Two Nested Inversions in the X Chromosome Differentiate the Dominant Malaria Vectors in Europe, and .

The Maculipennis subgroup of malaria mosquitoes includes both dominant malaria vectors and non-vectors in Eurasia. Understanding the genetic factors, particularly chromosomal inversions, that differentiate species can provide valuable insights for vector control strategies. Although autosomal inversions between the species in this subgroup have been characterized based on the chromosomal banding patterns, the number and positions of rearrangements in the X chromosome remain unclear due to the divergent banding patterns. Here, we identified two large X chromosomal inversions, approximately 13 Mb and 10 Mb in size, using fluorescence in situ hybridization. The inversion breakpoint regions were mapped by hybridizing 53 gene markers with polytene chromosomes of . The DNA probes were designed based on gene sequences from the annotated genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the genome, changed their position and orientation in the X chromosome. The analysis of the genome revealed an enrichment of gene ontology terms associated with immune system and mating behavior in the rearranged syntenic blocks. Additionally, the enrichment of DNA transposons was found in sequences homologous to three of the four breakpoint regions. This study demonstrates the successful application of the physical genome mapping approach to identify rearrangements that differentiate species in insects with polytene chromosomes.