Highlights • Simulated gastric acid and pepsin were used to digest LHRHa in vitro. • Two small fragments from in vitro digestion of LHRHa were quantitatively analyzed using UPLC–MS. • LHRHa was completely digested in the simulated human stomach. • LHRHa applied as a fish spawning aid would not pose a human food safety risk. Abstract Luteinizing hormone releasing hormone analog (LHRHa, des Gly10, [D-Ala6] ethylamide) is routinely applied for induced spawning of fish. Simulated gastric acid and pepsin were used to mimic human digestion in vitro to determine the stability of LHRHa upon possible ingestion. In vitro cleavage of LHRHa was quantified using UPLC–MS, ultra-performance liquid chromatography-mass spectrometry. LHRHa was broken down mainly into two fragments from Glp-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (LHRHa) to Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 1) and Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 2). By 24 h of incubation, LHRHa was completely digested or barely detectable if the starting material was at 250 ng/μl. If the starting concentration was at 5 ng/μl, LHRHa was completely digested by 5 h of incubation, or earlier. In both extreme scenarios, the results indicated that LHRHa would be digested completely in the human stomach and would pose no risk in human food consumption. This study supports the assertion that LHRHa used as a spawning aid would not pose a human food safety risk.

Adoption of the hybrid catfish (channel catfish, Ictalurus punctatus, female x blue catfish I. furcatus, male) is increasing in the catfish industry. The most effective way to produce fry is hormone induced spawning of females coupled with hand stripping and in vitro fertilization. The success of common carp pituitary extract (CPE), luteinizing hormone releasing hormone analog (LHRHa) injections and LHRHa implants for producing hybrid catfish fry was investigated over the past 15 years. Data from 65 studies, 25, 20 and 20 for CPE, LHRHa injections, and LHRHa implants, respectively, were evaluated. Mean fry/kg female body weight (all females) produced was 948, 2483 and 2765 for CPE, LHRHa injections, and LHRHa implants, respectively. LHRHa administered as an injection or implant produced more (P<0.001) fry/kg than CPE. LHRHa implants and CPE (CV=37.5) gave a more consistent result (CV=35.9) than LHRHA injections (CV=49.9). LHRHa is advantageous for ovulating channel catfish for producing hybrid catfish fry compared to CPE. This advantage is even more important if brood fish, space or time are limiting. (C) 2012 Published by Elsevier B.V.

Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

Common carp, Cyprinus carpi, is one of the most widely grown aquaculture species. Four types of knockdown constructs designed to transgenically sterilize fish were electroporated into common carp embryos to knockdown or eliminate expression of primordial germ cell (PGC) proteins, nanos or dead end. Embryos from different developmental stages were collected and tested using quantitative real-time PCR. Relative levels of gene expression were compared among control embryos and embryos exposed to these constructs during development. Two proteins, nanos and dead end, were targeted, each specific for a set of constructs, and a third related, but non-targeted PGC protein, vasa, was assayed. The effectiveness of the constructs varied depending upon the promoter. The expression levels of these proteins naturally decreased during development. In some, but not all cases, the expression of targeted mRNA was knocked down to minimal levels or degradation was accelerated. Up-regulation also was observed for dead end, possibly due to interferon responses. In other cases, the sterilization constructs had little effect on the targeted gene, but strongly down-regulated one or two of the alternative PGC genes examined in this study. At 19 months of age, common carp males exposed to these constructs had reduced rates of sexual maturity; however, reduction of female rates of sexual maturity was not conclusively shown. These constructs appear promising for transgenic sterilization of common carp. Published by Elsevier B.V.

Abstract Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3′ nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms.

Cathepsin F (CTSF) is a recently described papain-like cysteine protease and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin-inactivated V. vulnificus whole-cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot. (C) 2017 Elsevier Ltd. All rights reserved.

Putative spermatogonia A from a fresh-cell isolate or a density-gradient-centrifuged isolate from the testes of Blue Catfish Ictalurus furcatus were transplanted into the gonads of triploid Channel Catfish I. punctatus. The cells were introduced into gonads of the host via catheterization (2 x 10(4)-1.43 x 10(6) cells) or by surgically inserting the cells directly into the gonad (7 x 10(4)-1.25 x 10(5) cells). Ten months after implantation, DNA was analyzed from biopsies of the gonads and seven of eight males were found to be xenogenic, having Blue Catfish cells in their gonads. The xenogenic males successfully courted normal Channel Catfish that had been induced with hormones to ovulate, but none of the eggs hatched, indicating inadequate sperm production, an inability to ejaculate, and/or low sperm quality. Male xenogenic catfish treated with luteinizing hormone releasing hormone analog had well-developed testes, and sperm production was detected in three of seven xenogenic males examined 2 years after transplantation. Sperm were removed from a male that had been surgically transplanted with Blue Catfish cells and used to fertilize eggs from a hand-stripped Channel Catfish female. One percent of these eggs hatched. All seven surviving 6-month-old progeny of this male had the external morphology, swim bladder shape, nuclear DNA profile, and mitochondrial DNA profile of female Channel Catfish x male Blue Catfish F-1 hybrids, indicating that this triploid Channel Catfish male produced Blue Catfish sperm. This is the first report of successful production of xenogenic catfish and the first report of producing 100% hybrid progeny using xenogenesis in fish.

The mucosal surfaces of fish serve as the first line of defense against the myriad of aquatic pathogens present in the aquatic environment. The immune repertoire functioning at these interfaces is still poorly understood. The skin, in particular, must process signals from several fronts, sensing and integrating environmental, nutritional, social, and health cues. Pathogen invasion can disrupt this delicate homeostasis with profound impacts on signaling throughout the organism. Here, we investigated the transcriptional effects of virulent Aeromonas hydrophila infection in channel catfish skin, Ictalurus punctatus. We utilized a new 8 x 60 K Agilent microarray for catfish to examine gene expression profiles at critical early timepoints following challenge-2 h, 8 h, and 12 h. Expression of a total of 2,168 unique genes was significantly perturbed during at least one timepoint. We observed dysregulation of genes involved in antioxidant, cytoskeletal, immune, junctional, and nervous system pathways. In particular, A. hydrophila infection rapidly altered a number of potentially critical lectins, chemokines, interleukins, and other mucosal factors in a manner predicted to enhance its ability to adhere to and invade the catfish host. (C) 2012 Elsevier Ltd. All rights reserved.

Fish are covered by a watery gel-mucus, mainly secreted by the goblet cells, serving as the physical and biochemical barrier between the external environment and the interior milieu, playing more important roles in fish that without scale. Despite the important roles of mucus in fish immunity, the knowledge of detailed molecular events happened during infection process is still limited. While most studies were focused on characterizing the protein and enzyme activities in the mucus following challenge, no studies have examined the gene expression profiles in fish mucus. In this regard, herein we carried out the first gene profiling analysis in catfish mucus using real-time PCR. Ten important immune-related genes were selected according to our previous studies. Their expression levels were examined in the early timepoints (namely, 1 h, 2 h, 4 h, 8 h, and 24 h) following Flavobacterium columnare challenge. Notably, expression levels of most of the selected genes were rapidly altered by the challenge. Seven genes were down-regulated, while only three genes were up-regulated. In addition, the gene expression patterns in mucus were very different from the mucosal surfaces (skin, gill and intestine) and the classical immune organs (liver, spleen and kidney). The unique expression patterns obtained here may be resulted from the great advantage of the large amount of attached bacteria in the mucus than the internal tissues, and resulted from the bacteria virulent actors to suppress the host immune response. Taken together, our results can expand our knowledge of fish mucosal immunity, and the un-lethal mucus sampling can provide early insight for developing the strategies for selection of disease resistant families and strains in catfish as well as other fish species. Copyright =C2=A9 2015 Elsevier Ltd. All rights reserved.

Salinity tolerance of yolk-sac larvae and swim-up fry of channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and hybrid catfish (channel catfish female x blue catfish male) was investigated at 0, 3, 6, and 9 ppt salinity. One-hundred percent mortality occurred at 9 ppt. Survivals were different (P b 0.05) by day 3 post hatch at 3 ppt, (P b 0.05) with blue catfish N hybrid catfish N channel catfish for salinity tolerance. Long-term survival of hybrid fry was better than that of the parent species at 0 ppt salinity, but highest for channel catfish at 3 ppt and 6 ppt with the hybrid being intermediate. NaCl had a negative effect on survival rate of yolk sac larvae starting at 3 ppt and for swim-up fry at 6 ppt. Genotype x environment interactions occurred for growth as hybrids grew faster than channel catfish and blue catfish at 0 ppt. Raising salinity to 3 ppt greatly increased the growth rate (50-75%) of channel catfish and blue catfish, but only slightly (10%) for hybrid catfish, which were still larger than the parent species. 6 ppt was detrimental to yolk-sac larvae and swim-up fry. These results help identify the saline environments that might be suitable for long-term growth and disease resistance of ictalurid catfish, and reveals changes in salinity tolerance during development. Statement of relevance: The life stages of channel, blue and hybrid catfish were not previously studied for tolerance of sodiumchloride and genotype-environment interations were observed indicating the ideal environment for culture or disease treatment varies among these genetic groups. Heterosis of the hybrids almost disappeared at 3 ppt and growth of channel catfish was greatly enhanced at 3 ppt. Greater Ich resistance of hybrids compared to parents at 0 ppt contradicted earlier reports at older life stages. (C) 2016 Elsevier B. V. All rights reserved.

Channel catfish (Ictalurus punctatus) has been recognized as a dominant freshwater aquaculture species in the United States. It is also a suitable model for studying the mechanisms of sex determination and differentiation because of its sexual plasticity and exhibition of both genetic and environmental sex determination. The testicular differentiation in male channel catfish normally starts between 90 and 102 days postfertilization (dpf), while the ovarian differentiation starts early from 19 dpf. As such, efforts to better understand the postponed testicular development at the molecular level are needed. Toward that end, we conducted transcriptomic comparison of gene expression of male and female gonads at 90, 100, and 110 dpf using high-throughput RNA-Seq. Transcriptomic profiles of male gonads on 90 and 100 dpf exhibited high similarities except for a small number of significantly up-regulated genes that were involved in development of germ cell-supporting somatic cells, while drastic changes were observed during 100-110 dpf, with a group of highly up-regulated genes that were involved in germ cells development, including nanog and pou5f1. Transcriptomic comparison between testes and ovaries identified male-preferential genes, such as gsdf, cxcl12, as well as other cytokines mediated the development of the gonad into a testis. Co-expression analysis revealed highly correlated genes and potential pathways underlying germ cell differentiation and spermatogonia stem cell development. The candidate genes and pathways identified in this study set the foundation for further studies on sex determination and differentiation in catfish as well as other teleosts.

Luteinizing hormone releasing hormone analog (LHRHa, des Gly10, [D-Ala6] ethylamide) is routinely applied for induced spawning of fish. Simulated gastric acid and pepsin were used to mimic human digestion in vitro to determine the stability of LHRHa upon possible ingestion. In vitro cleavage of LHRHa was quantified using UPLC MS, ultra-performance liquid chromatography-mass spectrometry. LHRHa was broken down mainly into two fragments from Glp-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (LHRHa) to Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 1) and Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 2). By 24 h of incubation, LHRHa was completely digested or barely detectable if the starting material was at 250 ng/mu l. If the starting concentration was at 5 ng/mu l, LHRHa was completely digested by 5 h of incubation, or earlier. In both extreme scenarios, the results indicated that LHRHa would be digested completely in the human stomach and would pose no risk in human food consumption. This study supports the assertion that LHRHa used as a spawning aid would not pose a human food safety risk. (C) 2015 Elsevier Ltd. All rights reserved.