| Summary: | The Antarctic nematode Panagrolaimus davidi is the only organism known to survive extensive intracellular freezing throughout its tissues. While some animals can tolerate extracellular freezing, intracellular freezing is only documented in specific tissues of very few species. The extreme tolerance of P. davidi enables this nematode, together with tardigrades and rotifers, to survive in one of earth's coldest and driest environments. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. The presence of ice-active proteins has been suggested by the observation of recrystallization inhibition and hexagonal ice crystals. Furthermore, a recent transcriptomic study identified a number of genes up-regulated during acclimation including trehalose synthesis (tps) genes, late embryogenesis abundant (lea) proteins, heat shock proteins (hsp) and antioxidants. To approach P. davidi on a molecular level, the accessibility of this nematode towards high-throughput RNAi techniques has been investigated. The sensitivity of P. davidi towards RNAi-feeding was investigated compared with C. elegans as a positive technical control. Nematodes were fed dsRNA from one of two embryonic lethal genes (rps-2 and dhc) and one blister gene (duox) and their mRNA level measured using quantitative PCR. Pd-rps-2(RNAi) treated nematodes showed a significant decrease in larval hatching, but Pd-dhc(RNAi) and Pd-duox-42(RNAi) treated nematodes showed no phenotype. Furthermore, qPCR did not show a significant decrease in the mRNA level of Pd-rps-2(RNAi) treated animals, but showed a maximum down-regulation of Pd-rps-2 after 24 h of feeding. The dsRNA uptake of three different soaking protocols was investigated using the fluorescent dye FITC: soaking alone, octopamine-enhanced soaking and desiccation-enhanced soaking. Nematodes soaked with and without octopamine showed mainly localized fluorescence, whereas those desiccated prior to soaking showed fluorescence throughout the body. Gene expression analysis of desiccated and soaked nematodes showed a significant and consistent decreased mRNA level of two of four tested genes (rps-2 and tps-2a). These data suggest that desiccation prior to soaking enhances the uptake of dsRNA-containing solution by the nematode on a cellular level. In order to use this newly developed RNAi technique to screen for genes important in tolerating freezing, the expression of tps-2, lea-1, hsp-70 and gpx-1 (glutathione peroxidase 1) of acclimated and non-acclimated nematodes was analyzed using qPCR. Pd-tps-2 and Pd-lea-1 were significantly up-regulated after acclimation, indicating an inducible expression in the cold adaptation of P. davidi. The activity of tps-2 after acclimation was also confirmed by an increased amount of trehalose itself (measured by gas chromatography). In contrast, Pd-gpx-1 and Pd-hsp-70 remained nearly unchanged, suggesting constitutive expression in P. davidi. Finally, the role of trehalose as well as the genes involved in trehalose synthesis in P. davidi was investigated. Compared to the controls, Pd-tps-2a(RNAi) treated and acclimated nematodes showed a significantly decreased mRNA level, but no reduction in trehalose or in freezing survival. The involvement of two other trehalose synthesis genes (tps-2b and gob-1) were revealed by qPCR. However, Pd-tps-2b(RNAi) treated and acclimated nematodes showed no decrease in mRNA level compared to the controls. The fact that not only the two tps-2 genes, but also gob-1 is involved in trehalose synthesis, suggests the existence of a multiple backup system in P. davidi. Taken together, these findings provide the first functional genomic approach to freezing tolerance in P. davidi and provide a set of tools which can be used to screen for genes involved in its extreme adaptation. The testing of different RNAi techniques showed that desiccation enhanced soaking can be used - in conjunction with qPCR - to screen for candidate genes. This molecular approach could help to uncover the secret of intracellular freezing tolerance in P. davidi, providing new insights in evolution and new applications for cryopreservation.
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