Flatworm Transcriptomes Reveal Widespread Parasitism by Histophagous Ciliates.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These "histophages" feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A co-phylogenetic reconstruction provides strong evidence for the coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

FLATWORM TRANSCRIPTOMES REVEAL WIDESPREAD PARASITISM BY HISTOPHAGOUS CILIATES.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These 'histophages' feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A cophylogenetic reconstruction provides strong evidence for coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

A de novo reference transcriptome for Bolitoglossa vallecula, an Andean mountain salamander in Colombia.

The amphibian order Caudata, contains several important model species for biological research. However, there is need to generate transcriptome data from representative species of the primary salamander families. Here we describe a de novo reference transcriptome for a terrestrial salamander, Bolitoglossa vallecula (Caudata: Plethodontidae). We employed paired-end (PE) illumina RNA sequencing to assemble a de novo reference transcriptome for B. vallecula. Assembled transcripts were compared against sequences from other vertebrate taxa to identify orthologous genes, and compared to the transcriptome of a close plethodontid relative (Bolitoglossa ramosi) to identify commonly expressed genes in the skin. This dataset should be useful to future comparative studies aimed at understanding important biological process, such as immunity, wound healing, and the production of antimicrobial compounds.

HDAC Regulates Transcription at the Outset of Axolotl Tail Regeneration.

Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 172 compounds designed to target epigenetic mechanisms in an axolotl (Ambystoma mexicanum) embryo tail regeneration assay. A relatively large number of compounds (N = 55) inhibited tail regeneration, including 18 histone deacetylase inhibitors (HDACi). In particular, romidepsin, an FDA-approved anticancer drug, potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acted within a very narrow, post-injury window. Romidepsin treatment for only 1-minute post amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that are implicated in tumor cell death, as well as genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration.

Comparative transcriptomics of limb regeneration: Identification of conserved expression changes among three species of Ambystoma.

Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model - Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 h post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Transcriptomics using axolotls.

Microarray and RNA-sequencing technology now exists for the characterization of the Ambystoma mexicanum transcriptome. With sufficient replication, these tools give the opportunity to truly investigate gene expression in a variety of experimental paradigms. Analysis of data from the Amby002 array and RNA-sequencing technology can identify genes that change expression levels in concert with each other, which in turn may reveal mechanisms associated with biological processes and molecular functions.

Sal-Site: research resources for the Mexican axolotl.

Sal-Site serves axolotl research efforts by providing Web access to genomic data and information, and living stocks that are reared and made available by the Ambystoma Genetic Stock Center (AGSC). In this chapter, we detail how investigators can search for genes of interest among Sal-Site resources to identify orthologous nucleotide and protein-coding sequences, determine genome positions within the Ambystoma meiotic map, and obtain estimates of gene expression. In the near future, additional genomic resources will be made available for the axolotl, including a listing of genes that are partially or wholly contained within Bacterial Artificial Chromosome (BAC) vectors, a prioritized collection of deeply sequenced BAC clones, chromosome-specific assemblies of genomic DNA, and transgenic axolotls that are engineered using TALENs and CRISPRs. Also, services provided by the AGSC will be expanded to include microinjection of user constructs into single cell embryos and distribution of axolotl tissues, DNA, and RNA. In conclusion, Sal-Site is a useful resource that generates, shares, and evolves Ambystoma associated information and databases to serve research and education.

A Tale of Two Axolotls.

The Mexican axolotl (Ambystoma mexicanum) is an icon of culture, a revered aquarium pet, and a highly valued animal model in biomedical research. Unfortunately, Mexican axolotls are critically endangered in their natural Xochimilco habitat in Mexico City. If axolotls go extinct, current efforts to conserve the Xochimilico ecosystem will be undermined, as will efforts to genetically manage the laboratory populations that are needed to sustain research efforts around the world. A concerted global effort is needed to protect and manage this irreplaceable species in natural and laboratory environments.

Nested hierarchal organization of conservation for microRNAs and their putative targets to Drosophila melanogaster.

This study examined microRNA network properties traced through taxonomic hierarchy considering both the acquisition of potential network targets and regulators. Primary literature review and database analyses were conducted to establish modules of conserved microRNAs across metazoan taxonomy. A hierarchical schema for the conservation of microRNAs and their putative targets to Drosophila melanogaster was engineered through comprehensive meta-analysis, and conservation history of 90.39% of the total Drosophila dataset could be resolved through this hierarchical sampling regime; tracing from taxonomic order down to empire. The findings presented in this study represent a documentation of Drosophila microRNA regulatory network behavior thorough taxonomic hierarchy. MicroRNA regulatory network properties were found to transect taxonomic hierarchy. Newly acquired microRNAs from novel families reinforce the pre-existing regulatory network, while expanding the target list to include a small number of novel genes. Lineage specific microRNAs were found to exhibit far fewer conserved targets than do the more broadly conserved microRNAs; even when considering only more recently emerged targets. There was a dramatic expansion in network complexity with the expansion of the microRNA repertoire, and this corresponds to the expansion in biological complexity.