Platyfish Xiphophorus Maculatus Research Articles

Assessment of various standard fish diets on gut microbiome of platyfish Xiphophorus maculatus.

Diet is an external factor that affects the physiological baseline of research animals. It can shape gut microbiome, which can impact the host. As a result, dietary variation can challenge experimental reproducibility and data integration across studies when not appropriately considered. To control for diet-induced variation, reference diets have been developed for common biomedical models. However, such reference diets have not yet been developed for nontraditional model organisms, such as Xiphophorus species. In this study, we compared two diets designed for zebrafish, a commercial zebrafish diet (Gemma and GEM), and a proposed zebrafish reference diet developed by the Watts laboratory at the University of Alabama at Birmingham (WAT) to the Xiphophorus Genetic Stock Center custom diet (CON) to evaluate the influence of diet on the Xiphophorus gut microbiome. Xiphophorus maculatus were fed the three diets from 2 to 6 months of age. Feces were collected and the gut microbiome was assessed using 16S rRNA sequencing every month. We observed substantial diet-driven variation in the gut microbiome. Our results indicate that diets developed specifically for zebrafish can affect the gut microbiome composition and may not be optimal for Xiphophorus.

Do tanque de piscicultura para o córrego muitos anos atrás: dinâmicas da história de vida de poecilídeos ferais no Brasil

Abstract Aim We investigated trophic and reproductive traits of feral guppy Poecilia reticulata and southern platyfish Xiphophorus maculatus in a headwater creek located in the Paraíba do Sul River basin, southeastern Brazil. Methods Fish were collected bimonthly from January to December 2017 using sieve in Lopes Creek. Stomach items were analyzed and developmental stages of females were classified as 1 (Non-gravid), 2 (Yolked ova), 3 (Eyed embryos), and 4 (Mature embryos). The mean values of fecundity and population structure for each developmental stage were determined for females using six length classes. Bimonthly mean values of gonadosomatic index of females were overlayed with average bimonthly rainfall, water level/temperature, and the sex ratio was calculated. The juvenile-to-adult proportion was calculated bimonthly, and the phenotype (ornamental-type × wild-type) was also analyzed. Results The guppy and southern platyfish fed, primarily, on detritus, and both species showed reproductive activity during all the sampling period. The mean fecundity of P. reticulata and X. maculatus was high and the smallest gravid female of guppy measured 0.8 cm SL, and southern platyfish 1.0 cm. The GSI of both species showed peaks coinciding with the highest rainfall, water level/temperature, and females were significantly more frequent than males. Juveniles were found during all the sampling period, and both species were represented by wild-type forms. Conclusions P. reticulata and X. maculatus presented opportunistic feeding, long reproductive period, high fecundity and early maturity. The year-round presence of reproductive females, juveniles and wild-type phenotypes suggests establishment of populations in the study area. We hypothesize that the introduction of both species has occurred several years ago, and the continued exposure to predation seems to have molded their life-history traits and phenotype.

Toxicidad en Xiphophorus maculatus por cipermetrina

El Arrivo®6EC es un insecticida utilizado para el control de plagas, como hormigas y garrapatas. Su ingrediente activo es la cipermetrina, en este caso como agente contaminante que emulsifica en el agua. Este ingrediente presenta un riesgo ambiental de alta toxicidad en peces y organismos acuáticos. El objetivo de este experimento fue evaluar la toxicidad de peces platy Xiphophorus maculatus al ser expuestos a la cipermetrina. Dado que el Arrivo®6EC es utilizado comúnmente en Panamá, se realizó este estudio para comprobar las afectaciones causadas en los peces platys. Las pruebas se realizaron de manera duplicada para dos concentraciones de cipermetrina, las cuales fueron 1 mg/L y 2.5 mg/L. Los peces presentaban un tamaño y peso promedio de 28.5 cm y 0.33g respectivamente, y una edad de dos a tres semanas aproximadamente. En las observaciones realizadas en las diferentes horas, los síntomas en los peces variaban desde: poca habilidad para nadar, movimiento errático, movimiento solo con estimulación, disnea, natación erguida y en espiral, hasta la muerte. Después de morir, los aspectos más notados fueron: degeneración de tejidos superficiales y colas, lesiones hiperémicas, signos hemorrágicos y pigmentación en la superficie corporal. Se comprobó que incluso en concentraciones de 1 mg/L resultan en una alta tasa de mortalidad de este contaminante letal para la especie Xiphophorus maculatus. Se recomienda iniciar con valores de concentraciones por debajo de 1 mg/L, para obtener mejores resultados, y tomándose en cuenta la edad, el tamaño y el peso, pues de esto depende los efectos toxicológicos.

β-Carotene-enriched Artemia as a natural carotenoid improved skin pigmentation and enhanced the mucus immune responses of platyfish Xiphophorus maculatus

The present study investigated the effects of enriched Artemia franciscana with β-carotene of Dunaliella salina on color, skin carotenoid, and mucosal immune responses of platyfish, Xiphophorus maculatus. To this end, one hundred and eighty platyfish with an initial weight of 0.9 ± 0.03 g were randomly assigned into four groups in triple and were daily fed on three times. The first and second groups were fed with commercial food (control) and unenriched A. franciscana (Un-EA), respectively. In addition, the third and fourth groups were fed with enriched A. franciscana with castor oil (oil-EA) and enriched A. franciscana with β-carotene (β-carotene EA) for 8 weeks, respectively. Based on results, the highest amount of carotenoids (112.4 ± 22.79 μg g−1) was significantly observed in the group fed with β-carotene EA while the lowest amount (24.31 ± 2.36 μg g−1) was found in the group fed Un-EA. As regards lysozyme, total immunoglobulin, and alkaline phosphatase activity of the mucus, the highest amounts were detected in fish with β-carotene EA as 84.5 ± 19.09 mg dL−1, 6.4 ± 0.41 mg dL−1, and 477 ± 39.59 U L−1, respectively. Based on skin colorization results, the group fed with β-carotene EA had no significant difference regarding the transparency, yellow color, and opacity, while a* index significantly increased in this group in comparison with the control group. However, the Hue index significantly decreased in the β-carotene EA group compared with the control group. In conclusion, A. franciscana enriched with β-carotene of D. salina enhanced the mucosal immune responses and improved the skin carotenoids as well as a* index in platyfish X. maculatus.

Colorful invasion in permissive Neotropical ecosystems: establishment of ornamental non-native poeciliids of the genera Poecilia/Xiphophorus (Cyprinodontiformes: Poeciliidae) and management alternatives

ABSTRACT Headwater creeks are environments susceptible to invasion by non-native fishes. We evaluated the reproduction of 22 populations of the non-native livebearers guppy Poecilia reticulata, black molly Poecilia sphenops, Yucatan molly Poecilia velifera, green swordtail Xiphophorus hellerii, southern platyfish Xiphophorus maculatus, and variable platyfish Xiphophorus variatus during an annual cycle in five headwater creeks located in the largest South American ornamental aquaculture center, Paraíba do Sul River basin, southeastern Brazil. With few exceptions, females of most species were found reproducing (stages 2, 3, 4) all year round in the creeks and gravid females of all species showed small sizes indicating stunting. Juveniles were frequent in all sites. The fecundity of the six poeciliids was always low in all periods. The sex ratio was biased for females in most species, both bimonthly as for the whole period. Water temperature, water level and rainfall were not significantly correlated with reproduction in any species. Therefore, most populations appeared well established. The pertinence of different management actions, such as devices to prevent fish escape, eradication with rotenone and research about negative effects on native species, is discussed in the light of current aquaculture practices in the region.

A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon

BackgroundTeleost fish present a high diversity of sex determination systems, with possible frequent evolutionary turnover of sex chromosomes and sex-determining genes. In order to identify genes involved in male sex determination and differentiation in the platyfish Xiphophorus maculatus, bacterial artificial chromosome contigs from the sex-determining region differentiating the Y from the X chromosome have been assembled and analyzed.ResultsA novel three-copy gene called teximY (for testis-expressed in Xiphophorus maculatus on the Y) was identified on the Y but not on the X chromosome. A highly related sequence called texim1, probably at the origin of the Y-linked genes, as well as three more divergent texim genes were detected in (pseudo)autosomal regions of the platyfish genome. Texim genes, for which no functional data are available so far in any organism, encode predicted esterases/lipases with a SGNH hydrolase domain. Texim proteins are related to proteins from very different origins, including proteins encoded by animal CR1 retrotransposons, animal platelet-activating factor acetylhydrolases (PAFah) and bacterial hydrolases. Texim gene distribution is patchy in animals. Texim sequences were detected in several fish species including killifish, medaka, pufferfish, sea bass, cod and gar, but not in zebrafish. Texim-like genes are also present in Oikopleura (urochordate), Amphioxus (cephalochordate) and sea urchin (echinoderm) but absent from mammals and other tetrapods. Interestingly, texim genes are associated with a Helitron transposon in different fish species but not in urochordates, cephalochordates and echinoderms, suggesting capture and mobilization of an ancestral texim gene in the bony fish lineage. RT-qPCR analyses showed that Y-linked teximY genes are preferentially expressed in testis, with expression at late stages of spermatogenesis (late spermatids and spermatozeugmata).ConclusionsThese observations suggest either that TeximY proteins play a role in Helitron transposition in the male germ line in fish, or that texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.

Gene Amplification and Functional Diversification of Melanocortin 4 Receptor at an Extremely Polymorphic Locus Controlling Sexual Maturation in the Platyfish

In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus-pituitary-gonadal axis.

Zisupton--A Novel Superfamily of DNA Transposable Elements Recently Active in Fish

Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid- and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.

Mucus Cells in the Digestive Tract in Prenatal Platyfish Larvae (Poeciliidae, Teleostei)

The appearance and histochemical properties of mucus cells in various parts of digestive tract in prenatal larvae of platyfish (Xiphophorus maculatus), a stomachless teleost, are described and compared. Such cells occurred regularly throughout the entire epithelial cell layer in mouth, pharynx, oesophagus and intestine in 6 mm long larvae i.e. approximately 1 week before delivery, but were very scarce in 4 mm long larvae. The mucin in these cells was strongly stained by alcian blue at pH 2.5 and weakly coloured by this dye at pH 1.0. It displayed a clean blue colour after high iron diamine followed by alcian blue (pH 2.5), except in some very few cells which displayed a purple-brown colour after this treatment. The mucus cells displayed various colours between purple-blue and magenta when the alcian blue (pH 2.5) was followed by periodic acid-Schiff (PAS) solution; the magenta-coloured cells were mainly confined to oesophagus. Together these findings suggest that the mucin in the digestive tract of prenatal platyfish consists mainly of non-sulphated, carboxylated glycoconjugates. The observations from the present lectin histochemistry suggest that the amount of galactosamine and glucosamine in this mucin varies much from cell to cell. Also, the various colours after PAS suggest a significant cellular heterogeneity in the mucus cell population. This may reflect different stages in development and maturation or a true and permanent cellular heterogeneity, suggesting that this mucus is able to play a number of important roles even a week before the larvae start to feed and digest.

Evolution of melanocortin receptors in teleost fish: The melanocortin type 1 receptor

The melanocortin type 1 receptor (Mc1r) belongs to a family of G-protein-coupled receptors involved in various physiological processes in vertebrates. Melanocortins, the Mcr natural agonists, are pituitary peptide hormones including adrenocorticotropin and melanocyte-stimulating hormones. In mammals and birds, Mc1r is involved in pigmentation and expressed in melanocytes and melanoma. Activation of Mc1r leads to eumelanin production as well as to proliferation and survival of melanocytes in the epidermis. Here we report the molecular and evolutionary analysis of mc1r from three major fish models, the zebrafish Danio rerio, the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. In contrast to some other melanocortin receptor genes, mc1r has been conserved as a single copy gene in divergent fish species. Its expression was detected in all organs tested in platyfish and medaka but was restricted to eyes, skin, brain and testis in zebrafish, this possibly reflecting differences in the distribution of extracutaneous melanophores. The mc1r gene was found to be expressed during embryogenesis, as well as in Xiphophorus hybrid melanoma, similar to human tumours. Protein sequence comparison between fish and mammalian Mc1r revealed a remarkable concordance between evolutionary and functional analyses for the identification of residues and regions critical for receptor function.

Lipofuscin-like pigments in endocardial cells enveloping the heart muscle trabeculae in platyfish (Poeciliidae: Teleostei)

Large amounts of lipofuscin-like pigments were demonstrated by means of the Schmorl’s technique in endocardial cells enveloping the heart muscle trabeculae in adult platyfish (Xiphophorus maculatus). Such pigment granules occurred in a low number in the corresponding cell layers in the heart of 6-mm long prenatal platies. The endocardial cells contained numerous lysosome-like bodies, bristle-coated vesicles, and tubules of agranular endoplasmic reticulum, particularly in adult specimens. The lipofuscin-rich endocardial cell layers displayed deep blue precipitations in hearts from the ferritin-injected adult platies 6 h or more post-injection, when treated with acid ferrocyanide instead of Schmorl’s solution, i.e., this cell type is able to take up foreign particles. The occurrence of much lipofuscin-like pigments in endocardial cell layers in adult platies provides evidence that these cells have performed important scavenger functions at this stage of development.

Melano-macrophage centres and endocytic cells in kidney and spleen of pearl gouramy and platyfish (Anabantidae, Poeciliidae: Teleostei)

The structure and putative cellular content of melano-macrophage centres (MMCs) and single macrophages in kidney and spleen of two teleosts, pearl gouramy Trichogaster leeri (Bleeker) and platyfish Xiphophorus maculatus (Günther), are described and compared. The MMCs were confined to the haemopoetic tissue in both species, and were markedly more prominent in pearl gouramy than in platyfish. They were rich in iron-compounds, probably haemosiderin, in the former species, whereas they contained mainly iron-free pigments, probably lipofuscin, in the latter species. We suggest that the kidney and spleen MMCs are involved in the regular storage, relocation and recycling of iron-compounds of effete or damaged red blood cells in healthy pearl gouramies, whereas they function more or less as a deposit for undegradable and potentially harmful iron-free cell debris in the corresponding organs in healthy platies. Numerous single macrophages took up much intraperitoneally injected horse ferritin in the kidney and spleen haemopoetic tissue in platies, whereas these cells contain much iron-containing pigment, probably haemosiderin, in the control pearl gouramies. We suggest that these cells are able to play a role in cleansing the circulation of foreign macromolecules and particles in platies, whereas they are mainly involved in the regular uptake of scavenger iron-containing compounds in pearl gouramies.

Molecular Analysis of the Sex-Determining Region of the PlatyfishXiphophorus maculatus

Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determination region containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the best models for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametes and three different types of sex chromosomes have been described in the platyfish, with several loci involved in pigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Using the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linked molecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determining region of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of the sex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syntenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomes in platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequently undergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic variability affecting sex determination and other sex-linked traits in Xiphophorus.

A Structural Model of the Extracellular Domain of the Oncogenic EGFR Variant Xmrk

The epidermal growth factor receptor (EGFR) variant Xmrk of the Mexican fish Xiphophorus is one of the first oncogenes described. Its overexpression in pigment cells of the skin occurs after certain repeated cross-breedings between the platyfish Xiphophorus maculatus and the swordtail Xiphophorus hellerii. Xmrk overexpression results in melanoma with a high malignant potential. The Xiphophorus melanoma model provides a valuable tool for the understanding of melanoma development in general. The xmrk gene is the duplicated version of the proto-oncogene egfr-b. Compared to this, it contains 14 nonsynonymous mutations. Two of these mutations lead to a permanent protein dimerization which results in constitutive signaling. For a better understanding of the structural conditions responsible for this effect, the authors modeled the extracellular domain of Egfr-b, based on the crystal structure of the closely related human EGFR, and introduced the mutations G336R or C555S. The resulting protein models show that in either mutation the formation of an intramolecular disulfide bridge is very likely prevented, leading to an intermolecular disulfide bridge between released cysteine residues. In the G336R model, the distance between the membrane-proximal domains from both chains is smaller than in the C555S model, confirming the previous observation than Egfr-b-G336R is more tumorigenic than Egfr-b-C555S in vivo.

Tissue-Specific Expression ofdmrtGenes in Embryos and Adults of the PlatyfishXiphophorus maculatus

The vertebrate dmrt gene family encodes transcription factors with a characteristic DNA-binding motif called the DM domain. The best studied member is dmrt1, which is involved in sexual development in fish and tetrapods. The cloning of dmrt5 from the platyfish Xiphophorus maculatus and the expression pattern of dmrt1, dmrt2a, dmrt4, and dmrt5 in adults and embryos are reported. Consistent with a role in sexual development, platyfish dmrt1 is expressed exclusively in adult testis. Interestingly, dmrt1 expression was detected in both spermatogonia and Sertoli cells. This contrasts with the situation in other fish, where dmrt1 is not expressed in both types of cells, and is reminiscent of the expression observed in other vertebrates. Certain expression patterns in platyfish embryos were similar to those found in other vertebrates, suggesting conserved functions of dmrt genes in vertebrate development. This was the case for dmrt2a/terra and dmrt4, presenting expression patterns compatible with roles in somitogenesis and olfactory system development, respectively. However, differences in expression during embryogenesis and in adult tissues were observed not only between fish and tetrapods, but also between fish species, illustrating the possible functional divergence of this gene family in fish and other vertebrates.

Helitron Transposons on the Sex Chromosomes of the PlatyfishXiphophorus maculatusand Their Evolution in Animal Genomes

The sex-determining region of the sex chromosomes of the platyfish Xiphophorus maculatus contains several copies of a recently described class of DNA transposons called Helitrons, which probably transpose through a mechanism involving rolling circle replication. The unique open reading frame of platyfish elements encodes a 2816 amino-acid protein with helicase and replication initiator (Rep) domains, which are hallmarks of Helitrons. Like previously described elements from zebrafish but unlike sequences from plants, insects and nematodes, platyfish Helitrons also encode a C-terminal apurinic-apyrimidinic-like endonuclease probably captured from a non-long-terminal- repeat retrotransposon. A cysteine protease domain related to the Drosophila ovarian tumor (OTU) protein was identified in the N-terminal part of the platyfish sequence. Putative endonuclease and protease have been acquired sequentially at least 600 million years ago and maintained functional in elements from sea urchin, lancelet and teleost fish, implying an important role for these domains in the transposition mechanism. Apparently intact Helitron elements are transcribed in Xiphophorus, and insertion polymorphism was observed between related fishes in different poeciliids. These observations suggest that Helitron transposons are still active in the genome of platyfish and related species, where they might play a role in the evolution of sex chromosomes and other genomic regions.

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 μm and a diameter of 0.66 μm with an osmotically inactive cell volume ( V b) of 0.6 V 0, where V 0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 μm −1. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L pg or L pg [cpa] and the activation energy, E Lp or E Lp [cpa]) were found to range from: L pg or L pg [cpa] = 0.0053–0.0093 μm/min atm; E Lp or E Lp [cpa] = 9.79–29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, B opt = 1009.5 exp ( - 0.0546 E Lp ) ( L pg ) ( SA WV ) where the units of B opt are °C/min, E Lp or E Lp [cpa] are kcal/mol, L pg or L pg [cpa] are μm/min atm and SA/WV are μm −1), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 °C/min (in HBSS), 47 °C/min (in HBSS + 14% glycerol) and 36 ° C/min (in HBSS + 10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be ∼25 °C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.

Cytogenetic analysis of platyfish (Xiphophorus maculatus) shows location of major and minor rDNA on chromosomes

Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated in platyfish (Xiphophorus maculates) using conventional and molecular cytogenetic techniques. Chromomycin A3 (CMA3) staining of GC- rich heterochromatin provided four CMA3 cytotypes in studied platyfish. As in fish, CMA3 positive sites are related to NORs, chromosomes sequentially stained by CMA3 and AgNO3 showed bright blocks that were transcriptionaly active NORs. Additionally, PRINS with primers enabling amplification of 5S rDNA and CMA3 staining revealed existence of 5S rDNA sites located close to GC rich regions. The analysis could suggests that minor and major rDNAs may be linked.

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.

Melano-macrophage centres in the liver of platyfish, Xiphophorus maculatus, Poeciliidae: Teleostei

This paper describes the localization and structure of melano-macrophage centres (MMCs) in the liver of platyfish (Xiphophorus maculatus), compares these centres with corresponding structures in the spleen and kidney of various teleosts and gives suggestions about their functions. The MMCs were closely apposed to the endothelial layer of liver venules covered with pancreas tissue, i.e. portal venules, and contained numerous cells displaying large amounts of Prussian blue precipitations in tissue treated with acid ferrocyanide. Thus, these centres were rich in ferric compounds, probably haemosiderin, and are suggested to be involved in the normal storage, relocation and recycling of iron compounds from effete or damaged red blood cells of the portal venule system in this species.