Researches:

Article title: Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus
Authors: Michael Coogan, Veronica Alston, Baofeng Su, Karim Khalil, Ahmed Elaswad, Mohd Khan, Andrew Johnson, De Xing, Shangjia Li, Jinhai Wang, Rhoda M. C. Simora, Cuiyu Lu, Patrick Page-McCaw, Wenbiao Chen, Max Michel, Wenwen Wang, Darshika Hettiarachchi, Tasnuba Hasin, Ian A. E. Butts, Roger D. Cone & Rex A. Dunham
Publication title: Marine Biotechnology 24:843–855, 2022

Abstract:
Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.
Full text available upon request to the author/s

Article title: CRISPR/Cas9 - Mediate knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)
Authors: De Xing, Baofeng Su, Max Bangs, Shangjia Li, Jinhai Wang, Logan Bern, Rhoda Mae C Simora, Wenwen Wang, Xiaoli Ma, Michael Coogan, Andrew Johnson, Yi Wang, Zhenkui Qin, Rex Dunham
Publication title: Aquaculture 560:738531, 2022

Abstract:
Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.
Full text available upon request to the author/s

Article title: Response of cecropin transgenesis to challenge with Edwardsiella ictaluri in channel catfish Ictalurus punctatus
Authors: Nermeen Y. Abass, Rhoda Mae C. Simora, Jinhai Wang, Shangjia Li, De Xing, Michael Coogan, Andrew Johnson, David Creamer, Xu Wang, Rex A. Dunham
Publication title: Fish & Shellfish Immunology 126:311-317, 2022

Abstract:
Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (β-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catfish family transgenic for cecropin transgene driven by the common carp β-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.
Full text available upon request to the author/s

Article title: CRISPR/Cas-9 induced knockout of myostatin gene improves growth and disease resistance in channel catfish (Ictalurus punctatus)
Authors: Michael Coogan, Veronica Alston, Baofeng Su, Karim Khalil, Ahmed Elaswad, Mohd Khan, Rhoda M.C. Simora, Andrew Johnson, De Xing, Shangjia Li, Jinhai Wang, Cuiyu Lu, Wenwen Wang, Darshika Hettiarachchi, Tasnuba Hasin, Jeffery Terhune, Ian A.E. Butts, Rex A. Dunham
Publication title: Aquaculture 557:738290, 2022

Abstract:
Channel catfish, Ictalurus punctatus, is the most produced aquaculture species in the United States, although production levels have decreased since their peak in 2003. Myostatin (mstn) regulates skeletal muscle growth and has been identified as an important gene for increasing body weight in aquaculture species. In this study, the effects of CRISPR/Cas9 knockout of the mstn gene in channel catfish were investigated. Three sgRNAs targeting exon 1 of the channel catfish myostatin gene were microinjected with Cas9 protein in embryos. A total of 209 fish survived to 6 mph after microinjection of Cas9 and sgRNA targeting exon 1 of the mstn gene over the 3 yr experiment (2017–2019) with an average mutation rate of 58%. Successful generation of myostatin knockout (MSTN KO) F1 heterozygotes was achieved in 2019 by individually mating two pairs of control females with MSTN homozygous KO males. The offspring of both families inherited the mutation at a mean rate of 88%. Growth was generally higher in MSTN mutants than controls at all life stages and environments. P1 MSTN mutants were 88% larger than controls at the stocker stage (100 to 200 g) and 27% larger than controls at market size. Heterozygous F1 mutants were 218% larger than controls at the stocker stage. MSTN mutants had reduced overall expression levels of mstn compared to controls. When challenged with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, MSTN mutants performed equally or better than controls. With a high mutation rate and inheritance as well as improved growth and disease resistance, using MSTN gene-edited channel catfish could greatly benefit commercial farms.
Full text available upon request to the author/s

Article title: Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens
Authors: Rhoda Mae C. Simora, Wenwen Wang, Michael Coogan, Nour El Husseini, Jeffery S. Terhune, Rex A. Dunham
Publication title: Journal of Aquatic Animal Health 33(2), 2021

Abstract:
One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin-injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.
Full text available upon request to the author/s

Article title: CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
Authors: Rhoda Mae C. Simora, De Xing, Max R. Bangs, Wenwen Wang, Xiaoli Ma, Baofeng Su, Mohd G. Q. Khan, Zhenkui Qin, Cuiyu Lu, Veronica Alston, Darshika Hettiarachchi, Andrew Johnson, Shangjia Li, Michael Coogan, Jeremy Gurbatow, Jeffery S. Terhune, Xu Wang & Rex A. Dunham
Publication title: Scientific Reports 10(1), 2020

Abstract:
CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.
Full text available upon request to the author/s

Article title: Occurrence of Histamine and Histamine-forming Bacteria in Philippine Traditional Dried-salted Fish Products
Authors: Rhoda Mae C. Simora and Ernestina M. Peralta
Publication title: Asian Fisheries Science 3:73–88, 2018

Abstract:
Twenty-one dried-salted fish products sold at major retail markets in the province of Iloilo, Philippines were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, moisture content, water activity (Aw), total volatile basic nitrogen (TVBN), aerobic plate count (APC), Escherichia coli, Salmonella and Staphylococcus aureus in all samples ranged from 6.36 to 6.71, 2.55 % to 15.84 %, 21.12 % to 49.99 %, 0.67 to 0.87, 19.25 to 69.05 mg.100 g-1 , 3.0 to 7.39 log CFU.g-1 , <3 to 28 MPN.g-1 , absent in 25 g and 3.58 to 6.89 log CFU.g-1 , respectively. Ten (47.6 %) dried fish samples had histamine levels greater than the United States Food and Drug Administration guideline of 5 mg.100 g-1 for scombroid fish and/or scombroid products, whereas seven (33.3 %) samples contained histamine levels greater than 20 mg.100 g-1 which is sufficient to cause the symptoms of scombroid poisoning according to the Centers for Disease Control and Prevention. Seven histamine-forming bacterial strains were isolated and identified biochemically and morphologically as belonging to genus Vibrio, Salmonella, Staphylococcus, Bacillus and Pseudomonas. The presence of these bacteria in dried fish is indicative of poor standards of process hygiene and sanitation as well as mishandling during storage.
Full text available upon request to the author/s

Article title: Isolation and Identification of Protease-Producing Pseudomonas sp. PD14 in the Gut of Rabbitfish Siganus guttatus (Bloch 1787)
Authors: Cindy D. Armada and Rhoda Mae C. Simora
Publication title: Asian Fisheries Science 29:82-95, 2016

Abstract:
Bacterial enzymes associated with the gut of fish are known to aid in digestion and nutrition of the host. Isolation, identification and characterisation of protease-producing bacteria from the gut of rabbitfish Siganus guttatus (Bloch 1787) were carried out in the present study. Protease-producing bacteria were isolated in peptone gelatin agar (PGA) plates and the isolated strains were qualitatively and quantitatively screened for enzyme production. Highest protease activity, 25.32±1.06 U. mg-1 protein, was observed in bacterial isolate PD14. Biochemical and molecular analysis revealed that the isolate is 99% homologous to Pseudomonas sp. The 16S rRNA gene sequence of the isolate was deposited in GenBank with accession number KR779515. Qualitative tests on enzyme production through measurement of the zone of hydrolysis further suggest that optimum protease production was 36 h at 40 o C, pH 7-8 in a peptone gelatin agar with 2% NaCl.
Full text available upon request to the author/s

Article title: Deproteination and demineralization of shrimp waste using lactic acid bacteria for the production of crude chitin and chitosan
Authors: Francisco, Farramae C; Simora, Rhoda Mae C; Nuñal, Sharon N.  Aquaculture, Aquarium, Conservation & Legislation; Cluj-Napoca
Publication title: AACL Bioflux 33(1):9, 2015

Abstract:
Deproteination and demineralization efficiencies of shrimp waste using two Lactobacillus species treated with different carbohydrate sources for chitin production, its chemical conversion to chitosan and the quality of chitin and chitosan produced were determined. Using 5% glucose and 5% cassava starch as carbohydrate sources, pH slightly increased from the initial pH of 6.0 to 6.8 and 7.2, respectively after 24 h and maintained their pH at 6.7 to 7.3 throughout the treatment period. Demineralization (%) in 5% glucose and 5% cassava was highest during the first day of treatment which was 82% and 83%, respectively. Deproteination (%) was highest in 5% cassava starch on the 3rd day of treatment at 84.4%. The obtained chitin from 5% cassava and 5% glucose had a residual ash and protein below 1% and solubility of 59% and 44.3%, respectively. Chitosan produced from 5% cassava and 5% glucose had protein content below 0.05%; residual ash was 1.1% and 0.8%, respectively. Chitosan solubility and degree of deacetylation were 56% and 33% in 5% glucose and 48% and 29% in 5% cassava, respectively. The advantage this alternative technology offers over that of chemical extraction is large reduction in chemicals needed thus less effluent production and generation of a protein-rich liquor, although the demineralization process should be improved to achieve greater degree of deacetylation.
Full text available upon request to the author/s

Article title: Characterization of extracellular enzymes from culturable autochthonous gut bacteria in rabbitfish (Siganus guttatus) 1
Authors: Rhoda Mae C. Simora, Rex Ferdinand M. Traifalgar, Francis S. Legario
Publication title: ELBA Bioflux 7(1), 2015

Abstract:
The intestinal microenvironment of bacteria in fish influences the host in many ways, including the metabolism of several nutrients. Isolation, enzymatic activities, and molecular identification of culturable bacteria associated with the gastrointestinal tract of rabbitfish, Siganus guttatus Bloch, were investigated in the present study. Occurrence and distribution of enzyme-producing bacteria in the proximal (PI), middle (MI), and distal (DI) gut segments were determined and data were presented as log viable counts g-1 intestine (LVC). The heterotrophic bacterial population (LVC = 6.95) as well as the amylolytic, proteolytic, cellulolytic, and carrageenolytic populations had the highest occurrence in the DI region (LVC = 5.73, 5.46, 3.21 and 5.00, respectively). The isolates were qualitatively screened on the basis of their extracellular enzyme producing ability. The selected strains were further quantitatively assayed for amylase, protease, and cellulase activities. Results of enzymatic studies revealed that three most promising bacterial isolates possess multienzyme activities and were studied through 16S rRNA gene sequence for identification. Isolates CP6,CM8 and PM14 showed high similarity to Bacillus cereus, Bacillus megaterium and Bacillus flexus, respectively. The NCBI Genbank accession numbers of the 16S rRNA gene sequences for isolates CP6, CM8, and PM14 were KR779513, KR779512, and KR779516, respectively. To date, this is the first time that characterization and enzymatic activities of gut bacteria in rabbitfish have been reported. The present study might offer scope for further research to evaluate prospects for application of the gut-associated extracellular enzyme-producing bacteria in fish nutrition.
Full text available upon request to the author/s

Article title: Molecular cloning and antiviral activity of IFN-? promoter stimulator-1 (IPS-1) gene in Japanese flounder, Paralichthys olivaceus
Authors: Rhoda Mae C. Simora, Maki Ohtani, Jun-ichi Hikima, Hidehiro Kondo, Ikuo Hirono, Tae Sung Jung, Takashi Aoki
Publication title: Fish & Shellfish Immunology 29(6):979-86, 2010

Abstract:
The mitochondrial adaptor, IFN-β promoter stimulator-1 (IPS-1), also known as MAVS/VISA/Cardif, plays a key role in the signal transduction of the RIG-1/MDA5 pathway to induce the production of interferons (IFNs) and other cytokines. In the present study, Japanese flounder (Paralichthys olivaceus) IPS-1 cDNA was cloned from Japanese flounder spleen using PCR-based methods. The full-length cDNA has 2235 nucleotides and encodes a polypeptide of 641 amino acids. The putative Japanese flounder IPS-1 protein contains an N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to other teleost counterparts ranging from 20% to 34%. Semi-quantitative RT-PCR showed that Japanese flounder IPS-1 mRNA was expressed in all tissues examined. The expression level of flounder IPS-1 gene was unchanged in viral hemorrhagic septicemia virus (VHSV)-infected kidney as measured by quantitative real-time PCR (Q-PCR). In addition, Japanese flounder IPS-1-overexpressing cells were protected against hirame rhabdovirus (HIRRV) and VHSV infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral titers. Expression of IFN-inducible genes including Mx, ISG15 and IRF3 were also induced in the IPS-1-overexpressing cells. These results suggest that Japanese flounder IPS-1 is involved in the antiviral immunity as a one of the adaptors in fish IFN-activation pathway.
Full text available upon request to the author/s