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© 2019, The Author(s). 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.

© 2018, The Author(s). The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to overcome fatal desiccation stress. The Pv11 cell line, derived from P. vanderplanki embryo, shows desiccation tolerance when treated with trehalose before desiccation and resumes proliferation after rehydration. However, the molecular mechanisms of this desiccation tolerance remain unknown. Here, we performed high-throughput CAGE-seq of mRNA and a differentially expressed gene analysis in trehalose-treated, desiccated, and rehydrated Pv11 cells, followed by gene ontology analysis of the identified differentially expressed genes. We detected differentially expressed genes after trehalose treatment involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction that were upregulated. In the desiccation phase, L-isoaspartyl methyltransferase and heat shock proteins were upregulated and ribosomal proteins were downregulated. Analysis of differentially expressed genes during rehydration supported the notion that homologous recombination, nucleotide excision repair, and non-homologous recombination were involved in the recovery process. This study provides initial insights into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells.

© 2016, Springer Science+Business Media New York.Anhydrobiotic larvae of an African chironomid Polypedilum vanderplanki are known to be highly resistant to various abiotic stress factors, including ultraviolet radiation. The comparison of survival rates after different doses of UV irradiation between P. vanderplanki larvae and closely related non-anhydrobiotic Polypedilum nubifer larvae showed strongly enhanced resistance of P. vanderplanki to UV irradiation, especially in completely desiccated state. Plasmid-based assay showed an evidence of contribution of LEA4 protein to the protection of the larvae’s DNA against UV damage.

© 2016, Springer Science+Business Media New York.L-isoaspartyl O-methyltransferases (PIMTs) are known mostly because of their protein chain reparation activity in plant seeds, but also in many other organisms, from bacteria to mammals, including human. The main function of PIMT is recognition and methylation of spontaneously isomerized aspartate residue (Asp) in the amino acid sequences of cellular proteins, and thus preventing their conformation shifts and subsequent inactivation or loss of particular functions. The correlation between PIMT activity and accumulation of isomerized aspartate residues is well studied and used as a marker of isoAsp presence, whereas regulation pathways of PIMT genes are not clear yet. In our study, we propose a possible model for multiple paralogous PIMT gene regulation in the genome of an anhydrobiotic insect Polypedilum vanderplanki. Analysis of promoter regions revealed that PIMT genes possess enriched motif nearby +1 site, associated with GO term heat-shock polytene chromosome puffing. Poly(ADP-ribose) polymerase (PARP) is involved in this process of chromatin loosening and subsequent transcription activation of related genes. This result implied that expression of PIMT genes in P. vanderplanki could be regulated by chromatin modification.

© 2015 Informa UK Limited, trading as Taylor & Francis Group.In this work, we describe a complete mitochondrial genome of chironomid Polypedilum vanderplanki. In spite of unique ecology of this species, the larvae are able to survive years of complete desiccation; the circular mitochondrial genome keeps basic structure and consists of 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and has a total length of 16 060 bp. Low GC level, small regulatory region, and unusually high number and length of non-coding spacers are the main determined features.

© 2016 Elsevier Inc.Water is an essential molecule for living organisms. However, some organisms can survive in environments which receive no rainfall for months and in which ordinary life cannot survive. How do they endure the extended dry season? The sleeping chironomid Polypedilum vanderplanki, which inhabits sub-Saharan Africa, exhibits extreme tolerance to complete desiccation, a process termed anhydrobiosis. During anhydrobiosis these organisms dry up and entirely shut down their metabolism. However, when the dried larvae are immersed in water, their metabolism is resumed. Interestingly, anhydrobiosis allows these organisms to tolerate not only desiccation but also high and low temperatures, the absence of oxygen, radiation, and chemical stresses. Here, we describe the mechanisms by which P. vanderplanki achieves anhydrobiosis revealed in our recent research.

© 2016, Springer Science+Business Media New York.Polipedilum vanderplanki is the most complex known organism able to survive body desiccation via entering a state of suspended metabolism called anhydrobiosis. This unique ability is based on the specific molecular machinery involving a synthesis of non-reducing sugar trehalose and a variety of protective proteins. Genes encoding these protective proteins are extensively duplicated in the P. vanderplanki genome and become hugely upregulated in response to desiccation. Some of these highly expressed genes encode substitutions of amino acids crucial for the function of corresponding proteins. An intriguing group of protective proteins in P. vanderplanki are thioredoxins (TRX). These antioxidant proteins are important for P. vanderplanki anhydrobiosis since desiccation is tightly related to the elevated production of free radicals and oxidative damage. The TRX set is unprecedentedly expanded in the P. vanderplanki genome up to 25 TRX genes. Genomes of congeneric midge Polipedilum nubifer, Apis mellifera, Drosophila melanogaster, and Anopheles gambiae encode only 3–7 TRX genes. Moreover, all three P. vanderplanki thioredoxin genes most expressed at 24 h of P. vanderplanki larva desiccation encode for proteins missing the typical CxxC motif.

© 2016, Springer Science+Business Media New York.Recent intensive studies showed that ability of several groups of organisms to withstand complete desiccation (anhydrobiosis state) is largely based on the activity of several groups of key proteins, including representatives of intrinsically disordered ones. In the largest known anhydrobiotic animal, the sleeping chironomid, both proteomic and genome-wide/targeted mRNA expression analysis suggest that several groups of the novel proteins greatly contribute to the formation of the molecular shield. Among them, 114 aa-long protein, dryporin. Being one of the most abundant in the desiccated larvae on protein level and showing one of the highest expression on mRNA level, this protein shows lack of the obvious homology with other known proteins. In this study, we successfully produced recombinant dryporin protein using BY-2 cell free lysate and showed the evidence of post-translational modification of the protein in the anhydrobiotic larvae.

© 2016, Springer Japan.It is assumed that resistance to ionizing radiation, as well as cross-resistance to other abiotic stresses, is a side effect of the evolutionary-based adaptation of anhydrobiotic animals to dehydration stress. Larvae of Polypedilum vanderplanki can withstand prolonged desiccation as well as high doses of ionizing radiation exposure. For a further understanding of the mechanisms of cross-tolerance to both types of stress exposure, we profiled genome-wide mRNA expression patterns using microarray techniques on the chironomid larvae collected at different stages of desiccation and after exposure to two types of ionizing radiation—70 Gy of high-linear energy transfer (LET) ions (4He) and the same dose of low-LET radiation (gamma rays). In expression profiles, a wide transcriptional response to desiccation stress that much exceeded the amount of up-regulated transcripts to irradiation exposure was observed. An extensive group of coincidently up-regulated overlapped transcripts in response to desiccation and ionizing radiation was found. Among this, overlapped set of transcripts was indicated anhydrobiosis-related genes: antioxidants, late embryogenesis abundant (LEA) proteins, and heat-shock proteins. The most overexpressed group was that of protein-L-isoaspartate/D-aspartate O-methyltransferase (PIMT), while probes, corresponding to LEA proteins, were the most represented. Performed functional analysis showed strongly enriched gene ontology terms associated with protein methylation. In addition, active processes of DNA repair were detected. We assume that the cross-tolerance of the sleeping chironomid to both desiccation and irradiation exposure comes from a complex mechanism of adaptation to anhydrobiosis.

© 2016, Springer Japan.Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5–34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3-4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by de

© Ninel A. Petrova et al. The African chironomid Polypedilum vanderplanki Hinton, 1951 is the only chironomid able to withstand almost complete desiccation in an ametabolic state known as anhydrobiosis. The karyotypes of two allopatric populations of this anhydrobiotic chironomid, one from Nigeria and another from Malawi, were described according to the polytene giant chromosomes. The karyotype from the Nigerian population was presented as the reference chromosome map for P. vanderplanki. Both populations, Nigerian and Malawian, showed the same number of chromosomes (2n=8), but important differences were found in the band sequences of polytene chromosomes, and in the number and the arrangement of active regions between the two populations. Such important differences raise the possibility that the Malawian population could constitute a distinct new species of anhydrobiotic chironomid.

© 2014 Macmillan Publishers Limited. All rights reserved. Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

© 2015, Springer-Verlag Berlin Heidelberg. Main conclusion: In the anhydrobiotic midgePolypedilum vanderplanki, LEA family proteins are likely to play distinct temporal and spatial roles in the larvae throughout the process of desiccation and rehydration. The larvae of the anhydrobiotic midge, P. vanderplanki, which can tolerate almost complete desiccation, accumulate late embryogenesis abundant (LEA) proteins in response to drying. Using complete genome data of the midge, we have identified 27 PvLea1-like genes based on the similarity to previously characterized PvLea1 gene belonging to group 3 LEA proteins. Generally, group 3 LEA proteins are characterized by several repetitions of an 11-mer motif. However, some PvLea genes lack the canonical motif in their sequences. We performed the detailed characterization of all 27 PvLea genes in terms of biochemical and biophysical properties and conserved motifs. The motif analysis among their amino acid sequences revealed that all 27 PvLEA proteins have at least one of two types of motifs (motif 1: G AKDTTKEKLGE AKDATAEKLG or motif 2: KD ILExAKDKLxD AKDAVKEKL), indicating the presence of at least two repeated 11-mer LEA motifs. Most of PvLEA proteins were localized to the cytosol. We also performed quantitative real-time PCR of all 27 PvLea genes in detail during the process of desiccation and rehydration. The expression of these genes was upregulated at the beginning of dehydration, the latter phase of the desiccation process and on rehydration process. These data suggested that each LEA protein is likely to play distinct temporal and spatial roles in the larvae throughout the process of desiccation and rehydration.

© 2015 Taylor & Francis In this work, we describe a complete mitochondrial genome of chironomid Polypedilum vanderplanki. In spite of unique ecology of this species, the larvae are able to survive years of complete desiccation; the circular mitochondrial genome keeps basic structure and consists of 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and has a total length of 16 060 bp. Low GC level, small regulatory region, and unusually high number and length of non-coding spacers are the main determined features.

© 2015 Zoological Society of Japan. Chironomid midges (Diptera; Chironomidae) are found in various environments from the high Arctic to the Antarctic, including temperate and tropical regions. In many freshwater habitats, members of this family are among the most abundant invertebrates. In the present study, the genome sizes of 25 chironomid species were determined by flow cytometry and the resulting C-values ranged from 0.07 to 0.20 pg DNA (i.e. from about 68 to 195 Mbp). These genome sizes were uniformly very small and included, to our knowledge, the smallest genome sizes recorded to date among insects. Small proportion of transposable elements and short intron sizes were suggested to contribute to the reduction of genome sizes in chironomids. We discuss about the possible developmental and physiological advantages of having a small genome size and about putative implications for the ecological success of the family Chironomidae.

© 2017 The Author(s). Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic midge embryo endure complete desiccation in an ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells could preserve an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase (Luc), preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including preincubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pv11-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pv11-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation tolerance-related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

© 2017 The Royal Entomological Society The sleeping chironomid (Polypedilum vanderplanki Hinton) lives on temporary rock pools in the semi-arid tropical regions of Africa. Its larvae are able to survive the dry season in a completely desiccated ametabolic state known as anhydrobiosis. So far, P. vanderplanki was the only species among all insects showing demonstrated anhydrobiotic ability. Here, we show that a new related species originating from Malawi, Polypedilum pembai sp.n., is also anhydrobiotic and that its desiccation tolerance mechanism is probably similar to what is observed in P. vanderplanki. The new species, P. pembai sp.n., is described with special attention to the common and different morphological features, compared with P. vanderplanki. Phylogenetic analysis showed that both species are closely related, suggesting that anhydrobiosis evolved only once in their common ancestor about 49 Ma somewhere in Africa, before the divergence of two species, one in the sub-Saharan area and another in southeastern Africa.

© 2017 The Royal Entomological Society The sleeping chironomid (Polypedilum vanderplanki Hinton) lives on temporary rock pools in the semi-arid tropical regions of Africa. Its larvae are able to survive the dry season in a completely desiccated ametabolic state known as anhydrobiosis. So far, P. vanderplanki was the only species among all insects showing demonstrated anhydrobiotic ability. Here, we show that a new related species originating from Malawi, Polypedilum pembai sp.n., is also anhydrobiotic and that its desiccation tolerance mechanism is probably similar to what is observed in P. vanderplanki. The new species, P. pembai sp.n., is described with special attention to the common and different morphological features, compared with P. vanderplanki. Phylogenetic analysis showed that both species are closely related, suggesting that anhydrobiosis evolved only once in their common ancestor about 49 Ma somewhere in Africa, before the divergence of two species, one in the sub-Saharan area and another in southeastern Africa.