The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level in fruit flies.

BACKGROUND: Multiple studies have demonstrated a negative correlation between gene expression and positioning of genes at the nuclear envelope (NE) lined by nuclear lamina, but the exact relationship remains unclear, especially in light of the highly stochastic, transient nature of the gene association with the NE. RESULTS: In this paper, we ask whether there is a causal, systematic, genome-wide relationship between the expression levels of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be in contact with the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning of Drosophila melanogaster TADs at the NE does not, by itself, systematically affect the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses regarding the underlying mechanism for the presence of the correlation without causality are discussed. These include the possibility that the epigenetic marks and affinity to the NE of a TAD are determined by various non-mutually exclusive mechanisms and remain relatively stable during interphase. CONCLUSIONS: At the level of TADs, the probability of chromatin being in contact with the nuclear envelope has no systematic, causal effect on the transcription level in Drosophila. The conclusion is reached by combining model-derived time-evolution of TAD locations within the nucleus with their experimental gene expression levels.

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

The Maculipennis subgroup of malaria mosquitoes includes both dominant malaria vectors and non-vectors in Eurasia. Understanding the genetic factors, particularly chromosomal inversions, that differentiate Anopheles species can provide valuable insights for vector control strategies. Although autosomal inversions between the species in this subgroup have been characterized based on the chromosomal banding patterns, the number and positions of rearrangements in the X chromosome remain unclear due to the divergent banding patterns. Here, we identified two large X chromosomal inversions, approximately 13 Mb and 10 Mb in size, using fluorescence in situ hybridization. The inversion breakpoint regions were mapped by hybridizing 53 gene markers with polytene chromosomes of An. messeae. The DNA probes were designed based on gene sequences from the annotated An. atroparvus genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the An. atroparvus genome, changed their position and orientation in the X chromosome. The analysis of the An. atroparvus genome revealed an enrichment of gene ontology terms associated with immune system and mating behavior in the rearranged syntenic blocks. Additionally, the enrichment of DNA transposons was found in sequences homologous to three of the four breakpoint regions. This study demonstrates the successful application of the physical genome mapping approach to identify rearrangements that differentiate species in insects with polytene chromosomes.

Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection.

A novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection was investigated in Anopheles stephensi. The presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway and is proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Silencing components of key signaling pathways through RNA interference (RNAi) that enhance proliferation of progenitor cells significantly decreased oocyst numbers, while limiting proliferation of progenitors increased oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts.

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.

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.

Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection.

A novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection was investigated in Anopheles stephensi. The presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway, and proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Enhancing proliferation of progenitors significantly decreases oocyst numbers, while limiting proliferation increases oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts.

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Strong interactions between highly dynamic lamina-associated domains and the nuclear envelope stabilize the 3D architecture of Drosophila interphase chromatin.

BACKGROUND: Interactions among topologically associating domains (TADs), and between the nuclear envelope (NE) and lamina-associated domains (LADs) are expected to shape various aspects of three-dimensional (3D) chromatin structure and dynamics; however, relevant genome-wide experiments that may provide statistically significant conclusions remain difficult. RESULTS: We have developed a coarse-grained dynamical model of D. melanogaster nuclei at TAD resolution that explicitly accounts for four distinct epigenetic classes of TADs and LAD-NE interactions. The model is parameterized to reproduce the experimental Hi-C map of the wild type (WT) nuclei; it describes time evolution of the chromatin over the G1 phase of the interphase. The simulations include an ensemble of nuclei, corresponding to the experimentally observed set of several possible mutual arrangements of chromosomal arms. The model is validated against multiple structural features of chromatin from several different experiments not used in model development. Predicted positioning of all LADs at the NE is highly dynamic-the same LAD can attach, detach and move far away from the NE multiple times during interphase. The probabilities of LADs to be in contact with the NE vary by an order of magnitude, despite all having the same affinity to the NE in the model. These probabilities are mostly determined by a highly variable local linear density of LADs along the genome, which also has the same strong effect on the predicted positioning of individual TADs -- higher probability of a TAD to be near NE is largely determined by a higher linear density of LADs surrounding this TAD. The distribution of LADs along the chromosome chains plays a notable role in maintaining a non-random average global structure of chromatin. Relatively high affinity of LADs to the NE in the WT nuclei substantially reduces sensitivity of the global radial chromatin distribution to variations in the strength of TAD-TAD interactions compared to the lamin depleted nuclei, where a small (0.5 kT) increase of cross-type TAD-TAD interactions doubles the chromatin density in the central nucleus region. CONCLUSIONS: A dynamical model of the entire fruit fly genome makes multiple genome-wide predictions of biological interest. The distribution of LADs along the chromatin chains affects their probabilities to be in contact with the NE and radial positioning of highly mobile TADs, playing a notable role in creating a non-random average global structure of the chromatin. We conjecture that an important role of attractive LAD-NE interactions is to stabilize global chromatin structure against inevitable cell-to-cell variations in TAD-TAD interactions.

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.

Molecular characterization and genetic authentication assay for Anopheles 'hemocyte-like' cell lines 4a-3A and 4a-3B.

BACKGROUND: Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. METHODS: Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B 'hemocyte-like' cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. RESULTS: The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. CONCLUSIONS: Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results.

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.

Retrogene Duplication and Expression Patterns Shaped by the Evolution of Sex Chromosomes in Malaria Mosquitoes.

Genes that originate during evolution are an important source of novel biological functions. Retrogenes are functional copies of genes produced by retroduplication and as such are located in different genomic positions. To investigate retroposition patterns and retrogene expression, we computationally identified interchromosomal retroduplication events in nine portions of the phylogenetic history of malaria mosquitoes, making use of species that do or do not have classical sex chromosomes to test the roles of sex-linkage. We found 40 interchromosomal events and a significant excess of retroduplications from the X chromosome to autosomes among a set of young retrogenes. These young retroposition events occurred within the last 100 million years in lineages where all species possessed differentiated sex chromosomes. An analysis of available microarray and RNA-seq expression data for Anopheles gambiae showed that many of the young retrogenes evolved male-biased expression in the reproductive organs. Young autosomal retrogenes with increased meiotic or postmeiotic expression in the testes tend to be male biased. In contrast, older retrogenes, i.e., in lineages with undifferentiated sex chromosomes, do not show this particular chromosomal bias and are enriched for female-biased expression in reproductive organs. Our reverse-transcription PCR data indicates that most of the youngest retrogenes, which originated within the last 47.6 million years in the subgenus Cellia, evolved non-uniform expression patterns across body parts in the males and females of An. coluzzii. Finally, gene annotation revealed that mitochondrial function is a prominent feature of the young autosomal retrogenes. We conclude that mRNA-mediated gene duplication has produced a set of genes that contribute to mosquito reproductive functions and that different biases are revealed after the sex chromosomes evolve. Overall, these results suggest potential roles for the evolution of meiotic sex chromosome inactivation in males and of sexually antagonistic conflict related to mitochondrial energy function as the main selective pressures for X-to-autosome gene reduplication and testis-biased expression in these mosquito lineages.

Anopheles mosquitoes reveal new principles of 3D genome organization in insects.

Chromosomes are hierarchically folded within cell nuclei into territories, domains and subdomains, but the functional importance and evolutionary dynamics of these hierarchies are poorly defined. Here, we comprehensively profile genome organizations of five Anopheles mosquito species and show how different levels of chromatin architecture influence each other. Patterns observed on Hi-C maps are associated with known cytological structures, epigenetic profiles, and gene expression levels. Evolutionary analysis reveals conservation of chromatin architecture within synteny blocks for tens of millions of years and enrichment of synteny breakpoints in regions with increased genomic insulation. However, in-depth analysis shows a confounding effect of gene density on both insulation and distribution of synteny breakpoints, suggesting limited causal relationship between breakpoints and regions with increased genomic insulation. At the level of individual loci, we identify specific, extremely long-ranged looping interactions, conserved for ~100 million years. We demonstrate that the mechanisms underlying these looping contacts differ from previously described Polycomb-dependent interactions and clustering of active chromatin.

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.

Chromosome-level genome assemblies of the malaria vectors Anopheles coluzzii and Anopheles arabiensis.

BACKGROUND: Anopheles coluzzii and Anopheles arabiensis belong to the Anopheles gambiae complex and are among the major malaria vectors in sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species. FINDINGS: In this study, we produced de novo chromosome-level genome assemblies for A. coluzzii and A. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 and 256.8 Mb of the total assemblies for A. coluzzii and A. arabiensis, respectively. Each assembly consists of 3 chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomic coordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in A. coluzzii and A. arabiensis. CONCLUSION: The new chromosome-level assemblies will facilitate functional and population genomic studies in A. coluzzii and A. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.

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.

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.