Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach.

BACKGROUND: Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. METHODS: To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. RESULTS: Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. CONCLUSIONS: A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing.

BACKGROUND: Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. RESULTS: A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. CONCLUSIONS: This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

New assessment of Anopheles vector species identification using MALDI-TOF MS.

BACKGROUND: Anopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used. METHODS: To determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal. RESULTS: Using the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS. CONCLUSIONS: The using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.

Potential Roles of Environmental and Socio-Economic Factors in the Distribution of Insecticide Resistance in Anopheles gambiae sensu lato (Culicidae: Diptera) Across Togo, West Africa.

Vector control strategies recommended by the World Health Organization are threatened by resistance of Anopheles mosquitoes to insecticides. Information on the distribution of resistant genotypes of malaria vectors is increasingly needed to address the problem. Ten years of published and unpublished data on malaria vector susceptibility/resistance and resistance genes have been collected across Togo. Relationships between the spatial distribution of resistance status and environmental, socio-economic, and landscape features were tested using randomization tests, and calculating Spearman rank and Pearson correlation coefficients between mosquito mortality and different gridded values. Anopheles gambiae sensu lato was resistant to DDT, pyrethroids, and the majority of carbamates and organophosphates. Three sibling species were found (i.e., An. gambiae, Anopheles coluzzii, and Anopheles arabiensis) with four resistance genes, including kdr (L1014F, L1014S, and N1575Y) and ace1 (G119S). The most frequent resistance gene was L1014F. Overall, no association was found between the susceptibility/resistance status and environmental features, suggesting that evolution of resistance may be most closely related to extreme selection from local insecticide use. Nevertheless, further research is necessary for firm conclusions about this lack of association, and the potential role of landscape characteristics such as presence of crops and percentage of tree cover.

Lessons learned, challenges and outlooks for decision-making after a decade of experience monitoring the impact of indoor residual spraying in Benin, West Africa.

BACKGROUND: Since 2008, Indoor Residual Spraying (IRS) has been performed in Benin in 19 districts, including 4 in southern Benin, 9 in Atacora, and 8 in Atacora, Alibori and Donga in northern Benin. However, Benin still struggles with questions about IRS cost-benefit and epidemiological impact. Lessons learned and challenges from 10 years of IRS in Benin to be shared with the stakeholders involved in vector control implementation for decision-making. METHODS: Entomological parameters have been assessed entomological parameters in IRS communes since 2008. In all IRS intervention communes, decreases in human biting rate (HBR) of Anopheles gambiae, blood feeding inhibition and entomological inoculation rate (EIR) as compared to control district have been measured. RESULTS: EIR was reduced by 80-90%, which is encouraging, but should be observed with caution because: (i) the reduction may be insufficient to decrease epidemiological indicators given that the residual EIR in IRS districts is still higher than it is in some regions of stable malaria; (ii) the reduction in EIR is based on comparisons with control communes, but it is difficult to select control areas with the same environmental characteristics as intervention areas; (iii) despite the reduction, half of all mosquitoes that entered IRS-treated houses succeeded in taking human blood meals. Further, there are behaviours among Benin's population that limit IRS efficacy, including recent data showing that > 90% of people are not protected by IRS between 7 and 10 p.m. This is due to the fact that they remain outdoors and that most people are not protected from mosquito bites after 10 p.m. because they either sleep outdoors without IRS protection or indoors without an ITN. Moreover, people have large amounts of clothing hanging on walls where mosquitoes can rest instead of IRS-treated walls. Finally, other components are important to consider in implementing IRS among which: (i) Vector resistance management strategies are sometimes poorly understood; this is actually different from the need to replace one insecticide with another after the emergence of resistance; (ii) African countries should prepare to finance IRS themselves. CONCLUSION: To curtail residual malaria transmission, additional interventions able to target vectors escaping IRS should be prioritized.

Species abundance, composition, and nocturnal activity of female Anopheles (Diptera: Culicidae) in malaria-endemic villages of Papua New Guinea: assessment with barrier screen sampling.

BACKGROUND: Community composition of Anopheles mosquitoes, and their host-seeking and peridomestic behaviour, are important factors affecting malaria transmission. In this study, barrier screen sampling was used to investigate species composition, abundance, and nocturnal activity of Anopheles populations in villages of Papua New Guinea. METHODS: Mosquitoes were sampled from 6 pm to 6 am in five villages from 2012 to 2016. The barrier screens were positioned between the village houses and the perimeter of villages where cultivated and wild vegetation ("the bush") grew thickly. Female Anopheles that rested on either village or bush side of the barrier screens, as they commuted into and out of the villages, were captured. Similarity in species composition among villages was assessed. Mosquitoes captured on village and bush sides of the barrier screens were sorted by feeding status and by hour of collection, and their numbers were compared using negative binomial generalized linear models. RESULTS: Females of seven Anopheles species were present in the sample. Species richness ranged from four to six species per village, but relative abundance was highly uneven within and between villages, and community composition was similar for two pairs of villages and highly dissimilar in a fifth. For most Anopheles populations, more unfed than blood-fed mosquitoes were collected from the barrier screens. More blood-fed mosquitoes were found on the side of the barrier screens facing the village and relatively more unfed ones on the bush side, suggesting commuting behaviour of unfed host-seeking females into the villages from nearby bush and commuting of blood-fed females away from villages towards the bush. For most populations, the majority of host-seeking mosquitoes arrived in the village before midnight when people were active and unprotected from the mosquitoes by bed nets. CONCLUSION: The uneven distribution of Anopheles species among villages, with each site dominated by different species, even among nearby villages, emphasizes the importance of vector heterogeneity in local malaria transmission and control. Yet, for most species, nocturnal activity patterns of village entry and host seeking predominantly occurred before midnight indicating common behaviours across species and populations relative to human risk of exposure to Anopheles bites.

Stress dependent infection cost of the human malaria agent Plasmodium falciparum on its natural vector Anopheles coluzzii.

Unraveling selective forces that shape vector-parasite interactions has critical implications for malaria control. However, it remains unclear whether Plasmodium infection induces a fitness cost to their natural mosquito vectors. Moreover, environmental conditions are known to affect infection outcome and may impact the effect of infection on mosquito fitness. We investigated in the laboratory the effects of exposition to and infection by field isolates of Plasmodium falciparum on fecundity and survival of a major vector in the field, Anopheles coluzzii under different conditions of access to sugar resources after blood feeding. The results evidenced fitness costs induced by exposition and infection. When sugar was available after blood meal, infected and exposed mosquitoes had either reduced or equal to survival to unexposed mosquitoes while fecundity was either increased or decreased depending on the blood donor. Under strong nutritional stress, survival was reduced for exposed and infected mosquitoes in all assays. We therefore provide here evidence of an environmental-dependant reduced survival in mosquitoes exposed to infection in a natural and one of the most important parasite-mosquito species associations for human malaria transmission.

Taxonomic assessment of Anopheles crawfordi and An. dangi of the Hyrcanus Group of subgenus Anopheles in Vietnam.

Anopheles dangi, introduced as a new species of the Hyrcanus Group of subgenus Anopheles in an illustrated dichotomous key for the identification of the Anopheles mosquitoes of Vietnam published in 1987, was distinguished from Anopheles crawfordi based on the presence of a humeral pale spot on the base of the costal vein of the wing. However, this character has been known to occur occasionally in An. crawfordi. To determine whether An. dangi is distinct from An. crawfordi, we analyzed nucleotide sequences of the COI, COII and Cyt-b genes of mtDNA and the D3 gene of rDNA obtained from specimens collected in south-central Vietnam that were identified as An. dangi and An. crawfordi based on the presence or absence, respectively, of a humeral pale spot. Maximum Likelihood and Bayesian analyses of the sequences showed a low mean genetic distance of 0.004 for specimens identified as An. crawfordi and 0.008 for those identified as An. dangi. The mean genetic distance between the two nominal species was 0.006, compared with 0.077 for any group versus the outgroup taxa Anopheles dirus and Anopheles minimus, and the specimens of the two forms clustered in a single strongly supported clade. Consequently, An. dangi is merely a morphological variant of An. crawfordi and is deemed to be a synonym of that nominal species.

Wing geometric morphometrics and molecular assessment of members in the Albitarsis Complex from Colombia.

Malaria parasites are transmitted to humans by female mosquitoes of the genus Anopheles. The Albitarsis Complex harbours at least eight species not readily differentiable by morphology. This complicates the determination of those species involved in malaria transmission and the implementation of targeted and effective vector control strategies. In Colombia, there is little information about the identity and distribution of the Albitarsis Complex members. In this work, COI DNA barcoding was used to assign specimens Anopheles albitarsis s.l. to any of the previously designated species of the Albitarsis Complex. Two molecular operational taxonomic units (MOTUs), differentially distributed in Colombia, were detected, A. albitarsis I in the NW and NE, and A. albitarsis F, E and NE Colombia. In contrast, nuclear white gene and ITS2 sequence analyses did not allow differentiating between the MOTUs. Wing landmark-based geometric morphometrics applied to explore intertaxa phenotypic heterogeneity showed a subtle but significant difference in size, while shape did not allow the separation of the MOTUs. In general, the multiple marker analysis was not supportive of the existence in Colombia of more than one species of the Albitarsis Complex.

Integrated vector management targeting Anopheles darlingi populations decreases malaria incidence in an unstable transmission area, in the rural Brazilian Amazon.

BACKGROUND: Studies on vector behaviour should be conducted in order to evaluate the effectiveness of vector control measures on malaria protection in endemic areas of Latin America, where P. vivax predominates. This work aims to investigate the fauna of anopheline mosquitoes and verify the impact of integrated vector management in two colonization projects in the Careiro Municipality, Western Brazilian Amazon. METHODS: Four mosquitoes' captures were carried out from August 2008 to March 2010, with an interval of six months between each collection. Since September 2009 a large programme to reduce the burden of malaria has started in the two communities by distribution of insecticide-treated bed nets (ITN) and intensification of indoor residual spraying (IRS). Human biting rates (HBRs), entomological inoculation rates (EIRs), malaria incidence rate (MIR) and Plasmodium carrier's prevalence were used as outcomes to estimate the impact of the control measures. RESULTS: A total of 3,189 anophelines were collected, belonging to 13 species. Anopheles darlingi was the predominant species in the period (42.6%), followed by Anopheles albitarsis (38.4%). An. darlingi HBRs showed a notable decreasing trend from the start to the end of the study. Conversely, An. albitarsis increased its contribution to overall HBRs throughout the study. For An. darlingi there was a significant positive correlation between HBRs and MIR (p = 0.002). Anopheles albitarsis HBRs showed a significant negative correlation with the corresponding MIR (p = 0.045). EIR from total anophelines and from An. darlingi and An. albitarsis presented decreasing patterns in the successive collections. Four species of anophelines (An. darlingi, An. albitarsis, Anopheles braziliensis and Anopheles nuneztovari) were naturally infected with Plasmodium, albeit at very low infection rates. There were a decrease in the MIR for both vivax and falciparum malaria and in the prevalence of Plasmodium vivax and Plasmodium falciparum carriers during the period of study. CONCLUSIONS: There is strong evidence of association between the density of An. darlingi and the incidence of malaria in the studies sites, further highlighting the importance of this vector in malaria transmission in this region. An. darlingi susceptibility to control using ITN and IRS is likely to be high in the rural settlements studied.

Assessment of the effect of insecticide-treated nets and indoor residual spraying for malaria control in three rural kebeles of Adami Tulu District, South Central Ethiopia.

BACKGROUND: In the Adami Tulu District, indoor residual spraying (IRS) and insecticide-treated nets (ITNs) has been the main tool used to control malaria. The purpose of this study was to assess the effect of IRS and ITNs control strategies in Aneno Shisho kebele (lowest administrative unit of Ethiopia) compared with Kamo Gerbi (supplied ITN only) and Jela Aluto (no IRS and ITNs), with regards to the prevalence of malaria and mosquito density. METHODS: Cross-sectional surveys were conducted after heavy rains (October/November, 2006) and during the sporadic rains (April, 2007) in the three kebeles of Adami Tulu District. Malaria infection was measured by means of thick and thin film. Monthly collection of adult mosquitoes from October-December 2006 and April-May 2007 and sporozoite enzyme-linked immunosorbent assay (ELISA) on the collected mosquitoes were detected. Data related to the knowledge of mode of malaria transmission and its control measures were collected. Data collected on parasitological and knowledge, attitude and practice (KAP) surveys were managed and analysed using a statistical computer program SPSS version 13.0. A P-value <0.05 was considered to be statistically significant. RESULTS: The overall prevalence of malaria was 8.6% in Jela Aluto, 4.4% in Kamo Gerbi and 1.3% in Aneno Shisho in the two season surveys. The vector, Anopheles gambiae s.l., Anopheles pharoensis and Anopheles coustani were recorded. However, sporozoite ELISA on mosquito collections detected no infection. The difference in overall malaria prevalence and mosquito density between the three kebeles was significant (P<0.05). CONCLUSIONS: The present study has provided some evidence for the success of ITNs/IRS combined malaria control measures in Aneno Shisho kebele in Adami Tulu District. Therefore, the combined ITNs/IRS malaria control measures must be expanded to cover all kebeles in the District of Ethiopia.

Complete mtDNA genomes of Anopheles darlingi and an approach to anopheline divergence time.

BACKGROUND: The complete sequences of the mitochondrial genomes (mtDNA) of members of the northern and southern genotypes of Anopheles (Nyssorhynchus) darlingi were used for comparative studies to estimate the time to the most recent common ancestor for modern anophelines, to evaluate differentiation within this taxon, and to seek evidence of incipient speciation. METHODS: The mtDNAs were sequenced from mosquitoes from Belize and Brazil and comparative analyses of structure and base composition, among others, were performed. A maximum likelihood approach linked with phylogenetic information was employed to detect evidence of selection and a Bayesian approach was used to date the split between the subgenus Nyssorhynchus and other Anopheles subgenera. RESULTS: The comparison of mtDNA sequences within the Anopheles darlingi taxon does not provide sufficient resolution to establish different units of speciation within the species. In addition, no evidence of positive selection in any protein-coding gene of the mtDNA was detected, and purifying selection likely is the basis for this lack of diversity. Bayesian analysis supports the conclusion that the most recent ancestor of Nyssorhynchus and Anopheles+Cellia was extant ~94 million years ago. CONCLUSION: Analyses of mtDNA genomes of Anopheles darlingi do not provide support for speciation in the taxon. The dates estimated for divergence among the anopheline groups tested is in agreement with the geological split of western Gondwana (95 mya), and provides additional support for explaining the absence of Cellia in the New World, and Nyssorhynchus in the Afro-Eurasian continents.

Development of multiplex real-time PCR assays for identification of members of the Anopheles funestus species group.

BACKGROUND: The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR). One disadvantage of AS-PCR is the requirement for post-PCR processing by gel electrophoresis of PCR products. In this study, three new high-throughput 'closed-tube' assays were developed and compared with the previously described AS-PCR technique. METHODS: Protocols for three fluorescence-based assays based on Melt Curve Analysis (MCA), High Resolution Melt (HRM) and TaqMan SNP genotyping were developed to detect and discriminate Anopheles parensis, Anopheles leesoni, Anopheles vaneedeni, Anopheles rivulorum and An. funestus s.s. The sensitivity and specificity of these assays were compared with the widely used AS-PCR in a blind trial using DNA extracted from wild-caught mosquitoes. RESULTS: The TaqMan assay proved to be the most sensitive and specific of the three new assays. The MCA and HRM assays initially gave promising results, but were more sensitive to both DNA quality and quantity and consequently showed a higher rate of incorrect identifications. CONCLUSION: The TaqMan assay proved to be the most robust of the three protocols tested in this study. This assay very effectively identified all five members of the An. funestus group using fluorescently-labeled probes with distinct emission and excitation spectra allowing their independent detection in a single reaction. This method is at least as sensitive and specific as the gold standard AS-PCR approach and because it has no requirement for post-PCR processing is simpler and more rapid to run. The one disadvantage of the TaqMan assay is the cost of this assay, both in terms of initial capital outlay and running cost per sample, which is higher than AS-PCR. However, the cost of both the real-time PCR machine and fluorescently labelled probes required is falling and in the future the cost of this assay is likely to become closer to that of standard PCR.

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness.

BACKGROUND: The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes. METHOD: While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph. RESULTS: This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 mu SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1-100 muM) and from the extracted standards (1-100 muM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology. CONCLUSION: HPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.

Morphological assessment and molecular phylogenetics of the Funestus and Minimus groups of Anopheles (Cellia).

A morphological comparison and molecular study of the Afrotropical Funestus and Afro-Oriental Minimus groups within the Myzomyia series of Anopheles (Cellia) was conducted to determine their phylogenetic affinities. Relationships were investigated using morphological characters and ribosomal (D3) and mitochondrial (COII) nucleotide sequences. Cross-identification of specimens from one group by using keys for the other group confirmed their morphological similarity, i.e., members of one group shared the key characters with members of the other group. Molecular analyses recognized five clades, not strictly related to geographical distribution: the Aconitus, Culicifacies, Funestus, Minimus, and Rivulorum subgroups. Morphological observations were congruent with the results of molecular analyses. Anopheles leesoni, an Afrotropical species, is closely related to the Oriental Minimus complex, and these taxa share a close relationship with the Fluviatilis complex that occurs from the Arabian Peninsula through India. The immature and adult stages of An. rivulorum in Africa bear morphological characters that distinguish this species from members of the Afrotropical Funestus subgroup. A composite scheme of classification based on the results and previously published information is proposed for the two groups. It is noted that An. fluviatilis species S is conspecific with An. minimus species C.

Maps of the Sri Lanka malaria situation preceding the tsunami and key aspects to be considered in the emergency phase and beyond.

BACKGROUND: Following the tsunami, a detailed overview of the area specific transmission levels is essential in assessing the risk of malaria in Sri Lanka. Recent information on vector insecticide resistance, parasite drug resistance, and insights into the national policy for malaria diagnosis and treatment are important in assisting national and international agencies in their control efforts. METHODS: Monthly records over the period January 1995-October 2004 of confirmed malaria cases were used to perform an analysis of malaria distribution at district spatial resolution. Also, a focused review of published reports and routinely collected information was performed. RESULTS: The incidence of malaria was only 1 case per thousand population in the 10 months leading up to the disaster, in the districts with the highest transmission. CONCLUSION: Although relocated people may be more exposed to mosquito bites, and their capacity to handle diseases affected, the environmental changes caused by the tsunami are unlikely to enhance breeding of the principal vector, and, given the present low parasite reservoir, the likelihood of a malaria outbreak is low. However, close monitoring of the situation is necessary, especially as December-February is normally the peak transmission season. Despite some losses, the Sri Lanka public health system is capable of dealing with the possible threat of a malaria outbreak after the tsunami. The influx of foreign medical assistance, drugs, and insecticides may interfere with malaria surveillance, and the long term malaria control strategy of Sri Lanka, if not in accordance with government policy.

Molecular characterization of the Anopheles maculipennis complex in the Islamic Republic of Iran.

A diagnostic polymerase chain reaction (PCR) assay using species-specific primers and direct sequencing was used to identify members of the Anopheles maculipennis complex in the north-west and central regions of the Islamic Republic of Iran. Specimens were collected from 9 provinces during 2 seasonal activities in 2001-2002, identified morphologically and subjected to PCR assay and direct sequencing. Results showed that only 2 species, An. maculipennis Meigen, and An. sacharovi Faver, were present in the area of study. This was confirmed by the high similarity (99.2%-100%) of their sequences with those available in GenBank. The molecular data and relative distribution of these species in relation to their vectorial capacity and the epidemiology of malaria in the region are discussed.

Ribosomal ITS2 sequence data for Anopheles maculipennis and An. messeae in northern Greece, with a critical assessment of previously published sequences.

DNA sequences were generated for eight specimens of the Anopheles maculipennis complex from Florina in NW Greece, and identified to species on the basis of comparison with ITS2 sequences for members of the complex already in GenBank. The sequences revealed the presence of An. maculipennis and An. messeae in Florina. Problems with sequence reliability and accessibility of sequences generated in earlier studies of Palaearctic members of the complex are discussed.

Promotion of insecticide-treated mosquito nets in Myanmar.

A simple health promotion message administered by village midwives raised bednet usage to over 60% in trial hamlets in north Shan State, Myanmar. Treatment of the nets in the study villages produced a reduction in malaria cases. Most villagers were prepared to buy their nets at market prices and were willing to pay for the cost of re-treatment of nets, but very poor, members of the Wa ethnic group required a half-price subsidy for them to afford them. The use of insecticide treated bednets was felt to be appropriate for undeveloped and remote areas of the country where malaria control was difficult.