Spatial-temporal heterogeneity in malaria receptivity is best estimated by vector biting rates in areas nearing elimination.

BACKGROUND: Decisions on when vector control can be withdrawn after malaria is eliminated depend on the receptivity or potential of an area to support vector populations. To guide malaria control and elimination programmes, the potential of biting rates, sporozoite rates, entomological inoculation rates and parity rates to estimate malaria receptivity and transmission were compared within and among geographically localised villages of active transmission in the Western Province of the Solomon Islands. RESULTS: Malaria transmission and transmission potential was heterogeneous in both time and space both among and within villages as defined by anopheline species composition and biting densities. Biting rates during the peak biting period (from 18:00 to 00:00 h) of the primary vector, Anopheles farauti, ranged from less than 0.3 bites per person per half night in low receptivity villages to 26 bites per person in highly receptive villages. Within villages, sites with high anopheline biting rates were significantly clustered. Sporozoite rates provided evidence for continued transmission of Plasmodium falciparum, P. vivax and P. ovale by An. farauti and for incriminating An. hinesorum, as a minor vector, but were unreliable as indicators of transmission intensity. CONCLUSIONS: In the low transmission area studied, sporozoite, entomological inoculation and parity rates could not be measured with the precision required to provide guidance to malaria programmes. Receptivity and potential transmission risk may be most reliably estimated by the vector biting rate. These results support the meaningful design of operational research programmes to ensure that resources are focused on providing information that can be utilised by malaria control programmes to best understand both transmission, transmission risk and receptivity across different areas.

Reduced-representation sequencing identifies small effective population sizes of Anopheles gambiae in the north-western Lake Victoria basin, Uganda.

BACKGROUND: Malaria is the leading cause of global paediatric mortality in children below 5 years of age. The number of fatalities has reduced significantly due to an expansion of control interventions but the development of new technologies remains necessary in order to achieve elimination. Recent attention has been focused on the release of genetically modified (GM) mosquitoes into natural vector populations as a mechanism of interrupting parasite transmission but despite successful in vivo laboratory studies, a detailed population genetic assessment, which must first precede any proposed field trial, has yet to be undertaken systematically. Here, the genetic structure of Anopheles gambiae populations in north-western Lake Victoria is explored to assess their suitability as candidates for a pilot field study release of GM mosquitoes. METHODS: 478 Anopheles gambiae mosquitoes were collected from six locations and a subset (N = 96) was selected for restriction site-associated DNA sequencing (RADseq). The resulting single nucleotide polymorphism (SNP) marker set was analysed for effective size (Ne), connectivity and population structure (PCA, FST). RESULTS: 5175 high-quality genome-wide SNPs were identified. A principal components analysis (PCA) of the collinear genomic regions illustrated that individuals clustered in concordance with geographic origin with some overlap between sites. Genetic differentiation between populations was varied with inter-island comparisons having the highest values (median FST 0.0480-0.0846). Ne estimates were generally small (124.2-1920.3). CONCLUSIONS: A reduced-representation SNP marker set for genome-wide An. gambiae genetic analysis in the north-western Lake Victoria basin is reported. Island populations demonstrated low to moderate genetic differentiation and greater structure suggesting some limitation to migration. Smaller estimates of Ne indicate that an introduced effector transgene will be more susceptible to genetic drift but to ensure that it is driven to fixation a robust gene drive mechanism will likely be needed. These findings, together with their favourable location and suitability for frequent monitoring, indicate that the Ssese Islands contain several candidate field locations, which merit further evaluation as potential GM mosquito pilot release sites.

Using barrier screens to characterize mosquito composition, flight activity, and abdominal status in South Lampung, Indonesia.

BACKGROUND: Mosquito sampling methods target different aspects of mosquito behavior and are subject to trap and location specific biases. The barrier screen sampling method was developed and tested to sample free-flying, blood-fed, and host-seeking mosquitoes. During a pilot study, this method was useful in obtaining an unbiased sample of mosquitoes flying between outdoor larval habitats, and sites where blood meals were obtained. However, a relatively small number of blood-fed Anopheles mosquitoes were collected in Indonesia during the pilot study. The sampling method was extended in South Lampung, Indonesia, to enable the collection of blood-fed mosquitoes. This study aimed to intercept mosquitoes flying between human habitations and larval habitats with a barrier screen and to characterize mosquito composition, flight characteristics (direction, height and time), abdominal status, and parity. RESULTS: Barrier screens intercepted 15 different mosquito species in South Lampung: eight Anopheles spp. and seven Culex spp. Species compositions varied among the villages in South Lampung. About 15% of Anopheles spp. caught were blood-fed, of which 28.2% of those tested had fed on humans. This is the first time human blood-fed anophelines have been collected in Indonesia using barrier screens. Blood meals identified included cow, dog, goat, and human, as well as mixed blood meals. Activity of unfed An. subpictus, the primary vector collected, flying towards human habitations peaked between 20:00-12:00 h, with a slow decline in activity until 18:00 h. Unfed and fed An. sundaicus, had a different activity profile compared to An. subpictus. Other species demonstrated varied peak activity times, with earlier activity occurring as a general trend. For the Anopheles mosquitoes collected, 55.5% were collected below 0.5 m and 83.9% were captured resting < 1 m from the ground. Parity dissections enabled age structure by species, which revealed species-specific traits such as nulliparous An. subpictus being more active early in the night relative to An. sundaicus. CONCLUSIONS: This study demonstrates that barrier screens are an effective mosquito sampling method that can be used to gain insights into local mosquito species composition, flight characteristics (direction, height and time), abdominal status, and parity.

Pathway to Deployment of Gene Drive Mosquitoes as a Potential Biocontrol Tool for Elimination of Malaria in Sub-Saharan Africa: Recommendations of a Scientific Working Group†.

Gene drive technology offers the promise for a high-impact, cost-effective, and durable method to control malaria transmission that would make a significant contribution to elimination. Gene drive systems, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein, have the potential to spread beneficial traits through interbreeding populations of malaria mosquitoes. However, the characteristics of this technology have raised concerns that necessitate careful consideration of the product development pathway. A multidisciplinary working group considered the implications of low-threshold gene drive systems on the development pathway described in the World Health Organization Guidance Framework for testing genetically modified (GM) mosquitoes, focusing on reduction of malaria transmission by Anopheles gambiae s.l. mosquitoes in Africa as a case study. The group developed recommendations for the safe and ethical testing of gene drive mosquitoes, drawing on prior experience with other vector control tools, GM organisms, and biocontrol agents. These recommendations are organized according to a testing plan that seeks to maximize safety by incrementally increasing the degree of human and environmental exposure to the investigational product. As with biocontrol agents, emphasis is placed on safety evaluation at the end of physically confined laboratory testing as a major decision point for whether to enter field testing. Progression through the testing pathway is based on fulfillment of safety and efficacy criteria, and is subject to regulatory and ethical approvals, as well as social acceptance. The working group identified several resources that were considered important to support responsible field testing of gene drive mosquitoes.

Negligible Impact of Mass Screening and Treatment on Mesoendemic Malaria Transmission at West Timor in Eastern Indonesia: A Cluster-Randomized Trial.

Background: Mass screening and treatment (MST) aims to reduce malaria risk in communities by identifying and treating infected persons without regard to illness. Methods: A cluster-randomized trial evaluated malaria incidence with and without MST. Clusters were randomized to 3, 2, or no MST interventions: MST3, 6 clusters (156 households/670 individuals); MST2, 5 clusters (89 households/423 individuals); and MST0, 5 clusters (174 households/777 individuals). All clusters completed the study with 14 residents withdrawing. In a cohort of 324 schoolchildren (MST3, n = 124; MST2, n = 57; MST0, n = 143) negative by microscopy at enrollment, we evaluated the incidence density of malaria during 3 months of MST and 3 months following. The MST intervention involved community-wide expert malaria microscopic screening and standard therapy with dihydroartemisinin-piperaquine and primaquine for glucose-6 phosphate dehydrogenase-normal subjects. All blood examinations included polymerase chain reaction assays, which did not guide on-site treatment. Results: The risk ratios for incidence density of microscopically patent malaria in MST3 or MST2 relative to that in MST0 clusters were 1.00 (95% confidence interval [CI], .53-1.91) and 1.22 (95% CI, .42-3.55), respectively. Similar results were obtained with molecular analysis and species-specific (P. falciparum and P. vivax) infections. Microscopically subpatent, untreated infections accounted for 72% of those infected. Conclusions: Two or 3 rounds of MST within 3 months did not impact the force of anopheline mosquito-borne infection in these communities. The high rate of untreated microscopically subpatent infections likely explains the observed poor impact. Clinical Trials Registration: NCT01878357.

Comparative evaluation of anopheline sampling methods in three localities in Indonesia.

BACKGROUND: The effectiveness of vector control efforts can vary based on the interventions used and local mosquito behaviour and adaptability. In many settings, biting patterns of Anopheles mosquitoes can shift in response to interventions targeting indoor-biting mosquitoes, often resulting in higher proportions of mosquitoes feeding outside or at times when people are not protected. These behaviourally resistant mosquitoes have been shown to sustain residual malaria transmission and limit control efforts. Therefore, it is important to accurately sample mosquitoes to understand their behaviour. METHODS: A variety of traps were evaluated in three geographically diverse sites in malaria-endemic Indonesia to investigate local mosquito feeding behaviour and determine effective traps for surveillance. RESULTS: Eight traps were evaluated in three sites: Canti village, Lampung, Kaliharjo village, Purworejo, and Saketa village, Halmahera, Indonesia, including the gold standard human landing collection (HLC) and a variety of traps targeting host-seeking and resting mosquitoes both indoors and outdoors. Trapping, using indoor and outdoor HLC, the Ifakara tent trap C, goat and human-occupied tents, resting pots and boxes, and CDC miniature light traps was conducted for 16 nights in two sites and 8 nights in a third site, using a Latin square design. Trap efficacy varied by site, with outdoor HLC yielding the highest catch rates in Canti and Kaliharjo and a goat-baited tent trap proving most effective in Saketa. In Canti village, anthropophilic Anopheles sundaicus were caught indoors and outdoors using HLCs, peaking in the early morning. In Kaliharjo, a variety of mosquitoes were caught, mostly outdoors throughout the night. HLC was ineffective in Saketa, the only site where a goat-baited tent trap was tested. This trap was effective in catching zoophilic vectors outdoors before midnight. CONCLUSIONS: Different trapping methods were suitable for different species, likely reflecting differences in behaviour among species. The three villages, each located on a different island in the Indonesian archipelago, contained mosquito populations with unique behaviours. These data suggest that the effectiveness of specific vector monitoring and control measures may vary by location.

Modern Vector Control.

The rapid spread of mosquito resistance to currently available insecticides, and the current lack of an efficacious malaria vaccine are among many challenges that affect large-scale efforts for malaria control. As goals of malaria elimination and eradication are put forth, new vector-control paradigms and tools and/or further optimization of current vector-control products are required to meet public health demands. Vector control remains the most effective measure to prevent malaria transmission and present gains against malaria mortality and morbidity may be maintained as long as vector-intervention strategies are sustained and adapted to underlying vector-related transmission dynamics. The following provides a brief overview of vector-control strategies and tools either in use or under development and evaluation that are intended to exploit key entomological parameters toward driving down transmission.

An online analytical processing multi-dimensional data warehouse for malaria data.

Database URL: https://dw.vecnet.org/datawarehouse/.

Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia.

BACKGROUND: Indonesia is home to a variety of malaria vectors whose specific bionomic traits remain largely uncharacterized. Species-specific behaviours, such as host feeding preferences, impact the dynamics of malaria transmission and the effectiveness of vector control interventions. METHODS: To examine species-specific host attraction and feeding behaviours, a Latin square design was used to compare Anopheles mosquitoes attracted to human, cow, and goat-baited tents. Anopheles mosquitoes were collected hourly from the inside walls of each baited tent. Species were morphologically and then molecularly identified using rDNA ITS2 sequences. The head and thorax of individual specimens were analysed for Plasmodium DNA using PCR. Bloodmeals were identified using a multiplex PCR. RESULTS: A total of 1024, 137, and 74 Anopheles were collected over 12 nights in cow, goat, and human-baited tents, respectively. The species were identified as Anopheles kochi, Anopheles farauti s.s., Anopheles hackeri, Anopheles hinesorum, Anopheles indefinitus, Anopheles punctulatus, Anopheles tessellatus, Anopheles vagus, and Anopheles vanus, many of which are known to transmit human malaria. Molecular analysis of blood meals revealed a high level of feeding on multiple host species in a single night. Anopheles kochi, An. indefinitus, and An. vanus were infected with Plasmodium vivax at rates comparable to primary malaria vectors. CONCLUSIONS: The species distributions of Anopheles mosquitoes attracted to human, goat, and cow hosts were similar. Eight of nine sporozoite positive samples were captured with animal-baited traps, indicating that even predominantly zoophilic mosquitoes may be contributing to malaria transmission. Multiple host feeding and flexibility in blood feeding behaviour have important implications for malaria transmission, malaria control, and the effectiveness of intervention and monitoring methods, particularly those that target human-feeding vectors.

Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene.

BACKGROUND: Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming. METHODS: In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330-0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax. RESULTS: The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands. CONCLUSIONS: This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination.

Behaviour and molecular identification of Anopheles malaria vectors in Jayapura district, Papua province, Indonesia.

BACKGROUND: Members of the Anopheles punctulatus group dominate Papua, Indonesia and Papua New Guinea (PNG), with a geographic range that extends south through Vanuatu. An. farauti and An. punctulatus are the presumed major vectors in this region. Although this group of species has been extensively studied in PNG and the southern archipelagoes within their range, their distribution, ecology and vector behaviours have not been well characterized in eastern Indonesia. METHODS: Mosquitoes were collected in five villages in Jayapura province, Papua, Indonesia using human-landing collections, animal-baited tents and backpack aspirators. Mosquitoes were morphologically typed and then molecularly distinguished based on ribosomal ITS2 sequences and tested for Plasmodium falciparum and P. vivax infection using circumsporozoite ELISA and PCR. RESULTS: The presence and vector status of An. farauti 4 in Papua, Indonesia is confirmed here for the first time. The data indicate that this species is entering houses at a rate that increases its potential to come into contact with humans and act as a major malaria vector. An. farauti 4 was also abundant outdoors and biting humans during early evening hours. Other species collected in this area include An. farauti 1, An. hinesorum, An. koliensis, An. punctulatus, and An. tessellatus. Proboscis morphology was highly variable within each species, lending support to the notion that this characteristic is not a reliable indicator to distinguish species within the An. punctulatus group. CONCLUSIONS: The vector composition in Papua, Indonesia is consistent with certain northern areas of PNG, but the behaviours of anophelines sampled in this region, such as early and indoor human biting of An. farauti 4, may enable them to act as major vectors of malaria. Presumed major vectors An. farauti and An. punctulatus were not abundant among these samples. Morphological identification of anophelines in this sample was often inaccurate, highlighting the importance of using molecular analysis in conjunction with morphological investigations to update keys and training tools.

Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene.

BACKGROUND: Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis. However, they are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. Here the use of "direct PCR" was investigated with the aim of improving diagnosis in the malaria elimination era. METHODS: The performance of the Plasmodium cytochrome oxidase III gene (COX-III) based novel malaria detection strategies (direct nested PCR and direct single PCR) were compared using a 18s-rRNA direct nested PCR as a reference tool. Evaluations were based on sensitivity, specificity and the ability to detect mixed infections using control blood spot samples and field collected blood samples with final species diagnosis confirmation by sequencing. RESULTS: The COX-III direct PCR (limit of detection: 0.6-2 parasites/µL) was more sensitive than the 18s-rRNA direct nested PCR (limit of detection: 2-10 parasites/µL). The COX-III direct PCR identified all 21 positive controls (no mixed infections detected) while the 18s-rRNA direct nested PCR identified 18/21 (including four mixed infections). Different concentrations of simulated mixed infections (Plasmodium vivax and Plasmodium falciparum) suggest that the COX-III direct PCR detects only the predominant species. When the 18s-rRNA direct nested PCR was used to detect Plasmodium in field collected bloods spots (n = 3833), there was discrepancy in the results from the genus PCR (16 % positive) and the species-specific PCR (5 % positive). Further, a large portion of a subset of these positive samples (93 % for genus and 60 % for P. vivax), did not align with Plasmodium sequences. In contrast, the COX-III direct PCR clearly identified (single bands confirmed with sequencing) 2 % positive Plasmodium samples including P. vivax, P. falciparum, Plasmodium malariae and Plasmodium ovale wallikeri. CONCLUSIONS: The COX-III single direct PCR is an alternative method for accurate detection of Plasmodium microscopic and submicroscopic infections in humans, especially when a large number of samples require screening. This PCR does not require DNA isolation, is sensitive, quick, produces confident/clear results, identifies all the Plasmodium species infecting humans, and is cost-effective.

Anopheles farauti is a homogeneous population that blood feeds early and outdoors in the Solomon Islands.

BACKGROUND: In the 1970s, Anopheles farauti in the Solomon Island responded to indoor residual spraying with DDT by increasingly feeding more outdoors and earlier in the evening. Although long-lasting insecticidal nets (LLINs) are now the primary malaria vector control intervention in the Solomon Islands, only a small proportion of An. farauti still seek blood meals indoors and late at night where they are vulnerable to being killed by contract with the insecticides in LLINs. The effectiveness of LLINs and indoor residual spraying (IRS) in controlling malaria transmission where the vectors are exophagic and early biting will depend on whether the predominant outdoor or early biting phenotypes are associated with a subpopulation of the vectors present. METHODS: Mark-release-recapture experiments were conducted in the Solomon Islands to determine if individual An. farauti repeat the same behaviours over successive feeding cycles. The two behavioural phenotypes examined were those on which the WHO recommended malaria vector control strategies, LLINs and IRS, depend: indoor and late night biting. RESULTS: Evidence was found for An. farauti being a single population regarding time (early evening or late night) and location (indoor or outdoor) of blood feeding. Individual An. farauti did not consistently repeat behavioural phenotypes expressed for blood feeding (e.g., while most mosquitoes that fed early and outdoors, and would repeat those behaviours, some fed late at night or indoors in the next feeding cycle). CONCLUSIONS: The finding that An. farauti is a homogeneous population is significant, because during the multiple feeding cycles required to complete the extrinsic incubation period, many individual female anophelines will enter houses late at night and be exposed to the insecticides used in LLINs or IRS. This explains, in part, the control that LLINs and IRS have exerted against a predominantly outdoor feeding vector, such as An. farauti. These findings may be relevant to many of the outdoor feeding vectors that dominate transmission in much of the malaria endemic world and justifies continued use of LLINs. However, the population-level tendency of mosquitoes to feed outdoors and early in the evening does require complementary interventions to accelerate malaria control towards elimination.

Determinants of host feeding success by Anopheles farauti.

BACKGROUND: The proportion of blood meals that mosquitoes take from a host species is a function of the interplay of extrinsic (abundance and location of potential hosts) and intrinsic (innate preference) factors. A mark-release-recapture experiment addressed whether host preference in a population of Anopheles farauti was uniform or if there were anthropophilic and zoophilic subpopulations. The corresponding fitness associated with selecting different hosts for blood meals was compared by measuring fecundity. METHODS: The attractiveness of humans for blood meals by An. farauti in the Solomon Islands was compared to pigs using tent traps. Host fidelity was assessed by mark-release-recapture experiments in which different colour dusts were linked to the host to which the mosquito was first attracted. Outdoor resting An. farauti were captured on barrier screens and the human blood index (HBI) as well as the feeding index were calculated. The fecundity of individual An. farauti after feeding on either humans or pigs was assessed from blood-fed mosquitoes held in individual oviposition chambers. RESULTS: Anopheles farauti were more attracted to humans than pigs at a ratio of 1.31:1.00. The mark-release-recapture experiment found evidence for An. farauti being a single population regarding host preference. The HBI of outdoor resting An. farauti was 0.93 and the feeding index was 1.29. Anopheles farauti that fed on a human host laid more eggs but had a longer oviposition time compared to An. farauti that had blood fed on a pig. CONCLUSIONS: One of the strongest drivers for host species preference was the relative abundance of the different host species. Here, An. farauti have a slight preference for humans over pigs as blood meal sources. However, the limited availability of alternative hosts relative to humans in the Solomon Islands ensures a very high proportion of blood meals are obtained from humans, and thus, the transmission potential of malaria by An. farauti is high.

Frequent blood feeding enables insecticide-treated nets to reduce transmission by mosquitoes that bite predominately outdoors.

BACKGROUND: The effectiveness of vector control on malaria transmission by long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) depends on the vectors entering houses to blood feed and rest when people are inside houses. In the Solomon Islands, significant reductions in malaria have been achieved in the past 20 years with insecticide-treated bed nets, IRS, improved diagnosis and treatment with artemisinin combination therapies; despite the preference of the primary vector, Anopheles farauti, to feed outdoors and early in the evening and thereby avoid potential exposure to insecticides. Rational development of tools to complement LLINs and IRS by attacking vectors outdoor requires detailed knowledge of the biology and behaviours of the target species. METHODS: Malaria transmission in Central Province, Solomon Islands was estimated by measuring the components comprising the entomological inoculation rate (EIR) as well as the vectorial capacity of An. farauti. In addition, the daily and seasonal biting behaviour of An. farauti, was examined and the duration of the feeding cycle was estimated with a mark-release-recapture experiment. RESULTS: Anopheles farauti was highly exophagic with 72% captured by human landing catches (HLC) outside of houses. Three-quarters (76%) of blood feeding on humans was estimated to occur before 21.00 h. When the hourly location of humans was considered, the proportion of exposure to mosquito bites on humans occurring indoors (πi) was only 0.130 ± 0.129. Peak densities of host seeking An. farauti occurred between October and January. The annual EIR was estimated to be 2.5 for 2012 and 33.2 for 2013. The length of the feeding cycle was 2.1 days. CONCLUSIONS: The short duration of the feeding cycle by this species offers an explanation for the substantial control of malaria that has been achieved in the Solomon Islands by LLINs and IRS. Anopheles farauti is primarily exophagic and early biting, with 13% of mosquitoes entering houses to feed late at night during each feeding cycle. The two-day feeding cycle of An. farauti requires females to take 5-6 blood meals before the extrinsic incubation period (EIP) is completed; and this could translate into substantial population-level mortality by LLINs or IRS before females would be infectious to humans with Plasmodium falciparum and Plasmodium vivax. Although An. farauti is primarily exophagic, the indoor vector control tools recommended by the World Health Organization (LLINs and IRS) can still provide an important level of control. Nonetheless, elimination will likely require vector control tools that target other bionomic vulnerabilities to suppress transmission outdoors and that complement the control provided by LLINs and IRS.

Larval habitats of the Anopheles farauti and Anopheles lungae complexes in the Solomon Islands.

BACKGROUND: There is an urgent need for vector control tools to supplement long-lasting insecticidal nets (LLINs) and indoor residual spraying; particularly in the Solomon Islands where the primary vector, Anopheles farauti, is highly anthropophagic and feeds mainly outdoors and early in the evening. Currently, the only supplementary tool recommended by the World Health Organization is larval source management (LSM). The feasibility and potential effectiveness of LSM requires information on the distribution of anophelines, the productivity of larval habitats and the potential impacts of larval control on adult fitness. METHODS: The distribution of anophelines in Central and Western Provinces in the Solomon Islands was mapped from cross-sectional larval habitat surveys. The composition and micro-distribution of larval instars within a large permanent river-mouth lagoon was examined with a longitudinal survey. Density-dependent regulation of An. farauti larvae was investigated by longitudinally following the development and survival of different densities of first instars in floating cages in a river-mouth lagoon. RESULTS: Five anopheline species were molecularly identified from a range of fresh and brackish water habitats: An. farauti s.s., An. hinesorum, An. lungae, An. nataliae and An. solomonis. The most common habitats used by the primary malaria vector, An. farauti, were coastal lagoons and swamps. In the detailed study of lagoon micro-productivity, An. farauti was non-uniformly distributed with highest densities found at collections sites most proximal and distal to the mouth of the lagoon. The survival of An. farauti larvae was more than twofold lower when larvae were held at the highest experimental density (1 larva per 3.8 cm(2)) when compared with the lowest density (1 larva per 38 cm(2)). CONCLUSIONS: The only documented major malaria vector collected in larval surveys in both Central and Western Provinces was An. farauti. Lagoons and swamps, the most common, largest and (potentially) most productive larval sites of this malaria vector, were "few, fixed and findable" and theoretically, therefore, amenable to successful LSM. However, the immense scale and complexity of these ecosystems in which An. farauti larvae are found raises questions regarding the ability to effectively control the larvae, as incomplete larviciding could trigger density dependent effects resulting in increased larval survivorship. While LSM has the potential to significantly contribute to malaria control of this early and outdoor biting vector, more information on the distribution of larvae within these extensive habitats is required to maximize the effectiveness of LSM.

Molecular Characterization Reveals Diverse and Unknown Malaria Vectors in the Western Kenyan Highlands.

The success of mosquito-based malaria control is dependent upon susceptible bionomic traits in local malaria vectors. It is crucial to have accurate and reliable methods to determine mosquito species composition in areas subject to malaria. An unexpectedly diverse set of Anopheles species was collected in the western Kenyan highlands, including unidentified and potentially new species carrying the malaria parasite Plasmodium falciparum. This study identified 2,340 anopheline specimens using both ribosomal DNA internal transcribed spacer region 2 and mitochondrial DNA cytochrome oxidase subunit 1 loci. Seventeen distinct sequence groups were identified. Of these, only eight could be molecularly identified through comparison to published and voucher sequences. Of the unidentified species, four were found to carry P. falciparum by circumsporozoite enzyme-linked immunosorbent assay and polymerase chain reaction, the most abundant of which had infection rates comparable to a primary vector in the area, Anopheles funestus. High-quality adult specimens of these unidentified species could not be matched to museum voucher specimens or conclusively identified using multiple keys, suggesting that they may have not been previously described. These unidentified vectors were captured outdoors. Diverse and unknown species have been incriminated in malaria transmission in the western Kenya highlands using molecular identification of unusual morphological variants of field specimens. This study demonstrates the value of using molecular methods to compliment vector identifications and highlights the need for accurate characterization of mosquito species and their associated behaviors for effective malaria control.

Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools.

The understanding of malaria vector species in association with their bionomic traits is vital for targeting malaria interventions and measuring effectiveness. Many entomological studies rely on morphological identification of mosquitoes, limiting recognition to visually distinct species/species groups. Anopheles species assignments based on ribosomal DNA ITS2 and mitochondrial DNA COI were compared to morphological identifications from Luangwa and Nyimba districts in Zambia. The comparison of morphological and molecular identifications determined that interpretations of species compositions, insecticide resistance assays, host preference studies, trap efficacy, and Plasmodium infections were incorrect when using morphological identification alone. Morphological identifications recognized eight Anopheles species while 18 distinct sequence groups or species were identified from molecular analyses. Of these 18, seven could not be identified through comparison to published sequences. Twelve of 18 molecularly identified species (including unidentifiable species and species not thought to be vectors) were found by PCR to carry Plasmodium sporozoites - compared to four of eight morphological species. Up to 15% of morphologically identified Anopheles funestus mosquitoes in insecticide resistance tests were found to be other species molecularly. The comprehension of primary and secondary malaria vectors and bionomic characteristics that impact malaria transmission and intervention effectiveness are fundamental in achieving malaria elimination.

A standard cytogenetic map of Culex quinquefasciatus polytene chromosomes in application for fine-scale physical mapping.

BACKGROUND: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes. METHODS: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping. RESULTS: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups. CONCLUSIONS: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

Phlebotomus papatasi SP15: mRNA expression variability and amino acid sequence polymorphisms of field populations.

BACKGROUND: The Phlebotomus papatasi salivary protein PpSP15 was shown to protect mice against Leishmania major, suggesting that incorporation of salivary molecules in multi-component vaccines may be a viable strategy for anti-Leishmania vaccines. METHODS: Here, we investigated PpSP15 predicted amino acid sequence variability and mRNA profile of P. papatasi field populations from the Middle East. In addition, predicted MHC class II T-cell epitopes were obtained and compared to areas of amino acid sequence variability within the secreted protein. RESULTS: The analysis of PpSP15 expression from field populations revealed significant intra- and interpopulation variation.. In spite of the variability detected for P. papatasi populations, common epitopes for MHC class II binding are still present and may potentially be used to boost the response against Le. major infections. CONCLUSIONS: Conserved epitopes of PpSP15 could potentially be used in the development of a salivary gland antigen-based vaccine.