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PubMed1999

Differential susceptibilities of Anopheles albimanus and Anopheles pseudopunctipennis to infections with coindigenous Plasmodium vivax variants VK210 and VK247 in southern Mexico.

The susceptibilities to coindigenous Plasmodium vivax of colonized Anopheles albimanus and Anopheles pseudopunctipennis from southern Mexico were investigated by simultaneous feeding with infected blood obtained from patients. The genes encoding circumsporozoite protein variant types (VK210 and VK247) in blood samples were determined by PCR and oligonucleotide probe hybridization. A. albimanus was more susceptible to VK210, and A. pseudopunctipennis was more susceptible to VK247.

Animals↗
PubMed2006

IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

BACKGROUND: Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps. METHODS: Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: (1) an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; (2) detection of the Mopti and Savanna rDNA types; and (3) an assay to distinguish resistance to dieldrin (Rdl) alleles in Anopheles arabiensis. RESULTS: Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands. CONCLUSION: The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.

Animals↗
PubMed2006

Detection of the East and West African kdr mutation in Anopheles gambiae and Anopheles arabiensis from Uganda using a new assay based on FRET/Melt Curve analysis.

BACKGROUND: Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results. METHODS: Here, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays. RESULTS: The new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiae s.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously. CONCLUSION: The presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.

Alleles↗
PubMed2006

Efficacy of bifenthrin-impregnated bednets against Anopheles funestus and pyrethroid-resistant Anopheles gambiae in North Cameroon.

BACKGROUND: Recent field studies indicated that insecticide-treated bednets (ITNs) maintain their efficacy despite a high frequency of the knock-down resistance (kdr) gene in Anopheles gambiae populations. It was essential to evaluate ITNs efficacy in areas with metabolic-based resistance. METHODS: Bifenthrin was used in this experiment because it is considered a promising candidate for bednets impregnation. Nets were treated at 50 mg/m2, a dose that has high insecticidal activity on kdr mosquitoes and at 5 mg/m2, a dose that kills 95% of susceptible mosquitoes under laboratory conditions with 3 minutes exposure. Bednets were holed to mimic physical damage. The trial was conducted in three experimental huts from Pitoa, North-Cameroon where Anopheles gambiae displays metabolic resistance and cohabits with An. funestus. RESULTS: Bifenthrin at 50 mg/m2 significantly reduced anophelines' entry rate (>80%). This was not observed at 5 mg/m2. Both treatments increased exophily in An. gambiae, and to a lesser extent in An. funestus. With bifenthrin at high dosage, over 60% reduction in blood feeding and 75-90% mortality rates were observed in both vectors. Despite presence of holes, only a single An. gambiae and two An. funestus females were collected inside the treated net, and all were found dead. The same trends were observed with low dosage bifenthrin though in most cases, no significant difference was found with the untreated control net. CONCLUSION: Bifenthrin-impregnated bednets at 50 mg/m2 were efficient in the reduction of human-vector contact in Pitoa. Considerable personal protection was gained against An. funestus and metabolic pyrethroid resistant An. gambiae populations.

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PubMed2003

Isoenzymatic variability among five Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera of the Amazon region, Brazil.

An isoenzymatic comparative analysis of the variability and genetics differentiation among Anopheles species was done in populations of An. (Nys.) intermedius and An. (Ano.) mattogrossensis of the Anopheles subgenus, and of An. darlingi, An. albitarsis and An. triannulatus of the Nyssorhynchus subgenus, with the aim of detecting differences between both subgenera and of estimating the degree of genetic intere specific divergence. Samples from Macap , State of Amap and Janauari Lake, near Manaus, State of Amazonas, were analyzed for eight isoenzymatic loci. Analysis revealed differences in the average number of alleles per locus (1.6-2.3) and heterozygosity (0.060-0.284). However, the proportion of polymorphic loci was the same for An. (Nys.) darlingi, An. (Nys.) triannulatus and An. (Ano.) mattogrossensis (50%), but differed for An. (Nys.) albitarsis (62.5%) and An. (Ano.) intermedius (25%). Only the IDH1 (P > 0.5) locus in all species studied was in Hardy-Weinberg equilibrium. The fixation index demonstrated elevated genetic structuring among species, based on values of Fst = 0.644 and genetic distance (0.344-0.989). Genetic difference was higher between An. (Nys.) triannulatus and An. (Ano.) intermedius (0.989) and smaller between An. (Nys.) albitarsis sensu lato and An. (Nys.) darlingi (0.344). The data show interspecific genetic divergence which differs from the phylogenetic hypothesis based on morphological characters.

Alleles↗
PubMed2006

Insecticide resistance in Anopheles arabiensis and Anopheles gambiae from Mozambique.

Malaria control in the southern part of Mozambique is currently by indoor residual spraying with a carbamate insecticide and by pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles gambiae s.s. and Anopheles arabiensis Patton to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 17 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. gambiae s.s. from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A low level of pyrethroid resistance was detected in populations in southern Mozambique. Populations outside Maputo province were still susceptible to pyrethroids. Low level resistance to the carbamate propoxur also was detected in An. arabiensis from two localities. Mosquitoes from five of the localities had elevated p450 estimates, compared with the insecticide susceptible Durban strain. The lack of cross-resistance between pyrethroids and DDT in Mozambican populations suggests that a kdr-type target site resistance mechanism has not been selected. Increased frequencies of insecticide insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in 16 of the populations tested. Although vector control with bendiocarb is not being compromised by the presence of the acetylcholinesterase mechanism alone, the high level of insensitive acetylcholinesterase unless sensibly managed may have long-term implications for malaria control programs in Mozambique.

Acetylcholinesterase↗
PubMed2006

Sequence analysis of the rDNA internal transcribed spacer 2 and polymerase chain reaction identification of Anopheles fluminensis (Diptera: Culicidae: Anopheles) in Bolivia.

Anopheles fluminensis Root is a member of the Arribalzagia Series in the subgenus Anopheles. We report the first record of this species in the department of Cochabamba, Bolivia. This species was sampled from two locations in the foothills of the eastern Andes Mountains within the Chapare Valley. Larvae were collected in fast-flowing, shaded streams at the edges of rocky pools. We provide the first sequence data for the rDNA of An. fluminensis, a partial sequence of the 5.8S and the internal transcribed spacer 2 (ITS2). The ITS2 of An. fluminensis, sequenced from two individuals at one site, was at least 596 bp, had 56.5% GC, and included three large repeats (approximately equal to 125 bp each). We describe a polymerase chain reaction protocol and species-specific primers for identifying this species in the Chapare Valley, Bolivia.

Animals↗
PubMed2006

A simple method for sampling indoor-resting malaria mosquitoes Anopheles gambiae and Anopheles funestus (Diptera: Culicidae) in Africa.

Sampling indoor resting African malaria vectors is traditionally done by hand catches with oral or mechanical aspirators and pyrethrum spray catches (PSCs). In this study, we designed and briefly evaluated an inexpensive but practical alternative by using a cloth resting box or wicker resting basket and a ceiling net. Evaluations were performed in greenhouse and field situations in rural Kenya by comparing capture rates of Anopheles gambiae s.l. and Anopheles funestus (Giles) in these traps to hand collections and PSCs. A resting box and a ceiling net when used together collected more mosquitoes than a single collector using a hand-held aspirator but only one-third the number collected by PSCs. At sites where PSCs are impractical, a resting box and ceiling net can be effectively used as an alternative to hand catches in malaria surveillance.

Animals↗
PubMed2000

Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae), a rarely recorded member of the Anopheles punctulatus group.

Anopheline specimens collected in Papua New Guinea were morphologically identified as the rarely recorded Anopheles clowi Rozeboom & Knight. Amplification of the rDNA ITS2 region of this material revealed a fragment of 750 bp confirming its placement in the Anopheles punctulatus group. This group contains 12 species and includes the major malaria vectors in the islands of the southwest Pacific. Digestion of the ITS2 with the restriction enzyme MspI produced restriction fragment-length polymorphism with bands at 380, 300, and 150 bp, a pattern shared by no other members of this group. Phylogenetic analysis involving the sequencing of a 2 kb region of the rDNA 18S gene indicated that An. clowi was monophyletic and basal to the rest of the group and showed considerable independent evolution from the other members. This is the first record of An. clowi in Papua New Guinea and only the third collection of this species since its discovery in 1945.

Animals↗
PubMed2002

Detection of knockdown resistance mutations in Anopheles sacharovi (Diptera: Culicidae) and genetic distance with Anopheles gambiae (Diptera: Culicidae) using cDNA sequencing of the voltage-gated sodium channel gene.

The knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene (VGSCG), an important resistance mechanism against pyrethroids, was studied in Anopheles sacharovi Favre. It was found that the specific primers Agd1 and Agd2 used for polymerase chain reaction (PCR) amplification of Anopheles gambiae Giles VGSCG also amplified this genomic region in An. sacharovi. Comparison of the IIs4-IIs6 domain segments of the gene indicated 70% nucleotides common to both species and a genetic distance of 0.255 between them. Four different samples of pyrethroid-resistant An. sacharovi produced three types of amino acid, serine (TCG),leucine (TTG),and phenylalanine (TTT) at the kdr mutation point, whereas only two kdr mutations, leucine to phenylalanine and leucine to serine, occur in An. gambiae.

Animals↗
PubMed2004

Anopheles (Anopheles) pseudopunctipennis Theobald (Diptera: Culicidae): neotype designation and description.

Detailed morphological descriptions and illustrations are provided for the adult male and female, male genitalia, pupal, and larval stages of Anopheles (Anopheles) pseudopunctipennis Theobald, a major vector of human malaria in Central and South America. Taxonomic and related literature records, diagnostic features, distribution, and bionomics of the species are included. A neotype male for the species from the type locality of Grenada is designated.

Animals↗
PubMed2004

Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group.

In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. Identification of these two cryptic species would facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale field studies. In this study, we report on the development of a polymerase chain reaction (PCR)-restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-amplified product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identified morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 degrees N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.

Animals↗
PubMed2004

Analysis of reproductive isolation between sibling species anopheles Albitarsis sensu stricto and Anopheles deaneorum, two malaria vectors belonging to the Albitarsis complex (Diptera: Culicidae).

Complexes of sibling species are common among mosquitoes, and their existence within vector species can have important epidemiological consequences. Anopheles albitarsis sensu stricto and Anopheles deaneorum Rosa-Freitas are two putative vectors of malaria parasites belonging to the Albitarsis species complex (Diptera: Culicidae). Using an induced mating technique, we studied the reproductive isolation between these two closely related species and their reciprocal hybrids. Evidence for hybrid male sterility consistent with Haldane's rule was found. The results indicate that male hybrids show very low insemination rates, probably due to abnormalities in their reproductive organs. In addition, the data show that hybrid males carrying an X chromosome derived from An. deaneorum perform significantly worse than hybrid males carrying an An. albitarsis s.s. X chromosome.

Animals↗
PubMed2005

Anopheles triannulatus (Neiva and Pinto): a new Anopheles record from Trinidad, West Indies.

We report the first collection of Anopheles triannulatus from Trinidad, West Indies. Adults were captured using human bait while larvae and pupae were collected from a pond located in Valencia, north Trinidad. The associated species was An. oswaldoi. This new record increases the number of mosquito species belonging to the Genus Anopheles to 14 and the total species count from 162 to 163 in Trinidad, West Indies.

Animals↗
PubMed1991

Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae).

Approximately 460 base pairs (bp) of DNA sequence that included the second internal transcribed spacer (ITS2) and some flanking 5.8S and 28S ribosomal RNA coding regions were compared between the two closely related and morphologically indistinguishable mosquito species Anopheles freeborni and A. hermsi and a third related species, A. occidentalis. Sequences were determined from 14 clones of polymerase chain reaction (PCR)-amplified DNA obtained from four colonies of A. freeborni, two colonies of A. hermsi, and one individual A. occidentalis. Four clones showed independent single bp differences from the consensus for the relevant species. Eleven sites differed between the consensus sequences of A. hermsi and A. freeborni; 28 sites differed between A. hermsi and A. occidentalis. With the exception of a single bp mismatch in the 5.8S and two single bp mismatches near the undetermined junction of the ITS2 and 28S regions, all differences were confined to the ITS2 region. A PCR-based species-diagnostic assay for the cryptic species A. hermsi and A. freeborni was developed; it uses four synthetic oligonucleotides, two derived from areas of interspecies sequence difference in the ITS2, and two derived from highly conserved regions in the flanking coding sequences. Small amounts of mosquito DNA amplified in the presence of these four primers produce fragments of diagnostic size for each species: 900 bp for A. freeborni, 350 bp for A. hermsi, and approximately 1.2-1.4 kb for various other Anopheles species tested. We believe that this general approach to the development of species-diagnostic assays can be extended easily to other complexes of closely related, morphologically indistinguishable species.

Animals↗
PubMed2000

A new species of the Hyrcanus Group of Anopheles, subgenus Anopheles, a secondary vector of malaria in coastal areas of southern Vietnam.

Anopheles (Anopheles) nimpe, a new species of the Hyrcanus Group from the coastal areas of southern Vietnam, is described and illustrated in the adult, pupal, and larval stages. Partial DNA sequence data from paratypes are included for the mitochondrial cytochrome c oxidase I and 16S rRNA genes. The species is distinguished from other members of the group occurring in Southeast Asia.

Animals↗
PubMed2000

Studies on Anopheles fluviatilis and Anopheles culicifacies sibling species in relation to malaria in forested hilly and deforested riverine ecosystems in northern Orissa, India.

In a malaria-endemic region in northwestern Orissa, India, a longitudinal study was undertaken to delineate information on the sibling species of Anopheles fluviatilis and Anopheles culicifacies and their bionomics and role in malaria transmission in forested and deforested ecosystems. In forested villages, An. fluviatilis sibling species S (97.97%) and T (2.02%) were present. The former was highly anthropophagic (human blood index 0.88). Among the sibling species of An. culicifacies, species B (27.96%), C (71.1%), and B/C heterozygotes (0.94%) were present and were highly zoophagic. In deforested riverine villages An. fluviatilis was nearly absent and An. culicifacies sibling species A (0.48%), B (21.1%), C (77.94%), and B/C heterozygotes (0.48%) were present. In forested villages, the annual parasite incidence (269 cases/1,000) and the slide positivity rate (45%) were significantly higher than those in deforested areas, which had values of 39 cases/1,000 and 27%, respectively. The study showed that the high endemicity of malaria in the forested villages was due primarily to 2 vectors, the high rate of anthropophagy of An. fluviatilis species S, and also the more favorable ecological conditions for this vector.

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