A standard photomap of ovarian nurse cell chromosomes in the European malaria vector Anopheles atroparvus.

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.

A standard cytogenetic map for Anopheles sinensis and chromosome arm homology between the subgenera Anopheles and Cellia.

Anopheles sinensis (Diptera: Culicidae) is an important vector of Plasmodium vivax in Southeast Asia. To facilitate population genetic and genomic studies of An. sinensis, we developed a standard cytogenetic photomap for this species. The polytene chromosomes were straightened and divided into 39 numbered divisions and 116 lettered subdivisions. The chromosomal localizations of 13 DNA probes were determined by fluorescent in situ hybridization. A comparison of the physical map for An. sinensis with the genome map for Anopheles gambiae revealed a whole-arm autosomal translocation between the two species. Specifically, the 2R arm of An. gambiae corresponds to the 3R arm of An. sinensis and the pattern of correspondence of the other chromosome arms remains regular. We mapped the breakpoints of the polymorphic paracentric chromosomal inversion 3Ra to subdivisions 28A and 31A. The standard cytogenetic map developed in this study will be useful for detailed comparative genome mapping and population genetic studies of An. sinensis.

Polymorphic chromosomal inversions in Anopheles moucheti, a major malaria vector in Central Africa.

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.

Progress in mapping the yellow fever mosquito genome.

Mosquito-borne diseases cause significant problems for the human health. For this reason, the genomes of three most dangerous species of mosquitoes, including the yellow fever mosquito Aedes aegypti, were sequenced in last decade. The efficient vector of arboviruses. Ae. aegypti, is also a convenient model for laboratory research. The intensive genetic mapping of morphological and molecular markers conducted for this mosquito in the past was very successful. This mapping was also used as a tool to localize a number of quantitative trait loci related to the mosquito's ability to transmit various pathogens. However, physical mapping of the Ae. aegypti genome is difficult due to the lack of high-quality polytene chromosomes. Here, we review different mapping approaches that help improving genome sequence assembly and also integrate linkage, chromocome and genome maps the yellow fever mosquito.

Chromosomal localization of actin genes in the malaria mosquito Anopheles darlingi.

Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report the physical mapping of two actin genes on polytene chromosomes of the major malaria vector in the Amazon, Anopheles darlingi (Diptera: Culicidae). Clones with actin gene sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results, together with previous mapping data, provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors.

Transcription profiling associated with life cycle of Anopheles gambiae.

Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (< 30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

Chromosome evolution in malaria mosquitoes.

The genomic era offers excellent opportunities to improve our understanding of the genetic basis of mosquito adaptation, evolution, and competence to a pathogen. The availability of polytene chromosomes in anopheline mosquitoes makes them an excellent model system for studying genome organization, evolution, and function. Physical mapping facilitated the whole genome sequence assembly for the major malaria vector Anopheles gambiae and comparative genome mapping has determined types, patterns, and rates of chromosomal rearrangements in mosquito evolution. Together with sequencing projects, high-resolution physical mapping can shed light on mechanisms of chromosomal rearrangements and phylogenetic relationships among species.

High-resolution cytogenetic map for the African malaria vector Anopheles gambiae.

Cytogenetic and physical maps are indispensible for precise assembly of genome sequences, functional characterization of chromosomal regions, and population genetic and taxonomic studies. We have created a new cytogenetic map for Anopheles gambiae by using a high-pressure squash technique that increases overall band clarity. To link chromosomal regions to the genome sequence, we attached genome coordinates, based on 302 markers of bacterial artificial chromosome, cDNA clones, and PCR-amplified gene fragments, to the chromosomal bands and interbands at approximately a 0.5-1 Mb interval. In addition, we placed the breakpoints of seven common polymorphic inversions on the map and described the chromosomal landmarks for the arm and inversion identification. The map's increased resolution can be used to further enhance physical mapping, improve genome assembly, and stimulate epigenomic studies of malaria vectors.

Organization and evolution of heterochromatin in malaria mosquitoes.

The African malaria mosquito Anopheles gambiae was the first disease vector chosen for genome sequencing. Although its genome assembly has been facilitated by physical mapping, large gaps still pose a serious problem for accurate annotation and genome analysis. The majority of the gaps are located in regions of pericentromeric and intercalary heterochromatin. Genomic analysis has identified protein-coding genes and various classes of repetitive elements in the Anopheles heterochromatin. Molecular and cytogenetic studies have demonstrated that heterochromatin is a structurally heterogeneous and rapidly evolving part of the malaria mosquito genome.

[Localization of repetitive DNA sequences in the pericentromeric heterochromatin of malarial mosquitoes of the "Anopheles maculipennis" complex].

Distribution of eight fragments of conserved repetitive DNA from pericentromeric heterochromatin of chromosome 2 of Anopheles atroparvus has been investigated by in situ hybridization on polytene chromosomes of An. atroparvus and An. messeae. We have shown that heterochromatic regions of all chromosomes both in An. atroparvus and An. messeae vary in combinations of, at least, conserved repeats. Some repeats have been found only in pericentromeric heterochromatic regions of chromosomes 2 (clones Atr2R-46a, Atr2R-73, Atr2R-85a in An. atroparvus and Atr2R-25 in An. messeae). Others have been found in two (clones Atr2R-25a and Atr2R-90 in An. atroparvus, Atr2R-25a in An. messeae) and more (clones Atr2R-118, Atr2R-136 in An. atroparvus, Atr2R-73 in An. messeae) pericentromeric heterochromatic regions of chromosomes. DNA comparison of pericentromeric heterochromatic regions of chromosomes in species of the "Anopheles maculipennis" complex is species- and chromosome-specific, due, in particular, to different maintenance of conserved repeates.

[Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)]. / Kharakteristika i sravnitel'nyi analiz DNK iz pritsentromernogo geterokhromatina khromosomy 2 Anopheles atroparvus V Teil (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.

Linear and spatial organization of polytene chromosomes of the African malaria mosquito Anopheles funestus.

Anopheles funestus Giles is one of the major malaria vectors in Africa, but little is known about its genetics. Lack of a cytogenetic map characterized by regions has hindered the progress of genetic research with this important species. This study developed a cytogenetic map of An. funestus using ovarian nurse cell polytene chromosomes. We demonstrate an important application with the cytogenetic map for characterizing various chromosomal inversions for specimens collected from coastal Kenya. The linear and spatial organization of An. funestus polytene chromosomes was compared with the best-studied malaria mosquito, An. gambiae Giles. Comparisons of chromosome morphology between the two species have revealed that the most extensive chromosomal rearrangement occurs in pericentromeric heterochromatin of autosomes. Differences in pericentromeric heterochromatin types correlate with nuclear organization differences between An. funestus and An. gambiae. Attachments of chromosomes to the nuclear envelope strongly depend on the presence of diffusive beta-heterochromatin. Thus, An. funestus and An. gambiae exhibit species-specific characteristics in chromosome-linear and -spatial organizations.

[Pericentromeric and intercalating alpha-heterochromatin of polytene chromosomes of the malaria mosquito]. / Pritsentromernyi i interkaliarnyi alpha-geterokhromatin politennykh khromosom maliariinykh komarov.

The properties of heterochromatin from polytene chromosomes of the malaria mosquito Anopheles messeae Fal. and A. atroparvus V. Tiel. were studied by various methods of differential staining and by hybridization in situ with two repetitive DNA sequences of Drosophila melanogaster. In malaria mosquito, the heterochromatin was heterogeneous. Two forms of alpha-heterochromatin were revealed: pericentromeric and intercalary heterochromatin, which is localized within the internal chromosome regions.

[Polymorphism of pericentromeric heterochromatin in natural populations of Anopheles maculipennis Mg]. / Polimorfizm pritsentromernogo geterokhromatina v prirodnykh populiatsiiakh Anopheles maculipennis Mg.

Variations in pericentromeric heterochromatin quantity was found in polytene chromosomes X and 3 (in 3L arm) in the ovarian trophocytes in two natural populations of Anopheles meculipennis Mg. (Moscow, Sochi). The existence of block and non-block variants and of heterozygotes between them was shown. Northern population have more variants of blocks than the southern populations do.

[Spatial organization of polytene chromosomes in ovarian trophocyte nuclei of the malaria mosquito Anopheles labranchiae Fall]. / Prostranstvennaia organizatsiia politennykh khromosom v iadrakh trofotsitov iaichnikov maliariinogo komara Anopheles labranchiae Fall.

Spatial arrangement of polytene chromosomes in the ovarian trophocyte nuclei was studied in Anopheles labranchiae Fall. The system of chromosome attachment to the nuclear envelope in this species was found to differ from those in other species of the A. maculipennis complex. These results confirm that the spatial organization of nurse ovarian cell chromosomes is species-specific.

[Variability of pericentromeric chromatin in chromosome 2 of ovarian nurse cells during inbreeding in Anopheles atroparvus V.Tiel]. / Izmenchivost' pritsentromernogo geterokhromatina khromosomy 2 pitaiushchikh kletok iaichnikov pri inbridinge u Anopheles atroparvus V. Tiel.

We investigated the variability of pericentromeric chromatin of chromosome 2 in ovarian nurse cells (trophocytes) in two laboratory lines of malaria mosquito Anopheles atroparvus V. Tiel and in their hybrids. One line had been raised by means of sib inbreeding, the other kept at constantly high population density. The inbreeding was shown to result in an increased percentage of chromosomes bearing an achromatinic zone in the centromeric region, which resulted in chromosome breakage. Toxicological tests demonstrated an increase in the sensitivity of the progeny of females with abnormal morphotypes of chromosome 2 to the entomopathogenic bacterium Bacillus thuringiensis israelensis. The appearance of the achromatinic zone is attributed to local chromatin underreplication accompanying chromosome polytenization. Possible reasons for this phenomenon and its implication for adaptation are discussed.

[Chromocenter organization in salivary glands of the malaria mosquito Anopheles messeae Fall]. / Organizatsiia khromotsentra v sliunnykh zhelezkh maliariinogo komara Anopheles messeae Fall.

The chromocenter organization and the relationship with the nuclear envelope were studied in Anopheles messeae Fall. malaria mosquito. The interchromosomal contacts within the chromocenter were shown to be formed by both alpha- and beta-heterochromatin. In the pericentric regions of all chromosomes and in highly repeated sequences localized mostly in alpha-heterochromatin, the incorporation of the moderate He-T repeat of D. melanogaster was also found. Each chromosome was shown to have an independent contact to the nuclear envelope via certain beta-heterochromatin loci. The morphology of the Y chromosome, which is also capable of forming ectopic contacts within the chromocenter boundaries, was studied.

[Polymorphism of pericentromere heterochromatin of polytene chromosomes of ovarian trophocytes in natural populations of the malaria mosquito Anopheles messeae Fall]. / Polimorfizm pritsentromernogo geterokhromatina politennykh khromosom trofotsitov iaichnikov v prirodnykh populiatsiiakh maliariinogo komara Anopheles messeae Fall.

Pericentric heterochromatin polymorphism in chromosome 2 of ovarian trophocytes was studied in three natural populations of the malaria mosquito Anopheles messeae Fall. It is shown that pericentric heterochromatin blocks occur in four different variants whose sizes are at the 1:2:3:4 ratio (single, double, triple, and quadruple blocks). Mosquitoes with homo- and heterozygous variants of these blocks were identified. By analyzing frequency distribution of different size variants, interpopulation differences in this parameter were revealed. Mosquitoes with double heterochromatin blocks prevailed in the southern population, whereas those with triple blocks were more abundant in the northern population.

[Interspecies differences in the ovarian trophocyte precentromere heterochromatin structure and evolution of the malaria mosquito complex Anopheles maculipennis]. / Mezhvedivovye razlichiia struktury pritsentromernogo geterokhromatina trofotsitov iaichnikov i évoliutsiia maliariinykh komarov kompleksa Anopheles maculipennis.

Studies on polytene chromosomes of ovarian trophocytes in malaria mosquitoes revealed significant variation of the structure of pericentromeric heterochromatin (PH). Variations in the number and size of pericentromeric alpha-heterochromatin blocks, puffing and interaction with the nuclear envelope of beta-heterochromatin, and the degree of asynapsis between pericentromeric regions of homologues were detected. Interspecific differences are characteristics of all three chromosomes of Anopheles maculipennis and seem to be directly associated with systemic mutations determining speciation.

[Systemic reorganization of the architechtonics of polytene chromosomes in onto- and phylogenesis of malaria mosquitoes. Structural features regional of chromosomal adhesion to the nuclear membrane]. / Sistemnaia reorganizatsiia arkhitektoniki politennykh khromosom v onto- i filogeneze maiariinykh komarov. Strukturnye osobennosti zon prikrepleniia khromosom k iadernoi obolochke.

Principles of organization of chromocenter in salivary gland cells and zones of chromosome attachment to nuclear envelope in ovarian nurse cells were determined. It was shown that blocks of centromeric heterochromatin (alfa-heterochromatin) have no direct connection with nuclear envelope. Such connections are ensured by beta-heterochromatin. Homologous chromosome regions were shown to be of different morphology and nature of chromosome-membrane links in different mosquito species. A map of polytene chromosomes of ovarian nurse cells in Anopheles messeae Fall, was established. No differences were found in band quantity of these chromosomes as compared to salivary gland chromosomes.