Species Composition, Ecological Preferences and Chromosomal Polymorphism of Malaria Mosquitoes of the Crimean Peninsula and the Black Sea Coast of the Caucasus

1. Introduction

The environmental and ecological conditions of the Crimean Peninsula and the Black Sea coast of the Caucasus have changed dramatically over the last 20 years [1,2]. Human population growth, expansion of economic activities and increased anthropogenic pressure from resort development, have led to the destruction and further degradation of natural ecosystems and landscapes, and massive environmental pollution. In addition, global warming has an increasingly strong impact on ecosystems, thus constantly changing the epidemiologic situation in the world, including those at the Black Sea coast [3,4]. Although the implementation of antimalarial interventions led to the elimination of autochthonous malaria in Europe [5,6], the territories of Crimea and the Black Sea coast of the Caucasus are currently classified as high-risk areas for resurgence of malaria transmission [7]. Cases of imported malaria are reported annually in the Crimea and Krasnodar Krai [8,9,10]. Therefore, understanding the current state of epidemiologically important groups of insects, such as malaria mosquitoes of the genus Anopheles (Diptera, Culicidae), has become urgent.

Most of the data on species composition and geographical distribution of malaria mosquito in the southern European part of Russia were collected between 1920 and 1960 [11] and are largely out of date. Moreover, the taxonomic status of mosquitoes in the Maculipennis complex has changed. Originally, the sibling species of this complex were considered as subspecies of the broadly polytypic species An. maculipennis s. l. [12]. Hybridization experiments and numerous data on morphology, ecology, and cytogenetics supported the taxonomic status of the following species of the Maculipennis group: An. atroparvus Van Theil, 1927; An. maculipennis Meigen, 1818; An. messeae Falleroni, 1926; An. melanoon Hackett, 1934; An. sacharovi Favre, 1903; An. subalpinus Hackett & Lewis, 1937 [13,14,15,16,17,18]. Later, several sibling species of the Maculipennis group were diagnosed using cytogenetic analysis [13,19,20,21,22,23]. Two new species of Palearctic malaria mosquitoes have been distinguished by differences in banding patterns of polytene chromosomes: An. beklemishevi Stegniy & Kabanova, 1976 [24] and An. martinius Shingarev, 1926 [25]. The reproductive isolation of new species has been demonstrated by studying experimental interspecific hybrids [26] obtained by the method of forced mating [27].

Molecular genetic methods provided additional opportunities to discover new malaria mosquito species and became an important approach in determining their taxonomic status [28,29,30]. The most valuable data for Anopheles systematics have been obtained by studying the nucleotide composition of the second internal transcribed spacer (ITS2) of the ribosomal gene cluster that separates the 5,8S and 28S genes of ribosomal RNA [31]. Three new species have been identified in the Maculipennis complex based on ITS2 sequence: An. persiensis Linton, Sedaghat & Harbach, 2003 [32,33]; An. daciae Linton, Nicolescu & Harbach, 2004 [34]; An. artemievi Gordeev, Zvantsov, Goryacheva, Shaikevich & Yezhov, 2005 [35]. Anopheles subalpinus has been synonymized with An. melanoon because the ITS2 sequences were identical in both species [36]. In addition, the ITS2 region of ribosomal DNA served as a tool for reconstructing phylogenetic relationships in malaria mosquitoes [37,38,39,40,41,42]. Further advances in species recognition and phylogeny reconstruction in the Maculipennis group of the malaria mosquitoes have been achieved through whole-genome sequencing [43,44].

According to 2003 and 2008 studies, the list of malaria mosquitoes of the North Caucasus and the Southern Russian Plain included 10 species [45,46,47]. Nine of which belong to the subgenus Anopheles: An. algeriensis Theobald, 1903; An. atroparvus Van Theil, 1927; An. claviger Meigen, 1804; An. hyrcanus Pallas, 1771; An. maculipennis Meigen, 1818; An. melanoon (subalpinus) Hackett, 1934; An. messeae Falleroni, 1926; An. plumbeus Stephens, 1828; An. sacharovi Favre,1903. One species was a member of the subgenus Cellia – An. superpictus Grassi, 1899. However, the present geographical distribution of malaria mosquitoes on the Black Sea Coast of the Caucasus and in Crimea is poorly understood.

The aim of this work was to revisit and describe in detail the species composition, geographic distribution, breeding sites and chromosomal polymorphism of the malaria mosquitoes of the Crimean Peninsula and the Black Sea coast of the Caucasus focusing on the sibling species of the Maculipennis group. The dynamics of the epidemic situation under the conditions of global warming and urbanization in the study area was discussed.

2. Materials and Methods

2.1. Field Collection and Material Preservation

Malaria mosquito larvae were collected from 2009 to 2024 at 56 locations along the Black Sea coast of the Caucasus and the Crimean Peninsula. A total of 2229 individual mosquitoes were included in this study. The coordinates of the collection sites and the number of mosquitoes collected are shown in Table 1. Fourth instar Anopheles larvae were collected from the surface of water by dipping method [48] and then were placed in Clark's solution (glacial acetic acid and 95% ethanol in a 1:3 ratio). Each larva was removed from Clark's solution and was divided into two parts. The head and thorax were placed back into Clark's solution for inversion polymorphism analysis. The abdomen was fixed in 70% ethanol for molecular identification. All samples were placed in a 1.5 ml Eppendorf tube and stored at −20°C. The obtained samples were used for preliminary species identification based on morphological characters [49,50]. Morphologically identical species were identified by cytogenetic and molecular genetic markers.

Ecological characteristics of malaria mosquito breeding sites were determined in local habitats. Water temperature (T), potential of hydrogen (pH), and total salinity (ppt) were measured using a Hanna Combo HI 98129 (Hanna Instruments, Woonsocket, Rhode Island, USA) conductometer. Dissolved oxygen content in water was measured using an ExStik DO600 oximeter (Extech Instruments, Waltham, Massachusetts, USA). The density of larvae of 1-4 stages (number of individuals per m²) was estimated in natural habitats (ponds, lakes, river outfalls).

2.2. Karyotyping

Polytene chromosome preparations were obtained from the salivary glands of fourth instar larvae according to the standard technique [23,51]. Salivary glands were extracted from larval thorax with dissecting needles in Clark's solution. The glands were stained with lacto-aceto-orcein (2% orcein in 80% lactic acid and 100% acetic acid in a 1:1 ratio). The time for lacto-aceto-orsine staining was increased from 40 to 80-90 minutes. The stained glands were squoshed under a coverslip in 50% acetic acid for 15-20 min.. Mosquito karyotypes were analyzed using an Eclipse E200 light microscope (Nikon, BioVitrum, Moscow, Russia). Karyotypes were used to identify sibling species of malaria mosquitoes. Chromosomal inversions were determined by comparing banding patterns with cytogenetic maps of the studied species [52,53]. Differences in inversion frequencies between mosquito populations were assessed using the Chi-square (χ²) test [54]. A total of 2229 individual mosquitoes were karyotyped, of which 75 An. atroparvus, 158 An. claviger, 27 An. hyrcanus, 908 An. maculipennis, 750 An. daciae, 309 An. plumbeus and 2 An. melanoon larvae.

2.3. Genotyping by RLFP-RCR

Karyotyped mosquitoes were used for molecular identification of the sibling species An. daciae/An. messeae. All samples were analyzed individually. Total DNA was extracted from alcohol-fixed larval fragments (abdomen) using the phenol-chloroform method [55]. DNA concentration was determined spectrophotometrically using Implen NanoPhotometer NP80 (Implen, Munich, Germany). The concentration was adjusted to 30–60 ng/µl. Polymerase chain reaction (PCR) was performed in a final volume of 20 μl using EncycloPlus PCR kit (Eurogen, Moscow, Russia) according to the manufacturer's instructions.

Species An. daciae and An. messeae were distinguished by the ITS2 fragment of the rDNA. The ITS2 fragment was amplified with the forward primer its2_vdir: 5′-TGTGAACTGCAGGACACATG-3′, and reverse primer its2_nrev: 5′-ATGCTTAAATTTAGGGGGTA-3′, as described previously [53]. RsaI endonuclease (SibEnzyme, Novosibirsk, Russia) was used for PCR-restriction fragment length polymorphism (RFLP) analysis [53]. PCR product ITS2 in An. daciae has 3 restriction sites for RsaI, the length of restriction fragments is 10, 47, 71 and 307 bp. PCR product ITS2 in An. messeae has 4 restriction sites, the length of restriction fragments is 10, 47, 71, 72 and 235 bp. The obtained PCR products were stained with ethidium bromide and analyzed by electrophoresis in 1.5% agarose gel and TBE buffer.

2.4. Genotyping by Sequencing

The rDNA-ITS2 of mosquitoes from two Crimean and two Caucasian populations were sequenced. Each larval abdomen was homogenized separately in liquid nitrogen. Genomic DNA was extracted using a standard protocol from the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA). DNA elution was performed in 100 μl of water. ITS2 from An. maculipennis s. l. which do not differ significantly in electrophoretic mobility of PCR products (An. messeae, An. daciae, An. atroparvus, and An. maculipennis) was amplified with the forward universal primers: its2_ndir 5′-ATCACTCGGCTCGTGGATCG-3′, or its2_vdir 5′-TGTGAACTGCAGGACACATG-3′ and the reverse primer its2_nrev 5′-ATGCTTAAATTTAGGGGGTA-3′, or its2_rev 5′-ATGCTTAAATTTAGGGGGTAGTC-3′, with modifications [56,57]. The HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD, USA) was used for PCR amplification. The PCR mix consisted of a total volume of 20 µl of ~40 ng DNA, 0.5 µM of each forward and reverse primer, and 10 µl of 2× HotStarTaq Plus reaction mix. PCR was performed on thermocycler Applied Biosystems GeneAmp PCR system 2700 (Applied Biosystems, Waltham, Massachusetts, USA) under the following conditions: initial denaturation at 95°C for 5 min, followed by 25–35 cycles of 95°C for 15 s, 58°C for 30 s and 72°C for 30 s and a final elongation step at 72°C for 5 min. The resulting reaction mix was placed in a stand-by mode at 4°C. The amplicons were visualized by gel electrophoresis in a 2% agarose gel. The DNA amplicons were purified using the Wizard™ PCR Clean Up Kit (Promega, Fitchburg, WI, USA). PCR products were sequenced by Sanger using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) from suitable forward or reverse primers. The DNA products of the sequencing reactions have been purified by ethanol precipitation and analyzed at the Genomics Core Facility of the Siberian Branch of the Russian Academy of Sciences (http://sequest.niboch.nsc.ru). Nucleotide positions in ITS2 sequences of An. daciae and An. messeae were compared with the reference sequence AY648982 in An. messeae [43]. Only nucleotides AC in positions 412 and 432 were considered as species-specific for An. daciae, respectively [43].

Anopheles melanoon / An. maculipennis s. s. mosquitoes were identified by the BOLD fragment of the mitochondrial COI gene, using the following Folmer primers: 5‘-TTTCAACAAACCATAAGGATATTGG-3’ and 5‘-TATACTTCAGGATGACCAAAAAATCA-3’, which were adapted using the “Primer3” program [58]. Total DNA was extracted individually from alcohol-fixed larval fragments (abdomen) using the phenol-chloroform method [55]. PCR amplification was performed at an annealing temperature of 59°C. PCR was performed in a final volume of 20 μl using EncycloPlus PCR kit (Eurogen, Moscow, Russia). Elution of fragments from the gel was performed using Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Los Angeles, California, USA). The obtained fragments were sequenced by Sanger sequencing. The nucleotide sequence of PCR fragments was determined from forward and reverse primers on a 3500 Genetic Analyzer using BigDye®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, Massachusetts, USA).

3. Results

3.1. Species Composition, Geographical Distribution and Ecological Preferences

Our study found 7 species of malaria mosquito in the Crimea and the Black Sea coast of the Caucasus: An. atroparvus Van Thiel, 1923; An. claviger Meigan, 1904; An. daciae Linton, Nicolescu & Harbach, 2004; An. hyrcanus Pallas, 1771; An. maculipennis Meigan, 1818; An. melanoon Hackett 1934; An. plumbeus Stephens, 1828 (Table 1). Four of them were sibling species of the Maculipennis group: An. atroparvus, An. daciae, An. maculipennis s. s., and An. melanoon. The geographical distribution of the species is shown in Figure 1. Six of them were present in both regions, one species An. melanoon was found only in the Imereti Valley on the Black Sea coast of the Caucasus (Table 1, location number 51 in the vicinity of Sochi). The previously reported in this area species An. algeriensis, An. messeae s. s., An. sacharovi, An. superpictus were not found [47].

Anopheles maculipennis s. s. mosquitoes dominated everywhere along the Black Sea coast and in the mountainous forest zone of the Crimean Peninsula, as well as in the foothills of the northern Caucasus. This species was found in 28 out of 56 locations (50%). Anopheles maculipennis s. s. larvae were found in most typical Anopheles breeding sites, and mosquitoes of this species alone were found in 14 locations (Table 1). Immature stages developed in ponds, water reservoirs, drainage, and irrigation canals, as well as in temporary pools of water. Mass larval development occurred in habitats with pH 6.48-8.85, but predominantly in the pH range of 7.0-8.4 (Table 2). Total dissolved solids (TDS) of water in larval habitats ranges ranged from 0.15 to 2.56 ppt, rarely exceeding 1.5. Larvae are sensitive to dissolved oxygen in the water. The oxygen content varied in the range of 4.4-10.2 mg/l in most breeding sites. The density of An. maculipennis s. s. larvae was low at oxygen levels of 0.5-2.3 mg/l. Larval development occurred over a wide range of daytime temperatures from 16.5 to 33°C, but the highest density of 160 larvae per square meter was observed at a water temperature of 20.7°C and an oxygen level of 4.5 mg/l (Table 2; location 3).

Anopheles daciae was the most abundant species in the plains located north of the Greater Caucasus Range. Anopheles daciae mosquitoes were recorded on the Black Sea coast of the Caucasus near Novorossiysk and Gelendzhik (Figure 1; locations 41-43). A single population of An. daciae in Crimea was found near Sevastopol (Figure 1; location 10). The larvae of An. daciae developed separately (Table 1; locations 40, 41) or together with An. maculipennis s. s. (Table 1; locations 10, 34-37, 42, 43). In some breeding sites shared with An. maculipennis s. s., An. daciae larvae dominated. At An. daciae breeding sites, water pH ranged from 7.0 to 8.4; TDS varied between 0.20 and 1.58 ppt; dissolved oxygen content varied between 0.5 and 8.1 mg/L; and water temperature ranged from 20.5 to 33.3°C (Table 2).

Anopheles atroparvus mosquitoes were found in steppe biotopes in the Crimea and the North Caucasus. Only this species larvae were able to develop in a highly saline water reservoirs with a TDS of 5.99 ppt (Table 2; location 33). Anopheles atroparvus larvae were found together with An. maculipennis s. s. and An. daciae mosquitoes in ponds and lakes with less saline water with TDS range of 0.59-1.46 ppt (Table 2; locations 10, 32). pH value at An. atroparvus breeding sites ranged from 7.14 to 9.1; daytime water temperature ranged from 18.0 to 28.0°C; and dissolved oxygen content ranged from 7.7 to 16.5 mg/L.

We found a single breeding site of An. melanoon mosquitoes in the Imereti Valley near Sochi (Figure 1; location 51). Two larvae were caught in a temporary water reservoir such as a stream overflow. Anopheles melanoon larvae were identified by the BOLD fragment of the mitochondrial COI gene. Anopheles melanoon larvae developed together with An. maculipennis s. s. in permanent water reservoirs with abundant aquatic vegetation in the floodplain of the Psou River. Many natural breeding sites in the Psou River estuary are now lost due to large-scale construction. Anopheles maculipennis s. s. and An. plumbeus mosquitoes numerically outnumber An. melanoon in the Imereti Valley and neighboring in Abkhazia (locations 47-49, 52-56).

Anopheles hyrcanus breeding sites were found in lakes along the eastern zone of the southern Crimean coast. Larvae developed in dense sedge and reed thickets, perennial grasses of the Cyperaceae and Poaceae families, where other malaria mosquito species were absent (Figure 1; locations 28-30). An. hyrcanus mosquitoes were found together with An. maculipennis s. s. and An. daciae larvae in two biotopes in the south of the Azov-Kuban Plain (Figure 1; locations 36-37). Water pH ranged from 7,00 to 8,24 An. hyrcanus breeding places; daytime temperature varies between 22,4 and 33,3°C; TDS changed from 0,81 to 3.02 ppt; the dissolved oxygen content in the water reached 10.0 mg/L (Table 2).

Because Anopheles claviger is a highly specialized malaria mosquito species, its larvae were found in springs, ditches with running water or in water reservoirs fed by groundwater. Water temperatures ranged from 16.5-23.7°C at most of the breeding sites (Table 2). Higher water temperatures of up to 27.5°C were only recorded in one biotope, with flowing water and a low density of An. claviger larvae (Table 2; location 50). Water pH varied within a narrow range of 7.14-7.72, but was 9.26 in one biotope (Table 2; location 50). TDS in all locations varied between 0.24 and 2.08 ppt. The amount of dissolved oxygen in water was 2.5 and 7.6 mg/L (Table 2; locations 45 and 50).

Anopheles plumbeus mosquitoes were found in the mountainous forested part of the southern Crimean coast. Larvae developed in shallow forest lakes, overflow of small rivers, in temporary micro-habitats such as tree holes, condensation puddles between piles of stones (Table 1; locations 7, 9, 11-13, 15, 16, 18, 19). Breeding sites of this species were found in tree holes on the Black Sea coast of the Caucasus (locations 48, 53) and in mountains at altitudes up to 1700 m (Table 1; locations 54, 55). Breeding sites of An. plumbeus mosquitoes were found in old car tires in Caucasian resorts (Table 1; locations 44, 53, 56). Water composition in larval habitats was highly variable (Table 2). Water acidity correlated with organic matter content, and pH varied from 5.20 to 8.32 (in open biotopes mainly in the range from 7.14 to 7.62). TDS varied between 0.03 and 2.78 ppt. Daytime water temperatures at breeding sites ranged from 14.9 to 32.3°C. Dissolved oxygen content was measured in a number of micro-watersheds and ranged from 2.5 to 7.5 mg/L. Larval densities did not exceed 9 per square meter at most breeding sites, but in habitats with high saprobic conditions larval densities reached 32-90 mosquitoes of all instars per square meter. Anopheles plumbeus larvae developed together with An. maculipennis s. s. in the two largest of the listed breading sites - in a forest lake and in an ornamental pond of the Nikitsky Botanical Gardens (Table 1; locations 7, 18).

3.2. Chromosomal Inversion Polymorphism

The mitotic chromosome set in malaria mosquitoes includes three pairs of chromosomes [59]. Salivary gland cells of the larvae contain polytene chromosomes. Homologues chromosomes are paired during polytenization. The karyotype of polytene chromosomes consists of 5 arms (short arm of sex chromosome XL and arms of autosomes 2R, 2L, 3R, 3L). All 5 arms are connected by a common chromocenter. The long arm of the XR sex chromosome and both arms of the Y chromosome consist of heterochromatin and are not polytenized. Male karyotypes have only one polytene X chromosome that is thinner than the X chromosome of females.

Chromosomal inversions were studied in populations of An. atroparvus and An. daciae. We identified chromosomal inversions in accordance with the previously published photo maps of polytene chromosomes in An. atroparvus and An. daciae/An. messeae [52,53].

Anopheles daciae differed from other species by a high degree of chromosomal polymorphism (Table 3).

Homo- and heterozygotes for three paracentric inversions were common in populations of this species: XL₁ (2a-5b); 3R₁ (23c/24a-26c/27a); 3L₁ (34b/c-37a/b-38c/39a-39c/d), whereas the 3L₁ inversion consists of two overlapping inversions. Only one heterozygote for new inversion 2R₅ (11c-14a) was found in the Novorossiysk population (Figure 2; location 42). There were no inversions in the 2L arm.

We described, for the first time, the chromosomal polymorphism of marginal populations of An. daciae in the southern part of the species range. The only one population of An. daciae in the Crimean Peninsula (Figure 3; location 10) differed in chromosomal composition from three Caucasian populations (Figure 3; locations 36, 40, 42).

A high level of inversion polymorphism of sex chromosome XL is observed in all populations of An. daciae on the Black Sea coast (Figure 3), but the frequency of chromosomal variants with XL₀ inversion was significantly higher in the Crimean population in males (χ²=4.47; number of degrees of freedom df=1; p<0.05) and females (χ²=6.16; df=2; p<0.05). XL₀ inversion serves as a species marker of An. daciae. The alternative inversion XL₁ is considered ancestral to the two cryptic species An. daciae and An. messeae [53]. According to molecular genetic analyses, the separation of An. daciae and An. messeae happened about 2 Ma during the glaciation in Eurasia [44]. It is assumed that An. daciae mosquitoes were isolated in a refugium in southern Europe. The XL₀ inversion, which occurs with high frequency in populations of the steppe zone and broad-leaved forest zone, probably arose during this period [60].

Autosomal inversions 3R₁ and 3L₁ are present with low frequency in coastal populations of An. daciae (Figure 3). These inversions were probably derived from the common ancestor of An. daciae and An. messeae, since polymorphism on inversions 3R₁ and 3L₁ is present in populations of both species. Geographical gradients in longitude suggest the influence of climatic factors on the frequencies of these inversions [56]. The frequencies of homo- and heterozygotes with 3R₁ and 3L₁ inversions were significantly higher in Caucasian populations (χ²=4.48 and 7.40; df=1; p<0.05 and p<0.01, respectively). In general, the level of chromosomal polymorphism was significantly higher in the Caucasian populations than in the Crimean population.

The inversion polymorphism was observed in An. atroparvus populations. Heterozygotes for inversion 3L₁ (34b/c-38b) were found in two populations from Crimea and the Stavropol Region (Figure 4).

Heterozygotes 3L₁ occurred with a frequency of 33.3% in both populations (Table 1; locations 2, 19). No homozygotes for this inversion were detected. Chromosome 3L has an identical disc pattern in An. atroparvus and An. daciae. Interestingly, the breakpoints of the 3L₁ inversion of An. atroparvus (34b/c and 38b) are close to two breakpoints of the overlapping 3L₁ inversion of An. daciae (34b/c-38c/39a). Perhaps this localization of inversions determines similar adaptive effects, so individuals of two species with these inversions are found in the same regions.