Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion.

BACKGROUND: The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex. METHODS: The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex. RESULTS: Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers. CONCLUSIONS: The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

A decade of invasive Anopheles stephensi sequence-based identification: toward a global standard.

Anopheles stephensi is an invasive malaria vector in Africa that has been implicated in malaria outbreaks in the Horn of Africa. In 10 years, it has been detected as far east as Djibouti and as far west as Ghana. Early detections were mostly incidental, but now active surveillance in Africa has been updated to include An. stephensi. Morphological identification of An. stephensi from native vectors can be challenging, thus, sequence-based assays have been used to confirm identification during initial detections. Methods of sequence-based identification of An. stephensi have varied across initial detections to date. Here, we summarize initial detections, make suggestions that could provide a standardized approach, and discuss how sequences can inform additional genomic studies beyond species identification.

The Anopheles coluzzii range extends into Kenya: detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

BACKGROUND: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. The current study sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well as describe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzii populations. METHODS: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing (WGS) data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Comparisons were made with An. coluzzii cohorts from West and Central Africa. RESULTS: This study reports the detection of An. coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points from which samples were analyzed and its presence confirmed through taxonomic analysis. Additionally, there was a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies up to 64%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. CONCLUSIONS: These findings emphasize the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

Kenya must wake up to the threat of cryptic Anopheles species and their impact on residual malaria transmission.

Malaria remains a major health problem in Kenya despite the huge efforts put in place to control it. The non-relenting malaria threat has partly been attributed to residual malaria transmission driven by vectors that cannot effectively be controlled by the two popularly applied control methods: long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). Reports indicate that residual transmission is widely spread in areas where malaria is endemic. This could mean that the World Health Organization's vision of a world free of malaria remains a mirage as elimination and prevention of re-establishment of malaria are rendered unachievable. Amongst the major contributors to residual malaria transmission are cryptic rare species, species of mosquitoes that are morphologically indistinguishable, but isolated genetically, that have not been the focus of malaria control programs. Recent studies have reported extensive new Anopheles cryptic species believed to be involved in malaria transmission in Kenya. This underscores the need to understand these malaria vector species, their distribution and bionomics and their impact on malaria transmission. This article discusses reports of these cryptic species, their importance to malaria transmission, especially in the arid and semi-arid areas, and what can be done to mitigate the situation.

Diversity, distribution and relative abundance of the mosquito fauna (Diptera: Culicidae) of Malakand and Dir Lower, Pakistan / Diversidade, distribuição e abundância relativa da fauna de mosquitos (Diptera: Culicidae) de Malakand e Dir Lower, Paquistão

The present study was conducted to evaluate the diversity, distribution (C) and relative abundance (RA) of the mosquito fauna (Diptera: Culicidae) of Malakand and Dir Lower, Pakistan. Collection of specimens (n = 1087) was made during September 2018 to July 2019 at six different habitats including freshwater bodies, rice fields, animal sheds, indoors, drains and sewage waters. Specimens were collected through light traps, pyrethrum spray, aspirators and nets and subsequently killed, preserved and then arranged in entomological boxes for identification. Three genera were identified namely Culex, Anopheles and Aedes. A total of fourteen species were identified namely: Cx. quinquefasciatus (Say, 1823), An. stephensi (Liston, 1901), Cx. tritaeniorhynchus (Giles, 1901), Ae. vittatus (Bigot, 1861), An. maculatus (Theobald, 1901), An. fluviatilis (James, 1902), Cx. vishnui (Theobald, 1901), Ae. aegypti (Linnaeus, 1762) An. subpictus (Grassi, 1899), An. dthali (Patton, 1905), An. culicifascies (Giles, 1901), An. pallidus (Theobald, 1901), Ae. albopictus (Skuse, 1894) and An. annularis (van der Wulp, 1884). Cx. quinquefasciatus was found constantly distributed in the study area with RA = 16.5% and C = 100%. An. annularis was found as a satellite species, sporadically distributed in the study area having RA = 0.9% and C = 17%. Diversity indices of mosquitoes in the studied habitats were found as, Shannon-Wiener Index (2.415), Simpson Index (9.919), Fisher's Index (2.269) and Margalef's Index (1.859). A statistically significant difference was recorded in mosquito diversity in the six habitats (Kruskal-Wallis, chi-squared, H = 17.5, df = 5, P = 0.003 at α = 0.05). The present study encompasses [...].

O presente estudo foi conduzido para avaliar a diversidade, distribuição (C) e abundância relativa (RA) da fauna de mosquitos (Diptera: Culicidae) de Malakand e Dir Lower, Paquistão. A coleta de espécimes (n = 1087) foi feita durante o período de setembro de 2018 a julho de 2019 em seis habitats diferentes, incluindo corpos d’água, campos de arroz, galpões de animais, ambientes internos, ralos e águas residuais. Os espécimes foram coletados por meio de armadilhas luminosas, spray de piretro, aspiradores e redes e posteriormente mortos, preservados e depois dispostos em caixas entomológicas para identificação. Três gêneros foram identificados, nomeadamente Culex, Anopheles e Aedes. Um total de 14 espécies foi identificado, a saber: Cx. quinquefasciatus (Say, 1823), An. stephensi (Liston, 1901), Cx. tritaeniorhynchus (Giles, 1901), Ae. vittatus (Bigot, 1861), An. maculatus (Theobald, 1901), An. fluviatilis (James, 1902), Cx. vishnui (Theobald, 1901), Ae. aegypti (Linnaeus, 1762), An. subpictus (Grassi, 1899), An. dthali (Patton, 1905), An. culicifascies (Giles, 1901), An. pallidus (Theobald, 1901), Ae. albopictus (Skuse, 1894) e An. annularis (Van der Wulp, 1884). Cx. quinquefasciatus foi encontrado constantemente distribuído na área de estudo com AR = 16,5% e C = 100%. A. annularis foi encontrada como espécie satélite, distribuída esporadicamente na área de estudo com RA = 0,9% e C = 17%. Os índices de diversidade de mosquitos nos habitats estudados foram encontrados como índice de Shannon-Wiener (2,415), índice de Simpson (9,919), índice de Fisher (2,269) e índice de Margalef (1,859). Uma diferença estatisticamente significativa foi registrada na diversidade de mosquitos nos seis habitats (Kruskal-Wallis, qui-quadrado, H = 17,5, df = 5, P = 0,003 em α = 0,05). O presente estudo abrange a fauna de mosquitos de Malakand, Paquistão, com respeito à diversidade, abundância relativa e distribuição em [...].

Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence.

BACKGROUND: Anopheles stephensi Listen (1901) is a major vector of malaria in Asia and has recently been found in some regions of Africa. The An. stepehnsi species complex is suspected to have three sibling species: type, intermediate, and mysorensis, each with its own vector competence to the malaria parasite and ecology. To identify the members of the species complex in our An. stephensi insectary colony, we used the morphological features of eggs and genetic markers such as AnsteObp1 (Anopheles stephensi odorant binding protein 1), mitochondrial oxidases subunit 1 and 2 (COI and COII), and nuclear internal transcribed spacer 2 locus (ITS2). METHODS: Eggs were collected from individual mosquitoes (n = 50) and counted for the number of ridges under stereomicroscope. Genomic DNA was extracted from female mosquitoes. After the amplification of partial fragments of AnsteObp1, COI, COII and ITS2 genes, the PCR products were purified and sequenced. Phylogenetic analysis was performed after aligning query sequences against the submitted sequences in GenBank using MEGA 7. RESULTS: The range of ridges number on each egg float was 12-13 that corresponds to the mysorensis form of An. stephensi. The generated COI, COII and ITS2 sequences showed 100%, 99.46% and 99.29% similarity with the sequences deposited for Chinese, Indian and Iranian strains of An. stephensi, respectively. All the generated AnsteObp1 intron I region sequences matched 100% with the sequences deposited for An. stephensi sibling species C (mysorensis form) from Iran and Afghanistan. CONCLUSIONS: This manuscript precisely describes the morphological and molecular details of the 'var mysorensis' form of An. stephensi that could be exploited in elucidating its classification as well as in differentiation from other biotypes of the same or other anopheline species. Based on our findings, we recommend AnsteObp1 as a robust genetic marker for rapid and accurate discrimination (taxonomic identification) of the An. stephensi species complex, rather than the COI, COII, and ITS2 marker, which could only be utilized for interspecies (Anopheles) differentiation.

Adult Anopheles Mosquito Distribution at a Low and High Malaria Transmission Site in Tanzania.

Malaria parasites are only transmitted by female mosquitoes of the genus Anopheles; hence, the disease's distribution is linked to that of the vector mosquitoes. As such, the goal of this study was to find out the spatial and temporal distribution of Anopheles mosquito adults in the research sites. This was a repeated cross-sectional ecological study that took place in Morogoro and Dodoma, Tanzania. Vacuum aspiration was used to collect mosquitoes both outside and inside human dwellings. All mosquito-related data was collected and entered into appropriate data collection forms. Female mosquitoes were recognized morphologically using Gillies and Coetzee morphological criteria, followed by PCR. In total, about 2742 Anopheles mosquitoes with an average collection of 18.21 ± 1.12 per day were collected outside human houses of which 1717 (10.51 ± 1.17) and 1025 (8.42 ± 1.41) were collected from Morogoro and Dodoma, respectively. Of the captured mosquitoes, 89.0%, 10.0%, and 1.0% were recognized as Anopheles arabiensis, Anopheles gambiae s.s., and Anopheles quadrianulatus, respectively. The distribution varied significantly with seasons, whereby 302 (4.72 ± 1.04) and 2440 (12.96 ± 1.52) mosquitoes were captured in the cold-dry and warm-wet season, respectively (p < 0.0001). Of the captured mosquitoes, 42.33%, 16.33%, 14.96%, and 4.27 were found on the ceiling, stored junks, verandas, and barks/tree, respectively. In malaria-endemic countries, vector control forms an important component of the malaria control efforts. This study found significant variation of Anopheles mosquito abundance in time and space with Anopheles arabiensis being the most predominant malaria vector. This signifies the need to introduce mosquito control methods that will target the less anthropophilic Anopheles arabiensis or the immature aquatic stages. The study further found that underbeds, store room/piled bags, and undisturbed curtains were the most preferred resting places by mosquitoes signifying to be the most effective strategic sites for spraying insecticides during the implementation of indoor residual spraying (IRS).

Spatial patterns of West Nile virus distribution in the Volgograd region of Russia, a territory with long-existing foci.

Southern Russia remains affected by West Nile virus (WNV). In the current study, we identified the spatial determinants of WNV distribution in an area with endemic virus transmission, with special reference to the urban settings, by mapping probable points of human infection acquisition and points of virus detection in mosquitoes, ticks, birds, and mammals during 1999-2016. The suitability of thermal conditions for extrinsic virus replication was assessed based on the approach of degree-day summation and their changes were estimated by linear trend analysis. A generalized linear model was used to analyze the year-to-year variation of human cases versus thermal conditions. Environmental suitability was determined by ecological niche modelling using MaxEnt software. Human population density was used as an offset to correct for possible bias. Spatial analysis of virus detection in the environment showed significant contributions from surface temperature, altitude, and distance from water bodies. When indicators of location and mobility of the human population were included, the relative impact of factors changed, with roads becoming most important. When the points of probable human case infection were added, the percentage of leading factors changed only slightly. The urban environment significantly increased the epidemic potential of the territory and created quite favorable conditions for virus circulation. The private building sector with low-storey houses and garden plots located in the suburbs provided a connection between urban and rural transmission cycles.

Temporally consistent predominance and distribution of secondary malaria vectors in the Anopheles community of the upper Zambezi floodplain.

Regional optimisation of malaria vector control approaches requires detailed understanding both of the species composition of Anopheles mosquito communities, and how they vary over spatial and temporal scales. Knowledge of vector community dynamics is particularly important in settings where ecohydrological conditions fluctuate seasonally and inter-annually, such as the Barotse floodplain of the upper Zambezi river. DNA barcoding of anopheline larvae sampled in the 2019 wet season revealed the predominance of secondary vector species, with An. coustani comprising > 80% of sampled larvae and distributed ubiquitously across all ecological zones. Extensive larval sampling, plus a smaller survey of adult mosquitoes, identified geographic clusters of primary vectors, but represented only 2% of anopheline larvae. Comparisons with larval surveys in 2017/2018 and a contemporaneous independent 5-year dataset from adult trapping corroborated this paucity of primary vectors across years, and the consistent numerical dominance of An. coustani and other secondary vectors in both dry and wet seasons, despite substantial inter-annual variation in hydrological conditions. This marked temporal consistency of spatial distribution and anopheline community composition presents an opportunity to target predominant secondary vectors outdoors. Larval source management should be considered, alongside prevalent indoor-based approaches, amongst a diversification of vector control approaches to more effectively combat residual malaria transmission.

Phylogenetic analysis of the Neotropical Albitarsis Complex based on mitogenome data.

BACKGROUND: Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex. METHODS: Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees. RESULTS: Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species-Anopheles janconnae and An. albitarsis G-which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits. CONCLUSION: The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J.

Entomological survey in two communes with residual malaria transmission in Gia Lai Province in the central highlands of Vietnam.

BACKGROUND: In 2018, the National Malaria Control Programme in Vietnam switched from prioritizing malaria control to elimination. However, with the ongoing elimination programme, there are still areas where residual malaria transmission persists, including the central highlands. This entomological survey was conducted to evaluate Anopheles diversity and host-seeking activity of Anopheles vectors in two communes with very low malaria transmission in Gia Lai Province. METHODS: Anopheles species were collected in Ia DReh commune and Ia KDam commune, Gia Lai Province in the central highlands of Vietnam. Collections were conducted using human-baited double net trap, light trap and manual aspiration collections around cattle sheds, in the dry and rainy season. Mosquito specimens were identified morphologically, and members of species complexes were distinguished molecularly. Mosquito night-feeding patterns were investigated during the dry and rainy seasons. RESULTS: Overall, 18,835 specimens including 19 taxa were collected in Ia KDam and Ia DReh communes. These included the primary malaria vectors, Anopheles dirus and Anopheles minimus, and other secondary vector species. Anopheles dirus was observed to be an anthropophilic species, whereas An. minimus and a number of secondary vectors were observed to be zoophilic. Anopheles vagus was the dominant species, followed by Anopheles sinensis and Anopheles peditaeniatus. The majority of specimens were collected in the rainy season due to the relatively large number of An. vagus, while An. peditaeniatus, An. dirus, Anopheles kochi, Anopheles monstrosus and Anopheles tessellatus were collected in greater numbers during the dry season. The peak of host-seeking activity for An. dirus, An. sinensis, and An. vagus was between 18.00 and 19.00 h. CONCLUSION: This study provided information on the diversity, seasonal prevalence and behaviour of Anopheles at the study sites. Identifying the diverse mosquito fauna in the central highlands of Vietnam allows species-specific control measures to be implemented by the National Programme to reduce malaria in areas of very low malaria transmission. The peak Anopheles host-seeking activity observed in this study was between 18.00 and 23.00 h, which highlights the need to better characterize Anopheles behaviour in this region of Vietnam to inform on vector control strategies.

Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of Côte d'Ivoire.

BACKGROUND: Although malaria and Anopheles mosquito vectors are highly prevalent in Côte d'Ivoire, limited data are available to help understand the malaria vector density and transmission dynamics in areas bordering the country. To address this gap, the Anopheles mosquito species diversity, the members of the Anopheles gambiae complex and the transmission of malaria were assessed in four health districts along the borders of Côte d'Ivoire. METHODS: From July 2016 through December 2016 and July 2017 through December 2017, adult Anopheles mosquitoes were collected in four health districts of Côte d'Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. The collected mosquitoes were identified morphologically at species level and the members of the An. gambiae complex were separated using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l. and Anopheles nili specimens were analysed for malaria Plasmodium parasite detection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks. RESULTS: A total of 281 female Anopheles were collected in Aboisso, 754 in Bloléquin, 1319 in Odienné and 2443 in Ouangolodougou. Seven Anopheles species were recorded including An. gambiae s.l. (94.8-99.1%) as the main vector, followed by An. funestus s.l. (0.4-4.3%) and An. nili (0-0.7%). Among An. gambiae s.l., Anopheles coluzzii represented the predominant species in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae sensu stricto (s.s.) was the major species in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0%; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus s.l., Plasmodium falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus s.l. specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria incidence was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared to that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585). CONCLUSION: Anopheles vector species diversity, abundance and Plasmodium sporozoite infection were high within the health districts along the borders of the country of Côte d'Ivoire, resulting in high malaria transmission among the local populations. Anopheles gambiae s.l. and An. funestus s.l. were found to be highly infected with Plasmodium in the health districts of Bloléquin and Odienné where higher malaria incidence was observed than the other districts. This study provides important information that can be used to guide Côte d'Ivoire National Malaria Control Programme for vector control decision-making, mainly in districts that are at the country borders.

Molecular detection and maternal transmission of a bacterial symbiont Asaia species in field-caught Anopheles mosquitoes from Cameroon.

BACKGROUND: Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. METHODS: Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0 Asaia-positive females. RESULTS: A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). CONCLUSIONS: This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa.

Emergence of the invasive malaria vector Anopheles stephensi in Khartoum State, Central Sudan.

The emergence of the Asian invasive malaria vector, Anopheles stephensi, has been identified in Khartoum, the capital city of Sudan. This is the first report that confirms the geographical expansion of this urban mosquito into Central Sudan. We urgently recommend the launch of a national entomological survey to determine the distribution of this invasive disease vector and to generate essential information about its bionomics and susceptibility to available malaria control measures.

Chemical composition and the insecticidal activity of Aeollanthus pubescens leaf essential oil against Anopheles gambiae sensu stricto.

BACKGROUND: The excessive use of synthetic insecticides is responsible for many cases of resistance in insects. Therefore, the use of natural molecules of ecological interest with insecticidal properties is an alternative approach to the use of synthetic insecticides. The aim of this study is to investigating the larvicidal and adulticidal activity and the chemical composition of the essential oil of Aeollanthus pubescens on the major malaria vector, Anopheles gambiae. METHODS: Three reference strains of Anopheles gambiae sensu stricto (Kisumu, Kiskdr and Acerkis) were used in this study. The leaves of A. pubescens were collected in southern Benin. The standard World Health Organisation (WHO) guidelines for larvicide evaluation were used, and the chemical composition of the essential oil was analysed by gas chromatography coupled to mass spectrometry. Adult mosquitoes of each strain were exposed to pieces of net coated with the essential oil for 3 min using the WHO cone bioassay method. Probit regression analysis was used to determine the concentrations that would kill 50 and 95% of each test population (LC50, LC95) and the knockdown time for 50 and 95% of each test population (KDT50, and KDT95). The difference between the mortality-dose regressions for the different strains was analysed using the likelihood ratio test (LRT). The log-rank test was performed to evaluate the difference in survival between the strains. RESULTS: A total of 14 components were identified, accounting for 98.3% of total oil content. The major components were carvacrol (51.1%), thymyle acetate (14.0%) and É£-terpinene (10.6%). The essential oil showed larvicidal properties on the Kisumu, Acerkis and Kiskdr strains, with LC50 of 29.6, 22.9 and 28.4 ppm, respectively. With pieces of netting treated at 165 µg/cm2, the KDT50 of both Acerkis (1.71 s; Z = 3.34, P < 0.001) and Kiskdr (2.67 s; Z = 3.49, P < 0.001) individuals were significantly lower than that of Kisumu (3.8 s). The lifespan of the three mosquito strains decreased to 1 day for Kisumu (χ2 = 99, df = 1, P < 0.001), 2 days for Acerkis (χ2 = 117, df = 1, P < 0.001) and 3 days for Kiskdr (χ2 = 96.9, df = 1, P < 0.001). CONCLUSION: Our findings show that A. pubescens essential oil has larvicide and adulticide properties against the malaria vector An. gambiae sensu stricto, suggesting that this essential oil may be a potential candidate for the control of the resistant malaria-transmitting vectors.

Wild populations of malaria vectors can mate both inside and outside human dwellings.

BACKGROUND: Wild populations of Anopheles mosquitoes are generally thought to mate outdoors in swarms, although once colonized, they also mate readily inside laboratory cages. This study investigated whether the malaria vectors Anopheles funestus and Anopheles arabiensis can also naturally mate inside human dwellings. METHOD: Mosquitoes were sampled from three volunteer-occupied experimental huts in a rural Tanzanian village at 6:00 p.m. each evening, after which the huts were completely sealed and sampling was repeated at 11:00 p.m and 6 a.m. the next morning to compare the proportions of inseminated females. Similarly timed collections were done inside local unsealed village houses. Lastly, wild-caught larvae and pupae were introduced inside or outside experimental huts constructed inside two semi-field screened chambers. The huts were then sealed and fitted with exit traps, allowing mosquito egress but not entry. Mating was assessed in subsequent days by sampling and dissecting emergent adults caught indoors, outdoors and in exit traps. RESULTS: Proportions of inseminated females inside the experimental huts in the village increased from approximately 60% at 6 p.m. to approximately 90% the following morning despite no new mosquitoes entering the huts after 6 p.m. Insemination in the local homes increased from approximately 78% to approximately 93% over the same time points. In the semi-field observations of wild-caught captive mosquitoes, the proportions of inseminated An. funestus were 20.9% (95% confidence interval [CI]: ± 2.8) outdoors, 25.2% (95% CI: ± 3.4) indoors and 16.8% (± 8.3) in exit traps, while the proportions of inseminated An. arabiensis were 42.3% (95% CI: ± 5.5) outdoors, 47.4% (95% CI: ± 4.7) indoors and 37.1% (CI: ± 6.8) in exit traps. CONCLUSION: Wild populations of An. funestus and An. arabiensis in these study villages can mate both inside and outside human dwellings. Most of the mating clearly happens before the mosquitoes enter houses, but additional mating happens indoors. The ecological significance of such indoor mating remains to be determined. The observed insemination inside the experimental huts fitted with exit traps and in the unsealed village houses suggests that the indoor mating happens voluntarily even under unrestricted egress. These findings may inspire improved vector control, such as by targeting males indoors, and potentially inform alternative methods for colonizing strongly eurygamic Anopheles species (e.g. An. funestus) inside laboratories or semi-field chambers.

The effect of irrigation on malaria vector bionomics and transmission intensity in western Ethiopia.

BACKGROUND: Irrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia. METHODS: Comparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package. RESULTS: A total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P < 0.0001). The overall sporozoite rate of anopheline species from the nearby clusters was 10-fold higher compared to the faraway clusters. CONCLUSIONS: Significantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area.

Predicting the spread and persistence of genetically modified dominant sterile male mosquitoes.

BACKGROUND: Reproductive containment provides an opportunity to implement a staged-release strategy for genetic control of malaria vectors, in particular allowing predictions about the spread and persistence of (self-limiting) sterile and male-biased strains to be compared to outcomes before moving to (self-sustaining) gene-drive strains. METHODS: In this study, we: (i) describe a diffusion-advection-reaction model of the spread and persistence of a single cohort of male mosquitoes; (ii) elicit informative prior distributions for model parameters, for wild-type (WT) and genetically modified dominant sterile strains (DSM); (iii) estimate posterior distributions for WT strains using data from published mark-recapture-release (MRR) experiments, with inference performed through the Delayed-Rejection Adaptive Metropolis algorithm; and (iv) weight prior distributions, in order to make predictions about genetically modified strains using Bayes factors calculated for the WT strains. RESULTS: If a single cohort of 5000 genetically modified dominant sterile male mosquitoes are released at the same location as previous MRR experiments with their WT counterparts, there is a 90% probability that the expected number of released mosquitoes will fall to < 1 in 10 days, and that by 12 days there will be a 99% probability that no mosquitoes will be found more than 150 m from the release location. CONCLUSIONS: Spread and persistence models should form a key component of risk assessments of novel genetic control strategies for malaria vectors. Our predictions, used in an independent risk assessment, suggest that genetically modified sterile male mosquitoes will remain within the locality of the release site, and that they will persist for a very limited amount of time. Data gathered following the release of these mosquitoes will enable us to test the accuracy of these predictions and also provide a means to update parameter distributions for genetic strains in a coherent (Bayesian) framework. We anticipate this will provide additional insights about how to conduct probabilistic risk assessments of stage-released genetically modified mosquitoes.

Updated distribution of anopheline mosquitoes in Hokkaido, Japan, and the first evidence of Anopheles belenrae in Japan.

BACKGROUND: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. METHODS: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. RESULTS: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. CONCLUSION: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.