Hologenomic Analysis of Anopheles plumbeus Reveals Genomic Complementarity with Its Endosymbiont Candidatus Tisiphia sp.

Anopheles plumbeus is a widespread forest mosquito species and an effective vector of human, animal, and avian malaria pathogens. In this study, we sequenced and assembled the genome of female An. plumbeus mosquitoes obtained from a laboratory culture. The mosquitoes were collected at the larval stage in the vicinity of Sochi (Black Sea coast of the Caucasus) and reared to adulthood on an artificial diet. The genome was generated using Oxford Nanopore Technologies long-read sequencing. The resulting hologenome comprises 1,449 contigs with a total length of 188,457,274 bp. It has been deposited in the European Nucleotide Archive (ENA) and is publicly available as a metagenome-assembled genome. The high quality of the eukaryotic metagenome-assembled genome (eMAG) assembly was confirmed by BUSCO analysis, demonstrating 97.5% complete BUSCOs. A single intracellular bacterial symbiont, Candidatus Tisiphia sp., was identified within the hologenome. The symbiont genome was assembled via a Whole-Genome Shotgun (WGS) approach using the Flye assembler (v. 2.9-b1774), yielding a size of 1.7 Mb. The registered assembly of Candidatus Tisiphia sp. was designated as a 'reference sequence'. Genome annotation revealed 1,761 genes, including 1,525 protein-coding genes, 4 rRNA genes, and 38 tRNA genes. A distinctive feature of this novel Candidatus Tisiphia genome is the high abundance of IS DNA transposons belonging to several families. In total, 361 IS elements were identified in the Candidatus Tisiphia sp. genome. Eight identified IS transposon families—IS6, IS701, IS4, IS630, ISNCY, IS110, IS5, and IS1634—are multicopy. Crucially, we identified for the first time in this bacterial species the biosynthetic genes for folic acid (vitamin B9), biotin (vitamin B7), and thiamine (vitamin B1), as well as a gene cluster for heme biosynthesis, which is essential for the functioning of mitochondrial cytochromes in the mosquito's respiratory chain. These data provide a foundational framework for developing biocontrol strategies against this potentially dangerous malaria vector.