Range expansion and complete mitochondrial genome of the highfin blenny (Lupinoblennius nicholsi).

BACKGROUND: The highfin blenny, Lupinoblennius nicholsi, is a marine fish species reported in reef and rocky inshore habitats with a disjunct distribution in the southern Gulf of Mexico. Overall, there are very few studies on this species and there is a scarcity of molecular resources for genetic comparisons. We set out to report the first mitochondrial genome for L. nicholsi and report a range expansion for the species. METHODS AND RESULTS: An individual of L. nicholsi was collected from the coast of Dauphin Island, Alabama. The mitochondrial genome was sequenced, assembled, and annotated. The fragment corresponding to cytochrome oxidase I (COI) was used to compare this sample to other cryptobenthic species of the Atlantic. Finding a mature individual in the coast of Alabama implies this species has a continuous distribution throughout the northern Gulf of Mexico. The mitochondrial genome of L. nicholsi is 16,416 bp in length and comprised of 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding D-loop. Comparisons using COI support the species is L. nicholsi and separate it from other cryptobenthic fishes found in the area. CONCLUSIONS: This study represents the first mitochondrial genome for this L. nicholsi, serving as a reference for future comparative studies with marine fishes. By reporting the range expansion of this species, this study provides insights on the fish diversity of the Gulf of Mexico.

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the nutritional value of channel catfish, the masou salmon Δ5-desaturase like gene (D5D) driven by the common carp beta-actin promoter (ßactin) was inserted into channel catfish. The objectives of this study were to determine the effectiveness of ßactin-D5D for improving n-3 fatty acid production in F1 transgenic channel catfish, as well as examine pleiotropic effects on growth, proximate analysis, disease resistance, and other performance traits. Transgenic F1 channel catfish showed a 33% increase in the relative proportion of n-3 fatty acids coupled with a 15% decrease in n-6 fatty acids and a 17% decrease in n-9 fatty acids when compared to non-transgenic full-siblings (P < 0.01, P < 0.01, P < 0.01). However, while the relative proportion of n-3 fatty acids was achieved, the total amount of fatty acids in the transgenic fish decreased resulting in a reduction of all fatty acids. Insertion of the ßactin-D5D transgene into channel catfish also had large effects on the body composition, and growth of channel catfish. Transgenic channel catfish grew faster, were more disease resistant, had higher protein and moisture percentage, but lower fat percentage than full-sib controls. There were sex effects as performance changes were more dramatic and significant in males. The ßactin-D5D transgenic channel catfish were also more uniform in their fatty acid composition, growth and other traits.

Deep Transcriptomic Analysis Reveals the Dynamic Developmental Progression during Early Development of Channel Catfish (Ictalurus punctatus).

The transition from fertilized egg to larva in fish is accompanied with various biological processes. We selected seven early developmental stages in channel catfish, Ictalurus punctatus, for transcriptome analysis, and covered 22,635 genes with 590 million high-quality RNA-sequencing (seq) reads. Differential expression analysis between neighboring developmental timepoints revealed significantly enriched biological categories associated with growth, development and morphogenesis, which was most evident at 2 vs. 5 days post fertilization (dpf) and 5 vs. 6 dpf. A gene co-expression network was constructed using the Weighted Gene Co-expression Network Analysis (WGCNA) approach and four critical modules were identified. Among candidate hub genes, GDF10, FOXA2, HCEA and SYCE3 were involved in head formation, egg development and the transverse central element of synaptonemal complexes. CK1, OAZ2, DARS1 and UBE2V2 were mainly associated with regulation of cell cycle, growth, brain development, differentiation and proliferation of enterocytes. IFI44L and ZIP10 were critical for the regulation of immune activity and ion transport. Additionally, TCK1 and TGFB1 were related to phosphate transport and regulating cell proliferation. All these genes play vital roles in embryogenesis and regulation of early development. These results serve as a rich dataset for functional genomic studies. Our work reveals new insights of the underlying mechanisms in channel catfish early development.

The chromosome-scale reference genome for the pinfish (Lagodon rhomboides) provides insights into their evolutionary and demographic history.

The pinfish (Lagodon rhomboides) is an ecologically, economically, and culturally relevant member of the family Sparidae, playing crucial roles in the marine food webs of the western Atlantic Ocean and Gulf of Mexico. Despite their high abundance and ecological importance, there is a scarcity of genomic resources for this species. We assembled and annotated a chromosome-scale genome for the pinfish, resulting in a highly contiguous 785 Mb assembly of 24 scaffolded chromosomes. The high-quality assembly contains 98.9% complete BUSCOs and shows strong synteny to other chromosome-scale genomes of fish in the family Sparidae, with a limited number of large-scale genomic rearrangements. Leveraging this new genomic resource, we found evidence of significant expansions of dietary gene families over the evolutionary history of the pinfish, which may be associated with an ontogenetic shift from carnivory to herbivory seen in this species. Estimates of historical patterns of population demography using this new reference genome identified several periods of population growth and contraction which were associated with ancient climatic shifts and sea level changes. This genome serves as a valuable reference for future studies of population genomics and differentiation and provides a much-needed genomic resource for this western Atlantic sparid.

High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species.

With populations of threatened and endangered species declining worldwide, efforts are being made to generate high quality genomic records of these species before they are lost forever. Here, we demonstrate that data from single Oxford Nanopore Technologies (ONT) MinION flow cells can, even in the absence of highly accurate short DNA-read polishing, produce high quality de novo plant genome assemblies adequate for downstream analyses, such as synteny and ploidy evaluations, paleodemographic analyses, and phylogenomics. This study focuses on three North American ash tree species in the genus Fraxinus (Oleaceae) that were recently added to the International Union for Conservation of Nature (IUCN) Red List as critically endangered. Our results support a hexaploidy event at the base of the Oleaceae as well as a subsequent whole genome duplication shared by Syringa, Osmanthus, Olea, and Fraxinus. Finally, we demonstrate the use of ONT long-read sequencing data to reveal patterns in demographic history.

Lacustrine speciation associated with chromosomal inversion in a lineage of riverine fishes.

Geographic isolation is the primary driver of speciation in many vertebrate lineages. This trend is exemplified by North American darters, a clade of freshwater fishes where nearly all sister species pairs are allopatric and separated by millions of years of divergence. One of the only exceptions is the Lake Waccamaw endemic Etheostoma perlongum and its riverine sister species Etheostoma maculaticeps, which have no physical barriers to gene flow. Here we show that lacustrine speciation of E. perlongum is characterized by morphological and ecological divergence likely facilitated by a large chromosomal inversion. While E. perlongum is phylogenetically nested within the geographically widespread E. maculaticeps, there is a sharp genetic and morphological break coinciding with the lake-river boundary in the Waccamaw River system. Despite recent divergence, an active hybrid zone, and ongoing gene flow, analyses using a de novo reference genome reveal a 9 Mb chromosomal inversion with elevated divergence between E. perlongum and E. maculaticeps. This region exhibits striking synteny with known inversion supergenes in two distantly related fish lineages, suggesting deep evolutionary convergence of genomic architecture. Our results illustrate that rapid, ecological speciation with gene flow is possible even in lineages where geographic isolation is the dominant mechanism of speciation.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Nanopore Amplicon Sequencing Reveals Molecular Convergence and Local Adaptation of Rhodopsin in Great Lakes Salmonids.

Local adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus Coregonus) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle and MinION. We sequenced five visual opsin genes for individuals of Coregonus artedi, Coregonus hoyi, Coregonus kiyi, and Coregonus zenithicus. Comparisons revealed species-specific differences in a key spectral tuning amino acid in rhodopsin (Tyr261Phe substitution), suggesting local adaptation of C. kiyi to the blue-shifted depths of Lake Superior. Ancestral state reconstruction demonstrates that parallel evolution and "toggling" at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.

Chromosome-Level Genome Assembly of Chinese Sucker (Myxocyprinus asiaticus) Reveals Strongly Conserved Synteny Following a Catostomid-Specific Whole-Genome Duplication.

Fishes of the family Catostomidae ("suckers"; Teleostei: Cypriniformes) are hypothesized to have undergone an allopolyploidy event approximately 60 Ma. However, genomic evidence has previously been unavailable to assess this hypothesis. We sequenced and assembled the first chromosome-level catostomid genome, Chinese sucker (Myxocyprinus asiaticus), and present clear evidence of a catostomid-specific whole-genome duplication (WGD) event ("Cat-4R"). Our results reveal remarkably strong, conserved synteny since this duplication event, as well as between Myxocyprinus and an unduplicated outgroup, zebrafish (Danio rerio). Gene content and repetitive elements are also approximately evenly distributed across homeologous chromosomes, suggesting that both subgenomes retain some function, with no obvious bias in gene fractionation or subgenome dominance. The Cat-4R duplication provides another independent example of genome evolution following WGD in animals, in this case at the extreme end of conserved genome architecture over at least 25.2 Myr since the duplication. The M. asiaticus genome is a useful resource for researchers interested in understanding genome evolution following WGD in animals.

Comparative Genomic and Transcriptomic Analyses Revealed Twenty-Six Candidate Genes Involved in the Air-Breathing Development and Function of the Bighead Catfish Clarias macrocephalus.

The bighead catfish (Clarias macrocephalus) and channel catfish (Ictalurus punctatus) are freshwater species in the Siluriformes order. C. macrocephalus has both gills and modified gill structures serving as an air-breathing organ (ABO), while I. punctatus does not possess such an organ, and cannot breathe in air, providing an excellent model for studying the molecular basis of ABO development in teleost fish. To investigate the critical time window for the development of air-breathing function, seven development stages were selected based on hypoxia challenge results, and RNA-seq was performed upon C. macrocephalus to compare with the non-air-breathing I. punctatus. Five-hundred million reads were generated and 25,239 expressed genes were annotated in C. macrocephalus. Among those, 8675 genes were differentially expressed across developmental stages. Comparative genomic analysis identified 1458 C. macrocephalus specific genes, which were absent in I. punctatus. Gene network and protein-protein interaction analyses identified 26 key hub genes involved in the air-breathing function. Three top candidate genes, mb, ngb, hbae, are mainly associated with oxygen carrying, oxygen binding, and heme binding activities. Our study provides a rich data set for exploring the genomic basis of air-breathing function in C. macrocephalus and offers insights into the adaption to hypoxic environments.

Comparative Transcriptome Analysis During the Seven Developmental Stages of Channel Catfish (Ictalurus punctatus) and Tra Catfish (Pangasianodon hypophthalmus) Provides Novel Insights for Terrestrial Adaptation.

Tra catfish (Pangasianodon hypophthalmus), also known as striped catfish, is a facultative air-breather that uses its swim bladder as an air-breathing organ (ABO). A related species in the same order (Siluriformes), channel catfish (Ictalurus punctatus), does not possess an ABO and thus cannot breathe in the air. Tra and channel catfish serve as great comparative models for investigating possible genetic underpinnings of aquatic to land transitions, as well as for understanding genes that are crucial for the development of the swim bladder and the function of air-breathing in tra catfish. In this study, hypoxia challenge and microtomy experiments collectively revealed critical time points for the development of the air-breathing function and swim bladder in tra catfish. Seven developmental stages in tra catfish were selected for RNA-seq analysis based on their transition to a stage that could live at 0 ppm oxygen. More than 587 million sequencing clean reads were generated, and a total of 21,448 unique genes were detected. A comparative genomic analysis between channel catfish and tra catfish revealed 76 genes that were present in tra catfish, but absent from channel catfish. In order to further narrow down the list of these candidate genes, gene expression analysis was performed for these tra catfish-specific genes. Fourteen genes were inferred to be important for air-breathing. Of these, HRG, GRP, and CX3CL1 were identified to be the most likely genes related to air-breathing ability in tra catfish. This study provides a foundational data resource for functional genomic studies in air-breathing function in tra catfish and sheds light on the adaptation of aquatic organisms to the terrestrial environment.

Effects of CRISPR/Cas9 dosage on TICAM1 and RBL gene mutation rate, embryonic development, hatchability and fry survival in channel catfish.

The current study was conducted to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo survival, embryonic development, hatchability and early fry survival in channel catfish, Ictalurus punctatus. Guide RNAs targeting two of the channel catfish immune-related genes, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) and rhamnose binding lectin (RBL) genes, were designed and prepared. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9, medium, 5 ng gRNA/15 ng Cas9 and high, 7.5 ng gRNA/22.5 ng Cas9) were microinjected into the yolk of one-cell embryos. Mutation rate increased with higher dosages (p < 0.05). Higher dosages increased the mutation frequency in individual embryos where biallelic mutations were detected. For both genes, microinjection procedures increased the embryo mortality (p < 0.05). Increasing the dosage of gRNA/Cas9 protein increased the embryo mortality and reduced the hatching percent (p < 0.05). Embryonic development was delayed when gRNAs targeting RBL gene were injected. Means of fry survival time were similar for different dosages (p > 0.05). The current results lay the foundations for designing gene editing experiments in channel catfish and can be used as a guide for other fish species.