Amphibian Genomes Research Articles

Comparative analysis of amphibian genomes: An emerging resource for basic and applied research.

Amphibians are the most threatened group of vertebrates and are in dire need of conservation intervention to ensure their continued survival. They exhibit unique features including a high diversity of reproductive strategies, permeable and specialized skin capable of producing toxins and antimicrobial compounds, multiple genetic mechanisms of sex determination and in some lineages, the ability to regenerate limbs and organs. Although genomic approaches would shed light on these unique traits and aid conservation, sequencing and assembly of amphibian genomes has lagged behind other taxa due to their comparatively large genome sizes. Fortunately, the development of long-read sequencing technologies and initiatives has led to a recent burst of new amphibian genome assemblies. Although growing, the field of amphibian genomics suffers from the lack of annotation resources, tools for working with challenging genomes and lack of high-quality assemblies in multiple clades of amphibians. Here, we analyse 51 publicly available amphibian genomes to evaluate their usefulness for functional genomics research. We report considerable variation in genome assembly quality and completeness and report some of the highest transposable element and repeat contents of any vertebrate. Additionally, we detected an association between transposable element content and climatic variables. Our analysis provides evidence of conserved genome synteny despite the long divergence times of this group, but we also highlight inconsistencies in chromosome naming and orientation across genome assemblies. We discuss sequencing gaps in the phylogeny and suggest key targets for future sequencing endeavours. Finally, we propose increased investment in amphibian genomics research to promote their conservation.

Genome assembly catalog for species in the Japanese Red List: unlocking endangered biodiversity through genomic inventory.

Improvements in DNA sequencing technology are allowing the dramatic increase of whole genome data for a wide variety of species. Such genome sequence data can assist the monitoring of intraspecific genetic diversity, but is often lacking for threatened species. In this project, we focused on the national Red List, a catalog of extinct and threatened species, issued by the Japanese government. We combined the data included in it with the record of genome assembly in NCBI and tabulated the assembly availability of the species in the list. The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List as well as a strong bias towards mammals and birds in Animalia and vascular plants in Plantae. Our data presentation highlights potential systematic limitations in genome sequencing (e.g., budget for sequencing large genomes of amphibians) and instructs future policies including which taxon needs more effort for genome sequencing. The resultant tables are available in the original website https://treethinkers.nig.ac.jp/redlist/ and are regularly updated.

The genomic evolution of visual opsin genes in amphibians

Among tetrapod (terrestrial) vertebrates, amphibians remain more closely tied to an amphibious lifestyle than amniotes, and their visual opsin genes may be adapted to this lifestyle. Previous studies have discussed physiological, morphological, and molecular changes in the evolution of amphibian vision. We predicted the locations of the visual opsin genes, their neighboring genes, and the tuning sites of the visual opsins, in 39 amphibian genomes. We found that all of the examined genomes lacked the Rh2 gene. The caecilian genomes have further lost the SWS1 and SWS2 genes; only the Rh1 and LWS genes were retained. The loss of the SWS1 and SWS2 genes in caecilians may be correlated with their cryptic lifestyles. The opsin gene syntenies were predicted to be highly similar to those of other bony vertebrates. Moreover, dual syntenies were identified in allotetraploid Xenopus laevis and X. borealis. Tuning site analysis showed that only some Caudata species might have UV vision. In addition, the S164A that occurred several times in LWS evolution might either functionally compensate for the Rh2 gene loss or fine-tuning visual adaptation. Our study provides the first genomic evidence for a caecilian LWS gene and a genomic viewpoint of visual opsin genes by reviewing the gains and losses of visual opsin genes, the rearrangement of syntenies, and the alteration of spectral tuning in the course of amphibians’ evolution.

Genome assembly catalog for species in the Japanese Red List: unlocking endangered biodiversity through genomic inventory

Improvements in DNA sequencing technology are allowing the dramatic increase of whole genome data for a wide variety of species. Such genome sequence data can assist the monitoring of intraspecific genetic diversity, but is often lacking for threatened species. In this project, we focused on the national Red List, a catalog of extinct and threatened species, issued by the Japanese government. We combined the data included in it with the record of genome assembly in NCBI and tabulated the assembly availability of the species in the list. The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List as well as a strong bias towards mammals and birds in Animalia and vascular plants in Plantae. Our data presentation highlights potential systematic limitations in genome sequencing (e.g., budget for sequencing large genomes of amphibians) and instructs future policies including which taxon needs more effort for genome sequencing. The resultant tables are available in the original website https://treethinkers.nig.ac.jp/redlist/ and are regularly updated.

Consequences of polyploidy and divergence as revealed by cytogenetic mapping of tandem repeats in African clawed frogs (Xenopus, Pipidae)

Repetitive elements have been identified in several amphibian genomes using whole genome sequencing, but few studies have used cytogenetic mapping to visualize these elements in this vertebrate group. Here, we used fluorescence in situ hybridization and genomic data to map the U1 and U2 small nuclear RNAs and histone H3 in six species of African clawed frog (genus Xenopus), including, from subgenus Silurana, the diploid Xenopus tropicalis and its close allotetraploid relative X. calcaratus and, from subgenus Xenopus, the allotetraploid species X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. Results allowed us to qualitatively evaluate the relative roles of polyploidization and divergence in the evolution of repetitive elements because our focal species include allotetraploid species derived from two independent polyploidization events — one that is relatively young that gave rise to X. calcaratus and another that is older that gave rise to the other (older) allotetraploids. Our results demonstrated conserved loci number and position of signals in the species from subgenus Silurana; allotetraploid X. calcaratus has twice as many signals as diploid X. tropicalis. However, the content of repeats varied among the other allotetraploid species. We detected almost same number of signals in X. muelleri as in X. calcaratus and same number of signals in X. pygmaeus, X. allofraseri, X. laevis as in the diploid X. tropicalis. Overall, these results are consistent with the proposal that allopolyploidization duplicated these tandem repeats and that variation in their copy number was accumulated over time through reduction and expansion in a subset of the older allopolyploids.

Chromosome-level genome assembly of a high-altitude-adapted frog (Rana kukunoris) from the Tibetan plateau provides insight into amphibian genome evolution and adaptation

BackgroundThe high-altitude-adapted frog Rana kukunoris, occurring on the Tibetan plateau, is an excellent model to study life history evolution and adaptation to harsh high-altitude environments. However, genomic resources for this species are still underdeveloped constraining attempts to investigate the underpinnings of adaptation.ResultsThe R. kukunoris genome was assembled to a size of 4.83 Gb and the contig N50 was 1.80 Mb. The 6555 contigs were clustered and ordered into 12 pseudo-chromosomes covering ~ 93.07% of the assembled genome. In total, 32,304 genes were functionally annotated. Synteny analysis between the genomes of R. kukunoris and a low latitude species Rana temporaria showed a high degree of chromosome level synteny with one fusion event between chr11 and chr13 forming pseudo-chromosome 11 in R. kukunoris. Characterization of features of the R. kukunoris genome identified that 61.5% consisted of transposable elements and expansions of gene families related to cell nucleus structure and taste sense were identified. Ninety-five single-copy orthologous genes were identified as being under positive selection and had functions associated with the positive regulation of proteins in the catabolic process and negative regulation of developmental growth. These gene family expansions and positively selected genes indicate regions for further interrogation to understand adaptation to high altitude.ConclusionsHere, we reported a high-quality chromosome-level genome assembly of a high-altitude amphibian species using a combination of Illumina, PacBio and Hi-C sequencing technologies. This genome assembly provides a valuable resource for subsequent research on R. kukunoris genomics and amphibian genome evolution in general.

State of the Amphibia 2020: A Review of Five Years of Amphibian Research and Existing Resources

Amphibians are a clade of over 8,400 species that provide unique research opportunities and challenges. With amphibians undergoing severe global declines, we posit that assessing our current understanding of amphibians is imperative. Focusing on the past five years (2016–2020), we examine trends in amphibian research, data, and systematics. New species of amphibians continue to be described at a pace of ∼150 per year. Phylogenomic studies are increasing, fueling a growing consensus in the amphibian tree of life. Over 3,000 species of amphibians are now represented by expert-curated accounts or data in AmphibiaWeb, AmphibiaChina, BIOWEB, or the Amphibian Disease Portal. Nevertheless, many species lack basic natural history data (e.g., diet records, morphological measurements, call recordings) and major gaps exist for entire amphibian clades. Genomic resources appear on the cusp of a rapid expansion, but large, repetitive amphibian genomes still pose significant challenges. Conservation continues to be a major focus for amphibian research, and threats cataloged on AmphibiaWeb for 1,261 species highlight the need to address land use change and disease using adaptive management strategies. To further promote amphibian research and conservation, we underscore the importance of database integration and suggest that other understudied or imperiled clades would benefit from similar assessments of existing data.

Avian surfactant protein (SP)-A2 first arose in an early tetrapod before the divergence of amphibians and gradually lost the collagen domain

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP -D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SP-A-like gene. Here we investigated the evolution of the SP-A and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SP-A analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SP-A. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SP-A. In fact, the CRD of SP-A is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

De novo assembly and analysis of the transcriptome of the Siberian wood frog Rana amurensis.

The Siberian wood frog Rana amurensis Boulenger, 1886 is the most hypoxia-tolerant amphibian. It can survive for several months in an almost complete absence of oxygen. Little is known about the mechanisms of this remarkable resilience, in part because studies of amphibian genomes are impeded by their large size. To make the Siberian wood frog more amenable for genetic analysis, we performed transcriptome sequencing and de novo assembly for the R. amurensis brain under hypoxia and normoxia, as well as for the normoxic heart. In order to build a de novo transcriptome assembly of R. amurensis, we utilized 125-bp paired-end reads obtained from the brain under normoxia and hypoxia conditions, and from the heart under normoxia. In the transcriptome assembled from about 100,000,000 reads, 81.5 % of transcripts were annotated as complete, 5.3 % as fragmented, and 13.2 % as missing. We detected 59,078 known transcripts that clustered into 22,251 genes; 11,482 of them were assigned to specific GO categories. Among them, we found 6696 genes involved in protein binding, 3531 genes involved in catalytic activity, and 576 genes associated with transporter activity. A search for genes encoding receptors of the most important neurotransmitters, which may participate in the response to hypoxia, resulted in a set of expressed receptors of dopamine, serotonin, GABA, glutamate, acetylcholine, and norepinephrine. Unexpectedly, no transcripts for histamine receptors were found. The data obtained in this study create a valuable resource for studying the mechanisms of hypoxia tolerance in the Siberian wood frog, as well as for amphibian studies in general.

Update of the keratin gene family: evolution, tissue-specific expression patterns, and relevance to clinical disorders

Intermediate filament (IntFil) genes arose during early metazoan evolution, to provide mechanical support for plasma membranes contacting/interacting with other cells and the extracellular matrix. Keratin genes comprise the largest subset of IntFil genes. Whereas the first keratin gene appeared in sponge, and three genes in arthropods, more rapid increases in keratin genes occurred in lungfish and amphibian genomes, concomitant with land animal-sea animal divergence (~ 440 to 410 million years ago). Human, mouse and zebrafish genomes contain 18, 17 and 24 non-keratin IntFil genes, respectively. Human has 27 of 28 type I “acidic” keratin genes clustered at chromosome (Chr) 17q21.2, and all 26 type II “basic” keratin genes clustered at Chr 12q13.13. Mouse has 27 of 28 type I keratin genes clustered on Chr 11, and all 26 type II clustered on Chr 15. Zebrafish has 18 type I keratin genes scattered on five chromosomes, and 3 type II keratin genes on two chromosomes. Types I and II keratin clusters—reflecting evolutionary blooms of keratin genes along one chromosomal segment—are found in all land animal genomes examined, but not fishes; such rapid gene expansions likely reflect sudden requirements for many novel paralogous proteins having divergent functions to enhance species survival following sea-to-land transition. Using data from the Genotype-Tissue Expression (GTEx) project, tissue-specific keratin expression throughout the human body was reconstructed. Clustering of gene expression patterns revealed similarities in tissue-specific expression patterns for previously described “keratin pairs” (i.e., KRT1/KRT10, KRT8/KRT18, KRT5/KRT14, KRT6/KRT16 and KRT6/KRT17 proteins). The ClinVar database currently lists 26 human disease-causing variants within the various domains of keratin proteins.

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

AbstractGenetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic‐gonadotropin (hCG) and luteinizing‐hormone‐releasing‐hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N‐dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa‐induced oviposition of eggs for in‐vitro fertilization (IVF). Post‐thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen–thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen–thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen–thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen–thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally‐induced wild males and captive females, introducing innovative methods that link in‐situ and ex‐situ populations of endangered amphibians to revolutionize genetic management.

The Visual Opsin Gene Repertoires of Teleost Fishes: Evolution, Ecology, and Function.

Visual opsin genes expressed in the rod and cone photoreceptor cells of the retina are core components of the visual sensory system of vertebrates. Here, we provide an overview of the dynamic evolution of visual opsin genes in the most species-rich group of vertebrates, teleost fishes. The examination of the rich genomic resources now available for this group reveals that fish genomes contain more copies of visual opsin genes than are present in the genomes of amphibians, reptiles, birds, and mammals. The expansion of opsin genes in fishes is due primarily to a combination of ancestral and lineage-specific gene duplications. Following their duplication, the visual opsin genes of fishes repeatedly diversified at the same key spectral-tuning sites, generating arrays of visual pigments sensitive to the ultraviolet to red spectrum of light. Species-specific opsin gene repertoires correlate strongly with underwater light habitats, ecology, and color-based sexual selection.

Quantification and evolution of mitochondrial genome rearrangement in Amphibians

BackgroundRearrangement is an important topic in the research of amphibian mitochondrial genomes ("mitogenomes" hereafter), whose causes and mechanisms remain enigmatic. Globally examining mitogenome rearrangements and uncovering their characteristics can contribute to a better understanding of mitogenome evolution.ResultsHere we systematically investigated mitogenome arrangements of 232 amphibians including four newly sequenced Dicroglossidae mitogenomes. The results showed that our new sequenced mitogenomes all possessed a trnM tandem duplication, which was not exclusive to Dicroglossidae. By merging the same arrangements, the mitogenomes of ~ 80% species belonged to the four major patterns, the major two of which were typical vertebrate arrangement and typical neobatrachian arrangement. Using qMGR for calculating rearrangement frequency (RF) (%), we found that the control region (CR) (RF = 45.04) and trnL2 (RF = 38.79) were the two most frequently rearranged components. Forty-seven point eight percentage of amphibians possessed rearranged mitogenomes including all neobatrachians and their distribution was significantly clustered in the phylogenetic trees (p < 0.001). In addition, we argued that the typical neobatrachian arrangement may have appeared in the Late Jurassic according to possible occurrence time estimation.ConclusionIt was the first global census of amphibian mitogenome arrangements from the perspective of quantity statistics, which helped us to systematically understand the type, distribution, frequency and phylogenetic characteristics of these rearrangements.

A large genome with chromosome-scale assembly sheds light on the evolutionary success of a true toad (Bufo gargarizans).

We present a high-quality genome assembly for the Asiatic toad (Bufo gargarizans) and explore the evolution of several large gene families in amphibians. With a large genome assembly size of 4.55Gb, the chromosome-scale assembly includes 747 scaffolds with an N50 of 539.8Mb and 1.79% gaps. Long terminal repeats (LTRs) constitute a high proportion of the genome and their expansion is a key contributor to the inflated genome size in this species. This is very different from other small amphibian genomes, but similar to that of the enormous axolotl genome. The genome retains a large number of duplicated genes, with tandem (TD) and proximal duplications (PD) the predominant mode of duplication. A total of 122 gene families have undergone significant expansion and were mainly enriched in sensory perception of smell and bitter taste. The CYP2C subfamily, which plays an important role in metabolic detoxification, specifically expanded via TD and PD in the Asiatic toad and the cane toad (true toads). Most of Na+ /K+ -ATPase genes experienced accelerated evolution along Bufonid lineages and two amino acid sites involving toad-toxin resistance were found to experience positive selection. We also revealed a dynamic evolution of olfactory and vomeronasal receptor gene families which was likely driven by the water-to-land transition. The high-quality genome of the Asiatic toad will provide a solid foundation to understand the genetic basis of its many biological processes.

Gigantic Genomes Provide Empirical Tests of Transposable Element Dynamics Models

Transposable elements (TEs) are a major determinant of eukaryotic genome size. The collective properties of a genomic TE community reveal the history of TE/host evolutionary dynamics and impact present-day host structure and function, from genome to organism levels. In rare cases, TE community/genome size has greatly expanded in animals, associated with increased cell size and changes to anatomy and physiology. Here, we characterize the TE landscape of the genome and transcriptome in an amphibian with a giant genome — the caecilianIchthyophis bannanicus, which we show has a genome size of 12.2 Gb. Amphibians are an important model system because the clade includes independent cases of genomic gigantism. The I. bannanicus genome differs compositionally from other giant amphibian genomes, but shares a low rate of ectopic recombination-mediated deletion. We examine TE activity using expression and divergence plots; TEs account for 15% of somatic transcription, and most superfamilies appear active. We quantify TE diversity in the caecilian, as well as other vertebrates with a range of genome sizes, using diversity indices commonly applied in community ecology. We synthesize previous models that integrate TE abundance, diversity, and activity, and test whether the caecilian meets model predictions for genomes with high TE abundance. We propose thorough, consistent characterization of TEs to strengthen future comparative analyses. Such analyses will ultimately be required to reveal whether the divergent TE assemblages found across convergent gigantic genomes reflect fundamental shared features of TE/host genome evolutionary dynamics.

Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae).

The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.

A draft genome assembly of the eastern banjo frog Limnodynastes dumerilii dumerilii (Anura:Limnodynastidae).

Amphibian genomes are usually challenging to assemble due to their large genome size and high repeat content. The Limnodynastidae is a family of frogs native to Australia, Tasmania and New Guinea. As an anuran lineage that successfully diversified on the Australian continent, it represents an important lineage in the amphibian tree of life but lacks reference genomes. Here we sequenced and annotated the genome of the eastern banjo frog Limnodynastes dumerilii dumerilii to fill this gap. The total length of the genome assembly is 2.38 Gb with a scaffold N50 of 285.9 kb. We identified 1.21 Gb of non-redundant sequences as repetitive elements and annotated 24,548 protein-coding genes in the assembly. BUSCO assessment indicated that more than 94% of the expected vertebrate genes were present in the genome assembly and the gene set. We anticipate that this annotated genome assembly will advance the future study of anuran phylogeny and amphibian genome evolution.

RNA-seq analysis provides insight into molecular adaptations of Andrias davidianus.

The Chinese giant salamander Andrias davidianus is regarded as an ideal model for studying local adaptations, such as longevity, tolerance to starvation, and cutaneous respiration. Transcriptome analysis is useful for studying the large and complex genomes of amphibians. Based on the coding gene set of adult A. davidianus, dozens of A. davidianus-specific genes were identified and three signaling pathway (JAK-STAT, HIF-1, and FoxO) genes were expanded as compared with other amphibians. The results of the pathway analysis of A. davidianus-specific genes indicated that the molecular adaptation of A. davidianus may have required a more rapid evolution of the immune system. Additionally, for the first time, the gene expressions in different parts of the skin tissue were compared. The results of the comparison analysis demonstrated that lateral skin could be more focused on mucus secretion, dorsal skin on immunity and melanogenesis, and abdominal skin on water and salt metabolism. This study provides the first insight into studying longevity and starvation tolerance in A. davidianus, and offers a basis for further investigation of the molecular mechanisms of adaptations in amphibians.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

BackgroundDirected DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa.ResultsHere, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background.ConclusionsAltogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

Molecular cloning and functional characterization of TRIF in large yellow croaker Larimichthys crocea

As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-β (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway.