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 is the ability of an organism to survive complete desiccation in the ametabolic state. Animals able to enter anhydrobiosis at least at some life stages are found in four invertebrate phyla, namely tardigrades, rotifers, nematodes and arthropods 1,2. Damaging effects of desiccation in these animals are mitigated via interplay of numerous protective mechanisms, including the formation of biological glass (vitrification), "molecular shield" and anti-aggregation activity of some proteins and enhanced antioxidant activity 3-5. Intrinsically disordered proteins (IDP's) frequently participate at least in some of the protective mechanisms related to desiccation tolerance 5-9. Anhydrobiotic animals share also such features as a small size which is typically less than 5 mm and an absence of internal skeletons, at least on anhydrobiotic life stages. This may be related to physical stresses associated with body shrinking during water loss 10. Larvae of the sleeping chironomid Polypedilum vanderplanki (Diptera) reaching 7 mm in length are the largest and the most complex organisms able to enter anhydrobiosis 11. This midge inhabits semi-arid rocks in Nigeria, and its larvae represent the only stage of the life cycle that is capable of enduring the desiccation at the onset of the dry season 12 .

Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.

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