Effects of Alkalinity Stress on Amino Acid Metabolism Profiles and Oxidative-Stress-Mediated Apoptosis/Ferroptosis in Hybrid Sturgeon (Huso dauricus ♀ × Acipenser schrenckii ♂) Livers.

Alkaline water is toxic to cultured aquatic animals that frequently live in pH-neutral freshwater. Overfishing and habitat destruction have contributed to the decline in the wild sturgeon population; consequently, the domestic hybrid sturgeon has become an increasingly important commercial species in China. Hybrid sturgeons are widely cultured in alkaline water, but little is known about the effects of alkalinity stress on hybrid sturgeon liver tissues. We exposed hybrid sturgeons to four alkaline concentrations (3.14 ± 0.02 mmol/L, 7.57 ± 0.08 mmol/L, 11.78 ± 0.24 mmol/L and 15.46 ± 0.48 mmol/L). Histopathology, biochemical index assessment, gene expression level detection and metabolomics analysis were used to investigate the negative effects on liver functions following exposure to NaHCO3. Livers exposed to alkaline stress exhibited severe tissue injury and clear apoptotic characteristics. With increased exposure concentrations, the hepatic superoxide dismutase, catalase, glutathione peroxidase and alkaline phosphatase activities significantly decreased in a dose-dependent manner. NaHCO3 exposure up-regulated the transcriptional levels of apoptosis/ferroptosis-related genes in livers. Similarly, the expression trends of interleukin-1ß and heat shock protein genes also increased in high-alkalinity environments. However, the expression levels of complement protein 3 significantly decreased (p < 0.05). Hepatic untargeted metabolomics revealed the alteration conditions of various metabolites associated with the antioxidant response, the ferroptosis process and amino acid metabolism (such as beta-alanine metabolism; alanine, aspartate and glutamate metabolism; and glycine, serine and threonine metabolism). These data provided evidence that NaHCO3 impaired immune functions and the integrity of hybrid sturgeon liver tissues by mediating oxidative-stress-mediated apoptosis and ferroptosis. Our results shed light on the breeding welfare of domestic hybrid sturgeons and promote the economic development of fisheries in China.

In Silico Screening and Development of Microsatellite Markers for Genetic Analysis in Perca fluviatilis.

Perca fluviatilis is an economically important species of freshwater fish. To understand the genetic structure of P. fluviatilis in China, 268 samples were collected from Wulungu Lake (WL), Jili Lake (JL), the Wulungu River (WR), and the Kalaeerqisi River (KR). These samples were then analyzed using microsatellite markers. A total of 98,425 microsatellite markers were developed based on the genomic data, and 29 polymorphic microsatellite markers were selected to analyze genetic diversity in this study. The number of alleles (Na) and observed heterozygosity (Ho) per population ranged from 4.621 (KR) to 11.172 (WL) and from 0.510 (KR) to 0.716 (JL), respectively. The results of the polymorphic information content (PIC) showed that the WL, JL, and WR populations were highly polymorphic (PIC≥ 0.5) and that the KR population was moderately polymorphic (0.25 ≤ PIC < 0.5). The genetic differentiation coefficient (Fst) among the four P. fluviatilis populations was 0.074, indicating moderate genetic differentiation among the populations in Xinjiang. The reason for the significant difference between the rivers and lakes could be the presence of a dam blocking the flow of P. fluviatilis. The development of microsatellite markers provides support for population genetics in the future. The evaluation of the genetic structure of P. fluviatilis in Xinjiang provides a reference for the reproduction and conservation of P. fluviatilis.

Transcriptome Analysis Reveals Molecular Underpinnings of Common Carp (Cyprinus carpio) Under Hypoxia Stress.

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp (Cyprinus carpio) is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp (C. carpio var wuyuanesis) and Songpu mirror carp (C. carpio var specularis). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet. This study tested the hypoxia tolerance of Songpu minor carp, Hebao red carp, and their hybrid F1 population by an acute hypoxia treatment. Muscle and liver tissues were used for transcriptome sequencing analysis to identify the key factors for hypoxia tolerance and explore the potential genetic mechanism for breeding high hypoxia tolerance in common carp. The comparative transcriptomic analysis revealed abundant hypoxia response-related genes and their differential regulation mechanism in these two tissues of different common carp strains under acute hypoxia, including immune response, cellular stress response, HIFs (hypoxia-inducible factors), MAP kinase, iron ion binding, and heme binding. Our findings will facilitate future investigation on the hypoxia response mechanism and provide a solid theoretical basis for breeding projects in common carp.

Effects of Luteinizing Hormone Releasing Hormone A2 on Gonad Development in Juvenile Amur Sturgeon, Acipenser schrenckii, Revealed by Transcriptome Profiling Analysis.

Acipenser schrenckii is an economically important aquatic species whose gonads require particularly long times to reach sexual maturity. Luteinizing hormone plays important roles in gonad development, and luteinizing hormone releasing hormone A2 (LH-A2) is used as an oxytocin to promote ovulation in aquaculture of A. schrenckii. In this study, we aimed to determine the effects of LH-A2 on gonad development in juvenile A. schrenckii through transcriptome profiling analysis of the pituitary and gonads after LH-A2 treatment at a dose of 3 µg/kg. The 17ß-estradiol (E2) levels gradually increased with LH-A2 treatment time, and significantly differed from those of the control group on days 5 and 7 (p < 0.01). However, the content of testosterone (Testo) gradually decreased with LH-A2 treatment time and showed significant differences on day 3 (p < 0.05), and on days 5 and 7 (p < 0.01), compared to those in the control group. Thus, LH-A2 promotes the secretion of E2 and inhibits the secretion of Testo. Transcriptome profiling analysis revealed a total of 2,883 and 8,476 differentially expressed genes (DEGs) in the pituitary and gonads, respectively, thus indicating that LH-A2 has more regulatory effects on the gonads than the pituitary in A. schrenckii. Signal transduction, global and overview maps, immune system, endocrine system and lipid metabolism were the main enriched metabolic pathways in both the pituitary and gonads. Sixteen important genes were selected from these metabolic pathways. Seven genes were co-DEGs enriched in both signal transduction and endocrine system metabolic pathways. The other co-DEGs were selected from the immune system and lipid metabolism metabolic pathways, and showed mRNA expression changes of >7.0. The expression of five DEGs throughout LH-A2 treatment was verified to show the same patterns of change as those observed with RNA-seq, indicating the accuracy of the RNA-seq in this study. Our findings provide valuable evidence of the regulation of gonad development of juvenile A. schrenckii by LH-A2 and may enable the establishment of artificial techniques to regulate gonad development in this species.

A High-Density Genetic Linkage Map and Fine Mapping of QTL For Feed Conversion Efficiency in Common Carp (Cyprinus carpio).

Feed conversion efficiency (FCE) is an economically crucial trait in fish, however, little progress has been made in genetics and genomics for this trait because phenotypes of the trait are difficult to measure. In this study, we constructed a high-density and high-resolution genetic linkage map with 28,416 SNP markers for common carp (Cyprinus carpio) based on high throughput genotyping with the carp 250K single nucleotide polymorphism (SNP) array in a full-sib F1 family of mirror carp (Cyprinus carpio) consisting of 141 progenies. The linkage map contained 11,983 distinct loci and spanned 3,590.09 cM with an average locus interval of 0.33 cM. A total of 17 QTL for the FCE trait were detected on four LGs (LG9, LG20, LG28, and LG32), explaining 8.9-15.9% of the phenotypic variations. One major cluster containing eight QTL (qFCE1-28, qFCE2-28, qFCE3-28, qFCE4-28, qFCE5-28, qFCE6-28, qFCE7-28, and qFCE8-28) was detected on LG28. Two clusters consisting of four QTL (qFCE1-32, qFCE2-32, qFCE3-32, and qFCE4-32) and three QTL (qFCE1-20, qFCE2-20, and qFCE3-20) were detected on LG32 and LG20, respectively. Nine candidate genes (ACACA, SCAF4, SLC2A5, TNMD, PCDH1, FOXO, AGO1, FFAR3, and ARID1A) underlying the feed efficiency trait were also identified, the biological functions of which may be involved in lipid metabolism, carbohydrate metabolism, energy deposition, fat accumulation, digestion, growth regulation, and cell proliferation and differentiation according to GO (Gene Ontology). As an important tool, high-density and high-resolution genetic linkage maps play a crucial role in the QTL fine mapping of economically important traits. Our novel findings provided new insights that elucidate the genetic basis and molecular mechanism of feed efficiency and the subsequent marker-assisted selection breeding in common carp.

Molecular characterization and expression patterns of two hormone-sensitive lipase genes in common carp Cyprinus carpio.

The hormone-sensitive lipase (HSL) gene plays an important role in mammals' lipid metabolism. Therefore, its function in fish is capturing increasing attention. In this study, two distinct cDNAs, designated HSL1 and HSL2, are firstly identified from common carp Cyprinus carpio. The full-length cDNA of HSL1 and HSL2 consists of 3379 bp and 2732 bp, encoding polypeptide of 693 and 847 amino acids, respectively, and shares 60.6% amino acid identity. Phylogenetic analysis suggests that HSL1 and HSL2 are derived from paralogous genes, which might have arisen during a teleost-specific genome duplication event. The two HSL mRNAs are differentially expressed, both in terms of distribution among tissues and in terms of abundance during embryogenesis. Moreover, both HSL mRNAs are expressed in various tissues, the highest in abdominal fat. Meanwhile, the two HSLs are detected at all stages of embryonic development, suggesting that they could be functional and involved in embryogenesis. In addition, the results show that the mRNA expression level of HSL2 in the high group of intramuscular fat content is significantly higher than that in the low group (P < 0.01). The research provides basic data for developing a further understanding of the function of HSL as well as molecular regulation mechanism in fat metabolism of common carp.

Quantitative trait loci for morphometric traits in multiple families of common carp (Cyprinus carpio).

Quantitative trait locus (QTL) mapping is frequently used to understand the genetic architecture of quantitative traits. Herein, we performed a genome scan for QTL affecting the morphometric characters in eight full-sib families containing 522 individuals using different statistical methods (Sib-pair and half-sib model). A total of 194 QTLs were detected in 25 different regions on 10 linkage groups (LGs). Among them, 37 QTLs on five LGs (eight, 13, 24, 40 and 45) were significant (5% genome-wide level), while the remaining 40 (1% chromosome-wide level) and 117 (5% chromosome-wide level) indicated suggestive effect on those traits. Heritabilities for most morphometric traits were moderate to high, ranging from 0.21 to 0.66, with generally strong phenotypic and genetic correlations between the traits. A large number of QTLs for morphometric traits were co-located, consistent with their high correlations, and may reflect pleiotropic effect on the same genes. Biological pathways were mapped for possible candidate genes on QTL regions. One significantly enriched pathway was identified on LG45, which had a P-value of 0.04 and corresponded to the "regulation of actin cytoskeleton pathway". The results are expected to be useful in marker-assisted selection (MAS) and provide valuable information for the study of gene pathway for morphometric and growth traits of the common carp.

Genome-Wide Association Study for Muscle Fat Content and Abdominal Fat Traits in Common Carp (Cyprinus carpio).

Muscle fat content is an important phenotypic trait in fish, as it affects the nutritional, technical and sensory qualities of flesh. To identify loci and candidate genes associated with muscle fat content and abdominal fat traits, we performed a genome-wide association study (GWAS) using the common carp 250 K SNP assay in a common carp F2 resource population. A total of 18 loci surpassing the genome-wide suggestive significance level were detected for 4 traits: fat content in dorsal muscle (MFdo), fat content in abdominal muscle (MFab), abdominal fat weight (AbFW), and AbFW as a percentage of eviscerated weight (AbFP). Among them, one SNP (carp089419) affecting both AbFW and AbFP reached the genome-wide significance level. Ten of those loci were harbored in or near known genes. Furthermore, relative expressions of 5 genes related to MFdo were compared using dorsal muscle samples with high and low phenotypic values. The results showed that 4 genes were differentially expressed between the high and low phenotypic groups. These genes are, therefore, prospective candidate genes for muscle fat content: ankyrin repeat domain 10a (ankrd10a), tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (tanc2), and four jointed box 1 (fjx1) and choline kinase alpha (chka). These results offer valuable insights into the complex genetic basis of fat metabolism and deposition.

The genetic map of goldfish (Carassius auratus) provided insights to the divergent genome evolutions in the Cyprinidae family.

A high-density linkage map of goldfish (Carassius auratus) was constructed using RNA-sequencing. This map consists of 50 linkage groups with 8,521 SNP markers and an average resolution of 0.62 cM. Approximately 84% of markers are in protein-coding genes orthologous to zebrafish proteins. We performed comparative genome analysis between zebrafish and medaka, common carp, grass carp, and goldfish to study the genome evolution events in the Cyprinidae family. The comparison revealed large synteny blocks among Cyprinidae fish and we hypothesized that the Cyprinidae ancestor undergone many inter-chromosome rearrangements after speciation from teleost ancestor. The study also showed that goldfish genome had one more round of whole genome duplication (WGD) than zebrafish. Our results illustrated that most goldfish markers were orthologous to genes in common carp, which had four rounds of WGD. Growth-related regions and genes were identified by QTL analysis and association study. Function annotations of the associated genes suggested that they might regulate development and growth in goldfish. This first genetic map enables us to study the goldfish genome evolution and provides an important resource for selective breeding of goldfish.

QTL variations for growth-related traits in eight distinct families of common carp (Cyprinus carpio).

BACKGROUND: Comparing QTL analyses of multiple pair-mating families can provide a better understanding of important allelic variations and distributions. However, most QTL mapping studies in common carp have been based on analyses of individual families. In order to improve our understanding of heredity and variation of QTLs in different families and identify important QTLs, we performed QTL analysis of growth-related traits in multiple segregating families. RESULTS: We completed a genome scan for QTLs that affect body weight (BW), total length (TL), and body thickness (BT) of 522 individuals from eight full-sib families using 250 microsatellites evenly distributed across 50 chromosomes. Sib-pair and half-sib model mapping identified 165 QTLs on 30 linkage groups. Among them, 10 (genome-wide P <0.01 or P < 0.05) and 28 (chromosome-wide P < 0.01) QTLs exhibited significant evidence of linkage, while the remaining 127 exhibited a suggestive effect on the above three traits at a chromosome-wide (P < 0.05) level. Multiple QTLs obtained from different families affect BW, TL, and BT and locate at close or identical positions. It suggests that same genetic factors may control variability in these traits. Furthermore, the results of the comparative QTL analysis of multiple families showed that one QTL was common in four of the eight families, nine QTLs were detected in three of the eight families, and 26 QTLs were found common to two of the eight families. These common QTLs are valuable candidates in marker-assisted selection. CONCLUSION: A large number of QTLs were detected in the common carp genome and associated with growth-related traits. Some of the QTLs of different growth-related traits were identified at similar chromosomal regions, suggesting a role for pleiotropy and/or tight linkage and demonstrating a common genetic basis of growth trait variations. The results have set up an example for comparing QTLs in common carp and provided insights into variations in the identified QTLs affecting body growth. Discovery of these common QTLs between families and growth-related traits represents an important step towards understanding of quantitative genetic variation in common carp.

The complete mitochondrial genome sequence of Silver Gudgeon (Gnathopogon argentatus).

The complete mitochondrial genome of Gnathopogon argentatus was determined to be 16,607 bp long circular molecule with a typical gene arrangement of vertebrate mitochondrial DNA. The complete mitochondrial genome of G. argentatus is 16,607 bp in length with 56.02% AT content, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. The complete mitochondrial genome of G. argentatus was obtained for the first time and would play an important role in population structure and conservation genetic studies.

Diversification of the duplicated Rab1a genes in a hypoxia-tolerant fish, common carp (Cyprinus carpio).

Common carp is a widely cultivated fish with longer than 2,000 years domestication history, due to its strong environmental adaptabilities, especially hypoxia tolerance. The common carp genome has experienced a very recent whole genome duplication (WGD) event. Among a large number of highly similar duplicated genes, a pair of Ras-associated binding-GTPase 1a (Rab1a) genes were found fast diverging. Four analogous Rab1a genes were identified in the common carp genome. Comparisons of gene structures and sequences indicated Rab1a-1 and Rab1a-2 was a pair of fast diverging duplicates, while Rab1a-3 and Rab1a-4 was a pair of less diverged duplicates. All putative Rab1a proteins shared conserved GTPase domain, which enabled the proteins serve as molecular switches for vesicular trafficking. Rab1a-1 and Rab1a-2 proteins varied in their C-terminal sequences, which were generally considered to encode the membrane localization signals. Differential expression patterns were observed between Rab1a-1 and Rab1a-2 genes. In blood, muscle, spleen, and heart, the mRNA level of Rab1a-1 was higher than that of Rab1a-2. In liver and intestine, the mRNA level of Rab1a-2 was higher. Expression of Rab1a-1 and Rab1a-2 showed distinct hypoxia responses. Under severe hypoxia, Rab1a-1 expression was down-regulated in blood, while Rab1a-2 expression was up-regulated in liver. Compared with the less diverged Rab1a-3/4 gene pair, common carp Rab1a-1/2 gene pair exhibited strong characteristics of sub-functionalization, which might contribute to a sophisticated and efficient Ras-dependent regulating network for the hypoxia-tolerant fish.

Screening of SSR markers associated with scale cover pattern and mapped to a genetic linkage map of common carp (Cyprinus carpio L.).

Fish scale is an attractive model in bone physiology research and is also a crucial character for breeding new varieties. Thus, it is important to identify loci in the genome associated with scale formation. In this study, 290 microsatellite markers in common carp (Cyprinus carpio L.) were selected and tested for their segregation in a full-sib mapping panel containing 96 individuals (population 1). Association analysis identified seven simple sequence repeats (SSRs) (HLJ2509, HLJ3227, HLJ3675, HLJ3766, HLJ3863, FGFR1, FGFR7) that showed significant correlation with scale cover pattern in population 1. When the seven SSRs were investigated in two other populations, seven and five SSRs were significantly correlated with scale cover pattern in population 2 (116 individuals) and population 3 (57 individuals), respectively. The exceptions were FGFR1 and HLJ3227. A genetic linkage map was constructed using the 290 SSRs and 241 SSRs were mapped into 47 linkage groups (LGs), with 2-15 markers per LG. The map spanned 2,241.7 cM, with LG sizes that varied from 1.1 to 124.9 cM. All seven markers associated with scale cover mapped into LG3. We considered that a gene cluster that affected the scale cover pattern possibly existed in LG3. By aligning the seven markers with the zebrafish (Danio rerio) genome, we identified six candidate genes (atoh1a, ptch1, bmp1a, fgfr1a, fgf17, wnt5a) that may be associated with scale formation. We propose that the seven markers could be used with marker-assisted selection to breed a new variety of common carp, and the six candidate genes could help in understanding the scale cover mechanism.

The complete mitochondrial genome sequence of Sarcochellichthys lacustris (Dybowski).

The complete mitochondrial genome of Sarcochellichthys lacustris was determined to be 16,683 bp long circular molecule with a typical gene arrangement of vertebrate mitochondrial DNA. The circular genome consists of 37 typical animal mitochondrial genes (13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes) and 1 control region (D-loop).

The complete mitochondrial genome sequence of Khanka spiny bitterling (Acanthorhodeus chankaensis).

The genus Acanthorhodeus chankaensis belongs to the family Cyprindea, subfamily Acheilognathinae. The complete mitochondrial genome of A. chankaensis is 16,774 bp in length with 57.65% AT content, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The mitogenome sequence of Acanthorhodeus chankaensis would play an important role in population structure and conservation genetic studies.

The complete mitochondrial genome sequence of pike perch (Sander canadensis).

Pike perch (Sander canadensis) is a member of the largest order of Osteichthyes, Perciformes, and is an important ecological and economic freshwater species, which distributes in Ili River and Ergis River of Xinjiang Province, China. In this study, we sequenced the whole mitochondrial genome of pike perch, and analyzed the similarity with its related species. The mitochondrial genome of S. canadensis is 16,542 bp in length with 55.05% AT content, contained 13 protein coding genes, 22 tRNA genes, 2 ribosomal genes and an 892 bp non-coding region. In control region, 6 CSBs (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F), one potential TAS and one poly-T region were identified. Comparing all protein-coding genes and whole genome sequence with 4 species of Perciformes (three species of Percidae, Perca flavescens. Percina macrolepida. Etheostoma radiosum and one outgroup Oreochromis sp. red tilapia), ND3 gene has the highest mutation rate, and S. canadensis has higher similarity with Perca flavescens than others. The mitochondrial genomic sequence will help us to study the conservation genetic and evolution of Percidae.

Genome sequence and genetic diversity of the common carp, Cyprinus carpio.

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.

Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance.

[Screening and body correlation analysis of microsatellite markers related to intermuscular bone number in common carp (Cyprinus carpio)].

In this study, 149 polymorphic markers were screened from 200 microsatellite markers. From a family of mirror carp, which included 107 individuals. All samples were analyzed for body correlation, and intermuscular bone number was tested using the General Linear Model (GLM) single marker regression. Determination of the threshold values by 10,000 permutation tests showed that eight markers had significant correlation (P<0.05), in which HLJ3086, HLJ3642 and HLJ3515 had very significant correlation with intermuscular bone number (P<0.01). In addition, the genotypes of the captured correlative loci were determined by Duncan's test using SPSS17.0 software. Markers were used to screen the protein and nucleotide database in the National Center for Biotechnology Information (NCBI). Blasting results showed that HLJ2891 was highly correlated (92%) with latrophilin-2-like and HLJ3515 was highly correlated (81%) with serine/threonine-protein kinase 32B-like of zebrafish. These functional markers and genotypes may provide an efficient basis for marker-assisted selection of intermuscular bone number in mirror carp.