S. Austin Hammond

Specific time of exposure during tadpole development influences biological effects of the insecticide carbaryl in green frogs (Lithobates clamitans).

The orchestration of anuran metamorphosis is initiated and integrated by thyroid hormones, which change dynamically during larval development and which may represent a target of disruption by environmental contaminants. Studies have found that some anurans experience increased rates of development when exposed to the insecticide carbaryl later in larval development, suggesting that this insecticide could affect thyroid hormone-associated biological pathways. However, the time in development when tadpoles are sensitive to insecticide exposure has not been clearly defined nor has the mechanism been tested. In two separate studies, we exposed recently hatched green frog (Lithobates clamitans) tadpoles to a single, three day carbaryl exposure in the laboratory at either 2, 4, 8, or 16 weeks post-hatching. We examined the impact of carbaryl exposure on mRNA abundance patterns in the brains of frogs following metamorphosis months after a single three day exposure (experiment 1) and in tadpole tails three days after exposure (experiment 2) using cDNA microarrays and quantitative real time polymerase chain reaction (QPCR) analyses. For tadpoles reared through metamorphosis, we measured tadpole growth and development, as well as time to, mass at, and survival to metamorphosis. Although carbaryl did not significantly impact tadpole development, metamorphosis, or survival, clear exposure-related alterations in both tail and brain transcript levels were evident when tadpoles were exposed to carbaryl, particularly in tadpoles exposed at weeks 8 and 16 post-hatching, indicating both short-term and long-term alterations in mRNA expression. These results indicate that carbaryl can have long-lasting effects on brain development when exposure occurs at sensitive developmental stages, which may have implications for animal fitness and function later in the life cycle.

De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes.

In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences.

The North American bullfrog draft genome provides insight into hormonal regulation of long noncoding RNA

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research.The globally-distributed Ranidae (true frogs) are the largest frog family. Here, Hammond et al. present a draft genome of the North American bullfrog, Rana (Lithobates) catesbeiana, as a foundation for future understanding of true frog genetics as amphibian species face difficult environmental challenges.

Characterization of Gene Expression Endpoints During Postembryonic Development of the Northern Green Frog (Rana clamitans melanota)

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue

Nanoparticles (NPs), materials that have one dimension less than 100 nm, are used in manufacturing, health, and food products, and consumer products including cosmetics, clothing, and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs), thyroxine, and 3,3′,5-triiodothyronine (T3), are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs). We used the cultured tailfin (C-fin) assay to examine the exposure effects of 0.1–10 nM (~8–800 ng/L) of three types of ~20 nm TiO2 NPs (P25, M212, M262) and micron-sized TiO2 (μ TiO2) ±10 nM T3. The actual Ti levels were 40.9–64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR) was used to measure the relative amounts of mRNA transcripts encoding TH-responsive THs receptors (thra and thrb) and Rana larval keratin type I (rlk1), as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30), superoxide dismutase (sod), and catalase (cat). The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25, and M212 while no effect was observed with M262 exposure. Therefore, the risk of adversely affecting amphibian tissue by disrupting TH-signaling or inducing cellular stress is low for these compounds relative to other previously-tested NPs.

Identification of organ-autonomous constituents of the molecular memory conferred by thyroid hormone exposure in cold temperature-arrested metamorphosing Rana (Lithobates) catesbeiana tadpoles.

Environmental temperature modulates thyroid hormone (TH)-dependent metamorphosis in some amphibian species. The North American bullfrog--Rana (Lithobates) catesbeiana - tadpole is naturally adapted to a wide range of temperatures over multiple seasons. Cold temperatures delay while warmer temperatures accelerate metamorphosis. Exogenous TH exposure of premetamorphic tadpoles results in a rapid precocious induction of metamorphosis at warm temperatures (20-25 °C). The same exposure at cold temperatures (4-5 °C) does not elicit an overt metamorphic response. However, a molecular memory of TH exposure is established such that cold, TH-exposed tadpoles returned to permissive warm temperatures will rapidly execute TH-induced genetic programs. Previous mRNA profiling has identified TH-regulated transcription factors encoded by thra, thrb, thibz, klf9, and cebp1 as components of the molecular memory after one week post-exposure. However, a further hierarchy may exist within the initiation phase since many gene transcripts demonstrated tissue-specific patterns. Whether the molecular memory is organ autonomous or requires additional modulating factors is unknown. Herein we examine tail fin and back skin and determine that thibz is the only transcript that is TH-responsive after 2 days post-exposure at low temperature in both tissues in the intact animal. In back skin, cebp1 is also TH-responsive under these conditions. Serum-free tail fin organ culture (C-Fin) reveals that the thibz response is organ autonomous whereas cultured back skin (C-Skin) results suggest that thibz and cebp1 require an additional factor for induction from elsewhere within the intact animal. Subsequent investigations are now possible to identify endogenous factors that modulate the molecular memory in intact animals.

Environmental influences on the epigenomes of herpetofauna and fish.

Herpetofauna (amphibians and reptiles) and fish represent important sentinel and indicator species for environmental and ecosystem health. It is widely accepted that the epigenome plays an important role in gene expression regulation. Environmental stimuli, including temperature and pollutants, influence gene activity, and there is growing evidence demonstrating that an important mechanism is through modulation of the epigenome. This has been primarily studied in human and mammalian models; relatively little is known about the impact of environmental conditions or pollutants on herpetofauna or fish epigenomes and the regulatory consequences of these changes on gene expression. Herein we review recent studies that have begun to address this deficiency, which have mainly focused on limited specific epigenetic marks and individual genes or large-scale global changes in DNA methylation, owing to the comparative ease of measurement. Greater understanding of the epigenetic influences of these environmental factors will depend on increased availability of relevant species-specific genomic sequence information to facilitate chromatin immunoprecipitation and DNA methylation experiments.

It′s okay to be green: Draft genome of the North American Bullfrog (Rana [Lithobates] catesbeiana)

Frogs play important ecological roles as sentinels, insect control and food sources. Several species are important model organisms for scientific research to study embryogenesis, development, immune function, and endocrine signaling. The globally-distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Consequently, the extensive Xenopus genomic resources are of limited utility for Ranids and related frog species. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. Herein, we report on the first genome sequence of a Ranid species, an adult male North American bullfrog (Rana [Lithobates] catesbeiana). We assembled high-depth Illumina reads (66-fold coverage), into a 5.8 Gbp (NG50 = 57.7 kbp) draft genome using ABySS v1.9.0. The assembly was scaffolded with LINKS and RAILS using pseudo-long-reads from targeted denovo assembler Kollector and Illumina Synthetic Long-Reads, as well as reads from long fragment (MPET) libraries. We predicted over 22,000 protein-coding genes using the MAKER2 pipeline and identified the genomic loci of 6,227 candidate long noncoding RNAs (IncRNAs) from a composite reference bullfrog transcriptome. Mitochondrial sequence analysis supported Lithobates as a subgenus of Rana. RNA-Seq experiments identified ~6,000 thyroid hormone– responsive transcripts in the back skin of premetamorphic tadpoles; the majority of which regulate DNA/RNA processing. Moreover, 1/6th of differentially-expressed transcripts were putative lncRNAs. Our draft bullfrog genome will serve as a useful resource for the amphibian research community.

Kollector: transcript-informed, targeted de novo assembly of gene loci

Motivation: Despite considerable advancements in sequencing and computing technologies, de novo assembly of whole eukaryotic genomes is still a time-consuming task that requires a significant amount of computational resources and expertise. A targeted assembly approach to perform local assembly of sequences of interest remains a valuable option for some applications. This is especially true for genecentric assemblies, whose resulting sequence can be readily utilized for more focused biological research. Here we describe Kollector, an alignment-free targeted assembly pipeline that uses thousands of transcript sequences concurrently to inform the localized assembly of corresponding gene loci. Kollector robustly reconstructs introns and novel sequences within these loci, and scales well to large genomes— properties that makes it especially useful for researchers working on non-model eukaryotic organisms. Results: We demonstrate the performance of Kollector for assembling complete or near-complete Caenorhabditis elegans and Homo sapiens gene loci from their respective, input transcripts. In a timeand memory-efficient manner, the Kollector pipeline successfully reconstructs respectively 99% and 80% (compared to 86% and 73% with standard de novo assembly techniques) of C.elegans and H.sapiens transcript targets in their corresponding genomic space using whole genome shotgun sequencing reads. We also show that Kollector outperforms both established and recently released targeted assembly tools. Finally, we demonstrate three use cases for Kollector, including comparative and cancer genomics applications. Availability and Implementation: Kollector is implemented as a bash script, and is available at https://github.com/bcgsc/kollector Contact: ibirol@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online.

De novo assembly of the ringed seal (Pusa hispida) blubber transcriptome: A tool that enables identification of molecular health indicators associated with PCB exposure

The ringed seal, Pusa hispida, is a keystone species in the Arctic marine ecosystem, and is proving a useful marine mammal for linking polychlorinated biphenyl (PCB) exposure to toxic injury. We report here the first de novo assembled transcriptome for the ringed seal (342,863 transcripts, of which 53% were annotated), which we then applied to a population of ringed seals exposed to a local PCB source in Arctic Labrador, Canada. We found an indication of energy metabolism imbalance in local ringed seals (n=4), and identified five significant gene transcript targets: plasminogen receptor (Plg-R(KT)), solute carrier family 25 member 43 receptor (Slc25a43), ankyrin repeat domain-containing protein 26-like receptor (Ankrd26), HIS30 (not yet annotated) and HIS16 (not yet annotated) that may represent indicators of PCB exposure and effects in marine mammals. The abundance profiles of these five gene targets were validated in blubber samples collected from 43 ringed seals using a qPCR assay. The mRNA transcript levels for all five gene targets, (Plg-R(KT), r2=0.43), (Slc25a43, r2=0.51), (Ankrd26, r2=0.43), (HIS30, r2=0.39) and (HIS16, r2=0.31) correlated with increasing levels of blubber PCBs. Results from the present study contribute to our understanding of PCB associated effects in marine mammals, and provide new tools for future molecular and toxicology work in pinnipeds.