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Background: Decisions on when vector control can be withdrawn after malaria is eliminated depend on the receptivity or potential of an area to support vector populations.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.

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.

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 (N e ), connectivity and population structure (PCA, F ST )., 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 F ST 0.0480-0.0846). N e 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 N e 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.

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.

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.

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.

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., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

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.

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.

Database Url: https://dw.vecnet.org/datawarehouse/., (© The Author(s) 2017. Published by Oxford University Press.)

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.

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.

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.

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.

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.

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.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

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., (© The American Society of Tropical Medicine and Hygiene.)

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.

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.

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.

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts., (Copyright © 2015, American Association for the Advancement of Science.)

VectorBase is a National Institute of Allergy and Infectious Diseases supported Bioinformatics Resource Center (BRC) for invertebrate vectors of human pathogens. Now in its 11th year, VectorBase currently hosts the genomes of 35 organisms including a number of non-vectors for comparative analysis. Hosted data range from genome assemblies with annotated gene features, transcript and protein expression data to population genetics including variation and insecticide-resistance phenotypes. Here we describe improvements to our resource and the set of tools available for interrogating and accessing BRC data including the integration of Web Apollo to facilitate community annotation and providing Galaxy to support user-based workflows. VectorBase also actively supports our community through hands-on workshops and online tutorials. All information and data are freely available from our website at https://www.vectorbase.org/., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Agent-based models (ABMs) have been used to model the behaviour of individual mosquitoes and other aspects of malaria. In this paper, a conceptual entomological model of the population dynamics of Anopheles gambiae and the agent-based implementations derived from it are described. Hypothetical vector control interventions (HVCIs) are implemented to target specific activities in the mosquito life cycle, and their impacts are evaluated., Methods: The core model is described in terms of the complete An. gambiae mosquito life cycle. Primary features include the development and mortality rates in different aquatic and adult stages, the aquatic habitats and oviposition. The density- and age-dependent larval and adult mortality rates (vector senescence) allow the model to capture the age-dependent aspects of the mosquito biology. Details of hypothetical interventions are also described., Results: Results show that with varying coverage and temperature ranges, the hypothetical interventions targeting the gonotrophic cycle stages produce higher impacts than the rest in reducing the potentially infectious female (PIF) mosquito populations, due to their multi-hour mortality impacts and their applicability at multiple gonotrophic cycles. Thus, these stages may be the most effective points of target for newly developed and novel interventions. A combined HVCI with low coverage can produce additive synergistic impacts and can be more effective than isolated HVCIs with comparatively higher coverages. It is emphasized that although the model described in this paper is designed specifically around the mosquito An. gambiae, it could effectively apply to many other major malaria vectors in the world (including the three most efficient nominal anopheline species An. gambiae, Anopheles coluzzii and Anopheles arabiensis) by incorporating a variety of factors (seasonality cycles, rainfall, humidity, etc.). Thus, the model can essentially be treated as a generic Anopheles model, offering an excellent framework for such extensions. The utility of the core model has also been demonstrated by several other applications, each of which investigates well-defined biological research questions across a variety of dimensions (including spatial models, insecticide resistance, and sterile insect techniques).

Using mobile devices, such as personal digital assistants (PDAs), smartphones, tablet computers, etc., to electronically collect malaria-related field data is the way for the field questionnaires in the future. This case study seeks to design a generic survey framework PDA-based geo-tagged malaria-related data collection tool (PGMS) that can be used not only for large-scale community-level geo-tagged electronic malaria-related surveys, but also for a wide variety of electronic data collections of other infectious diseases. The framework includes two parts: the database designed for subsequent cross-sectional data analysis and the customized programs for the six study sites (two in Kenya, three in Indonesia, and one in Tanzania). In addition to the framework development, we also present our methods used when configuring and deploying the PDAs to 1) reduce data entry errors, 2) conserve battery power, 3) field install the programs onto dozens of handheld devices, 4) translate electronic questionnaires into local languages, 5) prevent data loss, and 6) transfer data from PDAs to computers for future analysis and storage. Since 2008, PGMS has successfully accomplished quite a few surveys that recorded 10,871 compounds and households, 52,126 persons, and 17,100 bed nets from the six sites. These numbers are still growing., (© The American Society of Tropical Medicine and Hygiene.)

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

Anopheles farauti is the primary malaria vector throughout the coastal regions of the Southwest Pacific. A shift in peak biting time from late to early in the night occurred following widespread indoor residue spraying of dichlorodiphenyltrichloro-ethane (DDT) and has persisted in some island populations despite the intervention ending decades ago. We used mitochondrial cytochrome oxidase I (COI) sequence data and 12 newly developed microsatellite markers to assess the population genetic structure of this malaria vector in the Solomon Archipelago. With geographically distinct differences in peak A. farauti night biting time observed in the Solomon Archipelago, we tested the hypothesis that strong barriers to gene flow exist in this region. Significant and often large fixation index (FST) values were found between different island populations for the mitochondrial and nuclear markers, suggesting highly restricted gene flow between islands. Some discordance in the location and strength of genetic breaks was observed between the mitochondrial and microsatellite markers. Since early night biting A. farauti individuals occur naturally in all populations, the strong gene flow barriers that we have identified in the Solomon Archipelago lend weight to the hypothesis that the shifts in peak biting time from late to early night have appeared independently in these disconnected island populations. For this reason, we suggest that insecticide impregnated bed nets and indoor residue spraying are unlikely to be effective as control tools against A. farauti occurring elsewhere, and if used, will probably result in peak biting time behavioural shifts similar to that observed in the Solomon Islands., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As global efforts to eliminate malaria intensify, accurate information on vector populations and transmission dynamics is critical for directing control efforts, developing new control tools, and predicting the effects of these interventions under various conditions. Currently available sampling tools for mosquito population monitoring suffer from well-recognized limitations. As reported in this workshop summary, a recent gathering of medical entomologists, modelers, and malaria experts reviewed these issues and agreed that efforts are needed to improve methods to monitor key transmission parameters. Identified needs include standardized methods for sampling of both mosquito adults and larvae, improved tools for mosquito species identification and age-grading, and a better means for determining the entomological inoculation rate.

Background: Agent-based models (ABMs) have been used to estimate the effects of malaria-control interventions. Early studies have shown the efficacy of larval source management (LSM) and insecticide-treated nets (ITNs) as vector-control interventions, applied both in isolation and in combination. However, the robustness of results can be affected by several important modelling assumptions, including the type of boundary used for landscapes, and the number of replicated simulation runs reported in results. Selection of the ITN coverage definition may also affect the predictive findings. Hence, by replication, independent verification of prior findings of published models bears special importance., Methods: A spatially-explicit entomological ABM of Anopheles gambiae is used to simulate the resource-seeking process of mosquitoes in grid-based landscapes. To explore LSM and replicate results of an earlier LSM study, the original landscapes and scenarios are replicated by using a landscape generator tool, and 1,800 replicated simulations are run using absorbing and non-absorbing boundaries. To explore ITNs and evaluate the relative impacts of the different ITN coverage schemes, the settings of an earlier ITN study are replicated, the coverage schemes are defined and simulated, and 9,000 replicated simulations for three ITN parameters (coverage, repellence and mortality) are run. To evaluate LSM and ITNs in combination, landscapes with varying densities of houses and human populations are generated, and 12,000 simulations are run., Results: General agreement with an earlier LSM study is observed when an absorbing boundary is used. However, using a non-absorbing boundary produces significantly different results, which may be attributed to the unrealistic killing effect of an absorbing boundary. Abundance cannot be completely suppressed by removing aquatic habitats within 300 m of houses. Also, with density-dependent oviposition, removal of insufficient number of aquatic habitats may prove counter-productive. The importance of performing large number of simulation runs is also demonstrated. For ITNs, the choice of coverage scheme has important implications, and too high repellence yields detrimental effects. When LSM and ITNs are applied in combination, ITNs' mortality can play more important roles with higher densities of houses. With partial mortality, increasing ITN coverage is more effective than increasing LSM coverage, and integrating both interventions yields more synergy as the densities of houses increase., Conclusions: Using a non-absorbing boundary and reporting average results from sufficiently large number of simulation runs are strongly recommended for malaria ABMs. Several guidelines (code and data sharing, relevant documentation, and standardized models) for future modellers are also recommended.

We report the imminent completion of a set of reference genome assemblies for 16 species of Anopheles mosquitoes. In addition to providing a generally useful resource for comparative genomic analyses, these genome sequences will greatly facilitate exploration of the capacity exhibited by some Anopheline mosquito species to serve as vectors for malaria parasites. A community analysis project will commence soon to perform a thorough comparative genomic investigation of these newly sequenced genomes. Completion of this project via the use of short next-generation sequence reads required innovation in both the bioinformatic and laboratory realms, and the resulting knowledge gained could prove useful for genome sequencing projects targeting other unconventional genomes.

Background: The control of vector-borne diseases, such as malaria, dengue fever, and typhus fever is often achieved with the use of insecticides. Unfortunately, insecticide resistance is becoming common among different vector species. There are currently no chemical alternatives to these insecticides because new human-safe classes of molecules have yet to be brought to the vector-control market. The identification of novel targets offer opportunities for rational design of new chemistries to control vector populations. One target family, G protein-coupled receptors (GPCRs), has remained relatively under explored in terms of insecticide development., Methods: A novel classifier, Ensemble*, for vector GPCRs was developed. Ensemble* was validated and compared to existing classifiers using a set of all known GPCRs from Aedes aegypti, Anopheles gambiae, Apis Mellifera, Drosophila melanogaster, Homo sapiens, and Pediculus humanus. Predictions for unidentified sequences from Ae. aegypti, An. gambiae, and Pe. humanus were validated. Quantitative RT-PCR expression analysis was performed on previously-known and newly discovered Ae. aegypti GPCR genes., Results: We present a new analysis of GPCRs in the genomes of Ae, aegypti, a vector of dengue fever, An. gambiae, a primary vector of Plasmodium falciparum that causes malaria, and Pe. humanus, a vector of epidemic typhus fever, using a novel GPCR classifier, Ensemble*, designed for insect vector species. We identified 30 additional putative GPCRs, 19 of which we validated. Expression of the newly discovered Ae. aegypti GPCR genes was confirmed via quantitative RT-PCR., Conclusion: A novel GPCR classifier for insect vectors, Ensemble*, was developed and GPCR predictions were validated. Ensemble* and the validation pipeline were applied to the genomes of three insect vectors (Ae. aegypti, An. gambiae, and Pe. humanus), resulting in the identification of 52 GPCRs not previously identified, of which 11 are predicted GPCRs, and 19 are predicted and confirmed GPCRs.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Determining the proportion of blood meals on humans by outdoor-feeding and resting mosquitoes is challenging. This is largely due to the difficulty of finding an adequate and unbiased sample of resting, engorged mosquitoes to enable the identification of host blood meal sources. This is particularly difficult in the south-west Pacific countries of Indonesia, the Solomon Islands and Papua New Guinea where thick vegetation constitutes the primary resting sites for the exophilic mosquitoes that are the primary malaria and filariasis vectors., Methods: Barrier screens of shade-cloth netting attached to bamboo poles were constructed between villages and likely areas where mosquitoes might seek blood meals or rest. Flying mosquitoes, obstructed by the barrier screens, would temporarily stop and could then be captured by aspiration at hourly intervals throughout the night., Results: In the three countries where this method was evaluated, blood-fed females of Anopheles farauti, Anopheles bancroftii, Anopheles longirostris, Anopheles sundaicus, Anopheles vagus, Anopheles kochi, Anopheles annularis, Anopheles tessellatus, Culex vishnui, Culex quinquefasciatus and Mansonia spp were collected while resting on the barrier screens. In addition, female Anopheles punctulatus and Armigeres spp as well as male An. farauti, Cx. vishnui, Cx. quinquefasciatus and Aedes species were similarly captured., Conclusions: Building barrier screens as temporary resting sites in areas where mosquitoes were likely to fly was an extremely time-effective method for collecting an unbiased representative sample of engorged mosquitoes for determining the human blood index.

Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (< 30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

Anopheles gambiae, the primary African vector of malaria parasites, exhibits numerous rhythmic behaviors including flight activity, swarming, mating, host seeking, egg laying, and sugar feeding. However, little work has been performed to elucidate the molecular basis for these daily rhythms. To study how gene expression is regulated globally by diel and circadian mechanisms, we have undertaken a DNA microarray analysis of An. gambiae under light/dark cycle (LD) and constant dark (DD) conditions. Adult mated, non-blood-fed female mosquitoes were collected every 4 h for 48 h, and samples were processed with DNA microarrays. Using a cosine wave-fitting algorithm, we identified 1,293 and 600 rhythmic genes with a period length of 20-28 h in the head and body, respectively, under LD conditions, representing 9.7 and 4.5% of the An. gambiae gene set. A majority of these genes was specific to heads or bodies. Examination of mosquitoes under DD conditions revealed that rhythmic programming of the transcriptome is dependent on an interaction between the endogenous clock and extrinsic regulation by the LD cycle. A subset of genes, including the canonical clock components, was expressed rhythmically under both environmental conditions. A majority of genes had peak expression clustered around the day/night transitions, anticipating dawn and dusk. Genes cover diverse biological processes such as transcription/translation, metabolism, detoxification, olfaction, vision, cuticle regulation, and immunity, and include rate-limiting steps in the pathways. This study highlights the fundamental roles that both the circadian clock and light play in the physiology of this important insect vector and suggests targets for intervention.

Background: Transposable elements (TEs) are mobile sequences found in nearly all eukaryotic genomes. They have the ability to move and replicate within a genome, often influencing genome evolution and gene expression. The identification of TEs is an important part of every genome project. The number of sequenced genomes is rapidly rising, and the need to identify TEs within them is also growing. The ability to do this automatically and effectively in a manner similar to the methods used for genes is of increasing importance. There exist many difficulties in identifying TEs, including their tendency to degrade over time and that many do not adhere to a conserved structure. In this work, we describe a homology-based approach for the automatic identification of high-quality consensus TEs, aimed for use in the analysis of newly sequenced genomes., Results: We describe a homology-based approach for the automatic identification of TEs in genomes. Our modular approach is dependent on a thorough and high-quality library of representative TEs. The implementation of the approach, named TESeeker, is BLAST-based, but also makes use of the CAP3 assembly program and the ClustalW2 multiple sequence alignment tool, as well as numerous BioPerl scripts. We apply our approach to newly sequenced genomes and successfully identify consensus TEs that are up to 99% identical to manually annotated TEs., Conclusions: While TEs are known to be a major force in the evolution of genomes, the automatic identification of TEs in genomes is far from mature. In particular, there is a lack of automated homology-based approaches that produce high-quality TEs. Our approach is able to generate high-quality consensus TE sequences automatically, requiring the user to only provide a few basic parameters. This approach is intentionally modular, allowing researchers to use components separately or iteratively. Our approach is most effective for TEs with intact reading frames. The implementation, TESeeker, is available for download as a virtual appliance, while the library of representative TEs is available as a separate download., (© 2011 Kennedy et al; licensee BioMed Central Ltd.)

Background: Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies., Methods: Sequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon., Results: The breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations., Conclusions: The complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Mosquitoes in the Anopheles gambiae complex show rapid ecological and behavioral diversification, traits that promote malaria transmission and complicate vector control efforts. A high-density, genome-wide mosquito SNP-genotyping array allowed mapping of genomic differentiation between populations and species that exhibit varying levels of reproductive isolation. Regions near centromeres or within polymorphic inversions exhibited the greatest genetic divergence, but divergence was also observed elsewhere in the genomes. Signals of natural selection within populations were overrepresented among genomic regions that are differentiated between populations, implying that differentiation is often driven by population-specific selective events. Complex genomic differentiation among speciating vector mosquito populations implies that tools for genome-wide monitoring of population structure will prove useful for the advancement of malaria eradication.

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification.