks and loss of variation due to inbreeding. All but one European population were fixed for a single composite mtDNA haplotype, which also dominated in Central Asia but was completely missing in East/South-East Asia, indicating a single origin of European carp in Central Asia. All three classes of genetic markers clustered populations into two highly divergent groups: Europe/Central Asia and East/South-East Asia. Hierarchical partition of genetic diversity showed that for microsatellite loci most of variation was due to the within-population component while the highest proportion of mtDNA variation and substantial proportion of allozyme variation was accounted for by differences between geographical regions. Genetic data support the subspecies status of C. c. carpio assigned to the European carp and C. c. haematopterus assigned to the East/South-East Asian carp but do not justify a separate subspecies status (C. c. aralensis) for the Central Asian carp. As demonstrated for a wild/feral carp population from R. Danube, Germany, the genetic markers used in our study may be effectively applied to detect mixing and introgression of intra-species units in the presence of sufficient genetic differentiation.

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.

Genetic variability of German and foreign common carp (Cyprinus carpio L.) populations

Abstract In order to describe their genetic variability the polymorphisms of 8 enzymatic systems representing 23 gene loci were studied in 11 German and 5 foreign common carp lines using horizontal starch gel electrophoresis. The highest variability measured by the mean number of alleles per locus and the percentage of polymorphic loci was observed in Vietnamese wild carp (1.9 and 50%, respectively) followed by carp from River Rhine (1.8 and 45%, respectively). The lowest variability was found in all domesticated stocks (1.4 to 1.5 and 25 to 40%, respectively) and could be attributed to a loss of rare alleles. The mean observed heterozygosities ranged from 0.112 to 0.256 for all populations examined. The calculation of Neis unbiased genetic distances [Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583-590] revealed three pairs of genetically identical populations. All of them were German mirror carp stocks. The largest genetic distance was found between Japanese Koi carp and German domesticated scaly carp (D=0.133). A dendrogram based on these genetic distance measures showed that all populations could be assigned to two clearly distinct groups. One consisted of the German stocks and the Israeli Dor-70 carp (European group). The other one was formed by Koi carp, Vietnamese and River Amur wild carp, and the Ropsha carp (Asian group). Within the European group the carp from River Rhine were the most distinct ones. Their relatively separate position was mainly caused by the presence of alleles at four loci which could not be found in the majority of the domesticated European stocks.

PCR–RFLP analysis of the mitochondrial ND-3/4 and ND-5/6 gene polymorphisms in the European and East Asian subspecies of common carp (Cyprinus carpio L.)

Abstract Polymorphism within the mitochondrial NADH-3,4 dehydrogenase ( ND-3/4 ) and NADH-5,6 dehydrogenase ( ND-5/6 ) gene regions was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis among common carp populations belonging to the European (two farmed strains and three wild populations) and East Asian (Amur wild carp, Vietnamese wild carp and Japanese Koi carp) subspecies, Cyprinus carpio carpio and C. c. haematopterus , respectively. Polymorphism was detected using eight and six restriction enzymes, respectively, and a total of seven composite haplotypes were identified. Both distance-based and maximum likelihood phylogenetic inference methods clustered the haplotypes into four distinct groups—the European (two haplotypes), Amur (two haplotypes), Vietnamese (two haplotypes) and Koi (one haplotype), and their distributions strictly follow the geographic origin of populations. The populations are clustered into two highly divergent groups (average net nucleotide divergence, 2.4%), the European and the East Asian populations, suggesting an ancient separation. Six enzymes ( Hin fI, Alu I, Hpa II and Taq I at ND-3/4 ; Eco 47I and Bsu RI at ND-5/6 ) yielded diagnostic restriction sites for discriminating between the European and East Asian maternal lineages that can be applied to monitoring of genetic purity of the European farmed strains.

Genetic characterization of wild and domesticated common carp (Cyprinus carpio L.) populations from Uzbekistan

Abstract One domesticated and five wild common carp populations from Uzbekistan have been genetically characterized by examining variability at 22 allozyme loci. The observed level of polymorphism was high (1.4–1.7 alleles per locus, 26.3–42.1% polymorphic loci and expected heterozygosities from 0.098 to 0.146) and similar to that previously found in German or East Asian populations. The Uzbek wild populations, with the exception of the paired populations of Lake Arnasaiskie and Syr-Darya channels, were genetically very similar to each other as reflected by nonsignificant F ST values and clustering into one distinct group with high bootstrap support. In contrast, the differentiation between all Uzbek wild and the Uzbek domesticated carp was highly significant as well as the differentiation between Uzbek and German, Uzbek and East Asian, and German and East Asian populations. The Uzbek domesticated carp clustering with River Amur wild carp could be explained by their proposed crossing with Ropsha carp (a derivative of Amur wild carp), which could have taken place in their breeding history. Allelic distribution at some loci is consistent with introgressive hybridization between Uzbek domesticated carp and Syr-Darya channels wild carp. Considering that wild common carp has already disappeared, is endangered or has mixed with domesticated carp in many other parts of its natural distribution area, it is very important to conserve the wild populations in Uzbekistan in order to maintain the genetic diversity of the species.

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).

Genetic variability and differentiation of wild and cultured tench populations inferred from microsatellite loci

Nine species-specific microsatellites were used to characterize 792 tench, Tinca tinca (L.), from 21 wild and cultured populations. Seven loci were polymorphic expressing four to 22 alleles. A Spanish cultured strain was homozygous at all loci for all individuals studied. Low variability was also observed in a wild population from Sapanca Lake, Turkey and a Chinese cultured strain. In contrast, the highest variabilities were found in wild tench from lake Felchowsee (average number of alleles), and the cultured strain from Königswartha (average heterozygosity), both from Germany. Genetic differentiation between populations was moderate to high. The smallest genetic distances were found between the geographically most distant populations. A Neighbor-Joining tree showed only two major clades consisting of 4 and 17 populations, respectively. Within the smaller clade the Turkish wild and Spanish and Chinese cultured tench formed a sub-cluster with 100% bootstrap support. Possible reasons for the latter unexpected grouping are discussed.

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.

Lack of reproductive isolation between the Western and Eastern phylogroups of the tench

The Eurasian range of the tench distribution is subdivided into deeply divergent Western and Eastern phylogroups evidenced by nuclear and mitochondrial DNA sequence markers. A broad zone of overlap exists in central and western Europe, suggesting post-glacial contact with limited hybridisation. We conducted a population genetic test of this indication that the two phylogroups may represent distinct species. We analysed variation at introns of nuclear genes, microsatellites, allozymes and mitochondrial DNA in populations from two postglacial lakes within the contact zone in Germany. The test is based on the expectation that in the presence of strong barriers to reproduction, a hybrid population will show genome-wide associations among alleles and genotypes from each phylogroup even after hundreds of generations of interbreeding. In contrast to this expectation, no consistent significant deviations from linkage and Hardy–Weinberg equilibria were found. Samples from both lakes did show significant disequilibria but they were limited to individual loci and were not concordant between populations, and were not robust to the method used. The single consistent association can be attributed to physical linkage between two microsatellite loci. Thus, results of our study support the hypothesis of free interbreeding between the two phylogroups of tench. Therefore, although the phylogroups may be considered as separate phylogenetic species, the present data suggest that they are a single species under the biological species concept.

Microsatellite loci in tench: isolation and variabilityin a test population

Because of their high variability and rapid evolution, microsatellites became increasingly important in genetic research, e.g. population structure and differentiation studies, gene mapping and parentage analysis. However, such loci have not been isolated in tench so far. Applying a PCR based method of generating microsatellite enriched DNA fragment libraries we were able to identify nine loci (MTT-1 to MTT-9). The variability of these microsatellite loci was determined in 50 tench individuals originating from a wild population of Lake Döllnsee, Germany. Three loci were found to be monomorphic. The remaining six loci segregated for two to nine alleles. The observed heterozygosities at polymorphic loci were high (0.500–0.959) with only one exception: locus MTT-8 (0.167). These polymorphic microsatellite loci showed a much higher level of genetic variability than the allozyme loci previously studied in the same individuals. Thus, they seem to be more suitable for genetic studies of tench. On the other hand, it remains to be checked in other populations if the three loci that did not show any variation in this population are generally monomorphic in this species.