Background
Anopheles sacharovi, Favr [
1] is one of the Palearctic members of the Maculipennis group of malaria mosquitoes that also includes
Anopheles atroparvus,
Anopheles artemievi, Anopheles beklemishevi,
Anopheles daciae,
Anopheles labranchiae,
Anopheles maculipennis,
Anopheles martinus,
Anopheles melanoon,
Anopheles messeae, and
Anopheles persiensis [
2]. Four of these 11 species,
An. atroparvus,
An. labranchiae,
An. messeae, and
An. sacharovi, are considered dominant malaria vectors in Eurasia [
3]. Among them,
An. sacharovi has the most southern distribution and is the most dangerous malaria vector that transmits malaria in South Europe and in the Middle East [
3]. Currently,
An. sacharovi is involved in transmission of vivax malaria in Iran [
4‐
6], Iraq [
7] and Turkey [
8]. After the collapse of the Soviet Union, this species became responsible for malaria re-emergence in Georgia [
9], Armenia [
10,
11] and Azerbaijan [
12]. The success of
An. sacharovi as a malaria vector is a result of a highly plastic adaptation of this species at both adult and larval stages [
3]. It can breed in different water reservoirs, such as swamps, marshes, river margins, streams, pools, and ditches. Female mosquitoes have opportunistic blood-feeding behaviour and can feed on any available host including human, cow, sheep, chicken, horse, and donkey. It has been demonstrated that
An. sacharovi is resistant to DDT [
13] and dieldrin [
5]. Such ecological and behavioural plasticity, together with the emerged insecticide resistance, prevented elimination of
An. sacharovi during several anti-vector campaigns conducted in Israel, Greece and Turkey. Additionally, global warming raises concerns about the possible spread of
An. sacharovi to the dry areas and territories with high altitudes [
14].
An importance of
An. sacharovi as a malaria vector stimulates studies of this species from different perspectives including cytogenetic analyses. The first cytogenetic map for this species was developed based on photo images of lacto-aceto-orcein-stained polytene chromosomes from salivary glands [
15]. This map was used for species identification of
An. sacharovi and for the cytotaxonomic validation of the species status of
An. martinius Shingarev originally described in Uzbekistan in 1926 [
16].
Anopheles martinius is identical to
An. sacharovi based on external characters including egg morphology, and its name for a long period of time was considered a synonym for
An. sacharovi [
17]. The cytogenetic analysis clearly demonstrated that
An. sacharovi and
An. martinius differ from each other by two fixed paracentric inversions on chromosomes X and 3L and, thus, represent two different species [
15]. Moreover, polytene chromosomes of laboratory hybrids between
An. sacharovi and
An. martinius demonstrated partial asynapsis of the homeologous chromosomes, which is typical for the interspecies hybrids in other species of Diptera [
18]. The importance of this finding was emphasized by G. B. White in his review of systematic reappraisal of the
An. maculipennis complex [
19]. Later, the species validity of both
An. sacharovi and
An. martinius was supported by fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) [
20]. Chromosome polymorphism has not been yet detected in natural populations of
An. sacharovi.
In addition to species diagnostics, the cytogenetic map of
An. sacharovi was utilized to study chromosome evolution and phylogeny of Palearctic members of the Maculipennis group [
21]. The analysis of overlapping chromosomal inversions in 7 sibling species from the
An. maculipennis complex revealed three branches of the phylogenetic tree:
An. atroparvus-
An. labranchiae;
An. melanoon-
An. maculipennis-
An messeae; and
An. sacharovi-
An martinius. Among them, the branch
An. atroparvus-
An. labranchiae was considered as the basal. Although the phylogeny based on ITS2 sequences supported the presence of 3 major clades in the Palearctic group, the molecular analysis suggested that
An. sacharovi is the basal lineage [
20,
22,
23]. Thus, the phylogeny of the Maculipennis group remains unresolved.
The genomics era offers new opportunities for the development of modern genome-based strategies for vector control [
24,
25] including the CRISPR/Cas9-based gene-drive technologies [
26]. Following the major malaria vector in Africa
Anopheles gambiae [
27], the genomes for other malaria mosquitoes have been sequenced [
28‐
30]. The availability of cytogenetic maps allows the development of high-quality genome assemblies anchored to the chromosomes. However, only 5 chromosome-based genome assemblies have been developed for malaria mosquitoes [
28,
29,
31,
32], including one for the dominant vector of malaria in Europe
An. atroparvus [
33]. The comparison of the chromosome-based assemblies provided important insights into chromosomal evolution in the genus
Anopheles. A high rate of the sex chromosome evolution [
34], whole-arm translocations among autosomes [
35], and inter-arm rearrangements [
32] have been reported.
In this study, a standard cytogenetic photomap for An. sacharovi was developed. The suitability of this map for physical mapping was demonstrated by placing five PCR-amplified genes to the chromosomes of this species. In addition, cytogenetic landmarks that can be used for cytotaxonomic identification of An. sacharovi were described. This new map will assist in the development of the chromosome-based genome assembly for this important malaria vector.
Discussion
This study developed a high-resolution map for unstained polytene chromosomes from the ovarian nurse cells of
An. sacharovi. Chromosome images were developed using phase-contrast microscopy and high-resolution digital photography that provide a detailed banding pattern of the polytene chromosomes. The first chromosome map of
An. sacharovi was developed for the polytene chromosomes from the chromosomes of salivary glands [
15]. Those chromosomes were stained with lacto-aceto-orcein and imaged using film photography. That technique provided too contrasted banding patterns of the chromosomes because of the overstaining of the large bands under staining of thin bands. Also, using ovarian nurse cell chromosomes in this study helped to avoid the loss of detail in the structure of the pericentromeric regions, usually affected by under-replication in salivary glands due to their heterochromatic nature [
44].
The cytogenetic map that was developed for
An. sacharovi is one of 12 chromosome maps constructed for anopheline species using digital imaging technology, since 2000 (Table
2). A special emphasis has been placed on cytogenetic map construction for the dominant vectors of malaria from different parts of the world [
3,
45]. Cytogenetic maps are now available for the malaria vectors in Africa:
An. gambiae [
36],
Anopheles funestus [
46] and
Anopheles nili [
35]; in Asia:
An. stephensi [
47] and
Anopheles sinensis [
48]; and in America:
Anopheles albimanus [
49] and
Anopheles darlingi [
50,
51]. Chromosome maps have been used to identify and describe inversion polymorphism in natural populations of
An. funestus [
46],
An. nili [
35] and
An. darlingi [
50,
51]. Physical maps of microsatellite markers have been used in population genetic studies of
An. funestus [
52],
An. stephensi [
53] and
An. nili [
54]. Although inversion polymorphism has not been reported for
An. sacharovi, the availability of the cytogenetic map may help to discover polymorphic inversions in this species in the future. Considering the limited number of population studies conducted for
An. sacharovi, as well as the large geographical range of this mosquito, spreading from southern Europe to the Middle East, such discovery is quite possible.
Table 2
The development of chromosome maps for malaria mosquitoes
An. albimanus
| Salivary glands | | | |
An. atroparvus
| Ovarian nurse cells | | | |
An. beklemishevi
| Ovarian nurse cells | | | |
An. darlingi
| Salivary glands | | | |
An. funestus
| Ovarian nurse cells | | | |
An. gambiae
| Ovarian nurse cells | | | 2002 [ 27], 2007 [ 31], 2010 [ 36] |
An. lesteri
| Salivary glands | | | |
An. nili
| Ovarian nurse cells | | | |
An. ovengensis
| Ovarian nurse cells | | | |
An. sacharovi
| Ovarian nurse cells | Current study | | |
An. sinensis
| Salivary glands | | | |
An. stephensi
| Ovarian nurse cells | | 2010 [ 39], 2010 [ 34], 2011 [ 53] | |
Another reason that stimulated the recent interest in cytogenetic research in mosquitoes is the need to develop chromosome-based physical genome maps to enhance the quality of genome assemblies (Table
2). A superior physical genome map has been developed based on polytene chromosomes for the African malaria vector
An. gambiae [
36]. The genome of this species has been mapped by in situ hybridization of ~ 2000 bacterial artificial chromosome (BAC) clones [
27]. The map has been improved by additional mapping of cDNA probes that allowed a better coverage of the heterochromatic regions [
31]. Finally, the genome coordinates have been placed on the cytogenetic photomap developed using a high-pressure technique for squashing chromosomal preparation [
36]. This map assigns 84.3% of the AgamP3 assembly to the chromosomes with coordinates spaced at 0.5–1 Mb intervals. A remarkably high-coverage genome map has been developed for the Neotropical vector of malaria
An. albimanus [
32] and for the European vector
An. atroparvus [
55]. These maps include 98.2 and 89.6% of their genome assemblies, respectively, and represent the most complete genome maps developed for mosquitoes to date. Lower coverage physical maps have been developed for dominant Asian malaria vectors,
An. stephensi [
29,
39] and
An. sinensis [
56], and for a major malaria vector in Africa,
An. funestus [
28]. In this study, physical mapping was performed for 4 orthologous genes on
An. sacharovi chromosomes, for which location in the
An. atroparvus genome and chromosomes is known. This study demonstrates that the
An. sacharovi map is suitable for physical mapping and can serve as a tool for the development of a high-quality genome assembly for this species.
In addition, chromosome banding patterns of the
An. sacharovi map were compared with other chromosome maps that were recently developed for the members of the Maculipennis group,
An. atroparvus [
33] and
An. beklemishevi [
43]. The comparison clearly demonstrated that the chromosome map of
An. sacharovi can be utilized as a tool for the cytotaxonomic diagnostic of these 3 species and for a sister taxon
An. martinius [
15]. This comparison of the chromosomal banding patterns and positions of the physically mapped DNA probes also indicated that chromosome-banding patterns are not always reliable in determining chromosomal re-arrangements between the species. For example, FISH data demonstrated that the gene AATE009010 of
An. atroparvus and its orthologue in
An. sacharovi localize in two different positions in the X chromosome; the difference is likely caused by paracentric inversions that are not evident cytogenetically (Fig.
4a). The position of AATE016383 in 3L arm was also different in
An. sacharovi and
An. atroparvus (Fig.
4d), suggesting a more complex pattern of chromosomal re-arrangements in the 3L arm between the species from the Maculipennis group than was previously thought based on chromosomal banding patterns [
21]. Thus, additional physical mapping is required to better understand chromosomal evolution in the Maculipennis group of malaria mosquitoes.
Authors’ contributions
IVS and MVS conceived and designed the experiments. GA, AIV, and SB performed the experiments. GA, SB, GHK, SAA, and MSA collected mosquitoes. VNS provided resources and made contributions to conception and discussion of the study. GA, IVS, and MVS conducted data analysis and wrote the manuscript. All authors read and approved the final manuscript.