Two New Types of Homodiploid Fish and Polyploid Hybrids Derived from the Distant Hybridization of Female Koi Carp and Male Bighead Carp.

Bighead carps  (Hypophthalmichthys nobilis) and silver carps (Hypophthalmichthys molitrix) represent an important component of freshwater ichthiofauna in its native range, though they might become mass propagation in other systems (North America) and the reason of concern for fisheries management. Therefore, understanding their reproductive traits and particularly in the context of hybridization with other cyprinids was of value to explain their rapid propagation as well as potential benefits for aquaculture due to their unique diet, behavior, growth potential, and tolerance to deteriorating environmental conditions in freshwater ecosystems. Distant hybridization is an effective tool to create different ploidy offspring with changed phenotypes and genotypes. In this study, we reported distant hybridization of female koi carp (Cyprinus carpio haematopterus, KOC, 2n = 100) × male bighead carp (Hypophthalmichthys nobilis, BIC, 2n = 48) and the spontaneous occurrence of two new "crucian" carp-like homodiploid fish (2nGCC-L; 2nCCC-L; 2n = 100), a new type of triploid hybrid (3nKB, 3n = 124), and a new type of tetraploid hybrid (4nKB, 4n = 148). The body color of 2nGCC-L and 2nCCC-L were gray and multicolor, respectively. Both phenotypes were similar to the crucian carp (Carassius auratus). The difference was that their heads were rounder than those of the crucian carp and they had higher backs. Compared with the KOC with two pairs of barbels and BIC without barbel, 2nGCC-L, 2nCCC-L, and 4nKB had no barbel, but 3nKB had one pair of barbels. Microsatellite patterns and 5S rDNA sequences confirmed that 2nGCC-L, 2nCCC-L, and 3nKB were of hybrid origin. In regard to feeding, KOC was omnivorous and BIC was a typical filter-feeder. However, the 2nGCC-L, 2nCCC-L, and 3nKB were omnivorous. The formation of four kinds of new offspring is a groundbreaking finding in fish genetic breeding and evolutionary biology.

Post-mortem quality changes of common carp (Cyprinus carpio) during chilled storage from two culture systems.

BACKGROUND: Omega-3 common carp (OCC) raised by patented culture systems have higher level of n-3 fatty acids and n-3/n-6 ratio than normal common carps (NCCs) from traditional culture system. Whether the patented farming system and modified fatty acid profile will influence OCC storage stability is unclear. This study aimed to expose the differences of post-mortem quality changes between NCC and OCC. RESULTS: NCC and OCC have similar rigor mortis patterns, only a higher level of lactic acid was observed in NCC after 96 h. Adenosine triphosphate (ATP) related compounds had no major differences, but slightly higher inosine monophosphate in OCC was found at 36 h. The K-value, Ki-value and Hx-index demonstrated high cohesiveness (Pearsons two-tailed, r = 0.968-0.984, P < 0.05) during storage, with statistically comparable (P > 0.05) temporal progress of change in NCC and OCC. The indices were lower in OCC than in NCC. Attenuation of myosin heavy chain in OCC was not as distinct as in NCC, coincided with its higher salt-soluble protein level at 144 h. Before 96 h, thiobarbituric acid value (TBA), total viable count (TVC), cooking loss (CL), drip loss (DL), and hardness in NCC and OCC were similar. However, at 144 h, higher TBA, TVC, CL and DL while lower hardness in NCC than in OCC were observed. Principle component analysis showed good separation of NCC and OCC in biplot at 0 and 144 h. CONCLUSION: Patented culture system has a slightly positive influence on post-mortem quality of common carp. It can be used for producing OCC without compromising storage stability. © 2020 Society of Chemical Industry.

Combined effects of dosage compensation and incomplete dominance on gene expression in triploid cyprinids.

Hybridization and polyploidy are pervasive evolutionary features of flowering plants and frequent among some animal groups, such as fish. These processes always lead to novel genotypes and various phenotypes, including growth heterosis. However, its genetic basis in lower vertebrate is still poorly understood. Here, we conducted transcriptome-level analyses of the allopolyploid complex of Carassius auratus red var. (R) (♀) × Cyprinus carpio L. (C) (♂), including the allodiploid and allotetraploid with symmetric subgenomes, and the two allotriploids with asymmetric subgenomes. The gradual changes of gene silencing and novel gene expression suggested the weakening of the constraint of polymorphic expression in genotypic changes. Then, analyses of the direction and magnitude of homoeolog expression exhibited various asymmetric expression patterns, which supported that R incomplete dominance and dosage compensation were co-regulated in the two triploids. Under these effects, various magnitudes of R-homoeolog expression bias were observed in growth-regulated genes, suggesting that they might contribute to growth heterosis in the two triploids. The determination of R incomplete dominance and dosage compensation, which might be led by asymmetric subgenomes and multiple sets of homologous chromosomes, explained why various expression patterns were shaped and their potential contribution to growth heterosis in the two triploids.

Cloning, functional analysis, and microRNA-induced negative regulation of growth arrest and DNA damage-inducible 45 γ (Gadd45g) in grass carp (Ctenopharyngodon idella).

Growth arrest and DNA damage-inducible 45 gamma (Gadd45g) is a member of Gadd45 gene family of immunological proteins in mammals. Herein, we identified and characterised Gadd45g from grass carp. The cDNA spans over 1189 bp, with an open reading frame of 480 bp encoding a 159 amino acid protein. CiGadd45g mRNAs were expressed in all tissues investigated, with abundant expression in liver, kidney, heart, brain, blood and skin. Following infection with Aeromonas hydrophila, CiGadd45g expression was upregulated in these immune-related tissues (gill, liver, spleen, intestine, kidney and head kidney). Immune-related cytokines (p38 and JNK) and proinflammatory cytokines (IL-8, IFN-1 and TNF-α) were activated by CiGadd45g. CiGadd45g and downstream genes were regulated by microRNA miR-429b. These results indicate that CiGadd45g plays an important immune role in the response to A. hydrophila infection in grass carp.

Morphologically indistinguishable hybrid Carassius female with 156 chromosomes: A threat for the threatened crucian carp, C. carassius, L.

The crucian carp Carassius carassius (Linnaeus, 1758), is native to many European freshwaters. Despite its wide distribution, the crucian carp is declining in both the number and sizes of populations across much of its range. Here we studied 30 individuals of a putative pure population from Helsinki, Finland. Despite clear external morphological features of C. carassius, an individual was of a higher ploidy level than the others. We therefore applied a set of molecular genetic (S7 nuclear and cytochrome b mitochondrial genes) and cytogenetic tools (sequential fluorescent 4', 6-diamidino-2-phenylindole [DAPI], Chromomycin A3 [CMA3], C-banding and in situ hybridization [FISH] with both 5S and 28S ribosomal DNA probes) to determine its origin. While all examined characteristics of a diploid representative male (CCAHe2Fi) clearly corresponded to those of C. carassius, a triploid individual (CCAHe1Fi) was more complex. Phylogenetic analysis revealed that the nuclear genome of CCAHe1Fi contained three haploid sets: two C. gibelio and one C. carassius. However the mitochondrial DNA was that of C. gibelio, demonstrating its hybrid origin. The FISH revealed three strong (more intensive) 5S rDNA loci, confirming the triploid status, and an additional 24 weak (less intensive) signals were observed in the chromosome complement of CCAHe1Fi. On the other hand, only two strong and 16 weak 5S rDNA signals were visible on the chromosomes of the CCAHe2Fi male. 28S rDNA FISH revealed four strong signals in both CCAHe1Fi and CCAHe2Fi individuals. CMA3 staining revealed four to six CMA3-positive bands of CCAHe1Fi, while that of diploids contained only two to four. The fact that a polyploid hybrid Carassius female with a strong invasive potential may share morphological characters typical for endangered C. carassius highlights a need to combine genetic investigations of Carassius cryptic diversity with conservation measures of C. carassius in Europe.

Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio.

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development.

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17ß-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio).

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.

Identification and functional characterization of tumor necrosis factor receptor 1 (TNFR1) of grass carp (Ctenopharyngodon idella).

Tumor necrosis factor-alpha (TNF-α) exerts its regulatory effects by binding one of two TNF receptors, TNF-α receptor 1 (TNFR1) or TNFR2. In this study, we isolated and identified the cDNA sequence of grass carp TNFR1 (gcTNFR1). Similar to its homologs in other fish species, the putative protein of gcTNFR1 possessed an extracellular region containing three TNF homology domains, a transmembrane region and a cytoplasmic region with a conserved death domain. Consistent with the widespread expression of mammalian TNFR1, gcTNFR1 transcripts ubiquitously expressed in spleen, thymus, liver, heart, gill, intestine, brain and head kidney with the highest expression levels in head kidney. To reveal its inductive expression patterns in inflammatory response, effect of in vivo bacterial infection on gcTNFR1 gene expression was examined, showing a rapid increase of gcTNFR1 expression in head kidney, gill, liver and intestine, which is consistent with the role of TNF-α as an early response gene during immune challenges. To define the functional role of gcTNFR1, recombinant extracellular region of gcTNFR1 (rgcTNFR1) was prepared and used to perform in vitro binding assay, demonstrating its ability to interact with recombinant grass carp TNF-α (rgcTNF-α). Furthermore, to characterize the function of gcTNFR1 in affecting rgcTNF-α actions, the effect of overexpressing gcTNFR1 on rgcTNF-α-induced grass carp IL-1ß (gcIL-1ß) promoter activity was determined in COS7 cells. Results showed that gcTNFR1 was involved in the regulation of rgcTNF-α on gcIL-1ß transcription. Consistently, rgcTNFR1 was effective in attenuating the effect of rgcTNF-α on IL-1ß mRNA expression in grass carp head kidney leukocytes, providing evidence for the involvement of TNFR1 in TNF-α signaling in grass carp. These data facilitate a better understanding of TNF-α receptor signaling in grass carp.

Identification and characterization of a LTR retrotransposon from the genome of Cyprinus carpio var. Jian.

A Ty3/gypsy-retrotransposon-type transposon was found in the genome of the Jian carp (Cyprinus carpio var. Jian) in a previous study (unpublished), and was designated a JRE retrotransposon (Jian retrotransposon). The full-length JRE retrotransposon is 5126 bp, which includes two long terminal repeats of 470 bp at the 5' end and 453 bp at the 3' end, and two open reading frames between them: 4203 bp encoding the group-specific antigen (GAG) and polyprotein (POL). The pol gene has a typical Ty3/gypsy retrotransposon structure, and the gene order is protease, reverse transcriptase, RNase H, and integrase (PR-RT-RH-IN). A phylogenetic analysis of the pol gene showed that it has similarities of 40.7, 40, and 32.8 %, to retrotransposons of Azumapecten farreri, Mizuhopecten yessoensis, and Xiphophorus maculatus, respectively. Therefore, JRE might belong to the JULE retrotransposon family. The copy number of the JRE transposon in the genome of the Jian carp is 124, determined with real-time quantitative PCR. The mRNA of the JRE retrotransposon is expressed in five Jian carp tissues, the liver, kidney, blood, muscle, and gonad, and slightly higher in the kidney and liver than in the other tissues.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481 bp along with 9 bp 5' UTR, 639 bp 3' UTR and the largest open reading frame (1833 bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C. idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414 bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. In vivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.

Identification and characterization of IKKε gene from grass carp Ctenopharyngodon idella.

IKKε is an IKK-related kinase implicated in antiviral immune response in higher vertebrates. To elucidate the function of IKKε in teleost fish, grass carp IKKε (gcIKKε) has been cloned and characterized in this paper. The full-length cDNA of gcIKKε is composed of 2529 nucleotides and encodes a polypeptide of 723 amino acids. The mRNA transcription of gcIKKε was constitutively detected in all the selected tissues and the gcIKKε mRNA level increased at 36 h after GCRV infection. Western blot data of both HEK293T cells and EPC cells demonstrated that gcIKKε was around 80 KDa; and immunofluorescence staining data of both NIH3T3 cells and EPC cells determined gcIKKε was a cytosolic protein. The mRNA level of gcIKKε in CIK cells was increased more than 150 times right after poly(I:C) treatment and PMA treatment triggered gcIKKε mRNA transcription in CIK cells more than 100 times. Over-expression of gcIKKε in EPC cells activated the promoter activity of both zebrafish IFN and fathead minnow IFN. gcIKKε mRNA transcription level in CIK cells was increased from 48 h post GCRV infection with different MOIs. All the data support the idea that gcIKKε is a novel teleost IκB kinase recruited in the IFN-mediated antiviral immunity of grass carp.

Stock assessment of fishery target species in Lake Koka, Ethiopia / Evaluación de las reservas pesqueras de especies objetivo en el lago Koka, Etiopia

Effective management is essential for small-scale fisheries to continue providing food and livelihoods for households, particularly in developing countries where other options are often limited. Studies on the population dynamics and stock assessment on fishery target species are thus imperative to sustain their fisheries and the benefits for the society. In Lake Koka (Ethiopia), very little is known about the vital population parameters and exploitation status of the fishery target species: tilapia Oreochromis niloticus,common carp Cyprinus carpióand catfish Clarias gariepinus.Our study, therefore, aimed at determining the vital population parameters and assessing the status of these target species in Lake Koka using length frequency data collected quarterly from commercial catches from 2007-2012. A total of 20 097 fish specimens (distributed as 7 933 tilapia, 6 025 catfish and 6 139 common carp) were measured for the analysis. Von Bertalanffy growth parameters and their confidence intervals were determined from modal progression analysis using ELEFAN I and applying the jackknife technique. Mortality parameters were determined from length-converted catch curves and empirical models. The exploitation status of these target species were then assessed by computing exploitation rates (E) from mortality parameters as well as from size indicators i.e., assessing the size distribution of fish catches relative to the size at maturity (L m),the size that provides maximum cohort biomass (Lopt) and the abundance of mega-spawners. The mean value of growth parameters L x, Kand the growth performance index 0' were 44.5 cm, 0.41/year and 2.90 for O. niloticus,74.1 cm, 0.28/year and 3.19 for C. carpioand 121.9 cm, 0.16/year and 3.36 for C. gariepinus,respectively. The 95 % confidence intervals of the estimates were also computed. Total mortality (Z) estimates were 1.47, 0.83 and 0.72/year for O. niloticus, C. carpioand C. gariepinus,respectively. Our study suggest that O. niloticusis in a healthy state, while C. gariepinusshow signs of growth overfishing (when both exploitation rate (E)and size indicators were considered). In case of C. carpio,the low exploitation rate encountered would point to underfishing, while the size indicators of the catches would suggest that too small fish are harvested leading to growth overfishing. We concluded that fisheries production in Lake Koka could be enhanced by increasing Etoward optimum level of exploitation (E opt)for the underexploited C. carpioand by increasing the size at first capture (Lc)toward the L opt range for all target species.

Un manejo pesquero eficiente es fundamental para que las pesquerías artesanales puedan continuar proveyendo alimento y sustento para los hogares, particularmente en los países en vía de desarrollo, en donde otras opciones a menudo son limitadas. Estudios sobre la dinámica poblacional de las especies objetivo de las pesquerías son, por lo tanto, imperativos para mantener las pesquerías y los beneficios para las sociedades. Esto también es válido para los recursos del Lago Koka (Etiopia) en donde hasta ahora se sabe muy poco sobre los parámetros poblacionales vitales y el estatus de las especies objetivo como la tilapia Oreochromis niloticus,la carpa Cyprinus carpioy el bagre Clarias gariepinus.El estudio aquí presentado tiene como objetivo determinar estos parámetros y evaluar el estado de la pesquería de estas especies en el lago Koka, utilizando los datos de frecuencia de tallas obtenidas de capturas trimestrales en el periodo 2007-2012. Un total de 20 097 especímenes fueron medidos (7 933 de tilapia, 6 025 de bagre y 6 139 de carpa). Los parámetros de crecimiento de von Bertalanffy fueron derivados del análisis de progresión de las modas usando ELEFAN I y aplicando la técnica de jackknife. Las tasas de mortalidad fueron estimadas de curvas de capturas basadas en longitudes y usando modelos empíricos. La tasa de explotación (E), se estimó con los parámetros de mortalidad y también considerando indicadores del tamaño a través de la distribución de tallas en las capturas y su relación con la talla de la primera madurez (L m),el tamaño que provee la biomasa máxima del cohorte (L opt)y la abundancia de los "mega-desovantes". Los valores calculados para los parámetros de crecimiento L m, Ky el índice de la capacidad de crecimiento 0' fueron: 44.5 cm, 0.41 año-1 y 2.90 para O. niloticus,74.1 cm, 0.28 año-1 y 3.19 para C. carpióy 121.9 cm, 0.16 año-1 y 3.36 para C. gariepinus,respectivamente. La tasa de mortalidad total (Z) fue estimada en 1.47, 0.83 y 0.72 año-1 para O. niloticus, C. carpioand C. gariepinus,respectivamente. Nuestros resultados sugieren que la población de O. niloticusse encuentra en un estado saludable, mientras C. gariepinusya muestra signos de sobrepesca por crecimiento. En el caso de C. carpiolas tasas de explotación tan bajas (E<0.5) que se encontraron, pueden apuntar a una sub-explotación, mientras que los indicadores de tallas sugieren que las pequeñas tallas obtenidas, pueden conducir a una sobrepesca por crecimiento. Concluimos que la producción pesquera en el lago Koka puede ser mejorada si se incrementa E al nivel E opt para C. carpioy aumenta el tamaño de la primera captura (Lc)al rango de L opt para todas las especies objetivo.

Molecular characterization, expression, and immunological response analysis of the TWEAK and APRIL genes in grass carp, Ctenopharyngodon idella.

TWEAK and APRIL are important members of the TNF superfamily, which play a crucial role in several diseases. Here, we describe the identification of grass carp (Ctenopharyngodon idella) homologs of TWEAK and APRIL (designated gcTWEAK and gcAPRIL, respectively) and their response to Aeromonas hydrophila and Aquareovirus infection. The gcTWEAK cDNA sequence contains 2273 bases with an open reading frame of 753 bases encoding 250-amino acid residues. The gcTWEAK protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 3 conserved cysteine residues, and a typical TNF homology domain. The gcAPRIL cDNA sequence contains 1408 bases with an open reading frame of 747 bases encoding 248-amino acid residues. The gcAPRIL protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 2 conserved cysteine residues, and a typical TNF homology domain corresponding to other, known APRIL homologs. Reverse transcription-polymerase chain reaction analysis shows that both gcTWEAK and gcAPRIL transcripts are predominantly expressed in the skin, spleen, and head kidney, and they are significantly upregulated in most immune tissues by A. hydrophila and Aquareovirus infections. Our results demonstrate that liver is the most responsive tissue against bacterial infection, whereas gill is the most responsive tissue against viral infection. The association of increased gcTWEAK and gcAPRIL expression after bacterial and viral infections suggests that they play a potentially important role in the immune system of fish.

Allopolyploidization is not so simple: evidence from the origin of the tribe Cyprinini (Teleostei: Cypriniformes).

The identification of allopolyploidization events benefits from molecular dating and divergence assessments of progenitor genomes. Information on gene duplications only, either orthologs or paralogs, provides incomplete information. Analyses of mitochondrial DNA yield insights into matrilineal history, which may differ from patrilineal evolution. Two important food and pet fishes, the common carp (Cyprinus carpio) and goldfish (Carassius sp.), appear to have experienced allotetraploidization sometime from 12 to 20 million years ago (Ma). However, much work is necessary to detail the initial polyploidization event. Herein, we use this group of fishes as a model system to investigate competing scenarios for allopolyploidization. We analyze both the nuclear genes encoding growth hormone (GH), recombination activating protein 1 (RAG1) and HOXA2B gene, and the maternal heredited 12 concatenated mitochondrial protein-coding gene in 19 species of cyprinids and use two species in Balitoridae as outgroup taxa. Our analyses clarify the phylogenetic position of the paternal and maternal ancestors for the common carp and goldfish. The estimation of matrilineal divergence (10.71-12.42 Ma) is significantly younger than the dates of the parental ancestor divergedthat obtained by nuclear genes (16.62-19.64 Ma). Analyses of both genomes date the allopolyploidization event of the common ancestor of Cy. carpio and Ca sp. to about 10.71-12.42 Ma, which is most likely represented by maternal divergent time. The divergence of the two copies of the nuclear genes which was more ancient than the maternal markers might have been included the divergence of the progenitors' genome divergence when the allopolyploidization event occurred. Thus, the scenarios of allopolyploidzation for this group of fish can be suggested as the following: the matrilineal common ancestor of species in tribe Cyprinini might have doubled its genome by mating with a paternal ancestor in the subfamily Cyprininae, which was a sister-group that diverged around 4.20-8.93 Ma. Our work provides new evidence for the divergence dates of allopolyploidization within the Cyprinini, and documents the necessity of considering both matrilineal and patrilineal histories when investigating allopolyploidization.

Complete mitochondrial genome of a natural triploid crucian carp mutant, Carassius auratus var. pingxiangnensis, and phylogenetic analysis of different ploidies in crucian carp.

Carassius auratus var. pingxiangnensis is a natural triploid crucian carp mutant. In order to understand its placement and genetic background at the gene level, the characteristics of mitochondrial DNA sequences and phylogenetic relationship were examined. The results showed that the mitochondrial DNA is a circular double-stranded DNA molecule that is 16,576 bp in length with 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding control region. Mitochondrial genes overlapped by a total of 40 bp in 11 different locations from 1 to 14 bp. The base composition of the C. auratus mitogenome was estimated to be 29.70% A, 26.74% C, 15.35% G, and 28.21% T. The central conserved blocks and the conserved blocks were compared and were similar among C. auratus var. pingxiangnensis and six other cyprinids with different ploidies. The origin of light strand replication was similar to that of other vertebrates; it was 33 bp, but the characteristic sequence motif 5ꞌ-GCCGG-3ꞌ at the base of the stem within tRNA(Cys) was mutated to 5ꞌ-GGCGG- 3ꞌ. Our phylogenetic analysis based on whole mitogenome sequences indicated that C. auratus var. pingxiangnensis was clustered with C. auratus and then sister-grouped with Carassius gibelio. The systemic developmental tree of crucian carp with different chromosome ploidies showed that diploid C. auratus auratus was clustered with triploid C. auratus auratus, sister-grouped with tetraploid C. auratus auratus, and clustered with other diploid, triploid, and tetraploid C. auratus.

Molecular cloning and functional characterization of the NFIL3/E4BP4 transcription factor of grass carp, Ctenopharyngodon idella.

NFIL3 (nuclear factor interleukin 3-regulated) is an important bZIP transcription factor in the immune response and immune cells' development. Here, we identified the NFIL3 gene from grass carp (Ctenopharyngodon idella; gcNFIL3). The deduced amino acid sequence of gcNFIL3 is 468 residues with a typical bZIP domain. Phylogenetics demonstrated that gcNFIL3 clustered closely with NFIL3 of zebrafish. Real-time PCR revealed gcNFIL3 is constitutively expressed in all tissues examined. Its expression was significantly upregulated in head kidney and trunk kidney after stimulation by bacteria. Immunofluorescence microscopy revealed that gcNFIL3 is mainly expressed in the nucleus. Overexpression of gcNFIL3 reduces Aeromonas hydrophila invasion and proliferation. In CIK cells, gcNFIL3 could induce the activation of NF-kappa B and upregulates the expression of IL10 and IFN. These results indicated that gcNFIL3 has immunoregulatory properties and might play a role in the immune response of fish.

Evolutionary history of two divergent Dmrt1 genes reveals two rounds of polyploidy origins in gibel carp.

Polyploidy lineages, despite very rare in vertebrates, have been proposed to play significant role in speciation and evolutionary success, but the occurrence history and consequences are still largely unknown. In this study, we used the conserved Dmrt1 to analyze polyploidy occurrence and evolutionary process in polyploid gibel carp. We identified two divergent Dmrt1 genes and respectively localized the two genes on three homologous chromosomes. Subsequently, the corresponding full-length cDNAs and genomic sequences of Dmrt1 genes were also characterized from the closely related species including Carassius auratus auratus and Cyprinus carpio, and their two Dmrt1 genes were respectively localized on two homologous chromosomes. Significantly, the evolutionary relationship analyses among cDNA and genomic DNA sequences of these Dmrt1 genes revealed two rounds of polyploidy origins in the gibel carp: an early polyploidy might result in an common tetraploid ancestor of Carassius auratus gibelio, Carassius auratus auratus and Cyprinus carpio before 18.49 million years ago (Mya), and an late polyploidy might occur from evolutionary branch of Carassius auratus at around 0.51 Mya, which lead to the occurrence of the hexaploid gibel carp. Therefore, this study provides clear genetic evidence for understanding occurrence time and historical process of polyploidy in polyploid vertebrates.

Identification and analysis of the jnk1 gene in polyploid hybrids of red crucian carp (Carassius auratus red var.) and common carp (Cyprinus carpio L.).

c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase superfamily. The allotetraploid crucian carp is a product of distant hybridization of female red crucian carp with male common carp. It is the first natural case of an allotetraploid with stable genetic characters, including fertility of both female and male animals. In this study, 2 jnk1 cDNAs (including jnk1a and jnk1b) have been cloned from the polyploid crucian carp system, consisting of the allotetraploid crucian carp, the triploid crucian carp, and their original parents (red crucian and common carp). We show that jnk1a and jnk1b represent 2 splice forms arising from the jnk1 gene. On the basis of the genetic structure of jnk1a gene in the polyploid crucian carp system, we demonstrated that the allotetraploid crucian carp is phylogenetically closer to its paternal parent (common carp) than to its maternal parent. We further show a similarity between the triploid crucian carp and its original female parent (red crucian carp). Comparisons of genetic structures indicated that the jnk1b genes of allotetraploid and triploid crucian carp are more similar to those of the original paternal parent rather than the original female parent (red crucian carp). RT-PCR analysis indicated that both the jnk1a and jnk1b genes are widely expressed in fish embryos and in the adult organs, displaying distinct features of embryonic-stage and organ specificity in the polyploid crucian carp system.

Chromosome studies of European cyprinid fishes: cross-species painting reveals natural allotetraploid origin of a Carassius female with 206 chromosomes.

A single female with 206 chromosomes and another 26 females with 156 chromosomes identified as Prussian carp, Carassius gibelio, and 5 individuals with 100 chromosomes identified as crucian carp, C. carassius, were sampled during field survey in one locality in the upper Elbe River. To identify the origin of females with high chromosome numbers, comparative karyotype analysis, GISH, with whole C. carassius DNA as probe and phylogenetic positions of sampled individuals revealed by cytochrome b mitochondrial marker were performed. GISH showed consistently bright labeling of 50 chromosomal elements out of 206, corresponding to the haploid chromosome number of C. carassius. The position of these females with high chromosome numbers in a reconstructed phylogenetic tree was within the clade of C. gibelio, documenting its affiliation to C. gibelio mitochondrial, i.e. maternal lineage. Our findings indicated that the mother of the female with high chromosome numbers was a gynogenetically reproducing 156-chromosome C. gibelio female and the father a bisexually reproducing C. carassius male. We, therefore, hypothesized that the C. gibelio × C. carassius allopolyploid female with 206 chromosomes arose by a mechanism of sperm genome addition to an unreduced egg of the mother.