Species Danio Research Articles

Identification of olfactory alarm substances in zebrafish

Escaping from danger is one of the most fundamental survival behaviors for animals. Most freshwater fishes display olfactory alarm reactions in which an injured fish releases putative alarm substances from the skin to notify its shoaling company about the presence of danger. Here, we identified two small compounds in zebrafish skin extract, designated as ostariopterin and daniol sulfate. Ostariopterin is a pterin derivative commonly produced in many freshwater fishes belonging to the Ostariophysi superorder. Daniol sulfate is a novel sulfated bile alcohol specifically present in the Danio species, including zebrafish. Ostariopterin and daniol sulfate activate distinct glomeruli in the olfactory bulb. Zebrafish display robust alarm reactions, composed of darting, freezing, and bottom dwelling, only when they are concomitantly stimulated with ostariopterin and daniol sulfate. These results demonstrate that the fish alarm reaction is driven through a coincidence detection mechanism of the two compounds along the olfactory neural circuitry.

Kcnj13 regulates pigment cell shapes in zebrafish and has diverged by cis-regulatory evolution between Danio species.

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

Thyroid hormone regulates proximodistal patterning in fin rays

Processes that regulate size and patterning along an axis must be highly integrated to generate robust shapes; relative changes in these processes underlie both congenital disease and evolutionary change. Fin length mutants in zebrafish have provided considerable insight into the pathways regulating fin size, yet signals underlying patterning have remained less clear. The bony rays of the fins possess distinct patterning along the proximodistal axis, reflected in the location of ray bifurcations and the lengths of ray segments, which show progressive shortening along the axis. Here, we show that thyroid hormone (TH) regulates aspects of proximodistal patterning of the caudal fin rays, regardless of fin size. TH promotes distal gene expression patterns, coordinating ray bifurcations and segment shortening with skeletal outgrowth along the proximodistal axis. This distalizing role for TH is conserved between development and regeneration, in all fins (paired and medial), and between Danio species as well as distantly related medaka. During regenerative outgrowth, TH acutely induces Shh-mediated skeletal bifurcation. Zebrafish have multiple nuclear TH receptors, and we found that unliganded Thrab-but not Thraa or Thrb-inhibits the formation of distal features. Broadly, these results demonstrate that proximodistal morphology is regulated independently from size-instructive signals. Modulating proximodistal patterning relative to size-either through changes to TH metabolism or other hormone-independent pathways-can shift skeletal patterning in ways that recapitulate aspects of fin ray diversity found in nature.

Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish

The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell−contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.

Hybrid between Danio reriofemaleandDanio nigrofasciatus male produces aneuploid sperm with limited fertilization capacity.

In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.

The geometry as an eyed fish feels it in spontaneous and rewarded spatial reorientation tasks

Disoriented human beings and animals, the latter both sighted and blind, are able to use spatial geometric information (metric and sense properties) to guide their reorientation behaviour in a rectangular environment. Here we aimed to investigate reorientation spatial skills in three fish species (Danio rerio, Xenotoca eiseni, Carassius auratus) in an attempt to discover the possible involvement of extra-visual senses during geometric navigation. We observed the fish’s behaviour under different experimental procedures (spontaneous social cued task and rewarded exit task), providing them different temporal opportunities to experience the environmental shape (no experience, short and prolonged experience). Results showed that by using spontaneous social cued memory tasks, fishes were not able to take advantage of extra-visual senses to encode the spatial geometry, neither allowing them short time-periods of environmental exploration. Contrariwise, by using a reference memory procedure, during the rewarded exit tasks, thus providing a prolonged extra-visual experience, fishes solved the geometric task, showing also differences in terms of learning times among species.

Decoding an organ regeneration switch by dissecting cardiac regeneration enhancers.

Heart regeneration in regeneration-competent organisms can be accomplished through the remodeling of gene expression in response to cardiac injury. This dynamic transcriptional response relies on the activities of tissue regeneration enhancer elements (TREEs); however, the mechanisms underlying TREEs are poorly understood. We dissected a cardiac regeneration enhancer in zebrafish to elucidate the mechanisms governing spatiotemporal gene expression during heart regeneration. Cardiac lepb regeneration enhancer (cLEN) exhibits dynamic, regeneration-dependent activity in the heart. We found that multiple injury-activated regulatory elements are distributed throughout the enhancer region. This analysis also revealed that cardiac regeneration enhancers are not only activated by injury, but surprisingly, they are also actively repressed in the absence of injury. Our data identified a short (22 bp) DNA element containing a key repressive element. Comparative analysis across Danio species indicated that the repressive element is conserved in closely related species. The repression mechanism is not operational during embryogenesis and emerges when the heart begins to mature. Incorporating both activation and repression components into the mechanism of tissue regeneration constitutes a new paradigm that might be extrapolated to other regeneration scenarios.

Structure, development, and functional morphology of the cement gland of the giant danio, Devario malabaricus.

Aquatic species in several clades possess cement glands producing adhesive secretions of various strengths. In vertebrates, transient adhesive organs have been extensively studied in Xenopus laevis, other anurans, and in several fish species. However, the development of these structures is not fully understood. Here, we report on the development and functional morphology of the adhesive gland of a giant danio species, Devario malabaricus. We found that the gland is localized on the larval head, is composed of goblet-like secretory cells framed by basal, bordering, and intercalated apical epithelial cells, and is innervated by the trigeminal ganglion. The gland allows nonswimming larvae to adhere to various substrates. Its secretory cells differentiate by 12 hours postfertilization and begin to disappear in the second week of life. Exogenous retinoic acid disrupts the gland's patterning. More importantly, the single mature gland emerges from fusion of two differentiated secretory cells fields; this fusion is dependent on nonmuscle myosin II function. Taken together, our studies provide the first documentation of the embryonic development, structure, and function of the adhesive apparatus of a danioninae. To our knowledge, this is also the first report of a cement gland arising from convergence of two bilateral fields.

Structural and Functional Characterization of the Adult Giant Danio Heart

Cardiovascular diseases remain that number one cause of death in the northern hemisphere and are increasing globally in developing economies. In spite of evolutionary separation between fishes and mammalian, the use of non‐mammalian model species has significantly increased our understanding of cardiac diseases. We have recently described the growth and cardiovascular development of the Devario malabaricus, a giant danio (GD) species closely related to the zebrafish. We hypothesize that the adult GD heart can serve as a robust non‐mammalian model for in vivo studies of cardiac biology. Using immunostaining, transmission electron microscopy, scanning block‐face electron microscopy, and Doppler, we have characterized the anatomical and functional characteristics of the adult GD heart. First, we found that the adult giant danio possesses a thick and highly vascularized compact heart. Second, the GD heart possesses a junctional region populated with a fibroblasts network similar to that observed in zebrafish. Third, multiple large coronary vessels investing the compact myocardium are connected to and continuous with atrioventricular (AV) canal lumen. In addition, the AV canal and bulbus are highly innervated, suggesting complex regulation in heart function. Moreover, we demonstrate that their cardiac function can be measured using Doppler flow velocity. These studies along with previous findings support the GD as a robust model for the study of cardiac biology.Support or Funding Information1R15HD084262‐01, Buehler Family FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Developmental Genetics of Vertebrate Color Pattern Formation: Lessons from Zebrafish.

Color patterns are prominent features of many animals; they are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio) has become an important model organism for developmental biology and biomedical research in general, and it is the model organism to study color pattern formation in vertebrates. The fish display a conspicuous pattern of alternating blue and golden stripes on the body and on the anal and tail fins. This pattern is produced by three different types of pigment cells (chromatophores) arranged in precise layers in the hypodermis of the fish. In this essay, we will summarize the recent advances in understanding the developmental and genetic basis for stripe formation in the zebrafish. We will describe the cellular events leading to the formation of stripes during metamorphosis based on long-term lineage imaging. Mutant analysis has revealed that a number of signaling pathways are involved in the establishment and maintenance of the individual pigment cells. However, the striped pattern itself is generated by self-organizing mechanisms requiring interactions between all three pigment cell types. The involvement of integral membrane proteins, including connexins and potassium channels, suggests that direct physical contacts between chromatophores are involved, and that the directed transport of small molecules or bioelectrical coupling is important for these interactions. This mode of patterning by transmitting spatial information between adjacent tissues within three superimposed cell layers is unprecedented in other developmental systems. We propose that variations in the patterns among Danio species are caused by allelic differences in the genes responsible for these interactions.

The complete mitochondrial genome of dwarf danio, Danio nigrofasciatus

In this study, we report the complete mitochondrial genome sequence of dwarf danio, Danio nigrofasciatus, for the first time. The genome is found to be 16 616 bp in length and has a base composition of A (31.97%), G (15.88%), C (23.99%), and T (28.16%), indicating that the percentage of A + T (60.13%) was higher than G + C (39.87%). The mitochondrial genome contains a typically conserved structure including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), and 1 control region (D-loop). Most of the genes were located on the H-strand except for the ND6 gene and 8 tRNA genes. To obtain a more complete understanding of the evolutionary history between the dwarf danio and other species, 11 mitochondrial genomes were used for phylogenetic analysis. This mitochondrial sequence reported here would be useful to uncover the D. nigrofasciatus evolution and add a new genetic resource for the genus Danio.

Phylogeny of zebrafish, a "model species," within Danio, a "model genus".

Zebrafish (Danio rerio) is an important model for vertebrate development, genomics, physiology, behavior, toxicology, and disease. Additionally, work on numerous Danio species is elucidating evolutionary mechanisms for morphological development. Yet, the relationships of zebrafish and its closest relatives remain unclear possibly due to incomplete lineage sorting, speciation with gene flow, and interspecies hybridization. To clarify these relationships, we first constructed phylogenomic data sets from 30,801 restriction-associated DNA (RAD)-tag loci (483,026 variable positions) with clear orthology to a single location in the sequenced zebrafish genome. We then inferred a well-supported species tree for Danio and tested for gene flow during the diversification of the genus. An approach independent of the sequenced zebrafish genome verified all inferred relationships. Although identification of the sister taxon to zebrafish has been contentious, multiple RAD-tag data sets and several analytical methods provided strong evidence for Danio aesculapii as the most closely related extant zebrafish relative studied to date. Data also displayed patterns consistent with gene flow during speciation and postspeciation introgression in the lineage leading to zebrafish. The incorporation of biogeographic data with phylogenomic analyses put these relationships in a phylogeographic context and supplied additional support for D. aesculapii as the sister species to D. rerio. The clear resolution of this study establishes a framework for investigating the evolutionary biology of Danio and the heterogeneity of genome evolution in the recent history of a model organism within an emerging model genus for genetics, development, and evolution.

Upper thermal tolerance of closely related Danio species

The main finding of this study was that measuring maximum heart rate during incremental warming was an effective tool to estimate upper thermal limits in three small cyprinid Danio species, which differed significantly. Arrhenius breakpoint temperature for maximum heart rate, purportedly an index of optimum temperature, was 21·2 ± 0·4, 20·1 ± 0·4 and 18·9 ± 0·8° C (mean ± s.e.) for zebrafish Danio rerio, pearl danio Danio albolineatus and glowlight danio Danio choprae, respectively. The temperature where cardiac arrhythmias were first induced during warming (T(arr)) was 36·6 ± 0·7, 36·9 ± 0·8 and 33·2 ± 0·8° C (mean ± s.e.) and critical thermal maximum (T(Cm)) was 39·9 ± 0·1, 38·9 ± 0·1 and 37·2 ± 0·1° C (mean ± s.e.) for D. rerio, D. albolineatus and D. choprae, respectively. The finding that T(arr) was consistently 3-4° C lower than T(Cm) suggests that collapse of the cardiac life support system may be a critical trigger for upper temperature tolerance. The upper thermal limits established here, which correlate well with a broad natural environmental temperature range for D. rerio and a narrow one for D. choprae, suggest that upper thermal tolerance may be a genetic trait even among closely related species acclimated to common temperatures.

Danio aesculapii, a new species of danio from south-western Myanmar (Teleostei: Cyprinidae)

Danio aesculapii, new species, is described from small rivers on the western slope of the Rakhine Yoma in south-western Myanmar. It is superficially similar to D. choprae from northern Myanmar in having a series of vertical bars anteriorly on the side, but differs from it and other species of Danio in having six instead of seven or more branched dorsal-fin rays, and from all other species of Danio except D. erythromicron and D. kerri in having 12 instead of 10 or 14 circumpeduncular scale rows.

Alarm substance alters early embryonic development in two Danio species

Alarm substance alters early embryonic development in two Danio species

The “Celestial Pearl danio” is a miniature Danio (s.s) (Ostariophysi: Cyprinidae): evidence from morphology and molecules

The osteology of the miniature cyprinid Celestichthys margaritatus Roberts, type species of Celestichthys Roberts, is described in detail and briefly compared with that of other members of the Rasborinae (notably Danio (s.s), Danionella, Devario, Esomus, Microrasbora, Paedocypris and Sundadanio). Celestichthys margaritatus possesses an “A” stripe on the anal fin and two pigment stripes on the caudal fin (apomorphic features of Danio sensu Fang, 2003). In addition, C. margaritatus exhibits a median projection on the outer arm of the os suspensorium, a derived feature, present only in species of Danio (including D. erythromicron) amongst the Cyprinidae, and a lateral projection on the lateral face of the dentary (present only in Danio, Sundadanio and Paedocypris). Phylogenetic analysis of 1,494 bp of the RAG1 nuclear gene for 31 rasborine taxa, including 5 species of Danio, places C. margaritatus as the sister group to D. erythromicron, and part of a larger monophyletic group including all other species of Danio included for analysis. Based on characters of morphology and its position in a molecular phylogeny of the Rasborinae it is proposed that Celestichthys be placed in the synonymy of Danio, its only member referred to as Danio margaritatus new combination.

Phylogenetic relationships of Danio within the order Cypriniformes: a framework for comparative and evolutionary studies of a model species

The evolutionary relationships of species of Danio and the monophyly and phylogenetic placement of the genus within the family Cyprinidae and subfamily Rasborinae provide fundamentally important phyloinformatics necessary for direct evaluations of an array of pertinent questions in modern comparative biology. Although the genus Danio is not one of the most diverse within the family, Danio rerio is one of the most important model species in biology. Many investigations have used this species or presumed close relatives to address specific questions that have lasting impact on the hypothesis and theory of development in vertebrates. Largely lacking from this approach has been a holistic picture of the exact phylogenetic or evolutionary relationships of this species and its close relatives. One thing that has been learned over the previous century is that many organismal attributes (e.g., developmental pathways, ecologies, behaviors, speciation) are historically constrained and their origins and functions are best explained via a phylogenetic approach. Herein, we provide a molecular evaluation of the phylogenetic placement of the model species Danio rerio within the genus Danio and among hypothesized closely related species and genera. Our analysis is derived from data using two nuclear genes (RAG1, rhodopsin) and five mitochondrial genes (ND4, ND4L, ND5, COI, cyt b) evaluated using parsimony, maximum likelihood, and Bayesian analyses. The family Cyprinidae is resolved as monophyletic but the subfamily Rasborinae (priority over Danioinae) is an unnatural assemblage. Danio is identified as a monophyletic group sister to a clade inclusive of the genera Chela, Microrasbora, Devario, and Inlecypris, not Devario nor Esomus as hypothesized in previous studies. Danio rerio is sister to D. kyathit among the species of Danio evaluated in this analysis. Microrasbora and Rasbora are non-monophyletic assemblages; however, Boraras is monophyletic.

Notes on the natural diet and habitat of eight danionin fishes, including the zebrafishDanio rerio

The diet and habitat of eight danionin species (DanioandDevario) from 18 sites in India and Thailand are reported. At every site, habitat characteristics, including pH, light penetration, water temperature, current speed, stream dimensions, canopy cover and substratum type, were recorded. Danionins were found primarily in warm (24–35° C), moving water of moderate clarity and pH (6·6–8·2) and there were significant differences among species in water temperature, pH and current speed. Deep‐bodiedDevariospecies were generally associated with faster water currents than more slender‐bodiedDaniospecies. Gut content analyses of 327 individuals representing 17 populations showed that insects were the primary food resource for the eightDanioandDevariospecies. Crustaceans, fish scales, algae and detritus were also important supplements for particular species. Stable isotope data from syntopic species indicated long‐term differences in consumption of terrestrialv.aquatic insects in one of two population pairs.

Zebrafish in comparative context: A symposium

The Symposium "Zebrafish in Comparative Context" was organized to bring together two largely separate but highly complementary research traditions in order to make developmental and genetic information about a model species (Danio rerio, the zebrafish) more accessible to the comparative biology community. The meeting focused on the relationship of this model organism to other vertebrates (particularly other fishes) using a comparative and evolutionary approach. Topics included the phylogeny of cypriniform fishes, genome evolution, the evolution of gastrulation, dentition, pigmentation, craniofacial development, and nervous system structure and function. Participants also met informally to discuss ways to facilitate collaborative projects in areas of common interest and determine priorities for the development of shared resources. Continuing interactions between comparative biologists, with their extensive body of knowledge of morphological variation among fish species, and developmental biologists and geneticists working with model species such as the zebrafish will facilitate our understanding of the evolution of developmental patterns and processes in vertebrates.

Temporal pattern in swimming activity of two fish species ( Danio rerio and Leucaspius delineatus ) under chemical stress conditions

Circadian periodicity of swimming activity was investigated in two fish species, the zebrafish (Danio rerio) and the sunbleak (Leucaspius delineatus) under sublethal long-term exposure to the cyanobacteria toxin microcystin-LR (nominal concentrations of 0.5 μg l − 1, 5 μg l − 1, 15 μg l − 1, 50 μg l − 1) in 15-litre tanks. Swimming activity of fish was monitored continuously by using an automated video-monitoring and object-tracing system over a period of 17 days. Influenced by long-term exposure to microcystin-LR, Leucaspius delineatus reversed their significant diurnal swimming activity and the fish became statistically significant nocturnal. Danio rerio remained diurnal active, but a significant phase shift was registered. In both Danio rerio and Leucaspius delineatus analysis of time series by cosinor regression revealed microcystin-LR induced dose-dependent alterations of the mean of oscillation, amplitude, acrophase and period length in a different extent. For Danio rerio the periodogram analysis revealed a significant circadian component of swimming activity for control as well as exposure groups, whereby the spectral amplitude clearly decreased at microcystin-LR concentrations of 15 and 50 μg l − 1. For Leucaspius delineatus the amplitude of circadian rhythm was decreased at all exposure concentrations of MC-LR. Furthermore the dominance of circadian rhythm was clearly reduced, whereas the rate of ultradian rhythms increased at elevated MC-LR concentrations of 5 μg l − 1, 15 μg l − 1 and 50 μg l − 1. The studied temporal aspects of behaviour clearly indicated stress symptoms in both fish species, therefore it proved to be a relevant method to characterise the impact of toxic substances in the environment and for biomonitoring.