The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

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.

Patterns of genetic differentiation imply distinct phylogeographic history of the mosquito species Anopheles messeae and Anopheles daciae in Eurasia.

Detailed knowledge of phylogeography is important for control of mosquito species involved in the transmission of human infectious diseases. Anopheles messeae is a geographically widespread and genetically diverse dominant vector of malaria in Eurasia. A closely related species, An. daciae, was originally distinguished from An. messeae based on five nucleotide substitutions in its ribosomal DNA (rDNA). However, the patterns of phylogeographic history of these species in Eurasia remain poorly understood. Here, using internal transcribed spacer 2 (ITS2) of rDNA and karyotyping for the species identification we determined the composition of five Anopheles species in 28 locations in Eurasia. Based on the frequencies of 11 polymorphic chromosomal inversions used as genetic markers, a large-scale population genetics analysis was performed of 1932 mosquitoes identified as An. messeae, An. daciae and their hybrids. The largest genetic differences between the species were detected in the X sex chromosome suggesting a potential involvement of this chromosome in speciation. The frequencies of autosomal inversions in the same locations differed by 13%-45% between the species demonstrating a restricted gene flow between the species. Overall, An. messeae was identified as a diverse species with a more complex population structure than An. daciae. The clinal gradients in frequencies of chromosomal inversions were determined in both species implicating their possible involvement in climate adaptations. The frequencies of hybrids were low ~1% in northern Europe but high up to 50% in south-eastern populations. Thus, our study revealed critical differences in patterns of phylogeographic history between An. messeae and An. daciae in Eurasia. This knowledge will help to predict the potential of the malaria transmission in the northern territories of the continent.

Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegyptiNix, 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.

The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis.

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.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies.

BACKGROUND: New 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. RESULTS: We evaluated and employed 3 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: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 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. CONCLUSIONS: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations 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.

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes.

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.

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

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.

A standard photomap of the ovarian nurse cell chromosomes for the dominant malaria vector in Europe and Middle East Anopheles sacharovi.

BACKGROUND: Anopheles sacharovi is a dominant malaria vector species in South Europe and the Middle East which has a highly plastic behaviour at both adult and larval stages. Such plasticity has prevented this species from eradication by several anti-vector campaigns. The development of new genome-based strategies for vector control will benefit from genome sequencing and physical chromosome mapping of this mosquito. Although a cytogenetic photomap for chromosomes from salivary glands of An. sacharovi has been developed, no cytogenetic map suitable for physical genome mapping is available. METHODS: Mosquitoes for this study were collected at adult stage in animal shelters in Armenia. Polytene chromosome preparations were prepared from ovarian nurse cells. Fluorescent in situ hybridization (FISH) was performed using PCR amplified probes. RESULTS: This study constructed a high-quality standard photomap for polytene chromosomes from ovarian nurse cells of An. sacharovi. Following the previous nomenclature, chromosomes were sub-divided into 39 numbered and 119 lettered sub-divisions. Chromosomal landmarks for the chromosome recognition were described. Using FISH, 4 PCR-amplified genic probes were mapped to the chromosomes. The positions of the probes demonstrated gene order reshuffling between An. sacharovi and Anopheles atroparvus which has not been seen cytologically. In addition, this study described specific chromosomal landmarks that can be used for the cytotaxonomic diagnostics of An. sacharovi based on the banding pattern of its polytene chromosomes. CONCLUSIONS: This study constructed a high-quality standard photomap for ovarian nurse cell chromosomes of An. sacharovi and validated its utility for physical genome mapping. Based on the map, cytotaxonomic features for identification of An. sacharovi have been described. The cytogenetic map constructed in this study will assist in creating a chromosome-based genome assembly for this mosquito and in developing cytotaxonomic tools for identification of other species from the Maculipennis group.

Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs.

BACKGROUND: An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito. RESULTS: Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624 Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti. CONCLUSIONS: The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.

Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females.

BACKGROUND: Y chromosomes are responsible for the initiation of male development, male fertility, and other male-related functions in diverse species. However, Y genes are rarely characterized outside a few model species due to the arduous nature of studying the repeat-rich Y. RESULTS: The chromosome quotient (CQ) is a novel approach to systematically discover Y chromosome genes. In the CQ method, genomic DNA from males and females is sequenced independently and aligned to candidate reference sequences. The female to male ratio of the number of alignments to a reference sequence, a parameter called the chromosome quotient (CQ), is used to determine whether the sequence is Y-linked. Using the CQ method, we successfully identified known Y sequences from Homo sapiens and Drosophila melanogaster. The CQ method facilitated the discovery of Y chromosome sequences from the malaria mosquitoes Anopheles stephensi and An. gambiae. Comparisons to transcriptome sequence data with blastn led to the discovery of six Anopheles Y genes, three from each species. All six genes are expressed in the early embryo. Two of the three An. stephensi Y genes were recently acquired from the autosomes or the X. Although An. stephensi and An. gambiae belong to the same subgenus, we found no evidence of Y genes shared between the species. CONCLUSIONS: The CQ method can reliably identify Y chromosome sequences using the ratio of alignments from male and female sequence data. The CQ method is widely applicable to species with fragmented genome assemblies produced from next-generation sequencing data. Analysis of the six Y genes characterized in this study indicates rapid Y chromosome evolution between An. stephensi and An. gambiae. The Anopheles Y genes discovered by the CQ method provide unique markers for population and phylogenetic analysis, and opportunities for novel mosquito control measures through the manipulation of sexual dimorphism and fertility.

Obtaining Polytene, Meiotic, and Mitotic Chromosomes from Mosquitoes for Cytogenetic Analysis.

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 for Anopheles mosquitoes. 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.

Visualization of Polytene Chromatin in Mosquito Cell Nuclei Using Three-Dimensional Fluorescence In Situ Hybridization.

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 of Anopheles mosquitoes. 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.

Visualization of the Linear and Spatial Organization of Chromosomes in Mosquitoes.

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.

Arm-specific dynamics of chromosome evolution in malaria mosquitoes.

BACKGROUND: The malaria mosquito species of subgenus Cellia have rich inversion polymorphisms that correlate with environmental variables. Polymorphic inversions tend to cluster on the chromosomal arms 2R and 2L but not on X, 3R and 3L in Anopheles gambiae and homologous arms in other species. However, it is unknown whether polymorphic inversions on homologous chromosomal arms of distantly related species from subgenus Cellia nonrandomly share similar sets of genes. It is also unclear if the evolutionary breakage of inversion-poor chromosomal arms is under constraints. RESULTS: To gain a better understanding of the arm-specific differences in the rates of genome rearrangements, we compared gene orders and established syntenic relationships among Anopheles gambiae, Anopheles funestus, and Anopheles stephensi. We provided evidence that polymorphic inversions on the 2R arms in these three species nonrandomly captured similar sets of genes. This nonrandom distribution of genes was not only a result of preservation of ancestral gene order but also an outcome of extensive reshuffling of gene orders that created new combinations of homologous genes within independently originated polymorphic inversions. The statistical analysis of distribution of conserved gene orders demonstrated that the autosomal arms differ in their tolerance to generating evolutionary breakpoints. The fastest evolving 2R autosomal arm was enriched with gene blocks conserved between only a pair of species. In contrast, all identified syntenic blocks were preserved on the slowly evolving 3R arm of An. gambiae and on the homologous arms of An. funestus and An. stephensi. CONCLUSIONS: Our results suggest that natural selection favors specific gene combinations within polymorphic inversions when distant species are exposed to similar environmental pressures. This knowledge could be useful for the discovery of genes responsible for an association of inversion polymorphisms with phenotypic variations in multiple species. Our data support the chromosomal arm specificity in rates of gene order disruption during mosquito evolution. We conclude that the distribution of breakpoint regions is evolutionary conserved on slowly evolving arms and tends to be lineage-specific on rapidly evolving arms.

An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi.

Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.

A Gene-Based Method for Cytogenetic Mapping of Repeat-Rich Mosquito Genomes.

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.

New Cytogenetic Photomap and Molecular Diagnostics for the Cryptic Species of the Malaria Mosquitoes Anopheles messeae and Anopheles daciae from Eurasia.

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.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Genome mapping and characterization of the Anopheles gambiae heterochromatin.

BACKGROUND: Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized. RESULTS: To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1) and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin. CONCLUSIONS: Our results demonstrate that Anopheles polytene chromosomes and whole-genome shotgun assembly render the mapping and characterization of a significant part of heterochromatic scaffolds a possibility. These results reveal the strong association between characteristics of the genome features and morphological types of chromatin. Initial analysis of the An. gambiae heterochromatin provides a framework for its functional characterization and comparative genomic analyses with other organisms.