High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Allometry and Scaling of the Intraocular Pressure and Aqueous Humour Flow Rate in Vertebrate Eyes

In vertebrates, intraocular pressure (IOP) is required to maintain the eye into a shape allowing it to function as an optical instrument. It is sustained by the balance between the production of aqueous humour by the ciliary body and the resistance to its outflow from the eye. Dysregulation of the IOP is often pathological to vision. High IOP may lead to glaucoma, which is in man the second most prevalent cause of blindness. Here, we examine the importance of the IOP and rate of formation of aqueous humour in the development of vertebrate eyes by performing allometric and scaling analyses of the forces acting on the eye during head movement and the energy demands of the cornea, and testing the predictions of the models against a list of measurements in vertebrates collated through a systematic review. We show that the IOP has a weak dependence on body mass, and that in order to maintain the focal length of the eye, it needs to be an order of magnitude greater than the pressure drop across the eye resulting from gravity or head movement. This constitutes an evolutionary constraint that is common to all vertebrates. In animals with cornea-based optics, this constraint also represents a condition to maintain visual acuity. Estimated IOPs were found to increase with the evolution of terrestrial animals. The rate of formation of aqueous humour was found to be adjusted to the metabolic requirements of the cornea, scaling as Vac0.67, where V_ac is the volume of the anterior chamber. The present work highlights an interdependence between IOP and aqueous flow rate crucial to ocular function that must be considered to understand the evolution of the dioptric apparatus. It should also be taken into consideration in the prevention and treatment of glaucoma.     

Goldfish's visual information processing patterns in food-reinforced discrimination learning between compound visual stimuli

Summary To examine how goldfish process and store information on compound visual stimuli, goldfish were trained with visual discriminative stimuli composed of varied colors and patterns using a Y-maze instrumental conditioning technique. The fish showed some very different types of information processing patterns depending upon the degree of discrimination difficulty of each constituent aspect (color and pattern). Those trained with compound stimuli with both a more easily discriminated aspect and a more difficult aspect learned the former selectively but did not learn the latter at all in spite of a high rate of training. Contrary to this, the fish trained with compound stimuli composed of two aspects with similar degrees of discrimination difficulty learned both aspects. In this case, only when fish were trained with compound stimuli composed of relatively more difficult aspects did they learn to discriminate between the stimuli more rapidly than the fish in each group trained with the constituent colored or patterned stimuli. These results were discussed in relation to visual processing patterns reported in other species and the mechanism of aspect selection.Abbreviations CP compound stimuli composed of VR and HB (7-mm ISBBs) - Cp compound stimuli composed of VR and HB (14-mm ISBBs) - cP compound stimuli composed of VG and HB (7-mm ISBBs) - cp compound stimuli composed of VG and HB (14-mm ISBBs) - HB horizontal patterns on blue background - HGra horizontal patterns on gray background - ISBBs interval spaces between bars - VG vertical patterns on green background - VGra vertical patterns on gray background - VR vertical patterns on red background     

Evolution of Duplicated <Emphasis Type="BoldItalic">reggie</Emphasis> Genes in Zebrafish and Goldfish

Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented. Received: 24 January 2001 / Accepted: 27 July 2001     

The effects of temperature on metabolic interaction between digestion and locomotion in juveniles of three cyprinid fish (Carassius auratus, Cyprinus carpio and Spinibarbus sinensis)

To test whether the effects of temperature on the metabolic mode changed among different fish species, we investigated the specific dynamic action (SDA) and swimming performance of fasting and fed fish at 15 and 25°C in three juvenile Cyprinidae fish species: goldfish (Carassius auratus), common carp (Cyprinus carpio) and qingbo (Spinibarbus sinensis). Both taxon and temperature had significant effects on the resting oxygen consumption rate (M˙O(rest)), SDA and swimming performance (p<0.05). In addition, the effect of temperature differed significantly among the different species (interaction effect, p<0.05). Under the low temperature condition, digestion had no effect on either critical swimming speed (U(crit)) or the active MO(2) (MO(active)) for all fish species (additive metabolic mode). When the temperature was increased from 15 to 25°C, the metabolic scope (MS) for digestion increased approximately 182, 49 and 17%, and the MS for locomotion increased approximately 129, 58 and 138% in goldfish, common carp and qingbo, respectively. The total metabolic demands for both digestion and locomotion (i.e., the sum of digestive MS and locomotive MS) increased approximately 143, 56 and 112% in goldfish, common carp and qingbo, respectively. The total MS for both digestion and locomotion (the difference between MO(active) in fed fish and MO(rest) in fasting fish) increased approximately 106, 58 and 78% in goldfish, common carp and qingbo, respectively. Thus, the MS for locomotion in fed goldfish decreased due to the large increase in digestive function at the high temperature, and the U(crit) of fed goldfish decreased by 11% compared to that of fasting fish (p<0.05) (digestion-priory metabolic mode). The metabolic mode of qingbo changed to locomotion-priority mode, as illustrated by the large increase in locomotive MS in response to the increase in temperature. In the common carp, temperature had no effect on metabolic mode as illustrated by the parallel increases in cardio-respiratory capacity and metabolic capacity of digestive and locomotive organs. A discussion on the changes in metabolic mode in response to temperature and its possible relationship with the metabolic characteristics of a given fish species was also documented in this paper.     

The Causes of Resource Monopolization: Interaction Between Resource Dispersion and Mode of Competition

The degree of monopolization of resources is thought to be higher in groups that compete by interference than by exploitation. However, the monopolization of resources will presumably depend on (1) whether the dispersion of resources is economically defendable, and (2) whether some competitors have the ability to defend these resource distributions and hence capitalize on this potential. We tested for an interaction between the effects of temporal resource dispersion and aggressiveness on the degree of resource monopolization in a foraging system. Two species of fish differing markedly in aggressiveness (high: convict cichlids, Archocentrus nigrofasciatus; low: goldfish, Carrasius auratus) were allowed to compete intra‐specifically in groups of four for food that was either potentially defendable (arrived asynchronously) or not (arrived synchronously). As predicted, the monopolization of food, measured as the coefficient of variation of food eaten within groups, was significantly higher in the defendable than in the undefendable treatment for convict cichlids but not for goldfish. However, the monopolization of food was higher in the non‐aggressive goldfish than in the aggressive convict cichlids. Future studies should quantify and compare the monopolization in species that compete primarily via scramble competition to those that use primarily resource defence.     

Sub-cellular Localisation of the White/Scarlet ABC Transporter to Pigment Granule Membranes Within the Compound Eye of Drosophila Melanogaster

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg⁻¹ body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 × 10⁷ cells ml⁻¹). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.     

Experiments on the Feeding Behavior of the Hematophagous Candiru, Vandellia cf. Plazaii

Hematophagous candirus (family Trichomycteridae Vandellia cf. plazaii) did not respond to potential chemical attractants (ammonia amino acids fresh fish slime and human urine) added to their aquarium water. They seemed to respond visually to live goldfish Carassius auratus and Amazonian cichlids Cichlasoma amazonarum. One hypothesis tested is that the goldfish (an ostariophysan) might be preferred to the cichlid (a perciform fish). Goldfish were attacked first significantly more often when both prey species were present (one-sided difference in proportions test, N = 15, p = 0.02). However neither the elapsed time to the first attack nor the duration of the attacks differed significantly (Mann-Whitney's U-test N = 32 p = 0.27 and p = 0.92). These two species seemed incapable (goldfish) or weakly capable (cichlid) of avoiding or repelling candiru attacks     

Genes encoding giant danio and golden shiner ependymin

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, trueepn genes have been characterized in one order of teleost fish,Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the completeepn genes,gd (1480 bp), andsh (2071 bp), fromCypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid, genome. Each gene was found to contain six exons and five introns. Genegd encodes a predicted 218-amino acid (aa) protein GD 93% conserved to goldfish EPN, whilesh encodes a predicted 214-aa protein SH 91% homologous to goldfish. Evidence is presented classifying proteins previously termed “EPNs” into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark features of true EPNs.     

Occurrence of partenites in mollusks and the influence that metacercaria of <Emphasis Type="Italic">Apophallus muehlingi</Emphasis> (Jagerskiold, 1898) and <Emphasis Type="Italic">Posthodiplostomum cuticola</Emphasis> (Nordmann, 1832) has on some biochemical parameters in fish

A heavy parasitic load from trematode partenites of Apophallus muehlingi (Jagerskiold, 1898) was observed in the mollusk population of Lithoglyphus naticoides (Pfeiffer) after its natural invasion into the Rybinsk Reservoir. An elevated concentration of glycogen was detected in the muscles of infected fish, both in the natural populations and under experimental conditions. Interspecific differences were found for the protein concentrations when elevated concentrations were observed in the muscles of the infected roach, and no differences were discovered for the goldfish (both infected and parasite-free). The ties between the biochemical parameters and motor activity are discussed for the infected fish.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Brain function in antarctic fish: frequency response analysis of central vestibular units

Summary Extracellular single-unit recordings were made from central vestibular units responding to horizontal head rotation in antarctic fish (Pagothenia borchgrevinki) at temperatures below 0 °C. The frequency of head rotation was varied between 0.05–16 Hz and the results were analysed in terms of the phase and gain of the response with respect to the stimulus. The model of the peripheral vestibular system derived by Hartmann and Klinke (1980) was fitted to the data from antarctic fish in order to obtain a quantitative comparison of vestibular function at two different temperatures. Despite the 20 °C temperature difference, and the different recording sites (primary afferents in the goldfish, and central vestibular units in the antarctic fish) vestibular function in antarctic fish is comparable to that previously reported in goldfish.     

Morphological and Physiological Correlates of Evolutionary Reduction of Metabolic Rate among Amblyopsid Cave Fishes

I report on multiple correlates of reduction in metabolic rate during evolutionary adaptation to caves in the fish family Amblyopsidae. The family includes six species with one surface-dweller, Chologaster cornuta, a facultative cave dweller, Chologaster agassizi, and four obligate cave-dwellers that, on the basis of eye degeneration, have been isolated in caves for increasing times: Typhlichthys subterraneus Amblyopsis spelaea Amblyopsis rosae Speoplatyrhinus poulsoni. Of those traits I examined, the strongest correlates of reduction in whole fishes metabolic rate were reduction in ventilation frequency and volume > brain metabolic rate > gill surface area. Relative amplitude declined and the estimated ventilation minute-volume decreased 5.9 fold from a value of 1.12 to 0.19 ml. Log brain rate of oxygen consumption was directly related to log body mass, b = + 0.75, and decreased from 1.5 to 0.06 per mg dry mass per hour among species. No single gill trait accounted for the reduction in total lamellar surface area from 228 to 103 mm² g^–1. There was no reduction in muscle metabolic rate or histological indices of thyroid activity. Log muscle rate of oxygen consumption was inversely related to log body mass, b = –0.60, but did not differ among species. The mean total thyroid follicle volume among species, from 0.010 to 0.020 mm³ g^–1 of adult fishes, showed no relation to interspecific differences in whole fish metabolic rate. In the general discussion I show that no trait that could contribute to a lowered metabolic rate is in the same rank order as metabolic rate, though collectively the reductions were in the same order as metabolic rates. I explain that this is as expected with convergent evolution of complex traits. Finally I discuss the literature about central nervous system control of metabolic rate.     

Whole-body metabolism of the organophosphorus pesticide, fenthion, in goldfish, Carassius auratus

The in vivo metabolism of fenthion, an organophosphorus pesticide, and its sulfoxide (fenthion sulfoxide) was examined in goldfish (Carassius auratus). When goldfish were administered fenthion i.p. at a dose of 100 mg/kg, two metabolites were isolated from the tank water. They were identified as fenthion sulfoxide and fenthion oxon, in which > P = S of fenthion is transformed to > P = O, by comparing their mass and UV spectra, and their behavior in HPLC and TLC, with those of authentic standards. However, fenthion sulfone was not detected as a metabolite. The amounts of fenthion, fenthion sulfoxide and fenthion oxon excreted within 4 days were 2.7, 3.4 and 2.5%, of the initial dose of fenthion, respectively. Unchanged fenthion was detected in the body of the fish to the extent of 42-50% of the dose after 10 days, but fenthion sulfoxide and fenthion oxon showed very low concentrations. When fenthion sulfoxide was administered to the fish, about 70% of the dose was excreted unchanged into the tank water within 24 h, but little of the reduced compound, fenthion, was found. In contrast, fenthion was detected at 2.1% of dose in the body of goldfish as a metabolite of fenthion sulfoxide. The fact that fenthion is metabolized to the toxic oxon form in fish presumably has environmental and health implication for its use as a pesticide.     

Phototropism in fishes,and its relation to the results obtained by eye-dislocation

Summary In the case of viviparous perch and goldfish, unilateral blinding causes a tilting on the anterior-posterior axis towards the normal eye, the amount of tilting being dependent upon the intensity of light. With a sudden change to a greater intensity of light, the fish circle toward the blind eye, and upon sudden reduction of the intensity they often circle toward the normal eye. The similarity of this reaction to the effect of unilateral blinding of insects is offered as an explanation.It is suggested that these results explain the experiments of Pearcy and Koppanyi. These workers dislocated one eye of a goldfish and removed the other one, explaining the consequent tilting of the fish as due to an attempt of the animal to regain its normal visual field. Since the fish was now totally blind, due to the effect of the operation, no tilting was present. As the dislocated eye. gradually recovered its vision, the fish began to tilt, reaching maximal tilting several weeks afterwards. However, since I have produced tilting by unilateral blinding alone, it seems evident that the results obtained by those two experimenters were also due to unilateral blinding, and not to dislocation of the eye.I am indebted to Dr. A. R. Moore both for the suggestion of the problem and for advice during the experiments.     

Evidence of cylindrospermopsin uptake and clearance in fish (Oreochromis niloticus) under laboratory conditions

Cylindrospermopsin (CYN) is a cyanotoxin that has raised serious concerns about public health in many parts of the world. It can bioaccumulate and affect the health of aquatic organisms, but despite this, few studies have been conducted on CYN uptake and clearance in fish. In this paper, the authors evaluate the uptake and clearance of CYN in the muscle tissue and viscera of juvenile tilapia (Oreochromis niloticus) after exposure to aqueous extracts and whole cells of Cylindrospermopsis raciborskii (CYN-producer). CYN blended with commercial fish food, and three experiments were conducted. In the first trial, fish food, and aqueous extracts containing 0.31 μg CYN g⁻¹ of food per day, was administered to tilapia for 15 days. In the second trial, fish were provided food and intact cells (5.4 μg CYN g⁻¹ of food per day) for 15 days. In the last trial, they were provided fish food and aqueous extracts (0.8 μg CYN g⁻¹ of food per day) for 12 days, and for the next 10 days, the animals were fed food without toxic cell extracts (to simulate a clearance period). The concentration of CYN in muscle tissue and viscera was analysed using ELISA. In the case of juvenile tilapia, the presence of CYN was higher in viscera than in muscle tissue, and the toxin remained in the tissues even after 10 days without the addition of contaminated food. The results suggest that tilapia represents a potential source of CYN transfer through the food web, and this shows the need for a continuous monitoring of this compound in organisms that are used for human and animal consumption.