Disease resistance and infectivity of virus susceptible and resistant common carp strains.

Infectious diseases challenge health and welfare of humans and animals. Unlike for humans, breeding of genetically resistant animals is a sustainable solution, also providing unique research opportunities. Chances to survive a disease are improved by disease resistance, but depend also on chances to get infected and infect others. Considerable knowledge exists on chances of susceptible and resistant animals to survive a disease, yet, almost none on their infectivity and if and how resistance and infectivity correlate. Common carp (Cyprinus carpio) is widely produced in aquaculture, suffering significantly from a disease caused by cyprinid herpes virus type 3 (CyHV-3). Here, the infectivity of disease-resistant and susceptible fish types was tested by playing roles of shedders (infecting) and cohabitants (infected) in all four type-role combinations. Resistant shedders restricted spleen viral load and survived more than susceptible ones. However, mortality of susceptible cohabitants infected by resistant shedders was lower than that of resistant cohabitants infected by susceptible shedders. Virus levels in water were lower in tanks with resistant shedders leading to lower spleen viral loads in cohabitants. Thus, we empirically demonstrated that disease resistant fish survive better and infect less, with implications to epidemiology in general and to the benefit of aquaculture production.

Revisiting the species list of freshwater fish in Israel based on DNA barcoding.

Israel's region forms a continental bridge; hence, the freshwater fish fauna in Israel consists of unique populations of species that originated from Africa, Asia, or Europe and are often endemic or at the edge of their distribution range. Worldwide, fish biodiversity suffers significantly from pressures and disturbances of freshwater habitats, especially in arid regions, such as in parts of Israel. Biodiversity conservation requires efficient tools for monitoring changes in populations. DNA barcoding, by complementing and enhancing species identification, provides such monitoring tools. In this study, over 200 specimens representing over 28 species were DNA barcoded and together with previously available records, a DNA barcoding database for freshwater fish of Israel was established. Of the 71 distinct barcodes generated, 37% were new, attesting to the uniqueness of fish populations in Israel. For most species, morphological and molecular species identifications agreed. However, discrepancies were found for five genera. Based on DNA barcoding, we propose Acanthobrama telavivensis as a junior synonym for Acanthobrama lissneri. In Garra spp., we propose splitting Garra nana into two species and assigning Garra rufa in the region to Garra jordanica, or possibly to two species. Israeli Pseudophoxinus kervillei is not the same species as in Syria and Lebanon. However, Pseudophoxinus syriacus might not be endangered since it is genetically very similar to Pseudophoxinus drusensis. In Israel, instead of five reported Oxynoemacheilus species, combining DNA barcoding with morphology suggests only three. Genetic and geographic separation suggested that Aphanius mento is likely a species complex. The study provides a thorough barcoding database, suggests significant species reconsiderations in the region, and highlights the Sea of Galilee and the Beit She'an valley streams as biodiversity "hotspots." This study will therefore promote further studying of the fish species in the region and their ecology, as well as the monitoring and conservation of freshwater fish biodiversity in Israel and the region.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Establishing and using a genetic database for resolving identification of fish species in the Sea of Galilee, Israel.

Freshwaters are a very valuable resource in arid areas, such as Mediterranean countries. Freshwater systems are vulnerable ecological habitats, significantly disturbed globally and especially in arid areas. The Sea of Galilee is the largest surface freshwater body in the Middle East. It is an isolated habitat supporting unique fish populations, including endemic species and populations on the edge of their distribution range. Using the Sea of Galilee for water supply, fishing and recreation has been placing pressure on these fish populations. Therefore, efficient monitoring and effective actions can make a difference in the conservation of these unique fish populations. To set a baseline and develop molecular tools to do so, in this study, DNA barcoding was used to establish a database of molecular species identification based on sequences of Cytochrome C Oxidase subunit I gene. DNA barcodes for 22 species were obtained and deposited in Barcode of Life Database. Among these, 12 barcodes for 10 species were new to the database and different from those already there. Barcode sequences were queried against the database and similar barcodes from the same and closely related species were obtained. Disagreements between morphological and molecular species identification were identified for five species, which were further studied by phylogenetic and genetic distances analyses. These analyses suggested the Sea of Galilee contained hybrid fish of some species and other species for which the species definition should be reconsidered. Notably, the cyprinid fish defined as Garra rufa, should be considered as Garra jordanica. Taken together, along with data supporting reconsideration of species definition, this study sets the basis for further using molecular tools for monitoring fish populations, understanding their ecology, and effectively managing their conservation in this unique and important habitat and in the region.

Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance.

BACKGROUND: Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. RESULTS: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. CONCLUSIONS: Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Multiple interacting QTLs affect disease challenge survival in common carp (Cyprinus carpio).

With the steady growth of the human population, food security becomes a prime challenge. Aquaculture is the fastest growing sector providing proteins from an animal source, but outbreaks of infectious diseases repeatedly hamper the production and further development of this sector. Breeding of disease-resistant strains is a desired sustainable solution to this problem. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus damaging production of common carp, an important food and ornamental fish. Previously, we have demonstrated successful introgression of CyHV-3 resistance from a feral strain to commercial strains. Here, we used genotyping by sequencing to identify two novel quantitative trait loci (QTLs) for disease survival that map to different linkage groups than two other QTLs that we previously identified. Effects of these four QTLs were validated and further studied in 14 families with various levels of disease resistance. CyHV-3 survival was found to be a quantitative trait conditioned by mild additive QTL effects and by intricate dominant allelic and epistatic QTL-QTL interactions. Both rare feral alleles and alleles common to feral and cultured strains contributed to survival. This and other advantages of feral alleles introgression were demonstrated. These QTLs, which affected survival of individuals within families, had no significant effect on variation in cumulative family % survival, suggesting that more between family variation remains to be explored. Unraveling the underlying genetics of survival is important for enhancing the breeding of resistant strains and our knowledge of disease resistance mechanisms.

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.).

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.

Resistance of Leishmania infantum to allopurinol is associated with chromosome and gene copy number variations including decrease in the S-adenosylmethionine synthetase (METK) gene copy number.

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL), a widespread, life-threatening disease. This parasite is responsible for the majority of human VL cases in Brazil, the Middle East, China, Central Asia and the Mediterranean basin. Its main reservoir are domestic dogs which, similar to human patients, may develop severe visceral disease and die if not treated. The drug allopurinol is used for the long-term maintenance of dogs with canine leishmaniasis. Following our report of allopurinol resistance in treated relapsed dogs, we investigated the mechanisms and markers of resistance to this drug. Whole genome sequencing (WGS) of clinical resistant and susceptible strains, and laboratory induced resistant parasites, was carried out in order to detect genetic changes associated with resistance. Significant gene copy number variation (CNV) was found between resistant and susceptible isolates at several loci, including a locus on chromosome 30 containing the genes LinJ.30.3550 through LinJ.30.3580. A reduction in copy number for LinJ.30.3560, encoding the S-adenosylmethionine synthetase (METK) gene, was found in two resistant clinical isolates and four induced resistant clonal strains. Using quantitative real time PCR, this reduction in METK copy number was also found in three additional resistant clinical isolates. Furthermore, inhibition of S-adenosylmethionine synthetase encoded by the METK gene in allopurinol susceptible strains resulted in increased allopurinol resistance, confirming its role in resistance to allopurinol. In conclusion, this study identified genetic changes associated with L. infantum resistance to allopurinol and the reduction in METK copy number identified may serve as a marker for resistance in dogs, and reduced protein activity correlated with increased allopurinol resistance.

Teleosts Genomics: Progress and Prospects in Disease Prevention and Control.

Genome wide studies based on conventional molecular tools and upcoming omics technologies are beginning to gain functional applications in the control and prevention of diseases in teleosts fish. Herein, we provide insights into current progress and prospects in the use genomics studies for the control and prevention of fish diseases. Metagenomics has emerged to be an important tool used to identify emerging infectious diseases for the timely design of rational disease control strategies, determining microbial compositions in different aquatic environments used for fish farming and the use of host microbiota to monitor the health status of fish. Expounding the use of antimicrobial peptides (AMPs) as therapeutic agents against different pathogens as well as elucidating their role in tissue regeneration is another vital aspect of genomics studies that had taken precedent in recent years. In vaccine development, prospects made include the identification of highly immunogenic proteins for use in recombinant vaccine designs as well as identifying gene signatures that correlate with protective immunity for use as benchmarks in optimizing vaccine efficacy. Progress in quantitative trait loci (QTL) mapping is beginning to yield considerable success in identifying resistant traits against some of the highly infectious diseases that have previously ravaged the aquaculture industry. Altogether, the synopsis put forth shows that genomics studies are beginning to yield positive contribution in the prevention and control of fish diseases in aquaculture.

Induction of allopurinol resistance in Leishmania infantum isolated from dogs.

Resistance to allopurinol in zoonotic canine leishmaniasis has been recently shown to be associated with disease relapse in naturally-infected dogs. However, information regarding the formation of resistance and its dynamics is lacking. This study describes the successful in-vitro induction of allopurinol resistance in Leishmania infantum cultured under increasing drug pressure. Allopurinol susceptibility and growth rate of induced parasites were monitored over 23 weeks and parasite clones were tested at selected time points and compared to their parental lines, both as promastigotes and as amastigotes. Allopurinol resistance was formed in strains from two parasite stocks producing a 20-fold rise in IC50 along three distinct growth phases. In addition, characteristic differential clustering of single nucleotide polymorphisms (SNP) was found in drug sensitive and resistant parasite clones. Results confirm that genetic polymorphism, as well as clonal heterogeneity, contribute to in-vitro resistance to allopurinol, which is likely to occur in natural infection.

Genomic and transcriptomic approaches to study immunology in cyprinids: What is next?

Accelerated by the introduction of Next-Generation Sequencing (NGS), a number of genomes of cyprinid fish species have been drafted, leading to a highly valuable collective resource of comparative genome information on cyprinids (Cyprinidae). In addition, NGS-based transcriptome analyses of different developmental stages, organs, or cell types, increasingly contribute to the understanding of complex physiological processes, including immune responses. Cyprinids are a highly interesting family because they comprise one of the most-diversified families of teleosts and because of their variation in ploidy level, with diploid, triploid, tetraploid, hexaploid and sometimes even octoploid species. The wealth of data obtained from NGS technologies provides both challenges and opportunities for immunological research, which will be discussed here. Correct interpretation of ploidy effects on immune responses requires knowledge of the degree of functional divergence between duplicated genes, which can differ even between closely-related cyprinid fish species. We summarize NGS-based progress in analysing immune responses and discuss the importance of respecting the presence of (multiple) duplicated gene sequences when performing transcriptome analyses for detailed understanding of complex physiological processes. Progressively, advances in NGS technology are providing workable methods to further elucidate the implications of gene duplication events and functional divergence of duplicates genes and proteins involved in immune responses in cyprinids. We conclude with discussing how future applications of NGS technologies and analysis methods could enhance immunological research and understanding.

Studying the Genetics of Resistance to CyHV-3 Disease Using Introgression from Feral to Cultured Common Carp Strains.

Sustainability and further development of aquaculture production are constantly challenged by outbreaks of fish diseases, which are difficult to prevent or control. Developing fish strains that are genetically resistant to a disease is a cost-effective and a sustainable solution to address this challenge. To do so, heritable genetic variation in disease resistance should be identified and combined together with other desirable production traits. Aquaculture of common carp has suffered substantial losses from the infectious disease caused by the cyprinid herpes virus type 3 (CyHV-3) virus and the global spread of outbreaks indicates that many cultured strains are susceptible. In this research, CyHV-3 resistance from the feral strain "Amur Sassan" was successfully introgressed into two susceptible cultured strains up to the first backcross (BC1) generation. Variation in resistance of families from F1 and BC1 generations was significantly greater compared to that among families of any of the susceptible parental lines, a good starting point for a family selection program. Considerable additive genetic variation was found for CyHV-3 resistance. This phenotype was transferable between generations with contributions to resistance from both the resistant feral and the susceptible cultured strains. Reduced scale coverage (mirror phenotype) is desirable and common in cultured strains, but so far, cultured mirror carp strains were found to be susceptible. Here, using BC1 families ranging from susceptible to resistant, no differences in resistance levels between fully scaled and mirror full-sib groups were found, indicating that CyHV-3 resistance was successfully combined with the desirable mirror phenotype. In addition, the CyHV-3 viral load in tissues throughout the infection of susceptible and resistant fish was followed. Although resistant fish get infected, viral loads in tissues of these fish are significantly lesser than in those of susceptible fish, allowing them to survive the disease. Taken together, in this study we have laid the foundation for breeding CyHV-3-resistant strains and started to address the mechanisms underlying the phenotypic differences in resistance to this disease.

Genes with a Combination of Over-Dominant and Epistatic Effects Underlie Heterosis in Growth of Saccharomyces cerevisiae at High Temperature.

Heterosis describes a phenotypic phenomenon of hybrid superiority over its homozygous parents. It is a genetically intriguing phenomenon with great importance for food production. Also called hybrid-vigor, heterosis is created by non-additive effects of genes in a heterozygous hybrid made by crossing two distinct homozygous parents. Few models have been proposed to explain how the combination of parental genes creates an exceptional hybrid performance. Over-dominant mode of inheritance is an attractive model since a single gene can potentially create the heterotic effect, but only a few such loci have been identified. To a collection of 120 hybrids, made by crossing 16 divergent Saccharomyces cerevisiae yeast strains, we applied a method for mapping heterozygous loci that non-additively contribute to heterotic growth at 37°. Among 803 candidate loci that were mapped, five were tested for their heterotic effect by analyzing backcrosses and F2 populations in a specific hybrid background. Consistently with the many mapped loci, specific analyses confirmed the minor heterotic effect of the tested candidate loci. Allele-replacement analyses of one gene, AEP3, further supported its heterotic effect. In addition to over-dominant effects, the contribution of epistasis to heterosis was evident from F2 population and allele-replacement analyses. Pairs of over-dominant genes contributed synergistically to heterosis. We show that minor over-dominant effects of multiple genes can combine to condition heterosis, similarly to loci affecting other quantitative traits. Furthermore, by finding of epistatic interactions between loci that each of them individually has an over-dominant effect on heterosis, we demonstrate how hybrid advantage could benefit from a synergistic combination of two interaction types (over-dominant and synergistic epistatic). Thus, by portraying the underlying genetic complexity, these findings advance our understanding of heterosis.

Allopurinol Resistance in Leishmania infantum from Dogs with Disease Relapse.

BACKGROUND: Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of Leishmania strains from dogs to allopurinol has not been described before in clinical studies. METHODOLOGY/PRINCIPAL FINDINGS: Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group) showed an average half maximal inhibitory concentration (IC50) of 996 µg/mL. A significantly lower IC50 (P = 0.01) was found for isolates from ten dogs before treatment (NT group, 200 µg/mL), as well as for five isolates obtained from treated dogs in remission (TA group, 268 µg/mL). Axenic amastigotes produced from isolates of the TR group also showed significantly higher (P = 0.002) IC50 compared to the NT group (1678 and 671 µg/mL, respectively). The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (P = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 µg/mL allopurinol). CONCLUSIONS: This is the first study to demonstrate allopurinol resistance in L. infantum and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.

Induced mutations in yeast cell populations adapting to an unforeseen challenge.

The modern evolutionary synthesis assumes that mutations occur at random, independently of the environment in which they confer an advantage. However, there are indications that cells facing challenging conditions can adapt rapidly, utilizing processes beyond selection of pre-existing genetic variation. Here, we show that a strong regulatory challenge can induce mutations in many independent yeast cells, in the absence of general mutagenesis. Whole genome sequencing of cell lineages reveals a repertoire of independent mutations within a single lineage that arose only after the cells were exposed to the challenging environment, while other cells in the same lineage adapted without any mutation in their genomes. Thus, our experiments uncovered multiple alternative routes for heritable adaptation that were all induced in the same lineage during a short time period. Our results demonstrate the existence of adaptation mechanisms beyond random mutation, suggesting a tight connection between physiological and genetic processes.

High-density tiling microarray analysis of the full transcriptional activity of yeast.

Understanding the relationship between DNA sequence variation and phenotypic variation in complex or quantitative traits is one of the major challenges in modern biology. We are witnessing a deluge of DNA sequence information and association studies of genetic polymorphisms with phenotypes of interest in families and populations. In addition, it has become clear that large portions of eukaryotic genomes beyond protein-coding genes are transcribed, generating numerous noncoding RNA (ncRNA) molecules whose functions remain mostly unknown.DNA oligonucleotide microarrays constitute a powerful technology for studying the expression of genes in different organisms. The Saccharomyces cerevisiae tiling array presents a significant advance over previous array-based platforms. It has a high density of overlapping probes that start on average every 8 bp along each strand of the genome, enabling precise definition of transcript structure. Furthermore, the array includes probes specific for the polymorphic positions of another, distantly related yeast strain, allowing accurate measurement of allele-specific expression in a hybrid of the two strains. This technology thus allows high-resolution, quantitative, strand- and allele-specific measurements of transcription from a full eukaryotic genome. In this chapter, we describe the methods for extracting RNA, synthesizing first-strand cDNA, fragmenting, and labeling of samples for hybridization to the tiling array. Combining genome-wide information on variation in DNA sequence with variation in transcript structure and levels promises to increase our understanding of the genotype-to-phenotype relationship.

DNA barcodes for the fishes of the Narmada, one of India's longest rivers.

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.

Multiple genomic changes associated with reorganization of gene regulation and adaptation in yeast.

Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.

Melanocortin receptor 1 and black pigmentation in the Japanese ornamental carp (Cyprinus carpio var. Koi).

Colors and their patterns are fascinating phenotypes with great importance for fitness under natural conditions. For this reason and because pigmentation is associated with diseases, much research was devoted to study the genetics of pigmentation in animals. Considerable contribution to our understanding of color phenotypes was made by studies in domesticated animals that exhibit dazzling variation in color traits. Koi strains, the ornamental variants of the common carp, are a striking example for color variability that was selected by man during a very short period on an evolutionary timescale. Among several pigmentation genes, genetic variation in Melanocrtin receptor 1 was repeatedly associated with dark pigmentation phenotypes in numerous animals. In this study, we cloned Melanocrtin receptor 1 from the common carp. We found that alleles of the gene were not associated with the development of black color in Koi. However, the mRNA expression levels of the gene were higher during dark pigmentation development in larvae and in dark pigmented tissues of adult fish, suggesting that variation in the regulation of the gene is associated with black color in Koi. These regulatory differences are reflected in both the timing of the dark-pigmentation development and the different mode of inheritance of the two black patterns associated with them. Identifying the genetic basis of color and color patterns in Koi will promote the production of this valuable ornamental fish. Furthermore, given the rich variety of colors and patterns, Koi serves as a good model to unravel pigmentation genes and their phenotypic effects and by that to improve our understanding of the genetic basis of colors also in natural populations.