Disruption of pituitary-ovarian axis by carbofuran in catfish, Heteropneustes fossilis (Bloch)

We investigated whether carbofuran (CF), a carbamate pesticide, at sub-lethal concentration had any adverse effects on reproductive function of the Indian catfish, Heteropneustes fossilis. 17β-Estradiol content of serum and ovary of pre-spawning (P) and spawning (S) fish was reduced after sub-lethal concentration of carbofuran treatment (0.5–2 mg/ml, 30 days). After 30 days of CF treatment, the serum and ovarian vitellogenin levels of fish at the P stage were also reduced but remained unaltered in the S stage. The staining intensity of the pituitary gonadotrophs of the pre-spawning fish was significantly higher in CF-treated fish compared to controls suggesting the inability of the pituitary gonadotrophs to release gonadotropin following CF treatment. CF thus acts as an antiestrogenic, endocrine-disrupting agent in fish, possibly targeting the pituitary-gonad axis.     

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     

Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration on target tissue mRNA expression. Analysis of sequence similarities indicated two genes putatively encoding GHS-R1 and GHS-R2, respectively, which have been known to be present in zebrafish. Organization and tissue expression of the GHS-R1 gene was similar to that reported for other species, and likewise yielded two detectable mRNA products as a result of alternative splicing. Expression of both full-length, GHS-R1a, and splice variant, GHS-R1b, mRNA was highest in the pituitary. Gene organization of GHS-R2 was similar to GHS-R1, but no splice variant was identified. Expression of GHS-R2a mRNA was highest in the Brockmann bodies. GHS-R1a mRNA was detected in unfertilized eggs and throughout embryogenesis, whereas GHR-R2a mRNA was not expressed in unfertilized eggs or early developing embryos and was the highest at the time of hatching. Catfish intraperitoneally injected with catfish ghrelin-Gly had greater mRNA expression of GHS-R1a in pituitaries at 2 h and Brockmann bodies at 4 h, and of GHS-R2a in Brockmann bodies at 6 h post injection. Amidated catfish ghrelin (ghrelin-amide) had no observable effect on expression of either pituitary receptor; however, GHS-R1a and GHS-R2a mRNA expression levels were increased 4 h post injection of ghrelin-amide in Brockmann bodies. This is the first characterization of GHS-R2a and suggests regulatory and functional differences between the two catfish receptors.     

Daily rhythms in the somatotropic axis of Senegalese sole (Solea senegalensis): The time of day influences the response to GH administration

Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase–activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28–0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture.     

Receptor sites for amino acids in the facial taste system of the channel catfish

1. Receptor sites for different amino acids in the facial taste system of the channel catfish, Ictalurus punctatus, were determined from in vivo electrophysiological cross-adaptation experiments. 2. Relatively independent receptor sites were indicated for L-proline, D-proline, D-arginine, L-histidine and L-lysine, as well as those previously reported for L-alanine, L-arginine and D-alanine. 3. The functional isolation of two nerve twigs that were more responsive to D-alanine than to L-alanine or to other test stimuli provided further evidence for the existence of D-alanine sites that are independent from those to L-alanine. 4. Under all cross-adaptation regimes, the taste responses to the majority of test stimuli were reduced. Various possible mechanisms accounting for this generalized reduction in action potential activity during adaptation are discussed.     

Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df

The complement system is important in both innate and adaptive host defense against microbial infection in vertebrates. It contains three pathways: the classical, alternative, and lectin pathways. Complement component factors B and D are two crucial proteases in the alternative pathway. In this study, the genes of complement factors Bf/C2 and Df from channel catfish, Ictalurus punctatus were identified and characterized. Two complement factor B-related genes, Bf/C2A and Bf/C2B, and factor D gene Df were identified. Phylogenetic analysis suggested that Bf/C2A and Bf/C2B is likely orthologous to factor B and factor C2, respectively. Southern blot results suggested that these three genes are all single-copy genes in the catfish genome. The catfish Bf/C2A, Bf/C2B and Df genes were genetically mapped on linkage group 3, 20 and 29, respectively. Bf/C2A and Bf/C2B are highly expressed in liver and kidney, while Df is highly expressed in gill and spleen. After infection with Edwardsiella ictaluri, the expression of Bf/C2A, Bf/C2B and Df genes were found to be remarkably induced in the gill, liver, spleen and kidney at some sampling times, indicating that these three complement factors play a pivotal role in immune responses after the bacterial infection in catfish.     

Resistance of the European catfish (Silurus glanis) to channel catfish virus

European catfish (Silurus glanis) fingerlings (2 to 4 g each) were tested for susceptibility to channel catfish virus (CCV). They had supported CCV replication at 2 days after intraperitoneal injection with 0.1 ml of saline containing 10⁵ TCID₅₀. Homogenized visceral organs (liver, kidney and spleen) contained 10⁴ TCID₅₀/0.1 ml at 2 days post inoculation (PI) but at 4 days the titer decreased to 10¹ TCID₅₀. Bathing European catfish in CCV yielded only one positive sample with à titer of 10^0.83 TCID₅₀ per 0.1 ml of tissue. No clinical signs of CCV developed and no virus related deaths occurred.     

Effects of pH and temperature on myocardial calcium-activation in Pterygoplichthys (catfish)

Fish are chronically exposed to a wide range of temperatures and acidic environments. Fish hearts have to therefore adapt to these changes in order to maintain contractility. Myofibrillar responsiveness to Ca²⁺ is exquisitely sensitive to both temperature and pH in mammalian myocardium. To evaluate myofilament calcium-activation, we chemically skinned ventricular myocardium from catfish (Pterygoplichthys). A decrease in pH from 7.5 to 6.8, irrespective of temperature change, shifted the calcium-force curve towards higher calcium concentrations without affecting maximal Ca²⁺-activated force. The contractile elements are therefore sensitive to changes in pH. In intact muscle preparations the active twitch force was decreased with increasing temperature (10–22 °C). However, the sensitivity of the myofilaments to Ca²⁺ was independent of temperature. These data suggest a possible role of the sarcoplasmic reticulum (SR) in mediating the effects of temperature. The response of intact muscle preparations to changes in temperature is therefore not likely due to temperature-dependent changes in myofilament calcium responsiveness. Accepted: 11 May 1998     

Humoral immune responses of channel catfish (Ictalurus punctatus) fry and fingerlings exposed toEdwardsiella ictaluri

The ability of channel catfish to develop antibody responses to Edwardsiella ictaluri was evaluated. Fish were produced and reared under specific pathogen free (SPF) conditions. At the ages of 1, 2, 3 and 4 weeks and 2, 3, 4, 5, and 6 months post-hatch, a group of these naive fish were given a primary immersion exposure to E. ictaluri (mean doses of 6·4×10⁴ cfu ml⁻¹of tank water). Each group received a secondary immersion exposure 4 weeks after the primary exposure and were sampled 2 weeks after each exposure. Control groups were exposed to sterile culture medium. Specific antibody titres were first detected in fish exposed at 4 weeks post-hatch at an average weight of 85 mg. A secondary response was first demonstrated by fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. However, a true boosting effect was first demonstrated in fish that received their primary exposure at 2 months of age. Fish given a primary exposure before 4 weeks of age and a secondary exposure failed to produce a significant antibody response, even though they were the same age at secondary exposure as fish that produced strong antibody responses upon primary exposure. This phenomenon suggests that immunological tolerance was induced and indicates that channel catfish may not be capable of generating a humoral immune response before four weeks of age.     

Identification and characterization of the tumor suppressor p53 in channel catfish (Ictalurus punctatus)

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

Afferent connections of the optic tectum in channel catfish Ictalurus punctatus

Summary Horseradish peroxidase was injected unilaterally into the optic tectum of the channel catfish, Ictalurus punctatus. The sources of tectal afferents were thereby revealed by retrogradely labeled neurons in various brain centers. Retrogradely labeled cells were seen in both the ipsilateral and contralateral telencephalon. The superficial pretectal area was labeled on both sides of the brain. Ipsilateral projections were also observed coming from the entopeduncular nucleus. Both the anterior thalamic nucleus and the ventro-medial thalamic nucleus projected to the ipsilateral optic tectum. Cells in the ipsilateral nucleus of the posterior commissure were seen to project to the tectum. Labeled fibers were visualized in the lateral geniculate nucleus ipsilateral to the injected tectum, however, no labeled cell bodies were observed. Therefore, tectal cells project to the lateral geniculate nucleus, but this projection is not reciprocal. No labeled cells were found in the cerebellum. Labeled cells occurred in both the ipsilateral and contralateral medial reticular formation; they were also observed in the ipsilateral nucleus isthmi. A projection was seen coming from the dorsal funicular nucleus. Furthermore, labeled cells were shown in the inferior raphe nucleus.Abbreviations AP Area pretectalis - C Cerebellum - DPTN Dorsal posterior tegmental nucleus - H Habenula - IRF Inferior reticular formation - LI Inferior lobe - LGN Lateral geniculate nucleus - LR Lateral recess - MB Mammillary body - MRF Medial reticular formation - MZ Medial zone of the telencephalon - NC Nucleus corticalis - NDL-M Nucleus opticus dorsolateralis/pars medialis - NI Nucleus isthmi - NPC Nucleus of the posterior commissure - OPT Optic tectum - OT Optic tract - PC Posterior commissure - PN Pineal organ - PrOP Preoptic nucleus - PT Pretectum - TBt Tectobulbar tract - TEL Telencephalon - TL Torus longitudinalis - TS Torus semicircularis - VC Valvula cerebelli - VLTN Ventrolateral thalamic nucleus - VMTN Ventromedial thalamic nucleus     

Retinofugal and retinopetal connections in the upside-down catfish (Synodontis nigriventris)

Summary The retinofugal and retinopetal connections in the upside-down catfish Synodontis nigriventris were studied by use of the horseradish-peroxidase (HRP) techniques, autoradiography, and degeneration-silver methods. An unusual retinal projection to the torus semicircularis as well as projections to the retina from three different sources in the brain are described. After intra-ocular injections of HRP, labeled cells were found in the optic tectum, the dorsomedial optic nucleus and one of the pretectal nuclei. These new findings support the basic hypothesis (i) that neuronal connections are more extensive in primitive brains, and (ii) that the evolutionary development of more complex brains involves the loss of some selected connections.     

Ultrastructure of cell types of the olfactory epithelium in a catfish,Heteropneustesfossilis (Bloch)

Transmission electron microscopical study of olfactory epithelium of a mud-dwelling catfish,Heteropneustes fossilis (Bloch) shows receptor, supporting, goblet and basal cells. The receptor cells are of ciliated and microvillous type. Both ciliated and microvillous receptor cells are provided with olfactory knob. The dendrite of all the receptor cells bears many longitudinally arranged microtubules. Occurrence of the rod cell and its function is quite debatable. Specialized juctional complexes between the receptor and adjacent cells are clearly noted. The supporting cells are both ciliated and nonciliated. The ciliated supporting cells are responsible for water ventilation in the olfactory chamber as well as in the inter-lamellar spaces. This facilitates better perception of odours by the receptor cells. In addition to providing mechanical support to other cells, the nonciliated supporting cells also have a secretory function which is evident from the present study. The different stages of maturity of goblet cells are well documented. The presence of white cells in the olfactory epithelium is a very rare finding.     

Persistence of gentian violet and leucogentian violet in channel catfish (ictalurus punctatus) muscle after water-borne exposure

Gentian violet is a triphenylmethane dye that is an antifungal/antiparastic agent. GV is similar to malachite green that has been used in the aquaculture industry for treatment or prevention of external fungal and parasitic infections in fish and fish eggs although it (MG) is not approved for this use. For these reasons, GV’s potential for misuse by the aquaculture industry is high. The uptake and depletion of gentian violet (GV) were determined in channel catfish (Ictalurus punctatus) after water-borne exposure (100 ng ml⁻¹, 1 h) under simulated aquaculture farming conditions. Leucogentian violet (LGV) was rapidly formed, concentrated in the muscle tissue, and very slowly eliminated from muscle tissue. An isocratic (60% acetonitrile–40% water; 0.05 M ammonium acetate buffer, pH 4.5) HPLC system consisting of a 5 μm LC–CN 250×4.6 mm I.D. column, a 20×2.0 mm I.D. PbO₂ oxidative post-column, and a UV–VIS detector set at 588 nm were used to determine uptake and depletion of tissue residues of GV and LGV with time. GV was rapidly depleted and converted to its major metabolite, LGV, which was detected out to 79 days. Therefore, LGV is the appropriate target analyte for monitoring exposure of channel catfish to GV.     

Distribution of γ-aminobutyric acid in catfish (Heteropneustes fossilis) forebrain in relation to season, ovariectomy and E2 replacement, and effects of GABA administration on plasma Gonadotropin-II level

In the catfish Heteropneustes fossilis, the hypothalamus and telencephalon showed seasonal variations in γ-aminobutyric acid (GABA) with high levels in prespawning and spawning phases and low levels in preparatory and postspawning phases. Ovariectomy for 4 and 5 weeks reduced significantly the GABA contents only in the hypothalamus. Replacement with E₂ (1 μg/g BW) restored the levels to that of sham ovariectomized or parallel control group. Treatment with GABA (i.p.; 10 or 50 μg/g body weight (BW)) alone did not produce any significant effect on plasma gonadotropin-II (GTH-II) level in any of the seasons. Injection of GABA, but not baclofen (a GABA_B agonist), stimulated GTH-II secretion in pimozide or GnRH analogue-pimozide pretreated fish at both 0.5 and 2 h in early prespawning phase except at 0.5 h in the pimozide—GABA (10 μg) group. This stimulatory effect was not evident in other seasons. The results of the present study suggest that Estradiol-17β (E₂) seems to stimulate GABA which may account for its high level in the recrudescent phase. GABA seems to have a permissive role in GTH-II secretion when dopamine receptor function is inhibited.     

Variations in hepatic estradiol-17beta receptor concentrations during the annual reproductive cycle of diploid and triploid female catfish, Heteropneustes fossilis (Bloch)

Concentrations of hepatic estradiol-17beta (E2) receptors (ER) in cytosolic and nuclear fractions were evaluated in diploid and triploid female catfish Heteropneustes fossilis (Bloch) during four different reproductive periods of a complete reproductive cycle. Basal level of ER concentration was noted in the resting period of both diploids and triploids. Receptor level gradually elevated through the preparatory period and reached a peak in the pre-spawning period in both diploids and triploids. However, ER concentrations were overall reduced in triploid to that of diploid females. In a single point assay, in diploids, ER concentration showed about a 3-fold rise (p<0.001) in the cytosolic and a 4-fold rise (p<0.001) in the nuclear extracts from resting to the pre-spawning period. In triploids, only a 2-fold rise was observed both in cytosolic (p<0.01) and nuclear (p<0.05) ER concentration during the same span. Finally, a sudden fall of receptor level was observed in the spawning period in both the ploidy groups with a lower concentration in the triploids. The K(d) value did not differ between the females of diploids (cytosolic 1.12+/-0.21 nM and nuclear 6.9+/-0.9 nM) and triploids (cytosolic--1.13+/-0.17 nM, nuclear--6.8+/-2 nM). However, B(max) of the diploid showed about double the value than triploid females both in the cytosolic (diploid--367.4+/-33.24 pmol/mg protein, triploid--187.3+/-13.20 pmol/mg protein, p<0.001) and nuclear extracts (diploid--946+/-66 pmol/mg DNA, triploid-558+/-98 pmol/mg DNA, p<0.01) of liver. Lower E2 binding capacity and lower amount of E2 receptors of triploid catfish liver with a stunted vitellogenic status could be one of the major causes for reduced gonadal development and sterility in female triploids.