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

Phylogenomics revealed migration routes and adaptive radiation timing of holarctic malaria mosquito species of the Maculipennis group

BackgroundPhylogenetic 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.ResultsTo 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.ConclusionsOur 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

Partial-arm translocations in evolution of malaria mosquitoes revealed by high-coverage physical mapping of the Anopheles atroparvus genome

Abstract Background Malaria mosquitoes have had a remarkable stability in the number of chromosomes in their karyotype (2n = 6) during 100 million years of evolution. Moreover, autosomal arms were assumed to maintain their integrity even if their associations with each other changed via whole-arm translocations. Here we use high-coverage comparative physical genome mapping of three Anopheles species to test the extent of evolutionary conservation of chromosomal arms in malaria mosquitoes. Results In this study, we developed a physical genome map for Anopheles atroparvus, one of the dominant malaria vectors in Europe. Using fluorescence in situ hybridization (FISH) of DNA probes with the ovarian nurse cell polytene chromosomes and synteny comparison, we anchored 56 genomic scaffolds to the An. atroparvus chromosomes. The obtained physical map represents 89.6% of the An. atroparvus genome. This genome has the second highest mapping coverage among Anophelinae assemblies after An. albimanus, which has 98.2% of the genome assigned to its chromosomes. A comparison of the An. atroparvus, An. albimanus, and An. gambiae genomes identified partial-arm translocations between the autosomal arms that break down the integrity of chromosome elements in evolution affecting the structure of the genetic material in the pericentromeric regions. Unlike An. atroparvus and An. albimanus, all chromosome elements of An. gambiae are fully syntenic with chromosome elements of the putative ancestral Anopheles karyotype. We also detected nonrandom distribution of large conserved synteny blocks and confirmed a higher rate of inversion fixation in the X chromosome compared with autosomes. Conclusions Our study demonstrates the power of physical mapping for understanding the genome evolution in malaria mosquitoes. The results indicate that syntenic relationships among chromosome elements of Anopheles species have not been fully preserved because of multiple partial-arm translocations

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

Correct expression and localization of collagen XIII are crucial for the normal formation and function of the neuromuscular system

Abstract Transmembrane collagen XIII has been linked to maturation of the musculoskeletal system. Its absence in mice (Col13a1−/−) results in impaired neuromuscular junction (NMJ) differentiation and function, while transgenic overexpression (Col13a1oe) leads to abnormally high bone mass. Similarly, loss‐of‐function mutations in COL13A1 in humans produce muscle weakness, decreased motor synapse function and mild dysmorphic skeletal features. Here, analysis of the exogenous overexpression of collagen XIII in various muscles revealed highly increased transcript and protein levels, especially in the diaphragm. Unexpectedly, the main location of exogenous collagen XIII in the muscle was extrasynaptic, in fibroblast‐like cells, while some motor synapses were devoid of collagen XIII, possibly due to a dominant negative effect. Concomitantly, phenotypical changes in the NMJs of the Col13a1oe mice partly resembled those previously observed in Col13a1−/− mice. Namely, the overall increase in collagen XIII expression in the muscle produced both pre‐ and postsynaptic abnormalities at the NMJ, especially in the diaphragm. We discovered delayed and compromised acetylcholine receptor (AChR) clustering, axonal neurofilament aggregation, patchy acetylcholine vesicle (AChV) accumulation, disrupted adhesion of the nerve and muscle, Schwann cell invagination and altered evoked synaptic function. Furthermore, the patterns of the nerve trunks and AChR clusters in the diaphragm were broader in the adult muscles, and already prenatally in the Col13a1−/− mice, suggesting collagen XIII involvement in the development of the neuromuscular system. Overall, these results confirm the role of collagen XIII at the neuromuscular synapses and highlight the importance of its correct expression and localization for motor synapse formation and function

The measurements of <i>Cx</i>. <i>quinquefasciatus</i> mitotic chromosomes from imaginal discs in comparison to <i>Ae</i>. <i>aegypti</i>.

<p>The measurements of <i>Cx</i>. <i>quinquefasciatus</i> mitotic chromosomes from imaginal discs in comparison to <i>Ae</i>. <i>aegypti</i>.</p

Two variants of sex-determining chromosome 1 at early- (A) and mid-metaphase (B).

<p>Position of 18S rDNA probe on chromosome 1 is indicated by arrow.</p