Integrative cytogenetic and genome size analyses reveal conserved karyotypes and repetitive DNA-driven genome variation in the mosquitoes Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti

Mosquitoes (Diptera: Culicidae) display a conserved chromosome number, yet their genomes exhibit substantial diversity in composition and organization. However, this diversity remains poorly understood in ecologically relevant but understudied taxa. Here, we investigate genome organization in the Neotropical mosquitoes Toxorhynchites theobaldi , Toxorhynchites violaceus , and Lutzia bigoti , revealing how conserved karyotypes can mask pronounced genomic divergence. Mitotic chromosomes were analyzed by Giemsa staining, C-banding, CMA₃/DAPI, and fluorescence in situ hybridization (FISH) with rDNA and microsatellite probes. Chromosome-specific probes were generated by micromanipulation and used in cross-species hybridization. Genome size was estimated by flow cytometry. All species shared the conserved karyotype (2 n = 6), but differed in chromosome architecture and genome size. Despite variation in chromosome length at the descriptive level, overall chromosome size did not differ significantly among species, whereas nuclear DNA content showed marked interspecific variation. This decoupling indicates that genome size evolution is not accompanied by proportional changes in chromosome number and length. Comparative analyses of repetitive DNA revealed a consistent concentration of heterochromatin in pericentromeric regions, alongside species-specific patterns of GC-rich sequences and rDNA loci. While Toxorhynchites species retained a conserved rDNA organization, no detectable signal was observed in L. bigoti , potentially reflecting divergence in rDNA structure. A chromosome-specific probe obtained by micromanipulation hybridized to pericentromeric regions in both T. theobaldi and T. violaceus , although the labeled chromosome pair differed between species. In addition, species-specific differences in microsatellite distribution and the partial conservation of repetitive DNA sequences between Toxorhynchites species highlight the dynamic nature of the mosquito repeatome. Together, these findings demonstrate that genome evolution in Culicidae is primarily driven by changes in repetitive DNA content and organization, rather than by alterations in chromosome number or large-scale morphology, revealing hidden genomic diversity.