Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus ), blue catfish ( I. furcatus) , and their F1, F2, and backcross hybrids

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish × blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish × blue catfish F1 hybrids (channel catfish female × blue catfish male; blue catfish female × channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection. Received: 10 September 1997 / Accepted: 10 December 1997     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Respiratory-cardiovascular physiology and chloroethane gill flux in the channel catfish, Ictalurus punctatus

A fish respirometer-metabolism chamber was used to obtain in vivo respiratory-cardiovascular and chloroethane gill flux data on transected channel catfish (Ictalurus punctatus). Methods used for spinal transection, attachment of an oral membrane (respiratory mast), placement and attachment of blood cannulas and urine catheters are described. Respiratory physiology, cardiac output and chemical extraction efficiencies for 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) were determined on 419–990 g catfish. The overall mean values (± s.d.) for ventilation volume (Q_v), effective respiratory volume (Q_w), oxygen consumption (Vo₂ and percentage utilization of oxygen (U) were 17-3 ±4–71 h^?1 kg^?1, 9·8±l·71 h^?1 kg^?1, 71·6±12·5mg h^?1 kg^?1, and 49± 10%, respectively, while cardiac output calculated via the Fick Method was 2·4±0·61 h^?1 kg^?1. Additional measurements were made on ventilation rate (V_r), total plasma protein, haematocrit (Hct), and urine volume; while both arterial and venous blood were analysed for pH, oxygen partial pressure (P02), carbon dioxide partial pressure (Pco₂), total oxygen (To₂), total carbon dioxide (Tco₂) and total ammonia (TAMM). Physiological measurements taken at 24 h were not significantly different from those taken at 48 h and indicated no deterioration of the in vivo preparation. All of these values agreed well with literature values on UTitransected channel catfish, except for Hct which was lower for cannulated animals used in this study. Overall, these data provide strong support for the use of transected channel catfish for in vivo collection of physiological and chemical gill flux data. The mean initial chemical extraction efficiencies for TCE, PCE and HCE were 41, 61 and 73%, respectively. Chemical clearances (Cl_X) for these same three chemicals were 5·9, 9·3 and 10·8 1 h^?1 kg^?1, respectively. The approximate 1: 1 relationship between effective respiratory volume (Q_w) and chemical clearance (Cl_x) indicated that branchial uptake of PCE and HCE was water flow-limited. Chemical gill flux observed for channel catfish and chloroethanes was similar to that observed for rainbow trout in previous studies and provided further support for the flow-limited model of chemical flux across fish gills.     

Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus)

Aim: To develop a method for conducting pulsed-field gel electrophoresis (PFGE) on Flavobacterium columnare, to use PFGE to characterize F. columnare channel catfish isolates, and to determine whether variation in pathogenic potential exists in F. columnare isolates from channel catfish. Methods and Results: On the basis of PFGE-derived profiles, similarity dendrograms constructed for more than 30 F. columnare isolates showed two major genetic groups with more than 60% similarity. Channel catfish fingerlings challenged with PFGE group A isolates by bath immersion had significantly higher average mortalities (>60%) than fish challenged with PFGE group B isolates (<9%). However, abrasion and skin mucus removal made channel catfish fingerlings susceptible to disease caused by group B isolates following immersion exposure. Conclusion: Our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, and that isolates in PFGE group A isolates tend to be more pathogenic to immunocompetent channel catfish fingerlings than PFGE group B isolates. Significance and Impact of the Study: PFGE is a potentially useful tool for determining whether F. columnare isolates are more likely to be primary or secondary pathogens. Pathogenesis research for columnaris disease in catfish should focus on pathogenic isolates from PFGE group A.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

Plasma corticosteroid dynamics in channel catfish, Ictalurus punctatus (Rafinesque), during and after oxygen depletion

Plasma corticosteroid concentrations in channel catfish, Ictalurus punctatus , (normally 1.0 ± 0.3 μg/100 ml) increased significantly (to 5.9 ± 1.2μg/100 ml) in response to acute oxygen depletion and then returned to control levels within 30 min after the dissolved oxygen concentration was increased; however, a secondary increase in plasma corticosteroid levels was observed 6 h after exposure. Corticosteroid levels also increased in fish exposed to dissolved oxygen concentration of <0.2 mg/1 for three days. Methylene blue was not effective in preventing interrenal response to low dissolved oxygen. No diurnal plasma corticosteroid rhythm was observed in fish exposed to diurnal chemical rhythms of culture ponds.     

Histophathology and electron microscopy of channel catfish virus in infected channel catfish,Ictalurus punctatus (Rafinesque)*

A histologic and electron microscopic study was made on selected organs from channel catfish Ictalurus punctatus (Rafinesque) fingerlings that were experimentally infected with channel catfish virus (CCV). Histopathology was characterized by necrosis and haemorrhage in kidney and liver, and haemorrhage in the spleen and gastrointestinal tract. Virus replication occurred in nuclei of cells in the kidney, liver and spleen. Intranuclear inclusion bodies consisting of geometric crystalline arrays and lamellar structures were associated with virus replication.     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resist-ant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866), DNA fingerprint technique, amplified ribo-somal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes ofAcinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high com-plementarity, and could be a useful tool in Acinetobacter genomic species identification.     

Vole preference for Salix caprea, S. repens, and their F1, F2, and backcross hybrids

In recent years, the influence of plant hybridization on plant–animal interactions has become an important issue; however, these studies have mainly focused on insects or slugs and to a large degree ignored mammalian herbivores despite their importance in many ecosystems. This study aims to determine the preference of voles for pure Salix caprea , S. repens , and their F1, F2, and backcross hybrids to evaluate whether voles select against hybrids in natural willow populations. To address this, we conducted two field studies and a cafeteria experiment in the laboratory with bank voles ( Clethrionomys glareolus ). We found no strong indications of reduced resistance in any of the hybrid taxa. Voles damaged more S. repens than S. caprea individuals. There was a general decreasing trend of utilization from pure S. repens to pure S. caprea , hybrid taxa being intermediate between the parents. Thus, voles seemingly do not have a more detrimental effect on hybrid fitness than on the fitness of pure individuals and probably will not select against hybrids in these willows populations.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae), Part 3: Analysis of the RAPD and ISSR molecular markers

The Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) molecular markers were used to complement the study of chromosomal polymorphism in Astyanax fasciatus (Teleostei, Characidae) from the Mogi-Guaçu River (Southeastern Brazil), analyzed in three collection sites along the river (Ouro Fino – MG, Cachoeira de Emas – SP and Barrinha – SP). Two cytotypes (or karyotypic types), denominated standard cytotypes, were previously characterized, one including 2n = 46 chromosomes and the other 2n = 48 chromosomes, where all the chromosomes of the complement form homologous pairs. Additionally, variant karyotypic forms with 2n = 45, 46 and 47 chromosomes were also detected, although with a lower frequency in relation to the standard cytotypes. RAPD turned out little informative in the analysis of the observed situation, indicating a high value of migrants per generation among the cytotypes. On the other hand, ISSR showed a small structure, especially among the standard cytotypes from the Barrinha region where the Nm was 0.4301 with a genetic identity of 0.6862 and genetic distance of 0.3765. However, the general results obtained do not discard the possibility of interbreeding between both standard cytotypes and/or their descendants as a source of chromosome variation. The association between the cytogenetic and molecular markers viabilized putative explanatory scenery for the origin and evolution of the forms seen in A. fasciatus.     

Inheritance of esterases in the white crappie (Pomoxis annularis), black crappie (P. nigromaculatus), and their F1 and F2 interspecific hybrids*

White crappie, Pomoxis annularis, and black crappie, P. nigromaculatus, were hybridized to produce Fi and F2 generations. There was considerable tissue specific esterase gene function within each parental species. Many of the esterases were homologous but possessed different electrophoretic mobilities between the two species which permitted their use as gene markers. The pattern of inheritance and expression of a number of esterase genes was determined in the Fi and F2 interspecific hybrids. Although certain key morphological phenotypes characteristic of the black crappie predominate in the Fi hybrid, the enzyme phenotypes reflect the contributions of both co-dominant parental genomes. There is a higher percentage of heterozygotes than expected in the F2 generation which may be indicative of heterosis. The genetic analysis also provided a better understanding of the polypeptide composition of these esterases.     

Effect of fasting on nychthemeral concentrations of plasma growth hormone (GH), insulin-like growth factor I (IGF-I), and cortisol in channel catfish (Ictalurus punctatus)

This experiment was conducted to characterize the effect of fasting versus satiety feeding on plasma concentrations of GH, IGF-I, and cortisol over a nychthemeron. Channel catfish fingerlings were acclimated for two weeks under a 12L:12D photoperiod, then fed or fasted for 21 d. On day 21, blood samples were collected every 2 h for 24 h. Weight of fed fish increased an average of 66.2% and fasted fish lost 21.7% of body weight on average. Average nychthemeral concentrations of plasma GH were not significantly different between fed (24.7 ng/mL) and fasted (26.8 ng/mL) fish, but average nychthemeral IGF-I concentrations were higher in fed (23.4 ng/mL) versus fasted (17.8 ng/mL) fish. An increase in plasma IGF-I concentrations was observed in fasted fish 2 h after a peak in plasma GH, but not in fed fish. Average nychthemeral plasma cortisol concentrations were higher in fed (14.5 ng/mL) versus fasted (11.0 ng/mL) fish after 21 d. Significant fluctuations and a postprandial increase in plasma cortisol were observed in fed fish and there was an overall increase in plasma cortisol of both fasted and fed fish during the scotophase. The present experiment indicates little or no effect of 21-d fasting on plasma GH levels but demonstrates fasting-induced suppression of plasma IGF-I and cortisol levels in channel catfish.     

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

Transgenic Research - 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...