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.

Analysis of Anopheles messeae s.l. intron gene polymorphism associated with imidacloprid resistance.

Due to their high solubility and stability, neonicotinoid insecticides are able to accumulate in water bodies, affecting aquatic organisms. The aims of this study were to evaluate resistance (LC50 ) of Anopheles messeae s.l. (Anopheles messeae and An. daciae) to the neonicotinoid imidacloprid and to search for genetic markers associated with insecticide resistance. The LC50 values of these species in the collections during 2017 and 2018 were indistinguishable and were in the range of 0.027-0.051 mg/l. In general, the LC50 values of the mosquitoes were comparable with values of other mosquito species of the Anopheles and Culex genera. Gene polymorphisms of the variations in intron lengths and the presence of restriction sites in introns that were potentially associated with the metabolism of insecticides were studied. Polymorphisms of the studied genes in the pair of closely related species considered overlapped, but allele frequencies were different. Part of the genetic variants arose due to insertions of repetitive elements of the genome. Two variants of the cytochrome P450 gene Cyp6AG1 in An. daciae were associated with increased resistance to imidacloprid. Possible side effects of selection on insecticide resistance in blood-sucking mosquitoes are discussed.

Improved reference genome of the arboviral vector Aedes albopictus.

BACKGROUND: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. RESULTS: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. CONCLUSION: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

Estimated resistance of the malaria mosquito Anopheles messeae s.l. to the insecticide malathion.

Resistance to agricultural pesticides is an important and insufficiently studied concern for pest and disease vector research. We determined the malathion resistance of species in the Anopheles maculipennis mosquito group in a habitat near Novosibirsk, Russia. Most of the 851 individuals we measured were members of the Anopheles messeae s.l. complex (An. messeae and An. daciae species). The LC50 value for malathion was 0.052 mg/L for the mixed specimens, and we failed to find any differences between species. The LC50 value was within the range of values for malathion resistance of Anopheles stephensi and Culex quinquefasciatus. As the main resistance mechanism to organophosphate and carbamate insecticides is a single mononucleotide substitution in the ace-1 gene, we searched for this mutation in An. messeae s.l. and An. beklemishevi by restriction analysis. This mutation was not found in 347 of the specimens. We sequenced the ace-1 gene fragment for 24 specimens from four species of the Anopheles maculipennis group, including An. messeae, An. daciae, An. atroparvus, and An. beklemishevi. These specimens harbored a nucleotide substitution in the triplet where a mutation can lead to insecticide resistance, but this substitution would make it difficult for the resistance to develop. Since the studied specimens belong to branches of the Palearctic portion of the Anopheles maculipennis group, we suspect that all other Palearctic species of this group would have difficulties harboring the ace-1 mutation that would lead to organophosphate and carbamate resistance.

GAGA protein is required for multiple aspects of Drosophila oogenesis and female fertility.

Investigation of Drosophila oogenesis provides the opportunity to understand conservative genetic mechanisms underlying fertile female gamete development. In this study, we showed that the Drosophila DNA-binding protein GAGA factor (GAF) had a multifunctional role in oogenesis and it is involved in the regulation of this process genetic program. We studied the influence on Drosophila oogenesis of a number of mutations in the 5' region of the Trl gene that encodes GAF. We found that our originally generated Trl mutations lead to a decrease in transcriptional gene activity and levels of GAF expression in both germline and follicular cells. Cytological (fluorescence and electron microscopy) analysis showed that GAF loss resulted in multiple oogenesis defects. Mutations affected the actin cytoskeleton, leading to decrease of cytoplasmic filaments in nurse cells and basal actin in follicular cells. GAF depletion also leads to abnormal follicular cells migration, both border and centripetal. In addition, mutant ovaries demonstrated abnormalities in germ cells, including mitochondria, endoplasmic reticulum, karyosome organization, yolk granule formation and selective transport. Loss of GAF also promoted excessive cell death and egg chamber degradation. In sum, these defects caused very high or full female sterility. Since one of the main GAF activities is regulation of transcription, the complex phenotypes of the Trl mutants might be the consequence of its multiple target genes misexpression.

Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP ß-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.