Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

Cas9-mediated Genome Editing Reveals a Significant Contribution of Calcium Signaling Pathways to Anhydrobiosis in Pv11

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin – calcineurin – NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin – calmodulin Kinase – CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose–induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

Population genomics of two closely related anhydrobiotic midges reveals differences in adaptation to extreme desiccation

The sleeping chironomid Polypedilum vanderplanki is capable of anhydrobiosis, a striking example of adaptation to extreme desiccation. Tolerance to complete desiccation in this species is associated with the emergence of multiple paralogs of protective genes. One of the gene families highly expressed under anhydrobiosis and involved in this process are protein-L-isoaspartate (D-aspartate) O-methyltransferases (PIMTs). Recently, a closely related anhydrobiotic midge from Malawi, P. pembai, showing the ability to tolerate complete desiccation similar to that of P. vanderplanki, but experiences more frequent desiccation-rehydration cycles due to differences in ecology, was discovered. Here, we sequenced and assembled the genome of P. pembai and performed a population genomics analysis of several populations of P. vanderplanki and a population of P. pembai. We observe positive selection and radical changes in the genetic architecture of the PIMT locus between the two species, including multiple duplication events in the P. pembai lineage. In particular, PIMT-4, the most highly expressed of these PIMTs, is present in six copies in the P. pembai; these copies differ in expression profiles, suggesting possible sub- or neofunctionalization. The nucleotide diversity (π) of the genomic region carrying these new genes is decreased in P. pembai, but not in the orthologous region carrying the ancestral gene in P. vanderplanki, providing evidence for a selective sweep associated with post-duplication adaptation in the former. Overall, our results suggest an extensive recent and likely ongoing, adaptation of the mechanisms of anhydrobiosis.

Combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect

Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called anhydrobiosis. The only known example of anhydrobiosis among insects is observed in larvae of the chironomidPolypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.

Transcriptome analysis of Pv11 cells infers the mechanism of desiccation tolerance and recovery

ABSTRACTThe larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to conquer fatal desiccation stress. The Pv11 cell line, derived from embryos of the midge, shows desiccation tolerance by pretreatment with trehalose before desiccation; they can resume proliferation after rehydration. To address the underlying molecular mechanisms, we desiccated Pv11 cells after pretreatment with the medium containing trehalose and induced proliferation by rehydration. We collected the cells at each before and after desiccation and rehydration step and performed CAGE-seq of mRNA of those cells. By analysing differentially expressed genes (DEGs) among the results of CAGE-seq, we detected 384 DEGs after trehalose treatment and 14 DEGs after rehydration. Hierarchical clustering of the identified DEGs indicated that rehydration returns their expression pattern to that in the control culture state. DEGs involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction were upregulated by trehalose treatment. DEGs for rehydration supported that DNA repair is one of the potential mechanisms involves recovery. This study provided initial insight into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells with a potential for proliferation following rehydration.