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

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 Anopheles 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 An. messeae. The DNA probes were designed based on gene sequences from the annotated An. atroparvus genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the An. atroparvus genome, changed their position and orientation in the X chromosome. The analysis of the An. atroparvus 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.

Phylogenomics revealed migration routes and adaptive radiation timing of Holarctic malaria mosquito species of the Maculipennis Group.

BACKGROUND: Phylogenetic analyses of closely related species of mosquitoes are important for better understanding the evolution of traits contributing to transmission of vector-borne diseases. Six out of 41 dominant malaria vectors of the genus Anopheles in the world belong to the Maculipennis Group, which is subdivided into two Nearctic subgroups (Freeborni and Quadrimaculatus) and one Palearctic (Maculipennis) subgroup. Although previous studies considered the Nearctic subgroups as ancestral, details about their relationship with the Palearctic subgroup, and their migration times and routes from North America to Eurasia remain controversial. The Palearctic species An. beklemishevi is currently included in the Nearctic Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics. RESULTS: To reconstruct historic relationships in the Maculipennis Group, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on sequences of 1271 orthologous genes. The analysis indicated that the Palearctic species An. beklemishevi clusters together with other Eurasian species and represents a basal lineage among them. Also, An. beklemishevi is related more closely to An. freeborni, which inhabits the Western United States, rather than to An. quadrimaculatus, a species from the Eastern United States. The time-calibrated tree suggests a migration of mosquitoes in the Maculipennis Group from North America to Eurasia about 20-25 million years ago through the Bering Land Bridge. A Hybridcheck analysis demonstrated highly significant signatures of introgression events between allopatric species An. labranchiae and An. beklemishevi. The analysis also identified ancestral introgression events between An. sacharovi and its Nearctic relative An. freeborni despite their current geographic isolation. The reconstructed phylogeny suggests that vector competence and the ability to enter complete diapause during winter evolved independently in different lineages of the Maculipennis Group. CONCLUSIONS: Our phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support the inclusion of An. beklemishevi into the Maculipennis Subgroup. Detailed knowledge of the evolutionary history of the Maculipennis Subgroup provides a framework for examining the genomic changes related to ecological adaptation and susceptibility to human pathogens. These genomic variations may inform researchers about similar changes in the future providing insights into the patterns of disease transmission in Eurasia.

A standard photomap of ovarian nurse cell chromosomes and inversion polymorphism in Anopheles beklemishevi.

BACKGROUND: Anopheles beklemishevi is a member of the Maculipennis group of malaria mosquitoes that has the most northern distribution among other members of the group. Although a cytogenetic map for the larval salivary gland chromosomes of this species has been developed, a high-quality standard cytogenetic photomap that enables genomics and population genetics studies of this mosquito at the adult stage is still lacking. METHODS: In this study, a cytogenetic map for the polytene chromosomes of An. beklemishevi from ovarian nurse cells was developed using high-resolution digital imaging from field collected mosquitoes. PCR-amplified DNA probes for fluorescence in situ hybridization (FISH) were designed based on the genome of An. atroparvus. The DNA probe obtained by microdissection procedures from the breakpoint region was labelled in a DOP-PCR reaction. Population analysis was performed on 371 specimens collected in 18 locations. RESULTS: We report the development of a high-quality standard photomap for the polytene chromosomes from ovarian nurse cells of An. beklemishevi. To confirm the suitability of the map for physical mapping, several PCR-amplified probes were mapped to the chromosomes of An. beklemishevi using FISH. In addition, we identified and mapped DNA probes to flanking regions of the breakpoints of two inversions on chromosome X of this species. Inversion polymorphism was determined in 13 geographically distant populations of An. beklemishevi. Four polymorphic inversions were detected. The positions of common chromosomal inversions were indicated on the map. CONCLUSIONS: The study constructed a standard photomap for ovarian nurse cell chromosomes of An. beklemishevi and tested its suitability for physical genome mapping and population studies. Cytogenetic analysis determined inversion polymorphism in natural populations of An. beklemishevi related to this species' adaptation.

Mitotic-chromosome-based physical mapping of the Culex quinquefasciatus genome.

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome.

Cyst geometry in the egg chambers of Calliphora erythrocephala Mg. (Diptera: Calliphoridae) ovaries.

In the germarium of polytrophic ovarioles of Calliphora erythrocephala (Mg.) fly, four mitotic divisions of cystoblasts give rise to 16-cell germ-line cysts. One cell differentiates into an oocyte, while the remaining 15 cells become nurse cells. Concomitantly actin-rich ring canals are formed at the intercellular junctions. The present study considers a mutual arrangement of the ring canals formed after the second to fourth mitoses relative to the ring canal formed after the first mitotic division in different regions of the germarium and egg chambers. During the cyst formation and its movement to the posterior end of the germarium, the ring canals are displaced relative to one another, thereby giving different branching variants of the cyst. The pattern of cell interconnections becomes stable in germarium region 2b and does not change during the cyst movement along the ovariole despite the cyst polarizes and increases in size.

The changes in chromosome 6 spatial organization during chromatin polytenization in the Calliphora erythrocephala Mg. (Diptera: Calliphoridae) nurse cells.

Localization of Calliphora erythrocephala chromosome 6 in a 3D nuclear space at different stages of nurse cell chromatin polytenization was analyzed by fluorescence in situ hybridization and 3D microscopy. The obtained results suggest a large-scale chromatin relocation in the C. erythrocephala nurse cell nuclei, which is accompanied by a change in the chromosome territory of chromosome 6 associated with the change in expression activity of the nucleus and formation of reticular chromatin structure. It was revealed that the relocation of chromosome 6 (nucleolus organizer chromosome) is accompanied by fragmentation of the single large nucleolus into micronucleoli, which are spread over the entire nuclear space being associated with their nucleolar organizer regions. Presumably, the chromosome 6 material during transition to a highly polytenized structure is redistributed in the nucleus so that the inactive pericentromeric regions are displaced to the nuclear periphery, while the chromosome regions carrying rDNA sequences loop out beyond the chromosome territory. Being dispersed over the entire nuclear space, rDNA sequences are likely to be amplified, thereby providing numerous small signals from the chromosome 6-specific DNA probe. Micronucleoli are formed around the actively transcribed nucleolar organizer regions.