Reference genomes of channel catfish and blue catfish reveal multiple pericentric chromosome inversions.

BACKGROUND: Channel catfish and blue catfish are the most important aquacultured species in the USA. The species do not readily intermate naturally but F1 hybrids can be produced through artificial spawning. F1 hybrids produced by mating channel catfish female with blue catfish male exhibit heterosis and provide an ideal system to study reproductive isolation and hybrid vigor. The purpose of the study was to generate high-quality chromosome level reference genome sequences and to determine their genomic similarities and differences. RESULTS: We present high-quality reference genome sequences for both channel catfish and blue catfish, containing only 67 and 139 total gaps, respectively. We also report three pericentric chromosome inversions between the two genomes, as evidenced by long reads across the inversion junctions from distinct individuals, genetic linkage mapping, and PCR amplicons across the inversion junctions. Recombination rates within the inversional segments, detected as double crossovers, are extremely low among backcross progenies (progenies of channel catfish female × F1 hybrid male), suggesting that the pericentric inversions interrupt postzygotic recombination or survival of recombinants. Identification of channel catfish- and blue catfish-specific genes, along with expansions of immunoglobulin genes and centromeric Xba elements, provides insights into genomic hallmarks of these species. CONCLUSIONS: We generated high-quality reference genome sequences for both blue catfish and channel catfish and identified major chromosomal inversions on chromosomes 6, 11, and 24. These perimetric inversions were validated by additional sequencing analysis, genetic linkage mapping, and PCR analysis across the inversion junctions. The reference genome sequences, as well as the contrasted chromosomal architecture should provide guidance for the interspecific breeding programs.

Production of viable homozygous, doubled haploid channel catfish (Ictalurus punctatus).

Production of doubled haploids via mitotic gynogenesis is a useful tool for the creation of completely inbred fish. In order to produce viable doubled haploid channel catfish, we utilized hydrostatic pressure or thermal treatments on eggs fertilized with sperm that had been exposed to ultraviolet light. At 1.5 h post-fertilization, the embryos were exposed to either 590 kg/cm(2) hydrostatic pressure for 3 min, 37 degrees C for 5 min, or 41 degrees C for 3 min. In the pressure-treated group, only 21 offspring hatched from five spawns with family sizes of one, two, two, four, and 12 offspring each. Eight embryos from the 37 degrees C treatment and 32 embryos from the 41 degrees C treatment survived to hatch. Genotype analysis using microsatellite loci demonstrated all 21 offspring resulting from pressure treatment were homozygous at the 64 loci tested, and none contained alleles unique to the donor male. Eleven of 32 offspring from the 41 degrees C treatment were homozygous at the 18 loci tested, while 21 offspring were heterozygous at six to 12 of these loci. Again, no offspring contained alleles unique to the donor male. However, all eight offspring from the 37 degrees C treatment were heterozygous at multiple loci, and one contained unambiguous paternal alleles. These experiments demonstrated our ability to produce viable homozygous, doubled haploid channel catfish. Doubled haploid catfish can be used to create completely inbred populations for genetic analyses, and homozygous genomic templates will be useful in gene identification and genome characterization.

New data on Henneguya pellis (Myxozoa: Myxobolidae), a parasite of blue catfish Ictalurus furcatus.

The original description of Henneguya pellis, a myxozoan parasitizing blue catfish Ictalurus furcatus, is supplemented with new data on histopathology, spore morphology, and 18S small subunit (SSU) ribosomal DNA (rDNA) sequence. Plasmodia presented as both internal and external, raised, cyst-like lesions on the body wall of the peritoneal cavity and on the skin. The cysts contained numerous elongate, lanceolate myxospores, flattened parallel to the suture line. The spore body was 14.8 ± 1.1 µm (range 13.0-17.1) long and 4.8 ± 0.8 µm (range 4.0-7.4) wide in frontal view. The caudal appendages were 77.7 ± 8.8 (range 57.4-96.4) in length. There were 2 pyriform polar capsules, unequal in length, with the longer capsule measuring 7.2 ± 0.6 µm (range 6.2-8.4) in length and the shorter capsule measuring 6.5 ± 0.5 µm (range 5.5-8.0). The polar capsules were not significantly different in width, measuring 1.7 ± 0.2 µm (range 1.4-1.9). There were 8 turns in the polar filament coil. The total length of the spore was 92.5 ± 9.2 µm (range 73.3-113.5). Spore morphology and site of development are similar to that of Henneguya sutherlandi from channel catfish; however, 18S rDNA sequence data support previous findings that identify H. pellis and H. sutherlandi as 2 distinct species.

Differences in mortality, growth, lysozyme, and toll-like receptor gene expression among genetic groups of catfish exposed to virulent Edwardsiella ictaluri.

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.

Response of toll-like receptors, lysozyme, and IGF-I in back-cross hybrid (F1 male (blue x channel) x female channel) catfish challenged with virulent Edwardsiella ictaluri.

Responses of toll-like receptors (TLR3 and TLR5), lysozyme, and insulin-like growth factor-I (IGF-I) to experimental challenge with virulent Edwardsiella ictaluri were measured in back-cross hybrid (F1 male (blue x channel) x female channel) catfish. The resistance levels to E. ictaluri and host response mechanisms of back-cross hybrids are unknown. Fish were challenged with virulent E. ictaluri and sampled pre-challenge, 2 h and 2, 5, 8, 14, and 21 days post-challenge. Levels of mRNA expression of two toll-like receptors (TLR3 and TLR5) in liver, kidney, spleen, and stomach, plasma lysozyme activity, and circulating IGF-I levels were measured at each timepoint. Throughout challenge, TLR3 was expressed at higher levels than TLR5 in liver (P=0.0011) and kidney (P=0.0007) whereas TLR5 was more highly expressed than TLR3 in stomach (P=0.0032). TLR3 was upregulated in comparison to non-exposed controls in liver (P=0.0015) and stomach (P<0.0001) on day 14 and TLR5 was upregulated in liver (P=0.0175) on days 2 through 8. Plasma lysozyme activity peaked on day 5 (P<0.001) and IGF-I levels significantly decreased on days 2 through 14 (P<0.0001). TLR expression patterns suggest that both TLR3 and TLR5 may play a role in host response to bacterial challenge. Plasma lysozyme activity also increased and circulating IGF-I decreased in response to the presence of the pathogen.

Differential gene expression of IGF-I, IGF-II, and toll-like receptors 3 and 5 during embryogenesis in hybrid (channel x blue) and channel catfish.

Insulin-like growth factors-I and-II (IGF-I and IGF-II) play important roles in growth and development of mammals. Toll-like receptors (TLRs) are pattern recognition molecules that orchestrate the induction of early innate immune response by recognition of specific sequences. Evidence is growing that suggests a relationship between growth and immune function. The objective of the study was to examine changes in gene expression of IGF-I, IGF-II, TLR3, and TLR5 during embryogenesis and early larval development in hybrid (channel catfishxblue catfish) and channel catfish. Egg samples were taken pre- and post-fertilization; embryos were collected at two stages of embryogenesis, at hatch, and at swim-up. All genes were detected in unfertilized catfish eggs. Expression levels of TLR5 and IGF-I mRNA in channel catfish and expression levels of TLR3, IGF-I, and IGF-II mRNA in hybrids increased over time (P<0.01). Effect of time was not significant for expression of IGF-II or TLR3 mRNA in channel catfish and for TLR5 mRNA in hybrid catfish. Results of this study suggest growth (IGF-I and IGF-II) and immune (TLR3 and TLR5) associated genes could be functional and play important roles during embryogenesis and early development of hybrid and channel catfish.

Lipopolysaccharide regulates myostatin and MyoD independently of an increase in plasma cortisol in channel catfish (Ictalurus punctatus).

The effects of lipopolysaccharide (LPS) on plasma cortisol and the expression of MyoD and myostatin (MSTN) mRNAs were evaluated in channel catfish. In addition, the effect of dexamethasone (Dex) on MyoD and MSTN mRNAs was examined. For the LPS injection experiments, juvenile channel catfish were injected intraperitoneally with 1.5 mg/kg LPS or sterile PBS. Blood was collected at 1, 3, 12, and 24 h post-injection for cortisol determination, and muscle samples were collected at 3, 12, and 24 h for mRNA analysis. For the Dex injection experiment, fish were injected with 1.0 mg/kg Dex or saline and muscle samples were collected at 12 and 24 h. There was no effect of LPS on plasma cortisol at any of the time points measured. Injection with LPS increased the abundance of MyoD mRNA at 3 and 12 h, and decreased the abundance of MSTN mRNA at 24 h. There was no effect of Dex injection on the abundance of MyoD mRNA. However, Dex injection decreased the abundance of MSTN mRNA at 12 h post-injection. These results suggest that LPS regulates the expression of MyoD and MSTN independently of an increase in plasma cortisol, and that the regulation of MyoD in the channel catfish differs from mammals in response to inflammatory stimuli. These results also confirm that exogenous glucocorticoids decrease the expression of MSTN as shown in other fish species.