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Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode Panagrolaimus sp. DAW1.

ABSTRACT: Panagrolaimus sp. DAW1 is a freeze-tolerant Antarctic nematode which survives extensive intracellular ice formation. The molecular mechanisms of this extreme adaptation are still poorly understood. We recently showed that desiccation-enhanced RNA interference (RNAi) soaking can be used in conjunction with quantitative polymerase chain reaction (qPCR) to screen for phenotypes associated with reduced expression of candidate genes in Panagrolaimus sp. DAW1. Here, we present the use of this approach to investigate the role of trehalose synthesis genes in this remarkable organism. Previous studies have shown that acclimating Panagrolaimus sp. DAW1 at 5°C before freezing or desiccation substantially enhances survival. In this study, the expression of tps-2 and other genes associated with trehalose metabolism, as well as lea-1, hsp-70 and gpx-1, in cold-acclimated and non-acclimated nematodes was analyzed using qPCR. Pd-tps-2 and Pd-lea-1 were significantly upregulated after cold acclimation, indicating an inducible expression in the cold adaptation of Panagrolaimus sp. DAW1. The role of trehalose synthesis genes in Panagrolaimus sp. DAW1 was further investigated by RNAi. Compared to the controls, Pd-tps-2a(RNAi)-treated and cold-acclimated nematodes showed a significant decrease in mRNA, but no change in trehalose content or freezing survival. The involvement of two other trehalose synthesis genes (tps-2b and gob-1) was also investigated. These findings provide the first functional genomic investigation of trehalose synthesis genes in the non-model organism Panagrolaimus sp. DAW1. The presence of several trehalose synthesis genes with different RNAi sensitivities suggests the existence of multiple backup systems in Panagrolaimus sp. DAW1, underlining the importance of this sugar in preparation for freezing.

SUBMITTER: Seybold AC

PROVIDER: S-EPMC5769639 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode Panagrolaimus sp. DAW1.

Seybold Anna C AC Wharton David A DA Thorne Michael A S MAS Marshall Craig J CJ

Biology open 20171215 12

Panagrolaimus sp. DAW1 is a freeze-tolerant Antarctic nematode which survives extensive intracellular ice formation. The molecular mechanisms of this extreme adaptation are still poorly understood. We recently showed that desiccation-enhanced RNA interference (RNAi) soaking can be used in conjunction with quantitative polymerase chain reaction (qPCR) to screen for phenotypes associated with reduced expression of candidate genes in Panagrolaimus sp. DAW1. Here, we present the use of ...[more]

PMID: 29175859

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Project description:BACKGROUND: Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics. RESULTS: We studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus. CONCLUSION: This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.

Project description:As exemplified by the famously successful model organism Caenorhabditis elegans, nematodes offer outstanding animal systems for investigating diverse biological phenomena due to their small genome sizes, short generation times and ease of laboratory maintenance. Nematodes in the genus Panagrolaimus have served in comparative development and anhydrobiosis studies, and the Antarctic species P. davidi offers a powerful paradigm for understanding the biological mechanisms of extreme cold tolerance. Panagrolaimus nematodes are also unique in that examples of gonochoristic, hermaphroditic and parthenogenetic reproductive modes have been reported for members of this genus. The evolutionary origins of these varying reproductive modes and the Antarctic species P. davidi, however, remain enigmatic.We collected nuclear ribosomal RNA gene and mitochondrial protein-coding gene sequences from diverse Panagrolaimus species and strains, including newly discovered isolates from Oregon, to investigate phylogenetic relationships in this nematode genus. Nuclear phylogenies showed that the species and strains historically identified as members of Panagrolaimus constitute a paraphyletic group, suggesting that taxonomic revision is required for Panagrolaimus and related nematode lineages. Strain-specific reproductive modes were mapped onto the molecular phylogeny to show a single origin of parthenogenesis from a presumably gonochoristic ancestor. The hermaphroditic strains were all placed outside a major monophyletic clade that contained the majority of other Panagrolaimus nematodes. Phylogenetic analyses of mitochondrial sequences showed that substantial molecular and geographic diversity exists within the clade of parthenogenetic strains. The Antarctic species P. davidi was found to be very closely related to two Panagrolaimus strains from southern California. Phylogenetic and molecular clock analyses suggested that P. davidi and the California strain PS1579 shared a common ancestor in the very recent evolutionary past.Our study provides a phylogenetic framework for understanding the evolutionary origins and diversification patterns of varying reproductive modes within Panagrolaimus and important insights into the origin of the Antarctic species P. davidi. Panagrolaimus offers a powerful nematode model for understanding diverse evolutionary phenomena including the evolution of asexuality and the adaptive evolution of extreme cold tolerance.

Dataset Information

Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode Panagrolaimus sp. DAW1.

Publications

Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode <i>Panagrolaimus</i> sp. DAW1.

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