Xiaojuan Cao

The complete mitochondrial genomes of natural diploid and tetraploid loaches Misgurnus anguillicaudatus (Cypriniformes: Cobitidae)

Abstract Dojo loach Misgurnus anguillicaudatus, a small-sized freshwater fish species, is not only one of the most important cultured fish in East Asia, but also a promising model animal to study evolutionary biology for polyploidy. We sequenced the complete mitochondrial genomes from the natural diploid and tetraploid individuals of this species. The two different cytotypes loaches share the same length (16,646 bp) and structural organization of mitochondrial genome. The two genomes have 98.3% nucleotide sequence similarity, and the biggest nucleotide sequence divergence between homologous genes is observed in CYTB gene.

The complete mitochondrial genome of natural Misgurnus bipartitus (Cypriniformes: Cobitidae)

Abstract Misgurnus bipartitus, a small sized freshwater fish species, is one of the most important cultured fish in East Asia. In this study, the complete mitochondrial genome of M. bipartitus is sequenced to be 16,636 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, a control region and the origin of the light strand replication. The overall base composition of M. bipartitus in descending order is A 29.79 %, T 27.95%, C 25.90% and G 16.36%, with a slight A + T bias. The mitogenome sequence data may provide useful information to the population genetics analysis of M. bipartitus and the elucidation of evolutionary mechanisms in Cobitidae.

High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus) and Their Response to Intestinal Air-Breathing Inhibition

MicroRNAs (miRNAs) exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs) of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus) under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group). Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

Hepatic transcriptome analysis and identification of differentially expressed genes response to dietary oxidized fish oil in loach Misgurnus anguillicaudatus

RNA sequencing and short-read assembly were utilized to produce a transcriptome of livers from loaches (Misgurnus anguillicaudatus) fed with three different diets respectively containing fresh fish oil (FO group), medium oxidized fish oil (MO group) and high oxidized fish oil (HO group). A total of 60,663 unigenes were obtained in this study, with mean length 848.74 bp. 50,814, 49,584 and 49,814 unigenes were respectively obtained from FO, MO and HO groups. There were 2,343 differentially expressed genes between FO and MO, with 855 down- and 1,488 up-regulated genes in the MO group. 2,813 genes were differentially expressed between FO and HO, including 1,256 down- and 1,552 up-regulated genes in the HO group. 2,075 differentially expressed genes were found in the comparison of MO and HO, including 1,074 up- and 1,001 down-regulated genes in the MO group. Some differentially expressed genes, such as fatty acid transport protein (fatp), fatty acid binding protein (fabp), apolipoprotein (apo), peroxisome proliferator activated receptor-gamma (ppar-γ), acetyl-CoA synthetase (acs) and arachidonate 5-lipoxygenase (alox5), were involved in lipid metabolism, suggesting these genes in the loach were responsive to dietary oxidized fish oil. Results of transcriptome profilings here were validated using quantitative real time PCR in fourteen randomly selected unigenes. The present study provides insights into hepatic transcriptome profile of the loach, which is a valuable resource for studies of loach genomics. More importantly, this study identifies some important genes responsible for dietary oxidized fish oil, which will benefit researches of lipid metabolism in fish.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C

Abstract Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full‐length cDNA sequences of HSP70, HSP90&agr; and HSP90&bgr; from loach Misgurnus anguillicaudatus were cloned. The full‐length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90&agr; and HSP90&bgr; were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90&agr; and HSP90&bgr; in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90&agr; and HSP90&bgr; were respectively present in the intestine, brain and kidney of loach. Quantitative real‐time PCR was employed to analyze the temporal expressions of HSP70, HSP90&agr; and HSP90&bgr; in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. HighlightsHSP70, HSP90&agr; and HSP90&bgr; were cloned and characterized in loach.Tissue expressions of the three HSP genes were investigated.Expressions of the three HSP genes in loach were sensitive to thermal stress.1000 mg/kg dietary vitamin C may decrease the body damages caused by thermal stress.

Developmental transcriptome analysis and identification of genes involved in formation of intestinal air-breathing function of Dojo loach, Misgurnus anguillicaudatus

Dojo loach, Misgurnus anguillicaudatus is a freshwater fish species of the loach family Cobitidae, using its posterior intestine as an accessory air-breathing organ. Little is known about the molecular regulatory mechanisms in the formation of intestinal air-breathing function of M. anguillicaudatus. Here high-throughput sequencing of mRNAs was performed from six developmental stages of posterior intestine of M. anguillicaudatus: 4-Dph (days post hatch) group, 8-Dph group, 12-Dph group, 20-Dph group, 40-Dph group and Oyd (one-year-old) group. These six libraries were assembled into 81300 unigenes. Totally 40757 unigenes were annotated. Subsequently, 35291 differentially expressed genes (DEGs) were scanned among different developmental stages and clustered into 20 gene expression profiles. Finally, 15 key pathways and 25 key genes were mined, providing potential targets for candidate gene selection involved in formation of intestinal air-breathing function in M. anguillicaudatus. This is the first report of developmental transcriptome of posterior intestine in M. anguillicaudatus, offering a substantial contribution to the sequence resources for this species and providing a deep insight into the formation mechanism of its intestinal air-breathing function. This report demonstrates that M. anguillicaudatus is a good model for studies to identify and characterize the molecular basis of accessory air-breathing organ development in fish.

The complete mitochondrial genome of natural Paramisgurnus dabryanus (Cypriniformes: Cobitidae).

Abstract Paramisgurnus dabryanus, a small-sized freshwater fish species, is one of the most important cultured fish in East Asia from Russia to China. In this study, the complete mitochondrial genome of P. dabryanus is sequenced to be 16,566 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, a control region and the origin of the light strand replication. The overall base composition of P. dabryanus in descending order is A 29.66 %, T 28.04%, C 25.81% and G 16.49%, with a slight A + T bias. The mitogenome sequence data may provide useful information to the population genetics analysis of P. dabryanus and the elucidation of evolutionary mechanisms in Cobitidae.

Production of Tetraploid Gynogenetic Loach Using Diploid Eggs of Natural Tetraploid Loach, Misgurnus anguillicaudatus, Fertilized with UV-Irradiated Sperm of Megalobrama amblycephala without Treatments for Chromosome Doubling

The gynogenesis phenomenon in nature mainly appears in the reproduction of fish and invertebrates. So far, gynogenesis has been successfully induced in many fish species with the aid of some physical or chemical methods for chromosome doubling. However, few fish can produce gynogenetic progenies, genetically identical or similar to the somatic cells of the mothers, without a treatment for the doubling of chromosomes, which may be related to apomixis, premeiotic endoreduplication, or premeiotic endomitosis. At present, no studies are available about fish with normal ovarian structures producing gynogenetic progenies that could spontaneously double their chromosomes. According to the analyses of flow cytometry, chromosome number, and microsatellites, we found that, with the use of UV-irradiated sperm of blunt snout bream Megalobrama amblycephala, tetraploid loach Misgurnus anguillicaudatus produced tetraploid gynogenetic progenies without any treatments for the doubling of chromosomes. To determine the genetic relationships of gynogenetic progenies and their maternal parent, microsatellite genotyping was conducted. The results indicated that the reason for spontaneous chromosome duplication in gynogenetic progenies may be cytokinesis or inhibition of the extrusion of the second polar body. This is the first report on fish with normal ovarian structures that can produce gynogenetic progenies which spontaneously double their chromosomes and which are genetically identical or similar to the somatic cells of the mothers.

Transcriptome Profile Analysis of Ovarian Tissues from Diploid and Tetraploid Loaches Misgurnus anguillicaudatus

RNA sequencing and short-read assembly was utilized to produce a transcriptome of ovarian tissues from three-year-old diploid and tetraploid loaches (Misgurnus anguillicaudatus). A total of 28,369 unigenes were obtained, comprising 10,546 unigenes with length longer than 1000 bp. More than 73% of the unigenes were annotated through sequence comparison with databases. The RNA-seq data revealed that 2253 genes were differentially expressed between diploid and tetraploid loaches, including 1263 up-regulated and 990 down-regulated genes in tetraploid loach. Some differentially expressed genes, such as vitellogenin (Vtg), gonadotropin releasing hormone receptor type A (GnRHRA), steroidogenic acute regulatory protein (StAR), mitogen-activated protein kinase 14a (MAPK14a), ATP synthase subunit alpha (atp5a), and synaptonemal complex protein 1 (Scp1), were involved in regulation of cell proliferation, division, gene transcription, ovarian development and energy metabolism, suggesting that these genes were related to egg diameter of the loach. Results of transcriptome profiling here were validated using real time quantitative PCR in ten selected genes. The present study provided insights into the transcriptome profile of ovarian tissues from diploid and tetraploid loaches Misgurnus anguillicaudatus, which was made available to the research community for functional genomics, comparative genomics, polyploidy evolution and molecular breeding of this loach and other related species.

Comparative analysis of three methods of making scale specimens for small fish

Scales are commonly used for investigations of fish age determination. However, there is a lack of appropriate methods on making scale specimens for small fish species. Three methods included the warm water immersion method, alkaline immersion method and a new method invented by us were introduced in this study. Effects of age determination for three kinds of small fishes containing Danio rerio, Pseudorasbora parva and Paramisgurnus dabryanus through using scale specimens made by the three methods were compared. Results obtained here showed that the warm water immersion method was not applicable for making scale specimens of small fish. However, the alkaline immersion method and the new method were more suitable, while the new method was especially suitable for making scale specimens of P. dabryanus whose scales presented complex structures.