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Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a ‘sandbox’ for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

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.

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes. Using a luciferase reporter assay, this strong transcriptional activity was shown to be conserved in cell lines from various insect species, including S2 (Drosophila melanogaster, Diptera), SaPe-4 (Sarcophaga peregrina, Diptera), Sf9 (Spodoptera frugiperda, Lepidoptera) and Tc81 (Tribolium castaneum, Coleoptera) cells. In conjunction with an appropriate selection maker gene, the 121 promoter was able to confer zeocin resistance on SaPe-4 cells and allowed the establishment of stable SaPe-4 cell lines expressing the fluorescent protein AcGFP1; this is the first report of heterologous gene expression in this cell line. These results show the 121 promoter to be a versatile tool for exogenous gene expression in a wide range of insect cell lines, particularly useful to those from non-model insect species.

Simple Summary Anhydrobiosis is a unique ametabolic state that is characterized by extreme body water loss due to desiccation, generally over 95%. The sleeping chironomid, Polypedilum vanderplanki, is an anhydrobiotic insect inhabiting semi-arid regions in Africa. Pv11 is an anhydrobiotic insect cell line established from the P. vanderplanki larvae, and the cell line also displays the extreme desiccation tolerance. This unique feature could be useful for developing a dry preservation technique. However, effective expression systems for protein production have not been available due to a limited number of experimental tools in Pv11 cells. To solve this problem, we identified a minimal promoter from the genome sequence of P. vanderplanki because promoters designed for model organisms are not available in Pv11 cells, although minimal promoters are commonly used for molecular biology experiments and biotechnology fields. The identification of the minimal promoter allowed us to develop an effectively inducible expression system in Pv11 cells. By using the system, several proteins including a functional protein with complex structures were successfully expressed in the cells. The successful establishment of the inducible system on Pv11 cells could contribute to bio-industrial protein production. Abstract The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

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.

Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called “anhydrobiosis”. The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with anhydrobiosis due to their common origin with some LEA genes and their induction during anhydrobiosis.

Anhydrobiosis, an adaptive ability to withstand complete desiccation, is in insects limited to a single species: the nonbiting midge Polypedilum vanderplanki (the sleeping chironomid). Evolution of anhydrobiosis in a single representative of a large genus is associated with drastic changes in genome structure, including the emergence of new multimember gene families directly involved in desiccation tolerance. Among them, Late Embryogenesis Abundant (LEA) proteins, which protect other proteins from aggregation caused by desiccation, are believed to originate via horizontal gene transfer from a bacterial donor. To obtain new insights on the biological background of the expanded 27-member LEA protein group in P. vanderplanki, we investigated the expression of corresponding genes in a P. vanderplanki-derived cell line, capable of anhydrobiosis, in a normal state and during induction of desiccation tolerance. We found that all LEA proteins genes identified in P. vanderplanki’s genome except PvLea16 and PvLea17 are also expressed in Pv11 cells. Their expression was elevated in response to anhydrobiosis-inducing trehalose treatment. Expression patterns of PvLea genes were well preserved in Pv11 cells in comparison to P. vanderplanki’s larvae both in the control group and during the anhydrobiosis cycle. We also investigated localization of LEA proteins in Pv11 cells and Sf9 cells and found a different level of conservation in intracellular localization of the protein expressed in mammalian and insect cells.

Significance Anhydrobiosis is an ametabolic state found in several organisms that can survive extreme desiccation. It is of practical interest because its application to other systems might allow room temperature preservation of cells, tissues, or organs in the dry state. The insect Polypedilum vanderplanki is the most complex animal that can enter anhydrobiosis. Proteins responsible for desiccation tolerance in P. vanderplanki are relatively well studied, but little is known about mechanisms underlying their induction during desiccation. Here, we show that the heat shock transcription factor regulatory network was coopted during the evolution of P. vanderplanki to activate many known desiccation-protective genes, including genes encoding late embryogenesis abundant (LEA) proteins., Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration–rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer. Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer. To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.