Gill remodeling increases the respiratory surface area of grass carp (Ctenopharyngodon idella) under hypoxic stress.

Seasonal changes, diurnal variations, and eutrophication result in periodic hypoxia in fish habitats, thus affecting the success of commercial aquaculture. In this study, the grass carp (Ctenopharyngodon idella) presented moderate hypoxia tolerance; they showed a medium critical oxygen tension during the loss of equilibrium. In response to 7 d of hypoxic exposure, the erythrocyte count and hemoglobin (Hb) concentration significantly increased (p < 0.01). To cope with the hypoxic environment, the grass carp underwent gill remodeling marked by reduction in the interlamellar cell mass (ILCM) and an increase in respiratory surface area. The gill remodeling under hypoxia was enabled by apoptosis induction. Although apoptotic signals were not found on ILCM cells, transferase dUTP nick end labeling (TUNEL) assay results indicated that after 1 d of hypoxic exposure, the number of TUNEL-positive cells per lamella increased until 4 d and then began to decrease. Consistent with the results of the TUNEL assay, the mRNA expression of apoptosis-related genes, caspase-3, Bax, and Bcl-2, increased at 1, 4, and 7 d of the hypoxia treatment. In addition, gill remodeling significantly (p < 0.01) decreased the concentration of sodium and chloride ions in the fish serum. These findings provide evidence that grass carps increase their respiratory surface area through gill remodeling by apoptosis in the gill filaments to acclimate to a hypoxic environment. This study expands our understanding of the morphological and physiological changes in grass carp in response to a hypoxic environment; therefore, it could be useful for maintaining grass carp production.

An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3' UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection.

N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3' UTR of Toll interacting protein gene (TOLLIP982T>C) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3' UTR, but TOLLIP982T>C mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3' UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor ß-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP982T>C mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP982T>C mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3' UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp.

Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV.

BACKGROUND: N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. RESULTS: This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal's responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. CONCLUSION: Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.