Identification of Conserved Repetitive Amino Acid Motifs in Antifreeze Proteins of Selected Helopini Species (Coleoptera: Tenebrionidae)

Antifreeze proteins (AFP) protect the organisms against freezing by increasing freezing tolerance. Insects inhabiting cold regions have been reported to utilize AFPs as part of their cold adaptation mechanisms. The tribe Helopini, a member of the Tenebrionidae family, which is distributed across cold environments ranging from lowlands to high-altitude areas. Cylindrinotus charlesi Nabozhenko, 2015, Odocnemis anatolica Pic, 1899, Nalassus clavicornis Allard, 1876, and Xanthomus ovulus Seidlitz, 1895 - four species belonging to the tribe Helopini (Coleoptera, Tenebrionidae) - are active during cold periods of the year and are known to inhabit cold environments such as under stones near snow or buried in cold dune areas in winter or early spring. Due to their cold habitat preferences, the presence of AFPs in these Helopini species was investigated. To begin, primers were designed. Then, genomic DNA was extracted from the specimens and amplified using polymerase chain reaction (PCR). All PCR samples were analyzed by agarose gel electrophoresis. The sequences were edited and aligned using the Geneious software. As a result, this study identified conserved repetitive amino acid sequences (TCTxSxxCxxAx) of antifreeze proteins in Cylindrinotus charlesi, Odocnemis anatolica, Nalassus clavicornis, and Xanthomus ovulus for the first time. Hypothetical 3D structures formed by these sequences were modeled and predicted using the Swiss-Model. The repeat motifs of Helopini AFPs whose sequences were determined, were analyzed using the MotifFinder. These AFPs were classified at the family levels using the SuperFamily database. In addition, the AFP sequences of Helopini species were compared to those of other beetle species available in the public database using BLAST analysis to assess sequence similarity.