Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.     

Intraspecific variation in sex heterochromatin of species B of the Anopheles dirus complex in Thailand

Cytological examination of F1 larval mitotic chromosomes from a total of 126 families of Anopheles dirus species B from southern Thailand populations has revealed a pronounced quantitative variation of constitutive heterochromatin in the two sex chromosomes. Five types of X chromosomes and four types of Y chromosomes have been identified in this study. Such gross variation in sex chromosomes is most likely due to a gradual acquisition of extra heterochromatin.     

Ribosomal DNA-probes differentiate five cryptic species in the Anopheles gambiae complex

This study describes the use of ribosomal DNA probes to identify the species of individual mosquitoes in the Anopheles gambiae complex, a group of six morphologically identical mosquito species among which are two of the principal vectors of malaria in Africa. The DNA probes are sequences of DNA derived from the ribosomal genes of An. gambiae. Each probe reveals a different sized restriction enzyme fragment specific to each of the five species in the complex that were examined in this study: An. gambiae, An. arabiensis, An. quadriannulatus, An. melas and An. merus. The probes detect highly repeated sequences of DNA, thus the method is sufficiently sensitive to be applied to a small portion of a mosquito. Furthermore, because the DNA can be extracted from desiccated or alcohol preserved specimens, the test is compatible with other mosquito assays performed on dried specimens such as blood meal and malaria sporozoite antigen ELISAs. Determination of the nucleotide sequences that underlie the species-specific restriction enzyme site differences detected by these probes will lead to the development of synthetic DNA probes that can be used to identify an individual mosquito to species on the basis of a simple dot-blot or squash-blot.     

SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in Southeast Asia

The Anopheles dirus Peyton & Harrison complex of mosquitoes (Diptera: Culicidae) comprises seven known species, including important malaria vectors in Southeast Asia. Specific identification of each species of the complex, which cannot be distinguished using morphological characters, is crucial for understanding vector ecology and implementing effective control measures. Derived from individual random amplified polymorphic DNA (RAPD) markers, sequence characterized amplified regions (SCAR) were analysed for the design of specific paired-primers. Combination of six SCAR primers resulted in the development of a simple, robust, single multiplex PCR able to identify three important malaria vectors among the four most common species (A, B, C, D) of the complex: species A from several Southeast Asian countries, species B from Perlis, Malaysia, and species C and D from Thailand.     

Population genetic evidence for species A, B, C and D of the Anopheles dirus complex in Thailand and enzyme electromorphs for their identification

Mixtures of chromosomal forms A, B, C and D in natural populations of Anopheles dirus Peyton & Harrison sensu lato in Thailand show significant positive values of Wright's fixation index for six enzyme-electromorph loci. The mean value of FIS over all loci was found to be +0.28 (SD 0.02), with a range of +0.57 (Odh) to +0.10 (Idh-2). Partitioning electromorph data for the chromosomal forms reduces the mean FIS to 0.03 (SD 0.01), which suggests that positive assortative mating is a characteristic of each form. This supports the hypothesis that the chromosomal/electrophoretic forms A, B, C and D represent four distinct biological species within the An. dirus complex. An example is given of the use of enzyme electromorphs as a means of vector identification during a malaria entomological field study involving a mixture of An. dirus species A and D. Electromorph identifications of 323 sp. A and 161 sp. D were more than 98% correct when cross-referenced to specific DNA probes.     

Pyrethroid induced behavioral responses of Anopheles dirus, a vector of malaria in Thailand

Contact and noncontact behavioral actions of wild-caught Anopheles dirus in response to the operational field dose of three synthetic pyrethroids (bifenthrin, α-cypermethrin and λ-cyhalothrin) were evaluated using an exito-repellency test chamber. DEET was used as the repellency standard for comparison with the other three synthetic pyrethroids. Results showed that test specimens rapidly escaped from the test chamber when exposed to direct contact with a surface treated with each of the three synthetic pyrethroids and DEET. Alpha-cypermethrin demonstrated the strongest irritant action (84.9% escape), followed by DEET (77.0%), λ-cyhalothrin (68.6%) and bifenthrin (68.3%). In the noncontact configuration, fewer mosquitoes escaped from the test chambers as compared to contact trials, although a significant escape response was still observed as compared to the controls (P<0.05). We conclude that An. dirus exhibits both irritant and repellent actions in response the three pyrethroids testing in this study. The information obtained will allow us to better understand the behavioral responses of vectors to various chemicals and provide guidance when designing control strategies for targeting specific disease vectors.     

The Anopheles dirus complex: spatial distribution and environmental drivers

Background

The Anopheles dirus complex includes efficient malaria vectors of the Asian forested zone. Studies suggest ecological and biological differences between the species of the complex but variations within species suggest possible environmental influences. Behavioural variation might determine vector capacity and adaptation to changing environment. It is thus necessary to clarify the species distributions and the influences of environment on behavioural heterogeneity.

Methods

A literature review highlights variation between species, influences of environmental drivers, and consequences on vector status and control. The localisation of collection sites from the literature and from a recent project (MALVECASIA) produces detailed species distributions maps. These facilitate species identification and analysis of environmental influences.

Results

The maps give a good overview of species distributions. If species status partly explains behavioural heterogeneity, occurrence and vectorial status, some environmental drivers have at least the same importance. Those include rainfall, temperature, humidity, shade, soil type, water chemistry and moon phase. Most factors are probably constantly favourable in forest. Biological specificities, behaviour and high human-vector contact in the forest can explain the association of this complex with high malaria prevalence, multi-drug resistant Plasmodium falciparum and partial control failure of forest malaria in Southeast Asia.

Conclusion

Environmental and human factors seem better than species specificities at explaining behavioural heterogeneity. Although forest seems essential for mosquito survival, adaptations to orchards and wells have been recorded. Understanding the relationship between landscape components and mosquito population is a priority in foreseeing the influence of land-cover changes on malaria occurrence and in shaping control strategies for the future.     

Intraspecific polymorphism of sex chromosome heterochromatin in two species of the Anopheles gambiae complex

The Hoechst 33258 banding pattern of the mitotic chromosomes of several laboratory and natural populations of the sibling species A. gambiae and A. arabiensis has been analyzed. A clear intraspecific polymorphism of sex chromosome heterochromatin has been observed. Nevertheless in each species heterochromatic variations fall within a characteristic species-specific pattern. Moreover, while laboratory populations tend to be monomorphic for a given heterochromatic variant, natural populations exhibit a high degree of intrapopulation polymorphism. The possible role of sex chromosome heterochromatin in controlling fertility and mating behaviour of Anopheles mosquitoes is discussed.     

Trophic behavior and biting activity of the two sibling species of the Anopheles minimus complex in western Thailand.

The trophic behavior and host preference of two sibling species, Anopheles minimus s.s. (= An. minimus species A) and species C, were observed during a two-year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Anopheles minimus s.s. and species C were more prevalent during the hot and wet periods of the year. Both species demonstrated exophagic and zoophilic activities. Feeding activity of An. minimus C was unique compared to An. minimus sensu lato from other localities in Thailand. Outdoor blood feeding by An. minimus C occurred throughout the night with one distinct feeding peak immediately after sunset (1800 h), whereas indoor feeding showed two small peaks at 2000 and 2400 h. The small number of An. minimus s.s collected during this study precluded a determination of peak activity patterns. A better understanding of mosquito behavior related to host and patterns of feeding activity will facilitate and improve the efficiency of vector control operations.     

Systematics of the Anopheles barbirostris species complex (Diptera: Culicidae: Anophelinae) in Thailand

This study deals with five species of the Barbirostris Complex of Anopheles subgenus Anopheles that are known to occur in Thailand. Three new species of the complex, A nopheles dissidens sp. nov. , A nopheles saeungae sp. nov. , and A nopheles wejchoochotei sp. nov. , are characterized and compared with Anopheles barbirostris van der Wulp and Anopheles campestris Reid based on specimens of molecularly identified progeny broods. For practical purposes, the five species are essentially isomorphic and can only be unequivocally identified from diagnostic mitochondrial and ribosomal DNA sequences. Based on overall morphological similarity, An. campestris is considered to be a member of the Barbirostris Complex rather than a separate member of the Barbirostris Subgroup. The molecular data, mitotic karyotypes, bionomics, and distributions of the species are reviewed and discussed. It is concluded that integrated molecular epidemiological studies of the complex throughout the Oriental Region are needed to unambiguously elucidate the individual species and their relation to disease. © 2015 The Linnean Society of London     

Multiple origins of cytologically identical chromosome inversions in the Anopheles gambiae complex.

For more than 60 years, evolutionary cytogeneticists have been using naturally occurring chromosomal inversions to infer phylogenetic histories, especially in insects with polytene chromosomes. The validity of this method is predicated on the assumption that inversions arise only once in the history of a lineage, so that sharing a particular inversion implies shared common ancestry. This assumption of monophyly has been generally validated by independent data. We present the first clear evidence that naturally occurring inversions, identical at the level of light microscopic examination of polytene chromosomes, may not always be monophyletic. The evidence comes from DNA sequence analyses of regions within or very near the breakpoints of an inversion called the 2La that is found in the Anopheles gambiae complex. Two species, A. merus and A. arabiensis, which are fixed for the "same" inversion, do not cluster with each other in a phylogenetic analysis of the DNA sequences within the 2La. Rather, A. merus 2La is most closely related to strains of A. gambiae homozygous for the 2L+. A. gambiae and A. merus are sister taxa, the immediate ancestor was evidently homozygous 2L+, and A. merus became fixed for an inversion cytologically identical to that in A. arabiensis. A. gambiae is polymorphic for 2La/2L+, and the 2La in this species is nearly identical at the DNA level to that in A. arabiensis, consistent with the growing evidence that introgression has or is occurring between these two most important vectors of malaria in the world. The parallel evolution of the "same" inversion may be promoted by the presence of selectively important genes within the breakpoints.     

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.     

Population-genetic evidence for two species in Anopheles minimus in Thailand

ABSTRACT. Sympatric occurrence of homozygotes for two electro-morphs controlled by a locus for octanol dehydrogenase, and the absence of heterozygotes, at two localities, indicates two isomorphic species within the taxon Anopheles minimus Theobald in Thailand. This view is supported by significant, relative deficiences of heterozygotes at other electromorphic loci. Gene frequency data are reported for seven electro-morphic loci in An.minimus sensu lato from eleven localities: one of the newly recognized species predominated in all but one locality and the second was confined to two localities. This species pair of An.minimus s.l. was clearly distinguished from An.aconitus Donitz, An.pampanai Biittiker & Beales and An.varuna Iyengar, three species closely related to An.minimus s.l. in the series Myzomyia of Anopheles subgenus Cellia.     

Polytene chromosome structure at the submicroscopic level

Nuclear DNA obtained by SDS treatment or phenol extraction of isolated polytene salivary gland nuclei of D. melanogaster and D. hydei was investigated electron-microscopically. All preparations contained only linear doublestranded DNA filaments of various length. The mean length of a sample of 52 DNA filaments of D. melanogaster produced by SDS treatment was 37.3 . For D. hydei a mean length of 24.2 was established on account of a sample of 51 filaments obtained by SDS treatment. In samples obtained by phenol extraction a mean length of 23.8 (26 filaments) was found. Pronase digestion following SDS treatment gave a mean length of 29.1 for D. melanogaster (46 filaments) and of 17.1 for D. hydei (57 filaments). — The mean length of DNA filaments from D. hydei sperm was 21.5 on the basis of 25 filaments measured. The length distribution of the DNA of the samples of filaments measured varied. Preparations of single-stranded DNA obtained by heat denaturation of samples of D. hydei nuclear DNA revealed very long filaments. An obvious increase in the number of filaments shorter than 30 as compared with double-stranded DNA could not be established.     

Polytene chromosome structure at the submicroscopic level

Electron microscopic observations on sections of squashed glutar-aldehyde-fixed salivary gland X-chromosomes ofDrosophila melanogaster provided an ultrastructural map of the band sequence of region 1A1 to 4E3. Comparison of this map with the map of Bridges (1938) revealed a significantly lower number of bands. Only 67% of the bands depicted in Bridges' map could be identified. This discrepancy seems to be a consequence mainly of the absence of doublet characters of bands on Bridges' map. — A possible relationship between bands and genes is discussed for some regions, including the white-Notch region. It appears that a one to one relationship between bands and genes does exist for two regions, whereas in region 3C1–3C7 more genes than bands are present. In this respect the structural organization of bands at the submicroscopic level is discussed. — The average DNA content of a band at the haploid level is calculated on the basis of the reduction in band number to be 4.5 × 10^?5 picogram.     

Cytological differences and chromosomal rearrangements in four members of the Anopheles dirus complex (Diptera: Culicidae)

A reference photomap of the larval salivary gland, polytene chromosomes of the Anopheles dirus complex (species A) is presented. Samples of species A, B, C, and D from natural populations in Thailand were compared to this standard map using the larval progeny of wild-caught females. All species show differences in their chromosome banding patterns involving band size, number, and shape, particularly at the free ends of the X, 2R, and 2L. These differences provide useful diagnostic characters for separating members of the species complex. However, overall banding patterns are conservative in the group: species A, B, and C are virtually homosequential. Species D is highly polymorphic for a single paracentric inversion in each of the four autosomal arms and has a fixed inversion on the X chromosome. This same X chromosome inversion occurs at low frequency in species A.     

Mosaic genome architecture of the Anopheles gambiae species complex

Background

Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission.

Methodology

We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas.

Principal Findings

We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors.

Conclusions

Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.     

Population genetic confirmation of species status of the malaria vectors Anopheles willmori and An.pseudowillmori in Thailand and chromosome phylogeny of the Maculatus group of mosquitoes

Among the Anopheles (Cellia) maculatus group of Oriental mosquitoes, positive assortative mating occurs within sympatric and synchronous populations of An.willmori and An.pseudowillmori in the presence of populations of An.maculatus and An.sawadwongporni, judged from the occurrence of inversion homozygotes and the absence of any heterozygotes in their distinctive, polytene chromosomes. Genotypic frequencies for enzyme electromorphs give additional evidence for the species status of An.pseudowillmori and a practical means of identification in field studies of malaria vectors. Autosomal rearrangements are referred to those of An.stephensi which is unique for 4y. An.willmori differs by a single inversion, 4x; An.pseudowillmori by three Arm 2 inversions; and An.dispar by one Arm 2 inversion and 4x. Fixed autosomal rearrangements in the Maculatus group are summarized and their phylogenetic distribution suggests some unknown, intrinsic mechanism by which genome structure is disrupted in association with speciation events. This could be relevant to the potential genetic manipulation of malaria vectors.     

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.     

Lactate dehydrogenase allozyme differentiation of species in the Anopheles culicifacies complex

Abstract Genetically controlled enzyme variation exists within and between four sibling species of the Anopheles culicifacies complex of malaria vectors in India. A study on electrophoretic variation of nine enzymes in An.culicifacies sibling species revealed that the lactate dehydrogenase ( Ldh ) locus has Fast (F) and Slow (S) allozymes distinguishing species A+D from species B+C with a probability of c . 95%.