Завершённая нуклеотидная последовательность полного генома хищного клеща Neoseiulus Californicus (Mesostigmata, Phytoseiidae) лабораторной линии (BioDefence)

Neoseiulus californicus McGregor, 1954 was initially discovered and named Typhlodromus californicus McGregor, 1954 in California. Today, N. californicus can be found in tropical and subtropical climates worldwide. This global distribution is likely due to human activities, as these mites are introduced into natural environments from greenhouses where they are imported as biocontrol agents. The reason for the widespread use of N. californicus in agriculture is its effectiveness in controlling both the common two-spotted spider mite Tetranychus urticae Koch, 1836, and the more harmful red tomato spider mite Tetranychus evansi Baker &Pritchard, 1960, particularly on solanaceous crops like tomatoes. N. californicus, a predator with limited specialization, not only preys on spider mites but also effectively controls thrips, whiteflies, tarsonemid mites, and other small pests that affect plants. In addition, N. californicus can survive without foraging mites by feeding on pollen grains, making it adaptable to varying conditions. Another advantage of N. californicus is its ability to thrive in both greenhouse and field environments, even in low humidity

Genomic DNA extraction. Adult predatory mites were transferred to a culture vial, where they were starved for two days to remove possible DNA contamination from food mites. The mites were then collected in a lysis solution. High molecular weight DNA for whole genome sequencing was isolated using the classic alkaline phenol-chloroform method, followed by treatment with Proteinase K enzyme. Most of the RNA was removed by T1 ribonuclease treatment. The quality and purity of the extracted DNA were assessed by employing a NanoDrop spectrophotometer (Thermo Scientific). The concentration of DNA was measured using a Qubit fluorometer (Invitrogen) and reagent kits (Qubit dsDNA HS Assay Kit, Invitrogen). For sequencing and basecalling purposes, a library for nanopore sequencing preparation was created using the SQK-LSK110 kit from Oxford Nanopore Technologies, following the instructions provided by the manufacturer. Additionally, a final step of long DNA selection was performed using the large fragment buffer (LFB). To prepare the library, the ends of the DNA molecules were repaired and then linked with adapters, utilizing the NEBNext Companion Module. Sequencing was performed in a MinION R9.4.1 streaming cell using MinKNOW v.5.1.0 without immediate base calling. Reads longer than 200 bp were retained. Base calling was performed on a server with two GeForce RTX 3090 graphics cards and 12 Intel Xeon processors running Guppy v6.0.1+652ffd179 software under the super-neat sup model

After base calling, we obtained 3507265 reads for the BioDefence lineage and 1769217 reads for the BioDefence2 lineage. The complete genomic sequence was then assembled from the reads using the canu v programme

GenBank currently has a contig level genome of N. contig. californicus from China [GenBank ID: GCA_028455905.1] and a raw read level genome from Japan [BioProject Accession ID: PRJNA966143]. The genome presented in this article adds a third genome of N. californicus from the Moscow region. In addition to the assembly of the nuclear genome, we assembled the complete mitochondrial genome of N. californicus, which has a unique mitochondrial gene sequence and an extended control region that varies in length between tick strains due to different numbers of direct incomplete repeats. Recently, the mitochondrial genome of N. californicus was obtained by Zu using Illumina sequencing technology and deposited in GenBank under accession no: NC_069213. This mitochondrial genome and two new mitochondrial genomes from N. californicus have the same gene order and very similar nucleotide sequence, but differ in the length of control regions due to different numbers of direct incomplete repeats. High variability in mitochondrial gene order is a characteristic feature of mites of the family Phytoseiidae. Mites belonging to the family Phytoseiidae possess a standard set of mitochondrial genes, with the exception of Metaseiulus occidentalis Nesbitt, 1951, which lacks the ND6 gene. However, these mites differ in the order of their mitochondrial genes

The genome sequence of the N. californicus strain named BioDefence has been submitted to the GenBank database under the accession number GCA_028659405.1 (version GCA_028659405.1). The corresponding BioProject accession number is PRJNA902297, and the BioSample accession number is SAMN32036229. The raw sequencing data are available from the SRA (Sequence Read Archive) under accession number SRP411367. The genome sequence of the strain N. californicus: BioDefence2 has been deposited in the GenBank database under accession number GCA_031001865.1 (version GCA_031001865.1). The corresponding BioProject accession number is PRJNA946627 and the BioSample accession number is SAMN33824953. The raw sequencing data are also available from the SRA (Sequence Read Archive) under accession number SRP428294. The N. californicus genome sequence presented here contains the genome sequences of the mite and its bacterial symbionts, obligate intracellular symbionts such as Wolbachia and Spiroplasma, and facultative intestinal symbionts that have not been removed by starvation for two days. The presented mitochondrial genome of N. californicus offers valuable insights for research on the genetic regulation of economically important characteristics of N. californicus and its hologenome, such as the makeup of beneficial and harmful bacteria that greatly influence the survival, reproductive capacity, and nutritional behavior of biocontrol mite populations belonging to the Phytoseiidae family.