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.     

Bionomics of Anopheles spp. (Diptera: Culicidae) in a malaria endemic region of Sukabumi,West Java,Indonesia

A 15‐month bionomic study of Anopheles species was conducted in two ecologically distinct villages (coastal and upland) of Sukabumi District, West Java, Indonesia from June 2006 to September 2007. Mosquitoes were captured using human‐landing collections at both sites. During the study, a total of 17,100 Anopheles mosquitoes comprising 13 Anopheles species were caught: 9,151 at the coastal site and 7,949 at the upland site. Anopheles barbirostris, Anopheles maculatus, and Anopheles vagus were the predominant species caught at the coastal site, and Anopheles aconitus, Anopheles barbirostris, and An. maculatus predominated in the upland site. Overall, species were exophagic at both sites, but there was variation between species. Anopheles aconitus was endophagic at the coastal site, exophagic at the upland site, collected most often in April 2007 and had a peak landing time between 22:00 and 23:00. Anopheles sundaicus was only collected at the coastal site, exophagic, collected most often in October 2006, and had a peak landing time between 19:00 and 20:00. Potential malaria vector species such An. aconitus, An. maculatus, and An. sundaicus were present throughout the year. None of the 7,770 Anopheles tested using CSP‐ELISA were positive for malaria, although the risk for malaria outbreaks in Sukabumi district remains high.     

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.     

Presence of the mosquito Anopheles hyrcanus in South Moravia, Czech Republic

During a survey of mosquitoes in the South Moravian lowland area, the mosquito Anopheles hyrcanus (Pallas) (Diptera: Culicidae) was found breeding in an ancient fishpond (Nesyt). It is not clear whether this southern Palaearctic species, a known vector of malaria in Asia which has not been recorded in the Czech Republic until this year, has gone undetected in the past or whether it has recently moved into the region as a result of climate change.     

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.     

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.     

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.     

我国赫坎按蚊复合体成员种的rDNA-ITS2区序列差异及系统发育分析

测定了我国赫坎按蚊复合体 9成员种的核糖体DNA第二内转录间隔区 (rDNA ITS2 )序列 ,根据序列差异分析各蚊种间的系统发育关系。结果显示 :( 1 )ITS2区序列最长的是中华按蚊 ( 4 6 8bp) ,最短的是克劳按蚊和赫坎按蚊 ( 4 36bp) ;GC含量为 4 4 9%～ 4 6 8% ;( 2 )发现该复合体 4成员种的ITS2区序列存在种内个体间差异 ,幅度为 0～ 3 8% ,明显小于种间差异 ;( 3)将各蚊种的ITS2区序列进行同源排序比较 ,发现其变异大多是简单重复单元的拷贝数不同 ;种间差异性最大的是克劳按蚊与嗜人按蚊( 32 3% ) ,最小的是贵阳按蚊与凉山按蚊 ( 9 0 % )平均差异率为 2 2 3% ;( 4 )根据ITS2区序列特征 ,用 3种方法构建的树状图拟合一致。以上结果表明赫坎按蚊复合体各成员种rDNA ITS2序列在种内非常保守 ,以种间序列差异分析为基础的分子鉴别技术是甄别蚊种分类地位混淆和错误的有效方法。     

Diversity of Anopheles species and trophic behavior of putative malaria vectors in two malaria endemic areas of northwestern Thailand

We determined the species diversity, blood‐feeding behavior, and host preference of Anopheles mosquitoes in two malaria endemic areas of Tak (Mae Sot District) and Mae Hong Son (Sop Moei District) Provinces, located along the Thai border with Myanmar, during a consecutive two‐year period. Anopheline mosquitoes were collected using indoor and outdoor human‐landing captures and outdoor cow‐baited collections. Mosquitoes were initially identified using morphological characters, followed by the appropriate multiplex AS‐PCR assay for the identification of sibling species within Anopheles (Cellia) complexes and groups present. Real‐time PCR was performed for parasite‐specific detection in mosquitoes (Plasmodium spp. and Wuchereria bancrofti). A total of 7,129 Anopheles females were captured, 3,939 from Mae Sot and 3,190 from Sop Moei, with 58.6% and 37% of all anophelines identified as An. minimus, respectively. All three malaria vector complexes were detected in both areas. One species within the Minimus Complex (An. minimus) was present along with two related species in the Funestus Group, (An. aconitus, An. varuna), two species within the Dirus Complex (An. dirus, An. baimaii), and four species within the Maculatus Group (An. maculatus, An. sawadwongporni, An. pseudowillmori, and An. dravidicus). The trophic behavior of An. minimus, An. dirus, An. baimaii, An. maculatus, and An. sawadwongporni are described herein. The highest An. minimus densities were detected from February through April of both years. One specimen of An. minimus from Mae Sot was found positive for Plasmodium vivax.     

Molecular characterization of the Anopheles maculipennis complex during surveillance for the 2004 Olympic Games in Athens

Specimens belonging to the Anopheles maculipennis complex were collected as larvae or resting adults from May 2003 to November 2004 in the area of the Athens 2004 Olympic Rowing Centre in Schinias, Attiki, Greece, and identified by morphological and molecular analyses. Of the 201 specimens collected, 199 were found to be Anopheles sacharovi Favre and two were An. maculipennis Meigen s.s. on the basis of similarity to published sequence data for the rDNA internal transcribed spacer (ITS2) region and the mitochondrial cytochrome c oxidase I gene (COI). Sequence data from a number of specimens were obtained for both genes and compared with corresponding GenBank data derived from diverse geographical areas. A high degree of homology in ITS2 sequences was found in both species, ranging from 99.5% to 100% in An. sacharovi and 99.4% to 100% in An. Maculipennis, with no intraspecific variation in either of the two species in our study. The degree of homology in the COI sequences was 94.8-99.8% in An. sacharovi and 95.0-99.8% in An. maculipennis. The 522-bp fragment produced a rather high degree of intrapopulation polymorphism for An. sacharovi, generating nine different haplotypes, five of which were singletons. Intraspecific variation for these sequences ranged from 0.2% to 1.4%, but was much lower (0.77%) for the two An. maculipennis sequences. These findings represent the first characterization of the An. maculipennis complex in the area of Schinias.     

DNA probes to identify members of the Anopheles farauti complex

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.     

Biting patterns and host preference of Anopheles epiroticus in Chang Island,Trat Province,eastern Thailand

A study of species diversity of Anopheles mosquitoes, biting patterns, and seasonal abundance of important mosquito vectors was conducted in two villages of Chang Island, Trat Province, in eastern Thailand, one located along the coast and the other in the low hills of the central interior of the island. Of 5,399 captured female anophelines, 70.25% belong to the subgenus Cellia and remaining specimens to the subgenus Anopheles. Five important putative malaria vectors were molecularly identified, including Anopheles epiroticus, Anopheles dirus, Anopheles sawadwongporni, Anopheles maculatus, and Anopheles minimus. Anopheles epiroticus was the most commonly collected species in the coastal site, whereas An. dirus was found to be most abundant in the forest‐hill site. From both locations, a greater number of mosquitoes was collected during the dry season compared to the wet. Anopheles epiroticus showed greater exophagic and zoophilic behavior with the highest blood feeding densities occurring between 18:00 and 19:00. In contrast, An. dirus demonstrated an activity peak between midnight and 01:00. We conclude that An. epiroticus and An. dirus, in coastal and inland areas, respectively, appear to be the most epidemiologically important malaria vectors on Chang Island. As no studies of vector competency specific to Chang Island have been conducted, our conclusions that these two species play a primary role in malaria transmission are based on evidence from other localities in Thailand and mainland Southeast Asia. This information serves as a basis for designing improved vector control programs that target specific species, and if integrated with other interventions could result in the elimination of malaria transmission on the island.     

Cytogenetic,hybridization and molecular evidence of four cytological forms of Anopheles nigerrimus (Hyrcanus Group) in Thailand and Cambodia

Thirteen isoline colonies of Anopheles nigerrimus were established from individual wild‐caught females collected from cow‐baited traps at locations in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and 4 types of Y (Y₁, Y₂, Y₃, Y₄) chromosomes were recovered, according to differing amounts of extra heterochromatin. Four karyotypic forms were designed depending upon apparently distinct figures of X and Y chromosomes, i.e., Form A (X₁, X₂, X₃, Y₁), B (X₂, X₃, Y₂), C (X₁, Y₃), and D (X₃, Y₄). Forms C and D were new metaphase karyotypes discovered in this study. Form A appeared to be common in both Thailand and Cambodia. Forms B and D were found to be rather specific to southern and northeastern Thailand, respectively, whereas Form C was confined to Cambodia. Hybridization experiments among the eight isoline colonies, which were representative of four karyotypic forms of An. nigerrimus, demonstrated genetic compatibility in giving viable progenies and synaptic salivary gland polytene chromosomes through F₂‐generations. These results elucidated the conspecific relationship, comprising four cytological forms within this taxon. Supportive evidence was confirmed further by very low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI) and subunit II (COII)].     

Chemically induced behavioral responses in Anopheles minimus and Anopheles harrisoni in Thailand

Behavioral responses of female mosquitoes representing two species in the Minimus Complex exposed to an operational field dose of bifenthrin or DEET (N,N-diethyl-m-toluamide) were described using an excito-repellency test system. Two test populations of An. minimus, one from the field (Tak Province, western Thailand), the other from a long-established laboratory colony, and Anopheles harrisoni collected from Kanchanaburi Province, western Thailand, were used. Results showed that all test populations rapidly escaped after direct contact with surfaces treated with either bifenthrin or DEET compared to match-paired untreated controls. Greater escape response by exposed females to bifenthrin and DEET were observed in the An. minimus colony compared to the two field populations. Field-collected An. minimus demonstrated a more rapid escape response to DEET than to bifenthrin, whereas An. harrisoni showed a converse response. Although fewer females escaped from test chambers without direct contact with treated surfaces compared to contact tests, the spatial repellency response was significantly pronounced in all test populations compared to match-paired controls (P < 0.05). DEET was found to perform as both a contact stimulant and moderate spatial repellent.     

Isolation and characterization of microsatellite markers from malaria vector,Anopheles culicifacies

Anopheles culicifacies, an important vector in the Indian subcontinent is a complex of five sibling species of which four are vectors. We describe the isolation of 31 microsatellite markers from the recently recognized isomorphic species A of which 13 were characterized in sympatric populations of Anopheles culicifacies isomorphic species A and B. The allele frequencies ranges from two to 12 in species A and two to seven in species B. Species A being a vector, and that these markers can be used in closely related species, makes the isolation of these markers important to study population structure of all sibling species in this complex.     

Molecular identification of the Anopheles nili group of African malaria vectors

Distinction between members of the Anopheles nili group of mosquitoes (Diptera: Culicidae), including major malaria vectors in riverside villages of tropical Africa, has been based mainly on doubtful morphological characters. Sequence variations of the ribosomal DNA second internal transcribed spacer (ITS2) and D3 28S region between morphological forms revealed four genetic patterns corresponding to typical An. nili (Theobald), An. carnevalei Brunhes et al., An. somalicus Rivola & Holstein and the newly identified variant provisionally named Oveng form. Primers were designed based on ITS2 fixed nucleotide differences between haplotypes to develop a multiplex PCR for rapid and specific identification of each species or molecular form. Specimens of the An. nili group from Cameroon, Burkina Faso, Ivory Coast and Senegal were successfully identified to species, demonstrating the general applicability of this technique based on criteria described in this paper.     

Molecular characterization of the malaria vector Anopheles gambiae s.s. in Madagascar

Abstract.  Anopheles gambiae s.s. Giles (Diptera: Culicidae), the primary African malaria vector, has been characterized at the subspecies level in Madagascar, where only the molecular form S and haplotype gIA occur. The haplotype gIC proposed by other authors was not observed amongst the 35 mosquito genomes sequenced. These S/gIA characteristics are also found on the Comoros archipelago and in continental Africa.     

Isozyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia

Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on UPGMA methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright's F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.     

Anopheles nili as the main vector of human malaria in villages of southern Cameroon

In villages near Sanaga river, in the forest zone of south Cameroon, Anopheles nili Theobald is the main species of mosquito regularly found biting man inside houses. Its densities are related to the flow level of the river. It is also the main malaria vector in terms of intensity and seasonal duration of transmission. The yearly malaria inoculation rate due to An. nili alone is 104 infective bites/man, varying between 3 infective bites/man in October and 20 in March. Anopheles gambiae Giles is a less important vector in the area but reached its peak in the dry season, when the Sanaga river is at its lowest level. These observations underline the influence of local ecology on malaria transmission and the need for entomological studies in each situation.