Medaka Fish Oryzias Latipes Research Articles

Direct effect of artificial warming on communities is stronger than its indirect effect through body mass reduction

Theory predicts that morphological and bioenergetic constraints due to temperature‐induced body size reduction can modulate the direct effects of warming on biotic interactions, with consequent effects on trophic cascades and biomass distribution. However, these theoretical predictions have rarely been tested empirically. Our aim was to distinguish the indirect effects of warming‐induced body size reductions from the direct effects of warming on community structure. We conducted a mesocosm experiment manipulating factorially 1) body size reduction in the medaka fish Oryzias latipes using two populations raised for several generations under contrasted climate conditions and 2) warming (+4°C), to test their independent and interactive effects on the structure of prey and primary resource communities, the predator–prey biomass ratio and the biomass size spectra. We further dissected the effects of seasonal temperature variation from the effects of constant artificial warming. We found that the indirect effects of warming (i.e. fish body size reduction) on composition and structure of communities as well as their biomass size spectra were of marginal amplitude compared to the direct effects of seasonal temperature variation and constant warming. There were no changes in community composition in response to fish body size reduction or constant warming. However, the density of macroinvertebrates and zooplankton were maximal at intermediate seasonal water temperatures and lower in constantly‐heated mesocosms. Contrastingly, phytoplankton was not strongly affected by seasonal temperature or warming, but rather responded to grazing effects of zooplankton. Finally, we found a reduction in predator–prey biomass ratio under warming and at the warmest seasonal temperature, inducing a steeper slope of the biomass size spectra under increasing seasonal (but not constant) temperature. We conclude that the direct effects of climate change on freshwater communities are stronger than its indirect effects mediated by body mass reduction.

Contribution of sox9b to pigment cell formation in medaka fish.

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a-/- ; sox10b-/- double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b-/- ; sox10a-/- and sox10a-/- ; sox10b-/- double mutants, but their numbers were significantly reduced in the sox9b-/- ; sox10a-/- mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.

Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka <i>Oryzias latipes</i>.

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.

Variation of thermal plasticity in growth and reproduction patterns: Importance of ancestral and developmental temperatures.

Temperature is an important ecological driver modulating life history traits of organisms, such as growth and reproduction. With the ongoing global warming, understanding the mechanisms underlying the effect of temperature on size and resource allocation trade-off is crucial. The temperature-size rule (TSR) describes plastic growth patterns in populations of ectothermic species under different thermal environments, whereby warming results in faster initial growth but lower size at maturity. However, the evolution of the TSR remains poorly understood. Here we conducted an experiment with populations of the medaka fish Oryzias latipes maintained at two temperatures for successive generations to investigate changes in the growth pattern of the TSR. After rearing six generations at cold (20 °C) and warm (30 °C) temperature, we conducted common garden experiments on the seventh generation where we compare growth trajectories and reproduction patterns in four different groups of fish: (i) fish reared at cold temperature over all seven generations (cold past and present), (ii) fish reared at warm temperature over six generations and at cold temperature at the seventh generation (warm past and cold present), (iii) fish reared at warm temperature over all seven generations (warm past and present) and (iv) fish reared at cold temperature over six generations and at warm temperature at the seventh generation (cold past and warm present). For each treatment, we monitored growth curves and reproduction, and investigated changes in model parameters and reproduction up to 350 days after hatching. Our study showed changes in TSR patterns according to ancestral and developmental temperatures. Developmental temperature mainly impacted age at maturity and asymptotic size, whereas size at maturity was driven more by the dissimilarity between developmental and ancestral temperatures. Our results also highlight a loss of plasticity in temperature-size and reproductive patterns for the fish that were reared under warm condition over six generations.

Correction to: Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.

Targeted gene knockin in zebrafish using the 28S rDNA-specific non-LTR-retrotransposon R2Ol

BackgroundAlthough most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. Most of the sequence-specific LINEs have been reported mainly among invertebrate species and shown to retrotranspose into the specific sequences in vivo and in vitro systems. Recenlty, 28S rDNA-specific LINE R2 elements are shown to be distributed among widespread vertebrate species, but the sequence-specific retrotransposition of R2 has never been demonstrated in vertebrates.ResultsHere we cloned a full length unit of R2 from medaka fish Oryzias latipes, named R2Ol, and engineered it to a targeted gene integration tool in zebrafish. By injecting R2Ol-encoding mRNA into zebrafish embryos, R2Ol retrotransposed precisely into the target site at high efficiency (98%) and was transmitted to the next generation at high frequency (50%). We also generated transgenic zebrafish carrying the enhanced green fluorescent protein (EGFP) reporter gene in 28S rDNA target by the R2Ol retrotransposition system.ConclusionsSequence-specific LINE retrotransposes into the precise sequence using target primed reverse transcription (TPRT), possibly providing an alternative and effective targeted gene knockin method in vertebrates.

Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.

Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development

Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene.

Frequency and size of micronuclei induced in gill cells of medaka fish (Oryzias latipes) after whole-body exposure to clastogenic chemicals

Medaka fish (Oryzias latipes) were whole-bodily treated with various doses of mitomycin C (MMC), ethylmethanesulfonate (EMS), cyclophosphamide (CP), diethylnitrosamine (DEN), or colchicine (COL) for 24 h, and the frequency of micronucleated cells (MNCs) was measured in the gills at 24 and 48 h after treatment. In the present experiments, MMC, CP, and DEN were recorded as efficient inducers of micronuclei at both sampling times, and none of the MNC frequencies recorded with these agents at 24 h significantly exceeded the corresponding frequency at 48 h. For EMS and COL, positive responses were recorded only 48 h after treatment. By comparison with the time-course data reported for radiation-induced MNCs in the same MN assay system, the clear responses observed at the 48-h time point for all the chemicals used were regarded as evidence of their delayed effects on micronucleus (MN) formation. The mean sizes of micronuclei induced after exposure to COL was significantly larger by a factor 2 as compared with that induced by X-irradiation, whereas those determined for the other four chemicals were almost equal to that induced by X-irradiation. These results demonstrate that the medaka gill-cell MN assay can detect chemically-induced chromosome damage, either directly or after metabolic activation, and spindle malfunction, and provide a basis for further development of the present assay system for testing cytogenetic activities of chemical agents.

Conserved regulatory modules in the Sox9 testis-specific enhancer predict roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination

While the primary sex determining switch varies between vertebrate species, a key downstream event in testicular development, namely the male-specific up-regulation of Sox9, is conserved. To date, only two sex determining switch genes have been identified, Sry in mammals and the Dmrt1-related gene Dmy ( Dmrt1bY) in the medaka fish Oryzias latipes. In mice, Sox9 expression is evidently up-regulated by SRY and maintained by SOX9 both of which directly activate the core 1.3 kb testis-specific enhancer of Sox9 (TESCO). How Sox9 expression is up-regulated and maintained in species without Sry (i.e. non-mammalian species) is not understood. In this study, we have undertaken an in-depth comparative genomics approach and show that TESCO contains an evolutionarily conserved region (ECR) of 180 bp which is present in marsupials, monotremes, birds, reptiles and amphibians. The ECR contains highly conserved modules that predict regulatory roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination/differentiation. Our data suggest that tetrapods share common aspects of Sox9 regulation in the testis, despite having different sex determining switch mechanisms. They also suggest that Sox9 autoregulation is an ancient mechanism shared by all tetrapods, raising the possibility that in mammals, SRY evolved by mimicking this regulation. The validation of ECR regulatory sequences conserved from human to frogs will provide new insights into vertebrate sex determination.

Medaka Proteome Study: Use of Cross-species Matching and Expressed Sequence Tag Data for Protein Identification

Proteomics aims to understand gene function and molecular processes of the living cell through a large-scale study of the expressed proteins. Although proteomics approach is largely applied on experimental models such as rodents, other animal models such as medaka fish (Oryzias latipes) also attracts considerable interest in proteomics field. Medaka is one of the most studied animal model in r eproductive or developmental biology. Although, its genome has been sequenced, only a few proteins are available in the databases. We pr esent the procedure used in one experiment as a model of identificatio n of proteins, in this case from liver of medaka treated by a hepatotoxic cyanotoxin, the microcystin. Because O. latipes is not listed in mascot search engine, the identification of proteins (selected because modified by the treatment) was done on closed related species. First, 15 spots were analyzed using peptide mass fingerprinting (PMF). However none could be reliably identified using mascot engine. The reason for low sequence coverage is due to the fact that the outcome of the research depends on the completness of the protein database obtained from NCBInr and the availability of cross-species for protein identification. In order to improve the identification, the PMF search was combined with a search in a medaka nucleotide specific database and confirmed by MS/MS ion searches which revealed successful. This identification procedure has been successfully applied in different experiments with the medaka model.

Distribution of complete and defective copies of the Tol1 transposable element in natural populations of the medaka fish Oryzias latipes

DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.

The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes

Tol1 is an active DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes. This element belongs to the hAT transposable element family, of which complete copies have relatively long sequences. In addition, we found that Tol1 elements as long as 18 and 20 kb occur in the medaka fish genome. These facts suggest that Tol1 is suitable for carrying large DNA fragments as a gene transfer vector. Focusing on this, we conducted two kinds of manipulations of the element. The first was to eliminate internal regions dispensable for transposition. It was revealed that a Tol1 element consisting of 157-bp left- and 106-bp right-terminal regions could be transposed without a loss of transposition efficiency. Next, we prepared long Tol1 elements by incorporating unrelated DNA fragments into this short Tol1 clone and examined their transposition efficiencies. The transposition frequency decreased as the element size increased. The longest Tol1 element we examined measured 22.1 kb, and its transposition frequency was approximately one fifth that of a 2.1-kb element. However, this frequency was still significantly higher than that of a random integration of DNA into the chromosomes. The element size of 22.1 kb is the longest ever reported for DNA-based elements currently used for mammals. Thus, Tol1 is a superior gene-transfer vector with a large cargo capacity.

The Tol1 transposable element of the medaka fish moves in human and mouse cells

DNA-based transposable elements can be used as tools for gene engineering and gene therapy. A great advantage over RNA-mediated elements and retroviruses is the simplicity and safety of usage. The Tol1 element of the medaka fish Oryzias latipes has structural features of DNA-based elements. Although its excision has already been demonstrated, de novo insertion has not been observed, and a transposase has not been hitherto identified. We first cloned, through in silico search alignments and genomic library screenings, a 4.4-kb Tol1 copy carrying open reading frames and then identified, by mRNA analysis, a 2.9-kb transcript coding for 851 amino acids. The protein product of this transcript catalyzed transposition of a nonautonomous Tol1 copy in human and mouse culture cells. This identification of a fully functional Tol1 transposase could lead to the development of new tools for basic and translational molecular biology applications in mammals.

INTER- AND INTRAPOPULATION VARIATION IN THERMAL REACTION NORMS FOR GROWTH RATE: EVOLUTION OF LATITUDINAL COMPENSATION IN ECTOTHERMS WITH A GENETIC CONSTRAINT

In ectotherms, lower temperatures in high-latitude environments would theoretically reduce the annual growth rates of individuals. If slower growth and resultant smaller body size reduce fitness, individuals in higher latitudes may evolve compensatory responses. Two alternative models of such latitudinal compensation are possible: Model I: thermal reaction norms for growth rates of high-latitude individuals may be horizontally shifted to a lower range of temperatures, or Model II: reaction norms may be vertically shifted so that high-latitude individuals can grow faster across all temperatures. Model I is expected when annual growth rates in the wild are only a function of environmental temperatures, whereas Model II is expected when individuals in higher latitudes can only grow during a shorter period of a year. A variety of mixed strategies of these two models are also possible, and the magnitude of horizontal versus vertical variation in reaction norms among latitudinal populations will be indicative of the importance of "temperature" versus "seasonality" in the evolution of latitudinal compensation. However, the form of latitudinal compensation may be affected by possible genetic constraints due to the genetic architecture of reaction norms. In this study, we examine the inter- and intrapopulation variations in thermal reaction norms for growth rate of the medaka fish Oryzias latipes. Common-environment experiments revealed that average reaction norms differed primarily in elevation among latitudinal populations in a manner consistent with Model II (adaptation to "seasonality"), suggesting that natural selection in high latitudes prefers individuals that grow faster even within a shorter growing season to individuals that have longer growing seasons by growing at lower temperatures. However, intrapopulation variation in reaction norms was also vertical: some full-sibling families grew faster than others across all temperatures examined. This tendency in intrapopulation genetic variation for thermal reaction norms may have restricted the evolution of latitudinal compensation, irrespective of the underlying selection pressure.

Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of the medaka fish Tol2 transposable element

The Tol2 element of the medaka fish Oryzias latipes is a member of the hAT (hobo/Activator/Tam3) transposable element family. There is evidence for rapid expansion in the genome and throughout the species in the past but a high spontaneous transposition rate is not observed with current fish materials, suggesting that the Tol2 element and its host species have already acquired an interactive mechanism to control the transposition frequency. DNA methylation is a possible contributing factor, given its involvement with many other transposable elements. We therefore soaked embryos in 5-azacytidine, a reagent that causes reduction in the DNA methylation level, and examined amounts of PCR products reflecting the somatic excision frequency, obtaining direct evidence that exposure promotes Tol2 excision. Our results thus suggest that methylation of the genome DNA is a factor included in the putative mechanisms of control of transposition of the Tol2 element.

Structural components of the synaptonemal complex, SYCP1 and SYCP3, in the medaka fish Oryzias latipes

The synaptonemal complex (SC) is a meiosis-specific structure essential for synapsis of homologous chromosomes. For the first time in any non-mammalian vertebrates, we have isolated cDNA clones encoding two structural components of the SC, SYCP1 and SYCP3, in the medaka, and investigated their protein expression during gametogenesis. As in the case of mammals, medaka SYCP1 and SYCP3 are expressed solely in meiotically dividing cells. In the diplotene stage, SYCP1 is diminished at desynaptic regions of chromosomes and completely lost on the chromosomes at later stages. SYCP3 is localized along the arm and centromeric regions of chromosomes at metaphase I, and its existence on the whole chromosomes persists up to anaphase I, a situation different from that reported in the mouse, in which SYCP3 is confined to the centromeric regions but lost on the arm regions at metaphase I. Thus, the expression patterns of SC components are different in mammals and fish despite the resemblance in morphological structure of the SC, suggesting divergence in the function of the SC in regulation of meiosis-specific chromosomal behavior. Since the antibody against medaka SYCP3 is cross-reactive to other fishes, it should be generally useful for a meiosis-specific marker in fish germ cells.

Organization and structure of hox gene loci in medaka genome and comparison with those of pufferfish and zebrafish genomes

We isolated BAC clones that cover the entire hox gene loci in the medaka fish Oryzias latipes. The BAC clones were characterized by the Southern hybridization with many hox gene probes isolated in our previous study and by PCR using primers designed for selective amplification of respective hox genes. Then, the BAC clones have been subjected to shotgun sequencing. The results revealed the organization of the entire hox gene loci. Forty-six hox genes in total are encoded in seven clusters as follows: 10 hox genes in Aa cluster; 5 in Ab; 9 in Ba; 4 in Bb; 10 in Ca; 6 in Da; and 2 in Db. Together with the information on the hox gene loci registered in the Fugu genome database and in the Danio genome database, the physical maps of three fish genomes were constructed and compared one another. Not only numbers of hox genes but also the distances between the neighboring hox genes are highly similar between medaka and fugu. As for six clusters, Aa, Ab, Ba, Bb, Ca and Da that are commonly present in the three fishes, only few or no differences were found in each cluster. Thus, the hox gene sets should have been well conserved once they had been established in respective species.

385. Tol2 Transposon as a Gene Therapy Vector: Characterization, Optimization and Correction of Hereditary Tyrosinemia Type 1 in Mouse

To expand the repertoire of transposable elements available for gene transfer in higher vertebrates, we investigated the properties of the Tol2 transposon in mammalian cells and in vivo. Tol2 is a member of the hAT family of DNA transposons and is derived from an active element found in the tyrosinase gene of medaka fish (Oryzias latipes). Tol2 can transpose in mouse ES cells, suggesting its potential use as a gene transfer reagent in higher vertebrates.