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Detailed knowledge of phylogeography is important for control of mosquito species involved in transmission of human infectious diseases.Anopheles messeaeis a geographically widespread and genetically diverse dominant vector of malaria in Eurasia. A closely related sibling species,An. daciae, was distinguished fromAn. messeaebased on a few nucleotide differences in its ribosomal DNA. However, the mechanisms of speciation and their evolutionary histories are poorly understood. Here, we performed a large-scale population genetics analysis of 3694 mosquitos from Eurasia to understand the species divergence, diversity, and population structure using the Internal Transcribed Spacer 2 of ribosomal DNA for species identification and frequencies of 11 polymorphic chromosomal inversions as genetic markers. The study revealed striking differences in the geographical distribution of the sibling species. The largest genetic differences betweenAn. messeaeandAn. daciaewere detected in the X sex chromosome suggesting that this chromosome plays a role in speciation. The frequencies of autosomal inversions differed significantly between the species, strongly supporting a restricted gene flow. The clinal variability of some inversion frequencies was revealed in both species implicating their possible involvement in climate adaptations. Statistical analysis of inversion polymorphism clearly distinguished two clusters associated with the two species and demonstrated much higher genetic diversity withinAn. messeae. Overall, the frequencies of hybrids in all locations were extremely low with the exception of several southeastern populations, where putative hybrids were abundant. Thus, the pattern of genetic differentiation implies dramatic differences in geographic distribution, population structure, and evolutionary histories of the sibling speciesAn. messeaeandAn. daciae.

Mosquitoes are vectors of dangerous human diseases such as malaria, dengue, Zika, West Nile fever, and lymphatic filariasis. Visualization of the linear and spatial organization of mosquito chromosomes is important for understanding genome structure and function. Utilization of chromosomal inversions as markers for population genetics studies yields insights into mosquito adaptation and evolution. Cytogenetic approaches assist with the development of chromosome-scale genome assemblies that are useful tools for studying mosquito biology and for designing novel vector control strategies. Fluorescence in situ hybridization is a powerful technique for localizing specific DNA sequences within the linear chromosome structure and within the spatial organization of the cell nucleus. Here, we introduce protocols used in our laboratories for chromosome visualization and their application in mosquitoes.

BackgroundUnderstanding the evolutionary relationships between closely related taxa is important for mosquitoes that transmit human diseases. Six out of 41 dominant malaria vectors in the world belong to the Maculipennis group, which is subdivided into two North American subgroups (Freeborni and Quadrimaculatus), and one Eurasian (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 Eurasian species An. beklemishevi is currently included in the North American Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics.ResultsTo reconstruct historic relationships between the North American and Eurasian mosquitoes, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on 1271 orthologous genes using their transcriptomic or genomic sequences. 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.ConclusionsOur phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support inclusion of An. beklemishevi into the Maculipennis subgroup. The vectorial capacity and the ability to diapause during winter evolved multiple times in Maculipennis evolution. Detailed knowledge of the evolutionary history in the Maculipennis subgroup will help us better understand the current and future patterns of malaria transmission in Eurasia.

Significance The presence of a dominant male-determining locus (M-locus) in one of a pair of autosomes establishes the male sex in the dengue fever mosquito Aedes aegypti. The Ae. aegypti M-locus contains 30 genes, including Nix, a previously reported male-determining factor. Here we show that the Nix transgene alone was sufficient to convert females into fertile males, which continued to produce sex-converted progeny. We also show that a second M-locus gene named myo-sex was needed for male flight. Nix-mediated sex conversion was 100% penetrant, heritable, and stable, indicating great potential for developing mosquito-control strategies to reduce vector populations by female-to-male conversion. This work also sheds lights into the molecular basis of the function of the M-locus., A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

Simple Summary The most dangerous vectors of malaria in the northern regions of the world belong to the Maculipennis group. Among the 22 species in this group, six are considered dominant vectors of malaria. Of these six, Anopheles messeae represents the most widely spread and genetically diverse species in Eurasia and Anopheles daciae, a cryptic species whose taxonomic status is still under debate, has been differentiated from An. messeae based on differences in their ribosomal DNA. However, genetic studies of these species are scarce. The availability of well-developed polytene chromosomes in malaria mosquitoes provides an opportunity to construct high-resolution cytogenetic photomaps that can be used to investigate the genetic divergence between these species. In this study, we created a standard universal cytogenetic map for the salivary gland polytene chromosomes of An. messeae and An. daciae and developed a simple and robust molecular approach for species diagnostics. The quality of the cytogenetic map was validated by studying inversion polymorphisms in populations of An. messeae and An. daciae from a location in the Asian part of Russia. The map will facilitate further investigation of the genetic diversity of these cryptic species. Abstract The Eurasian malaria vector Anopheles messeae is a widely spread and genetically diverse species. Five widespread polymorphic chromosomal inversions were found in natural populations of this mosquito. A cryptic species, Anopheles daciae, was differentiated from An. messeae by the presence of several nucleotide substitutions in the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA. However, because of the absence of a high-quality reference cytogenetic map, the inversion polymorphisms in An. daciae and An. messeae remain poorly understood. Moreover, a recently determined heterogeneity in ITS2 in An. daciae questioned the accuracy of the previously used Restriction Fragment Length Polymorphism (RFLP) assay for species diagnostics. In this study, a standard-universal cytogenetic map was constructed based on orcein stained images of chromosomes from salivary glands for population studies of the chromosomal inversions that can be used for both An. messeae and An. daciae. In addition, a new ITS2-RFLP approach for species diagnostics was developed. Both methods were applied to characterize inversion polymorphism in populations of An. messeae and An. daciae from a single location in Western Siberia in Russia. The analysis demonstrates that cryptic species are remarkably different in their frequencies of chromosomal inversion variants. Our study supports previous observations that An. messeae has higher inversion polymorphism in all autosomes than the cryptic species An. daciae.

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.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.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.

Chromosomes are intricately folded within the cell nucleus and interact with peripheral nuclear proteins. The chromatin architecture has a profound effect on how the genome is organized. 3D-FISH is a powerful technique that can reveal the structural and functional organization of chromosomes in the nuclear space. Here, we present a protocol for visualizing specific genomic regions in whole-mount paraformaldehyde-fixed cell nuclei ofAnophelesmosquitoes. This protocol was tested in our laboratories and has been showed to be effective and reliable for visualizing genomic regions of various lengths—from 1-kb gene-scale fragments to chromosome-scale segments of DNA.

Chromosome visualization is a key step for developing cytogenetic maps and idiograms, analyzing inversion polymorphisms, and identifying mosquito species. Three types of chromosomes—polytene, mitotic, and meiotic—are used in cytogenetic studies of mosquitoes. Here, we describe a detailed method for obtaining high-quality polytene chromosome preparations from the salivary glands of larvae and the ovaries of females forAnophelesmosquitoes. We also describe how to obtain mitotic chromosomes from imaginal discs of fourth-instar larvae and meiotic chromosomes from the testes of male pupae for all mosquitoes. These chromosomes can be used for fluorescence in situ hybridization (FISH), a fundamental technique in cytogenetic research that is used for physical genome mapping, detecting chromosomal rearrangements, and studying chromosome organization.

Simple Summary Mosquitoes from the family Culicidae are vectors of numerous human diseases such as dengue, Zika, and West Nile fevers, malaria, and lymphatic filariasis. The availability of high-quality genome assemblies for mosquitoes assists in identifying genes responsible for important epidemiological traits such as vector competence, insecticide resistance, and mosquito behavior and stimulates a better understanding of the genetic structure of mosquito populations. Thus, the development of genomic resources and tools is a necessary step for designing and implementing novel genome-based approaches to control mosquitoes. The emphasis of this study is the further development and optimization of a physical genome mapping approach. Physical mapping is placing genomic scaffolds to the chromosomes based on hybridization of the specific probes. This task is difficult for mosquitoes with large genome sizes which are enriched with repetitive DNA sequences and can be potentially misassembled. Here, we describe in detail a simple and robust technique for the physical mapping of such mosquito genomes. This method can be further used for physical genome mapping in other mosquitoes and insects. Abstract Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

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.

Understanding the population structure and mechanisms of taxa diversification is important for organisms responsible for the transmission of human diseases. Two vectors of West Nile virus, Culex pipiens pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The goal of this study is to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations but not in Cx. p. molestus populations. Comparison of mitochondrial genomes among the specimens showed a paraphyletic origin of the major haplogroups between the subspecies but a monophyletic structure between the continents. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. Russian Science FoundationRussian Science Foundation (RSF) [19-14-00130] We thank Igor V. Sharakhov for the productive discussion of the manuscript, Dmitri A. Karagodin for the help with formatting figures, and Janet Webster for proofreading the text. This project was supported by the Russian Science Foundation grant No 19-14-00130 to MVS. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the Anopheles gambiae complex of malaria mosquitoes, we analyzed metaphase chromosomes in An. arabiensis, An. coluzzii, An. gambiae, An. merus, and An. quadriannulatus. Using fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA), a highly repetitive fraction of DNA, and heterochromatic Bacterial Artificial Chromosome (BAC) clones, we established the correspondence of pericentric heterochromatin between the metaphase and polytene X chromosomes of An. gambiae. We then developed chromosome idiograms and demonstrated that the X chromosomes exhibit qualitative differences in their pattern of heterochromatic bands and position of satellite DNA (satDNA) repeats among the sibling species with postzygotic isolation, An. arabiensis, An. merus, An. quadriannulatus, and An. coluzzii or An. gambiae. The identified differences in the size and structure of the X chromosome heterochromatin point to a possible role of repetitive DNA in speciation of mosquitoes. We found that An. coluzzii and An. gambiae, incipient species with prezygotic isolation, share variations in the relative positions of the satDNA repeats and the proximal heterochromatin band on the X chromosomes. This previously unknown genetic polymorphism in malaria mosquitoes may be caused by a differential amplification of DNA repeats or an inversion in the sex chromosome heterochromatin.

Chromosomal inversions are important drivers of genome evolution. The Eurasian malaria vector Anopheles messeae has five polymorphic inversions. A cryptic species, An. daciae, has been discriminated from An. messeae based on five fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) of ribosomal DNA. However, the inversion polymorphism in An. daciae and the genome divergence between these species remain unexplored. In this study, we sequenced the ITS2 region and analyzed the inversion frequencies of 289 Anopheles larvae specimens collected from three locations in the Moscow region. Five individual genomes for each of the two species were sequenced. We determined that An. messeae and An. daciae differ from each other by the frequency of polymorphic inversions. Inversion X1 was fixed in An. messeae but polymorphic in An. daciae populations. The genome sequence comparison demonstrated genome-wide divergence between the species, especially pronounced on the inversion-rich X chromosome (mean Fst = 0.331). The frequency of polymorphic autosomal inversions was higher in An. messeae than in An. daciae. We conclude that the X chromosome inversions play an important role in the genomic differentiation between the species. Our study determined that An. messeae and An. daciae are closely related species with incomplete reproductive isolation. Published version

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.

BackgroundNew sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from ‘finished’. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.ResultsWe employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.ConclusionsExperimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

Chromosomal inversions are important drivers of genome evolution. The Eurasian malaria vector Anopheles messeae has five polymorphic inversions. A cryptic species, An. daciae, has been discriminated from An. messeae based on five fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) of ribosomal DNA. However, the inversion polymorphism in An. daciae and the genome divergence between these species remain unexplored. In this study, we sequenced the ITS2 region and analyzed the inversion frequencies of 289 Anopheles larvae specimens collected from three locations in the Moscow region. Five individual genomes for each of the two species were sequenced. We determined that An. messeae and An. daciae differ from each other by the frequency of polymorphic inversions. Inversion X1 was fixed in An. messeae but polymorphic in An. daciae populations. The genome sequence comparison demonstrated genome-wide divergence between the species, especially pronounced on the inversion-rich X chromosome (mean Fst = 0.331). The frequency of polymorphic autosomal inversions was higher in An. messeae than in An. daciae. We conclude that the X chromosome inversions play an important role in the genomic differentiation between the species. Our study determined that An. messeae and An. daciae are closely related species with incomplete reproductive isolation.

Female Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, has been slowed by the lack of a high-quality genome assembly. Here we combine diverse genome technologies to produce AaegL5, a dramatically improved and annotated assembly, and demonstrate how it accelerates mosquito science and control. We anchored the physical and cytogenetic maps, resolved the size and composition of the elusive sex-determining “M locus”, significantly increased the known members of the glutathione-S-transferase genes important for insecticide resistance, and doubled the number of chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites. Using high-resolution QTL and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. We predict that AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly arboviral vector.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services [U19AI110818]; USDA [2017-05741]; NIH Intramural Research Program; National Library of Medicine; National Human Genome Research Institute; NSF [PHY-1427654]; NIH [R01AI101112, R35GM118336, R21AI121853, R01AI123338, T32GM007739, NIH/NCATS UL1TR000043, DP2OD008540, U01AI088647, 1R01AI121211, D43TW001130-08, U01HL130010, UM1HG009375, 5K22AI113060, 1R21AI123937, R00DC012069]; Defence Advanced Research Project Agency [HR0011-17-2-0047]; Jane Coffin Childs Memorial Fund; Center for Theoretical Biological Physics postdoctoral fellowship; Robertson Foundation; McNair Foundation; Welch Foundation [Q-1866]; French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases [ANR-10-LABX-62-IBEID]; Agence Nationale de la Recherche [ANR-17-ERC2-0016-01]; European Union [734584]; Pew and Searle Scholars Programs; Klingenstein-Simons Fellowship in the Neurosciences; Verily Life Sciences We thank R. Andino; S. Emrich and D. Lawson (Vectorbase); A. A. James, M. Kunitomi, C. Nusbaum, D. Severson, N. Whiteman; T. Dickinson, M. Hartley and B. Rice (Dovetail Genomics) for early participation in the AGWG; C. Bargmann, D. Botstein, E. Jarvis and E. Lander for encouragement and facilitation. N. Keivanfar, D. Jaffe and D. M. Church (10X Genomics) prepared DNA for structural-variant analysis. We thank A. Harmon of the New York Times and acknowledge generous pro bono data and analysis from our corporate collaborators. This research was supported in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute (S.N.R. and D.E.N.); USDA 2017-05741 (E.L.A.); NSF PHY-1427654 Center for Theoretical Biological Physics (E.L.A.); NIH Intramural Research Program, National Library of Medicine and National Human Genome Research Institute (A.M.P. and S.K.) and the following extramural NIH grants: R01AI101112 (J.R.P.), R35GM118336 (R.S.M. and W.J.G.), R21AI121853 (M.V.S., I.V.S. and A. S.), R01AI123338 (Z.T.), T32GM007739 (M.H.), NIH/NCATS UL1TR000043 (Rockefeller University), DP2OD008540 (E.L.A.), U01AI088647, 1R01AI121211 (W.C.B. IV), Fogarty Training Grant D43TW001130-08, U01HL130010 (E.L.A.), UM1HG009375 (E.L.A), 5K22AI113060 (O.S.A.), 1R21AI123937 (O.S.A.), and R00DC012069 (C.S.M.); Defence Advanced Research Project Agency: HR0011-17-2-0047 (O.S.A.). Other support was provided by Jane Coffin Childs Memorial Fund (B.J.M.), Center for Theoretical Biological Physics postdoctoral fellowship (O.D.), Robertson Foundation (L.Z.), and McNair & Welch (Q-1866) Foundations (E.L.A.), French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID to L.L.), Agence Nationale de la Recherche grant ANR-17-ERC2-0016-01 (L.L.), European Union's Horizon 2020 research and innovation program under ZikaPLAN grant agreement no. 734584 (L.L.), Pew and Searle Scholars Programs (C.S.M.), Klingenstein-Simons Fellowship in the Neurosciences (C.S.M.). A.M.W., B.J.W., J.E.C. and S.N.M. were supported by Verily Life Sciences. L.B.V. is an investigator of the Howard Hughes Medical Institute.

The development of genomic resources and tools is an important step in designing novel approaches to genetic control of mosquitoes. Physical genome maps enhance the quality of the genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics studies in mosquitoes. In this chapter, we describe protocols for an important procedure in physical genome mapping-fluorescence in situ hybridization (FISH). We provide details on (1) dissection of salivary glands, ovaries, and imaginal discs for obtaining high-quality polytene or mitotic chromosome preparations; (2) DNA-labeling procedures and extraction of repetitive DNA fractions; and (3) approaches to FISH on polytene and mitotic chromosomes.

The development of genomic resources and tools is an important step in designing novel approaches to genetic control of mosquitoes. Physical genome maps enhance the quality of the genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics studies in mosquitoes. In this chapter, we describe protocols for an important procedure in physical genome mapping-fluorescence in situ hybridization (FISH). We provide details on (1) dissection of salivary glands, ovaries, and imaginal discs for obtaining high-quality polytene or mitotic chromosome preparations; (2) DNA-labeling procedures and extraction of repetitive DNA fractions; and (3) approaches to FISH on polytene and mitotic chromosomes.

Anopheles atroparvus (Diptera: Culicidae) is one of the main malaria vectors of the Maculipennis group in Europe. Cytogenetic analysis based on salivary gland chromosomes has been used in taxonomic and population genetic studies of mosquitoes from this group. However, a high-resolution cytogenetic map that could be used in physical genome mapping in An. atroparvus is still lacking. In the present study, a high-quality photomap of the polytene chromosomes from ovarian nurse cells of An. atroparvus was developed. Using fluorescent in situ hybridization, 10 genes from the five largest genomic supercontigs on the polytene chromosome were localized and 28% of the genome was anchored to the cytogenetic map. The study established chromosome arm homology between An. atroparvus and the major African malaria vector Anopheles gambiae, suggesting a whole-arm translocation between autosomes of these two species. The standard photomap constructed for ovarian nurse cell chromosomes of An. atroparvus will be useful for routine physical mapping. This map will assist in the development of a fine-scale chromosome-based genome assembly for this species and will also facilitate comparative and evolutionary genomics studies in the genus Anopheles.

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. Electronic supplementary material The online version of this article (10.1186/s13071-018-2657-3) contains supplementary material, which is available to authorized users.

Background The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. Results The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. Conclusions The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-4663-4) contains supplementary material, which is available to authorized users.

BackgroundThe 50-year old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome.ResultsThe C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells was mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of non-host reads.ConclusionsThe C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Manipulating M factor alters mosquito sex Female mosquitoes feed on blood and in so doing transmit pathogens to millions annually. Although the molecular mechanism for determining sex in many animals is known, the specific factors in mosquitoes have been elusive. This is because sex determination in insects involves a section of the genome that is highly repetitive. Hall et al. now identify a male-determining factor (M factor) in Aedes aegypti. Manipulation of the M factor produced sex-change phenotypes. Knocking out the gene Nix resulted in feminized males, and ectopic expression gave masculinized females. These findings should help to advance strategies for converting female mosquitoes into nonbiting males. Science , this issue p. 1268

Background Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors. Results Presented here is a standard cytogenetic map for An. lesteri, and a comparative analysis of chromosome structure and gene order between An. lesteri and An. sinensis. Our results demonstrate that much of the gene order on chromosomes X and 2 was reshuffled between the two species. However, the banding pattern and the gene order on chromosome 3 appeared to be conserved. We also found two new polymorphic inversions, 2Lc and 3Rb, in An. lesteri, and we mapped the breakpoints of these two inversions on polytene chromosomes. Conclusions Our results demonstrate the extent of structural divergence of chromosomes between An. lesteri and An. sinensis, and provide a new taxonomic cytogenetic tool to distinguish between these two species. Polymorphic inversions of An. lesteri could serve as markers for studies of the population structure and ecological adaptations of this major malaria vector. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1855-0) contains supplementary material, which is available to authorized users.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

Background Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes. Methods Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping. Results A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups. Conclusions This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0912-4) contains supplementary material, which is available to authorized users.

Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane’s rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was used to identify AT-rich regions, chromomycin A3 following pretreatment with barium hydroxide stained for GC-rich regions and stained the ribosomal RNA locus and YOYO-1 was used to test for differential staining. Chromosome patterns in SenAae strains revealed by these three stains differed from those in IB12. For FISH, 40 BAC clones previously physically mapped on Aaa chromosomes were used to test for chromosome rearrangements in SenAae relative to IB12. Differences in the order of markers identified two chromosomal rearrangements between IB12 and SenAae strains. The first rearrangement involves two overlapping pericentric (containing the centromere) inversions in chromosome 3 or an insertion of a large fragment into the 3q arm. The second rearrangement is close to the centromere on the p arm of chromosome 2. Linkage analysis of the SDL and the white-eye locus identified a likely chromosomal rearrangement on chromosome 1. The reproductive incompatibility observed within SenAae and between SenAae and Aaa may be generally associated with chromosome rearrangements on all three chromosomes and specifically caused by pericentric inversions on chromosomes 2 and 3., Author Summary Aedes aegypti is one of the best studied mosquito species and it is the principal vector of dengue, Zika, and yellow fever flaviviruses and the Chikungunya alphavirus. Aedes aegypti occurs throughout all tropical and subtropical regions of the world, and previous population genetic studies have shown that the highest genetic diversity occurs in Africa. Aedes aegypti from Senegal, West Africa (SenAae) have a low oviposition rate; those that do oviposit have a low fecundity and poor egg-to-adult survival. Furthermore rearrangements were detected on all three chromosomes in SenAae. These observations are consistent with the presence of at least two cryptic subspecies of Ae. aegypti in Senegal arising from reproductive isolation due to chromosome rearrangements. Genetic control strategies are being considered for the suppression of Ae. aegypti populations worldwide. Barriers to gene flow in African Ae. aegypti populations could compromise these future control efforts.

Polymorphic inversions in mosquitoes are distributed nonrandomly among chromosomes and are associated with ecological, behavioral, and physiological adaptations related to pathogen transmission. Despite their significance, the patterns and mechanism of genome rearrangements are not well understood. Recent sequencing and physical mapping of the genomes for 16 Anopheles mosquito species provided an opportunity to study chromosome evolution at the highest resolution. New studies revealed that fixed rearrangement accumulated [Formula: see text]3 times faster on the X chromosome than on autosomes. The highest densities of transposable elements (TEs) and satellites of different sizes have also been found on the X chromosome, suggesting a mechanism for the inversion generation. The high rate of X chromosome rearrangements is in sharp contrast with the paucity of polymorphic inversions on the X in the majority of anopheline species. This paper highlights the advances in understanding chromosome evolution in malaria vectors and discusses possible future directions in studying mechanisms and biological roles of genome rearrangements.