Hucho Bleekeri Research Articles

Interferon Regulatory Factors (IRF1, IRF4, IRF5, IRF7 and IRF9) in Sichuan taimen (Hucho bleekeri): Identification and Functional Characterization

Background/Objectives: Interferon regulatory factors (IRFs) are multifunctional transcription factors that play important roles in the transcriptional regulation of interferons and in the immune response to pathogens. Therefore, studying the interferon system in fish is highly relevant in the prevention and treatment of viral diseases. Methods: In this study, five IRF genes (IRF1, IRF4, IRF5, IRF7 and IRF9) were identified and characterized in Hucho bleekeri, and their expression profiles were determined after LPS and Poly(I:C) treatment. Results: These IRFs have typical DNA-binding domains and IRF-association domains. Amino acid sequence comparison revealed high homology between these IRFs and those of other vertebrates, with the highest homology being with other salmonid fish. Phylogenetic analysis revealed that these IRFs are divided into four subfamilies (IRF1, IRF3, IRF4 and IRF5), with both IRF4 and IRF9 belonging to the IRF4 subfamily. IRF genes were widely expressed in all of the tested tissues, with IRF1, IRF4 and IRF9 being highly expressed in the spleen and kidney and IRF5 and IRF7 highly expressed in the gonads. IRF1, IRF4 and IRF5 expression was induced at different time points post-LPS challenge. IRF7 and IRF9 expression in the spleen and head kidney was not significantly altered by LPS induction. Poly(I:C) treatment altered IRF expression more significantly than LPS treatment. Poly(I:C) significantly altered the spleen and head kidney expression of all five IRFs. Conclusions: These findings reveal the potential role of IRFs in the antiviral response of H. bleekeri and provide a reference for examining signal transduction pathways in the interferon system in fish.

Metagenomic Sequencing Elucidated the Microbial Diversity of Rearing Water Environments for Sichuan Taimen (Hucho bleekeri).

Sichuan taimen (Hucho bleekeri) is a fish species endemic to China's upper Yangtze River drainage and has significant value as an aquatic resource. It was listed as a first-class state-protected wild animal by the Chinese government due to its very limited distribution and wild population at present. To elucidate the diversity of microorganisms in rearing water environments for H. bleekeri, metagenomic sequencing was applied to water samples from the Maerkang and Jiguanshan fish farms, where H. bleekeri were reared. The results revealed that Pseudomonadota was the dominant phylum in the microbial communities of the water samples. Among the shared bacterial groups, Cyanobacteriota, Actinomycetota, Planctomycetota, Nitrospirota, and Verrucomicrobiota were significantly enriched in the water environment of Jiguanshan (p < 0.01), while Bacteroidota was more enriched in that of Maerkang (p < 0.01). Additionally, the Shannon diversity and Simpson index of the microbial community in the water environment of Maerkang were lower than in that of Jiguanshan. The present study demonstrated the similarities and differences in the microbial compositions of rearing water environments for H. bleekeri, which are expected to benefit the artificial breeding of H. bleekeri in the future.

Characterization, expression, and polymorphism of MHC II α and MHC II β in Sichuan taimen (Hucho bleekeri)

The major histocompatibility complex (MHC) is involved in antigen presentation and plays an essential role in regulating immune function. In the present study, we identified two MHC class II genes and investigated their potential roles in Hucho bleekeri. The MHC II α and MHC II β of H. bleekeri had typical leading peptides, extracellular domains, connecting peptides, transmembrane region, and cytoplasmic region. Amino acid sequence comparison revealed that MHC II of H. bleekeri has high homology with other vertebrates, among which homology with salmonid fish was the highest. Phylogenetic analysis showed that H. bleekeri MHC II clustered with salmonid fish; moreover they clustered with orthologous genes of other fish, whereas mammalian MHC II clustered into a separate branch. Tissue distribution analysis revealed MHC II was widely expressed in all tested tissues, with both MHC II α and MHC II β highly expressed in the spleen, gill, kidney, and hindgut. After lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)) stimulation, the expression of MHC II in the head kidney and spleen of H. bleekeri was significantly upregulated. Compared with MHC II α, MHC II β acted faster in response to the stimulation. Polymorphism analysis of MHC II revealed that all the different alleles belonged to the same major type, and very limited polymorphisms were found in H. bleekeri MHC II α and II β. Selection pressure analysis showed signs of weak and non-significant positive selection in the MHC II α and MHC II β extracellular region. Our study reveals the potential role of MHC II in the immune response of H. bleekeri and provides a reference for studying the evolutionary model of teleost MHC II.

Identification, expression and molecular polymorphism of T-cell receptors α and β from the glacial relict Hucho bleekeri

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β, were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.

Environmental DNA Detects Remaining Populations of Endangered Stream Salmon (Sichuan Taimen: Hucho bleekeri Kimura Salmonidae) in the Qinling Mountains

Sichuan taimen (Hucho bleekeri Salmonidae) populations are declining, and the status of remnant populations in the Qinling Mountains is poorly known. In this study, eDNA and creel netting were used to estimate the distributions of Sichuan taimen in two upper tributaries of the Hanjiang River. A qPCR assay was applied to detect the eDNA of H. bleekeri, and the presence of Sichuan taimen in the Taibai River, but not in the Xushui River, was confirmed. Sampling during summer achieved a relatively higher detection rate. The utility of eDNA techniques for detecting low-density Sichuan taimen was demonstrated in intricate stream ecosystems. The eDNA method can increase the efficiency of the management of endangered freshwater species, such as Sichuan taimen, by providing reliable distribution data.

Correlations between Environmental Factors and the Distribution of Juvenile Hucho bleekeri in the Taibai River, Shaanxi, China

Sichuan taimen (Hucho bleekeri) is a national highly protected wild animal that faces significant impacts from habitat degradation and loss. To study the habitat selection by H. bleekeri, data on the Taibai River and the distribution of juvenile H. bleekeri were recorded seasonally between February 2017 and January 2018, and habitat selection was assessed using the suitability curve method. The results indicate that the average distribution density of juvenile H. bleekeri in the Taibai River is 0.08 ± 0.09 ind./m, with an optimal elevation range from 1200 to 1600 m, river sinuosity range from 1.0 to 1.2, and vegetation coverage between 0.7 and 1.0. When choosing a microhabitat, juvenile H. bleekeri individuals tend to inhabit water areas with a water depth of 0.65 ± 0.33 m, flow velocity of 0.50 ± 0.24 m/s, and offshore distance of 7.66 ± 4.25 m. Furthermore, smaller juvenile fish prefer nearshore habitats with lower flow velocities and shallower water depths. The results provide technical support for the protection and restoration of the habitat of H. bleekeri.

Successful Ultrasonography-Assisted Artificial Reproduction of Critically Endangered Sichuan taimen (Hucho bleekeri)

Sichuan taimen (Hucho bleekeri), a critically endangered species, is listed as a first-class protected species in China. The natural population has been missing from most of its known distribution areas for many years but was rediscovered in the Taibai River in 2012, providing an important resource for the conservation stocking of the Sichuan taimen. In this study, 43 wild adult Sichuan taimen collected from the Taibai River were determined for gamete maturity for artificial reproduction using ultrasonography. The echogenicity of the gonads before and after hormonal induction was described to determine broodstock identification, injection dose of hormones, and spawning time. Four batches of artificial breeding were successfully achieved in 2013, 2014, and 2016. Broodstock (19 females and 16 males) spawned with average fertilization and hatching rates reaching 80.2% and 61.0%, respectively. This study demonstrates that ultrasonic imaging is a convenient and efficient method to track the state of gonads of mature Sichuan taimen during artificial reproduction, which will assist in broodstock cultivation, and further large-scale proliferation and effective resource restoration of the Sichuan taimen.

Phylogenetic analysis of Brachymystax and Hucho genera—Summary on evolutionary status within the Salmoninae subfamily

In this study comparative phylogenetic analyses of huchonid fishes (Brachymystax and Hucho genera) were carried out by application of various computational methods based on the supermatrix data retrieved from NCBI Gene Bank. For this purpose, the available mitochondrial (3575 bp) and nuclear (2037 bp) DNA data originated from all extant huchonid species were used. Additionally, molecular dating was carried out to estimate time background of huchonids divergence. Phylogenetic analyses were conducted using the Neighbor-joining (NJ), Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian approach (BA) methods. The ancestral position of Brachymystax/Hucho cluster within Salmoninae subfamily was resolved and dated to emerge c.a. 24.7–25.6 million years ago (Mya). The evolutionary age of the most recent ancestor of contemporary huchonids was estimated to 10.6–13.6 Mya, while the oldest lineage within huchonid fishes was the genus Brachymystax (5.5–6.1 Mya). All three extant Hucho species were dated to split about 2.5 Mya, suggesting their disjunctive distribution during speciation. The highest heterogeneity was observed within Hucho taimen and Hucho bleekeri species, which clustered into two distinct phylogeographic groups, implying their vicariant origin from different glaciation refugees. The lowest level of genetic differentiation was recorded in Hucho hucho species, which may reflect past low population size, bottlenecks, founder effect and/or low rate of molecular clock. The carried out comparative analyses evidenced the discordance between nuclear and mitochondrial DNA data, indicating probable introgression between H. bleekeri and Brachymystax lenok that was estimated to take place around 5.5 Mya. The monotypic Parahucho genus was resolved as evolutionary younger and separate from huchonid lineage, that emerged around 15.8–13.2 Mya.

Full-length transcriptome sequencing and identification of immune-related genes in the critically endangered Hucho bleekeri

Hucho bleekeri is a glacial relict and critically endangered fish restricted to the Yangtze River drainage in China. The lack of basic genomic information and immune characteristics will hinder the way toward protecting this species. In the present study, we conducted the first transcriptome analysis of H. bleekeri using the combination of SMRT and Illumina sequencing technology. Transcriptome sequencing generated a total of 93,330 non-redundant full-length unigenes with a mean length of 3072 bp. A total of 92,472 (99.08%) unigenes were annotated in at least one of the Nr protein, Swiss-Prot, KEGG, KOG, GO, Nt and Pfam databases. KEGG analysis showed that a total of 7240 unigenes belonging to 28 immune pathways were annotated to the immune system category. Meanwhile, differentially expressed genes between mucosa-associated tissues (skin, gill and hindgut) and systemic-immune tissues (spleen, head kidney and liver) were obtained. Importantly, genes participating in diverse immune signalling pathways and their expression profiles in H. bleekeri were discussed. In addition, a large number of long non-coding RNAs (lncRNAs) and simple sequence repeats (SSRs) were obtained in the H. bleekeri transcriptome. The present study will provide basic genomic information for H. bleekeri and for further research on analysing the characteristics of both the innate and adaptive immune systems of this critically endangered species.

Estimating the inbreeding level and genetic relatedness in an isolated population of critically endangered Sichuan taimen (Hucho Bleekeri) using genome-wide SNP markers.

Sichuan taimen (Hucho bleekeri) is critically endangered fish listed in The Red List of Threatened Species compiled by the International Union for Conservation of Nature (IUCN). Specific locus amplified fragment sequencing (SLAF‐seq)‐based genotyping was performed for Sichuan taimen with 43 yearling individuals from three locations in Taibai River (a tributary of Yangtze River) that has been sequestered from its access to the ocean for more than 30 years since late 1980s. Applying the inbreeding level and genetic relatedness estimation using 15,396 genome‐wide SNP markers, we found that the inbreeding level of this whole isolated population was at a low level (2.6 × 10−3 ± 0.079), and the means of coancestry coefficients within and between the three sampling locations were all very low (close to 0), too. Genomic differentiation was negatively correlated with the geographical distances between the sampling locations (p < .001), and the 43 individuals could be considered as genetically independent two groups. The low levels of genomic inbreeding and relatedness indicated a relatively large number of sexually mature individuals were involved in reproduction in Taibai River. This study suggested a genomic‐relatedness‐guided breeding and conservation strategy for wild fish species without pedigree information records.

Isolation and characterization of 34 SNP markers in Hucho bleekeri

Hucho bleekeri, also called Sichuan taimen, is a species endemic to Yangtze River drainage in China. Due to habitat destruction, heavy fishing and pollution, the wild population of H. bleekeri has declined rapidly in recent years. Insufficient molecular markers have limited the effective conservation of this species. In the present study, 34 novel single nucleotide polymorphism (SNP) markers were developed in H. bleekeri based on restriction site-associated DNA sequencing (RAD-seq). The minor allele frequency ranged from 0.059 to 0.486, and the observed and expected heterozygosities ranged from 0.118 to 0.861 and 0.112 to 0.507, respectively. Polymorphic information content ranged from 0.105 to 0.375. Among these SNPs, four loci showed significant departures from the Hardy–Weinberg equilibrium (P < 0.01). The novel polymorphic SNPs will be useful for future investigation of population structure and conservation genetics for this species.

Protocol optimization of eDNA analysis workflow for detecting Hucho bleekeri.

Environmental DNA (eDNA) analysis in water, consisting of water sample collection, DNA extraction and analysis, is increasingly important in detecting rare aquatic species and determining their distribution. Optimization of protocols used in an eDNA molecular workflow is critical to reduce its uncertainty in detecting target species, especially rare species. In this study, 250 mL, 500 mL, 1 L and 2 L water samples were collected by filtering method from a pond for Sichuan taimen (Hucho bleekeri) culture, followed by extraction and purification of eDNA using DNeasy Tissue and Blood DNA extraction kit or MoBio Power Water DNA extraction kit. A PCR procedure was then applied using specific primers from the D-loop region of mtDNA in Sichuan taimen. To explore an appropriate eDNA analysis protocol, the effect of filtering method, sample size and extraction protocols on the detection rate of target gene in water eDNA was studied. The results showed that the target gene was 100% detected from the water sample by using DNeasy Tissue and Blood DNA extraction kit, which was much better that the MoBio Power Water DNA extraction kit with a detection rate of 0%. Larger volume of water samples yielded better PCR products, with 2 L being the most appropriate volume. Sequence alignment results showed that target sequence from the D-loop region of mtDNA in Sichuan taimen was successfully amplified. The results indicated that DNA extracting method and water sample volume could strongly affect the detection rate. The protocol combining filtration, 2 L water sample, DNeasy Tissue and Blood DNA extraction kit was appropriate for eDNA analysis, and mtDNA D-loop region was recommended for detecting DNA of Sichuan taimen from water samples.

Conservation genetics assessment and phylogenetic relationships of critically endangeredHucho bleekeriin China

Hucho bleekeri is a critically endangered salmonid fish found in the Yangtze River drainage in China. In this study, the genetic diversity of a small population (n = 43) was first assessed with partial mitochondrial DNA sequences (D-loop region and a cytochrome b gene [CYTB] gene fragment) and 15 microsatellite markers. Low levels of nucleotide diversity (Pi) were demonstrated in the H. bleekeri population based on the two mitochondrial DNA markers. The number of haplotypes (h) and the haplotype diversity (Hd) in the D-loop region (12 haplotypes and Hd = 0.8208) were higher than in the CYTB gene fragment (three haplotypes and Hd = 0.0941). The number of microsatellite alleles (Na) ranged from 2 to 13 in these individuals. The mean observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.59719 and 0.44735, respectively. Analysis of molecular variance showed that the degree of differentiation in the population was low (FST = 0.04041) and the coefficient of inbreeding (FIS) was negative, indicating no obvious evidence of inbreeding in this population. A demographic assessment suggested that this species expanded a long time ago, but has suffered great losses in recent years. A molecular phylogenetic analysis clearly indicated that H. bleekeri is not introgressed by Brachymystax lenok tsinlingensis. The baseline population genetic information supplied by this study will be vital in monitoring this highly threatened species.

Development of 27 novel cross-species microsatellite markers for the endangered Hucho bleekeri using next-generation sequencing technology

In this study, we developed 27 polymorphic microsatellite loci in Hucho bleekeri, a glacial relict and freshwater resident salmonid fish in China. The number of alleles varied from 2 to 8 for each primer set. The observed and expected heterozygosity ranged from 0.250 to 0.906 and 0.508 to 0.845, respectively. The polymorphic information content ranged from 0.371 to 0.808. These microsatellite loci should be useful to study population genetics, paternity identification, speciation and adaptive evolution of this lineage.

The complete mitochondrial genome of the Endangered Hucho bleekeri (Salmonidae: huchen)

Hucho bleekeri, a glacial relict and freshwater resident salmonid fish, is endemic to the Yangtze River drainage in China. This species has important significances for studies of biogeography, evolution, phylogeny, ecology, reproduction and development. The complete mitochondrial genome of H. bleekeri was sequenced and characterized. The genome is 16,837 bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The gene arrangement and nucleotide composition of the mitochondrial genome are similar to those of H. taimen. An 82 bp tandem repeat was identified in the control region. We conclude that the control region is variable in length and copy number of repeat between and within species. The complete mitochondrial DNA of H. bleekeri should be useful to study population genetics, biogeography, speciation and adaptive evolution of this lineage.

Current global status of taimen and the need to implement aggressive conservation measures to avoid population and species-level extinction

An international effort carried out during 2011-2012 culminated in three completed IUCN status assessments of Hucho spp., thus completing assessments for all species in this and a related genus Parahucho. These species hold great ecological and evolutionary significance in the salmonid family, and are recognized as the largest salmonids in the world. Specialists have long recognized their precarious status. The reports conclude that all species of taimen are now listed as threatened or Data Deficient on The IUCN Red List of Threatened Species™, and point to a host of ongoing and emerging threats, including habitat loss and overharvest. I summarize the key data used to arrive at the status categories, and emphasize some key conservation measures that must be taken to avoid extinction at both the local population scale and at the species level. I provide some cautionary advice on the use of hatcheries to recover depressed populations and emphasize the need to undertake new research expeditions to assess the uncertain current status of Hucho bleekeri Kimura and Hucho ishikawae Mori.

The complete mitochondrial genome of the Sichuan taimen ( Hucho bleekeri): Repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen ( Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.

Threatened fishes of the world: Hucho bleekeri Kimura, 1934 (Salmonidae)

Conservation status, identification, distribution, habitat and ecology, reproduction, threats, conservation actions and recommendations of a critically endangered fish, Hucho bleekeri Kimura, which is endemic to China, was introduced.