Inversion polymorphism in the malaria mosquito Anopheles messeae. VII. Fertility and the population genetics structure of the species

The role of chromosomal inversional complexes of different fecundity has been studied in the process of formation of karyotypic populational structure. Females with homozygotes 2R11 and "north-est" inversional combinations are shown to have maximal total egg production, while females with three or four inversional heterozygotes from XL01, XL12, 2R01, 3R01 and 3L01 demonstrated lower level of fecundity. It is found that the seasonal conversion from r-selection to K-selection occurs in the period of population "prosperity" (July). The results of such conversion are destruction of "northest" genocomplexes and reduction of relative fecundity of inversional homozygotes 2R11-Alternative tendencies take place at the beginning of the reproductive period.     

Inversion polymorphism in the malaria mosquito Anopheles messeae. XI. The group effect of larval infection with Bacillus thuringiensis subsp. israeliensis bacteria]

Genetic consequences of the group and individual infections of Anopheles messeae larvae with bacteria Bacillus thuringiensis subsp. israelensis (Bti) were examined. The group effect of infections was manifested by increase in mortality and the speed-up of selection of chromosomal inversions resistant to Bti. Connection between the group and individual selections during the process of populations' adaptation is under discussion.     

Inversion polymorphism in malarial mosquito Anopheles messeae natural populations from the north-eastern region of the range

A cytogenetic study of eight natural populations of Anopheles messeae from the north-eastern part of the species areal was conducted. Complete predominance of homozygotes XL11 and 3R11 in the northern populations was observed. Change in the chromosome 2 inversion frequencies from south northwards was shown. The 2R11 variant, which was not observed in the southern region, was found in the northern populations. These results indicate the maintenance of chromosome frequency of the distribution of inversions XL1 and 3R (in longitude) and 2R1 (in latitude). The inversion frequency distribution in the examined part of the areal have been preserved for a long time.     

Inversion polymorphism in the malaria mosquito Anopheles messeae. VI. Local selection and 3-dimensional differentiation of the inversion gene complexes for fertility

A field experiment was carried on with the view of compulsory change of the frequency of chromosomal inversion (2R1) in the population of Anopheles messeae from the centre of the area. Quick return of the population to the original structure was observed. This confirmed that the local differentiation of chromosomal polymorphism is supported by natural selection. The fertility of the females was shown to be connected with the inversion polymorphism. The females with inversion complexes of genes of the "northern" type (with homozygotes 2R1) had higher fertility and lower dispersion of the fertility, in comparison with those having gene complexes which prevailed in the south part of the area.     

Inversion polymorphism in malaria mosquito Anopheles messeae. Part X. Resistance of larvae with different genotypes to toxins of crystal-forming bacteria Bacillus thuringiensis subsp. israelensis (serovar H14)

Insensibility of larvae with different chromosomal inversions to the toxins of Bacillus thuringiensis subsp. israelensis (Bti) was examined. It has been shown that larvae with inversion combinations XL0(XL1)2R0-3R0-3L0 had greater variability after treatment with Bti than larvae with inversions XL1(XL2)2R1-3R1-3L1(3L0). The former inversion combinations were previously shown to dominate in the south of the species area and to be supported by K-selection. The latter inversion combinations form "northest" karyotypes and are supported by r-selection. It is obvious that genetic effects of treatments with Bti depend on the population's structure and the directions of natural selection. The decrease in the level of heterozygotes after treatment of larvae with Bti reflects destruction of the system of genetic homeostasis in the natural populations of A. messeae.     

Chromosomal polymorphism in the populations of malaria vector mosquito Anopheles messeae at the south of Russian Plain

Zonal features of the geographic distribution of chromosomal inversions in the populations of An. messae at the south of the Russian Plain were examined. The An. messae was identified based on morphological characters and cytogenetically. The chromosomal inversions identified in populations of An. messae comprised XL₁, XL₄, 2R₁, 3R₁, and 3L₁. Inversion XL₄ was endemic and found with low frequency (2%) in Penza oblast and Moscow oblast. Based on the population karyotype structure similarity, on the territory of Russian Plain, three zones (southwestern, southeastern, and central) were identified. Central zone was characterized by higher levels of inversion polymorphism in all chromosomes, except arm 3L. In the two southern zones, high frequency of the XL₀ inversion, along with complete absence of homo- and heterozygotes for the 2R₁ inversion, and high proportion of the individuals with inversion 3L₁ was observed. Specific feature of southeastern zone was the increased frequency of hetero- and homozygotes for the 3R₁ and 3L₁ inversions. Zonal differences reflected adaptive character of chromosomal polymorphism and pointed to hierarchic organization of the species population structure.     

Inversion polymorphism of the malarial mosquito Anopheles messeae. V. The interaction of different chromosomal inversions in the spatial area

The relations between inversions of chromosome XL, 2R, 3R, 3L from 8 populations of the central part and periphery of the species area were studied. 2-, 3- and 4-factor analysis was carried out. Interaction of inversions XL1, 2R1, 3R1 in central populations (Riga, Moscow, Tomsk) was observed. The nonrandom association of definite inversional genotypes was stationary in spatial - temporary. The phylogenetic initial sequences XL0, 2R0, 3R0 display "+" interaction. The 3-factor analysis of populations of Tomsk shows: 1) interaction of inversions is more essential in males than in females; 2) the statistically significant interaction of 3-4 inversions is not always determined by the effect of 2-factor analysis; 3) the chromosome 3R has a dominance influence on the pattern of association of the multiinversional complex. These data support the hypothesis that differential selection for chromosomes with certain combinations of arrangements is mainly responsible for this phenomenon. In peripheral area populations (Syctyvkar, Irkutsk, Chita), no associations were observed (2R and 3R). When they do take place, their character is changed (Kiev - XL and 3R). This phenomenon may be explained by the influence of frequency-dependent selection.     

Estimated resistance of the malaria mosquito Anopheles messeae s.l. to the insecticide malathion

Resistance to agricultural pesticides is an important and insufficiently studied concern for pest and disease vector research. We determined the malathion resistance of species in the Anopheles maculipennis mosquito group in a habitat near Novosibirsk, Russia. Most of the 851 individuals we measured were members of the Anopheles messeae s.l. complex (An. messeae and An. daciae species). The LC₅₀ value for malathion was 0.052 mg/L for the mixed specimens, and we failed to find any differences between species. The LC₅₀ value was within the range of values for malathion resistance of Anopheles stephensi and Culex quinquefasciatus. As the main resistance mechanism to organophosphate and carbamate insecticides is a single mononucleotide substitution in the ace‐1 gene, we searched for this mutation in An. messeae s.l. and An. beklemishevi by restriction analysis. This mutation was not found in 347 of the specimens. We sequenced the ace‐1 gene fragment for 24 specimens from four species of the Anopheles maculipennis group, including An. messeae, An. daciae, An. atroparvus, and An. beklemishevi. These specimens harbored a nucleotide substitution in the triplet where a mutation can lead to insecticide resistance, but this substitution would make it difficult for the resistance to develop. Since the studied specimens belong to branches of the Palearctic portion of the Anopheles maculipennis group, we suspect that all other Palearctic species of this group would have difficulties harboring the ace‐1 mutation that would lead to organophosphate and carbamate resistance.     

Inversion polymorphism of the malarial mosquito Anopheles messeae. IV. The stability of the frequency distribution of the inversions by species area

Distribution of the frequencies of inversions XL1, 2R1, 3R1, 3L1, from geographically widely separated 8 populations had been studied during a long period of time. The geographical inclines of distribution of inversions XL1, 3R1 (a longitudinal) and 2R1 (a latitudinal) are stable in time. Stationarity of inversional polymorphism is not connected with superdominance of heterozygotes. It is supposed that the stable equilibrium of the frequencies of inversions is supported by variable selection.     

Single nucleotide polymorphism analysis of the ITS2 region of two sympatric malaria mosquito species in Sweden: Anopheles daciae and Anopheles messeae

Four species of the Anopheles maculipennis complex have previously been recorded in Sweden. A recent addition to the complex is Anopheles daciae, which is considered to be closely related to, but distinct from Anopheles messeae. The original designation of An. daciae was based on five genetic differences (161, 165, 167, 362 and 382) in the second internal transcribed spacer (ITS) 2 of the ribosomal RNA. Further studies have shown that only two nucleotide differences (362 and 382) robustly separate the species. Thirty-three An. maculipennis complex mosquitoes were collected in the province of Uppland, Sweden. All were An. daciae but showed double peaks for three variable positions (161, 165 and 167). When cloned, the intra-individual nucleotide variation was almost exclusively fixed with either TTC or AAT, originally diagnostic for An. messae and An. daciae, respectively. To further investigate the intra-individual variation, nine An. daciae and 11 An. messeae were collected in southern Sweden and their ITS2 fragments were amplified and sequenced using Illumina MiSeq sequencing (Illumina, Inc., San Diego, CA, USA). For the diagnostic nucleotide 382 no intra-individual variation could be detected. However, although each An. daciae specimen carried several ITS2 sequence variants for the four other nucleotides, there was no intra-individual variation in the An. messeae specimens.     

Genetic distance of the malarial mosquitoes, Anopheles beklemishevi and Anopheles messeae (Diptera, Culiccidae), and their intraspecific polymorphism

Allele frequencies at enzyme loci have been studied in Finnish populations of two species of the Anopheles maculipennis complex: A. beklemishevi and A. messeae. A. beklemishevi is spread over central and northern Finland, whereas A. messeae is found in southwestern and central Finland. The allele frequencies of these two species exhibit both similarities and differences. The results indicate that the two species do not interbreed in the nature. The allele frequencies at two loci--Hydroxybutyrate dehydrogenase (Hbdh) and Superoxide dismutase-2 (Su-2)--are almost totally different and adult individuals of the two species can be reliably diagnosed by these allelic differences. The genetic distance, D, between A. beklemishevi and A. messeae is 0.35. This value is compared with corresponding distances between other dipterans studied.     

Adaptive association of inversions in a natural population of the malaria mosquito Anopheles messeae Fall.]

The dependent combination of chromosomal variants by five paracentric inversions (IL1, IL2, IIR1, IIIR1, IIIL1) in Anopheles messeae population from the West Siberia was discovered. It is found that the inversion interactions interactions are the same at both larval and imaginal parts of the population, and they are stable during four years of studying. The inversion of hetero- and homozygotes demonstrates identical properties, that is, phylogenetic secondary sequencies dominate in heterozygotes. Two associative groups of chromosome variants were revealed. The association correlates with geographic distribution and temporal dynamics of inversions and on the whole, it reflects the evolutionary history of inversion polymorphism in A. messeae. It is supposed that the adaptive association of inversions found is the high degree polymorphism formed and functioned on the basis of the groups of inversion chromosome blocks.     

Oogenesis in the malaria mosquito Anopheles gambiae

Summary Oogenesis has been followed with the electron microscope in 2 strains of the malaria mosquito Anopheles gambiae, from the emergence of the adult (oocytes at leptonema) till shortly before the oocytes are ready for oviposition. After pachynema the chromosomes form a karyosphere and a fibrous capsule develops around it. Work on other mosquitoes suggests that the capsule may be related to the synaptonemal complexes. Both Anopheles strains contain at some time an extrachromosomal (not DNA-containing) body comparable to the karyosphere in size. Clusters of granules are present at the surface of the nucleolus and free in the nucleoplasm. Tentative results indicate that they may contain DNA. During oogenesis the nucleolus becomes very large, mainly because of proliferation of the nucleolonema. Towards the end of oocyte development the nucleus assumes the large canoe-shape also seen in Aedes and Culex. Nucleolonema traverse the entire nucleus, and modified granular clusters are found throughout.     

Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos