Proteomic evidence for the silk fibroin genes of spider mites (order Trombidiformes: family Tetranychidae)

Spider mites are a group of arachnids belonging in the family Tetranychidae, known to produce nano-scale silk fibers characterized by high Young’s modulus. Silk fibroin gene of spider mites has been computationally suggested through genomic analysis of Tetranychus urticae, but it is yet to be confirmed by proteomic evidences. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawaiand Panonychus citri, and combined with the silk proteomics of T. urticae and P. citrito characterize the fibroin genes through comparative genomics and multi-omics analysis. As a result, two fibroins were identified, that are different genes from the previous computational prediction. Amino acid composition and secondary structure suggests similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical property exhibiting high Young’s modulus. Availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that emerged in multiple lineages sometimes in convergent manner, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.

Sample preservation, RNA extraction, sequencing and assembly have been conducted for T. kanzawai, and P. citribased on methods previously described for spiders [18], with some modification. Briefly, a single specimen of each of the spider mite was preserved in RNAlater stored at -80 ˚C, and RNA was extracted using Direct-zol RNA Microprep Kit (Zymo Research). Illumina library was prepared using KAPA HyperPlus Kit (KAPA Biosystems) targeting 300bp fragments. Sequence library was then sequenced on NextSeq 500 (Illumina) with High Output Mode 300 cycles as paired-end reads. Sequences were basecalled, demultiplexed, and adaptors sequences were removed with bcls2fastq v.2 software (Illumina). Transcriptome assembly was performed using Bridger software with default parameters [19]. Expression levels of transcripts were quantified using kallisto v.0.44 [20]using the assembled transcriptome as well as previously published predicted coding sequences from the genome of T. urticae[16]. Assembly completeness was assessed using BUSCO v.3 [21]with Arthropoda dataset through gVolante server [22].