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

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

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

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

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

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

Comparison of climatic factors on mosquito abundance at US Army Garrison Humphreys, Republic of Korea.

INTRODUCTION: A number of studies have been conducted on the relationship between the distribution of mosquito abundance and meteorological variables. However, few studies have specifically provided specific ranges of temperatures for estimating the maximum abundance of mosquitoes as an empirical basis for climatic dynamics for estimating mosquito-borne infectious disease risks. METHODS: Adult mosquitoes were collected for three consecutive nights/week using Mosquito Magnet® Independence® model traps during 2018 and 2019 at US Army Garrison (USAG) Humphreys, Pyeongtaek, Gyeonggi Province, Republic of Korea (ROK). An estimate of daily mean temperatures (provided by the Korea Meteorological Administration) were distributed at the maximum abundance for selected species of mosquitoes using daily mosquito collection data after controlling for mosquito ecological cycles and environmental factors. RESULTS: Using the Monte-Carlo simulation, the overall mosquito population abundance peaked at 22.7°C (2.5th-97.5th: 21.7°C-23.8°C). Aedes albopictus, vector of Zika, chikungunya, dengue fever and other viruses, abundance peaked at 24.6°C (2.5th-97.5th, 22.3°C-25.6°C), while Japanese encephalitis virus (JEV) vectors, e.g., Culex tritaeniorhynchus and Culex pipiens, peaked at 24.3°C (2.5th-97.5th: 21.9°C-26.3°C) and 22.6°C (2.5th-97.5th: 21.9°C-25.2°C), respectively. Members of the Anopheles Hyrcanus Group, some of which are vivax malaria vectors in the ROK, abundance peaked at 22.4°C (2.5th-97.5th: 21.5°C-23.8°C). CONCLUSION: The empirical mean temperature ranges for maximum abundance were determined for each mosquito species collected at USAG Humphreys. These data contributed to the identification of relative mosquito abundance patterns for estimating mosquito-borne disease risks and developing and implementing disease prevention practices.

A potential global surveillance tool for effective, low-cost sampling of invasive Aedes mosquito eggs from tyres using adhesive tape.

BACKGROUND: The international movement of used tyres is a major factor responsible for global introductions of Aedes invasive mosquitoes (AIMs) (Diptera: Culicidae) that are major disease vectors (e.g. dengue, Zika, chikungunya and yellow fever). Surveillance methods are restricted by expense, availability and efficiency to detect all life stages. Currently, no tested method exists to screen imported used tyres for eggs in diapause, the life stage most at risk from accidental introduction. Here we test the efficiency of adhesive tape as an affordable and readily available material to screen tyres for eggs, testing its effect on hatch rate, larval development, DNA amplification and structural damage on the egg surface. RESULTS: We demonstrated that the properties of adhesive tape can influence pick up of dormant eggs attached to dry surfaces. Tapes with high levels of adhesion, such as duct tape, removed eggs with high levels of efficiency (97% ± 3.14). Egg numbers collected from cleaned used tyres were found to explain larval hatch rate success well, particularly in subsequent larval to adult emergence experiments. The strength of this relationship decreased when we tested dirty tyres. Damage to the exochorion was observed following scanning electron microscopy (SEM), possibly resulting in the high variance in the observed model. We found that five days was the optimal time for eggs to remain on all tested tapes for maximum return on hatch rate success. Tape type did not inhibit amplification of DNA of eggs from three, five or ten days of exposure. Using this DNA, genotyping of AIMs was possible using species-specific markers. CONCLUSIONS: We demonstrated for the first time that adhesive tapes are effective at removing AIM eggs from tyres. We propose that this method could be a standardised tool for surveillance to provide public health authorities and researchers with an additional method to screen tyre cargo. We provide a screening protocol for this purpose. This method has a global applicability and in turn can lead to increased predictability of introductions and improve screening methods at high risk entry points.

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

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

Assessment and an updated list of the mosquitoes of Saudi Arabia.

BACKGROUND: Mosquito-borne pathogens are important causes of diseases in the Kingdom of Saudi Arabia. Knowledge of the mosquito fauna is needed for the appropriate control of the vectors that transmit the pathogens and prevent the diseases they cause. An important first step is to have an up-to-date list of the species known to be present in the country. Original occurrence records were obtained from published literature and critically scrutinized to compile a list of the mosquito species that occur within the borders of the Kingdom. RESULTS: Fifty-one species have been recorded in the Kingdom; however, the occurrence of two of these species is unlikely. Thus, the mosquito fauna of the Kingdom comprises 49 species that include 18 anophelines and 31 culicines. Published records are provided for each species. Problematic records based on misidentifications and inappropriate sources are discussed and annotated for clarity. CONCLUSION: Integrated morphological and molecular methods of identification are needed to refine the list of species and accurately document their distributions in the Kingdom.

Species composition, seasonal abundance and distribution of potential anopheline vectors in a malaria endemic area of Iran: field assessment for malaria elimination.

BACKGROUND: Despite decreases in incidence, malaria remains a major public health challenge in south and southeast Iran, where vector control is considered one of the main strategies for elimination of the disease. The efficacy of this strategy depends on understanding malaria vector ecology, which varies by species. This study was conducted to determine the species composition, seasonal abundance and distribution of potential anopheline vectors in Bashagard County, one of the important malaria-endemic areas in south Iran. METHODS: In this cross-sectional study, four typical foothill and mountainous villages were selected by simple random sampling. Anopheline mosquitoes were collected by the standard dipping method for larvae and total catch for adults. Anopheline specimens were morphologically identified using taxonomic keys. Statistical analyses were performed using SPSS ver.20 software. RESULTS: In total, 1211 anopheline specimens, including 1055 (87.12%) larvae and 156 (12.88%) adults, were collected and identified. They consisted of 9 species: Anopheles moghulensis (27.89%), Anopheles dthali (18.91%), Anopheles culicifacies (16.60%), Anopheles stephensi (15.38%), Anopheles turkhudi (9.83%), Anopheles superpictus (9.66%), Anopheles apoci (1.40%), Anopheles fluviatilis (0.17%), and Anopheles sergentii (0.08%). The most prevalent species in adult catches were An. culicifacies (44.23%), An. dthali (21.15%) and An. stephensi (16.03%), and the most prevalent species caught as larvae were An. moghulensis (31.94%), An. dthali (18.85%) and An. stephensi (15.26%). Most of the anopheline species were distributed in different topographical areas and two proven malaria vectors, An. culicifacies and An. superpictus, were significantly associated with altitude and collected more frequently in the foothill regions. Most of the anopheline species were present almost throughout the year with a major peak in April and a smaller peak in October. CONCLUSION: The results of this study revealed that there are five malaria vectors in Bashagard County and some of them are more abundant in areas with special topographic features and are reproductively active throughout the year. These findings can be applied to successful planning and providing effective control measures in problematic areas during the malaria elimination phase in Iran.

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

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

Implementing a community vector collection strategy using xenomonitoring for the endgame of lymphatic filariasis elimination.

BACKGROUND: The global strategy for elimination of lymphatic filariasis is by annual mass drug administration (MDA). Effective implementation of this strategy in endemic areas reduces Wuchereria bancrofti in the blood of infected individuals to very low levels. This minimises the rate at which vectors successfully pick microfilariae from infected blood, hence requiring large mosquito numbers to detect infections. The aim of this study was to assess the feasibility of using trained community vector collectors (CVCs) to sample large mosquito numbers with minimal supervision at low cost for potential scale-up of this strategy. METHODS: CVCs and supervisors were trained in mosquito sampling methods, i.e. human landing collections, pyrethrum spray collections and window exit traps. Mosquito sampling was done over a 13-month period. Validation was conducted by a research team as quality control for mosquitoes sampled by CVCs. Data were analyzed for number of mosquitoes collected and cost incurred by the research team and CVCs during the validation phase of the study. RESULTS: A total of 31,064 and 8720 mosquitoes were sampled by CVCs and the research team, respectively. We found a significant difference (F(1,13) = 27.1606, P = 0.0001) in the total number of mosquitoes collected from southern and northern communities. Validation revealed similar numbers of mosquitoes sampled by CVCs and the research team, both in the wet (F(1,4) = 1.875, P = 0.309) and dry (F(1,4) = 2.276, P = 0.258) seasons in the southern communities, but was significantly different for both wet (F(1,4) = 0.022, P = 0.005) and dry (F(1,4 ) = 0.079, P = 0.033) seasons in the north. The cost of sampling mosquitoes per season was considerably lower by CVCs compared to the research team (15.170 vs 53.739 USD). CONCLUSIONS: This study revealed the feasibility of using CVCs to sample large numbers of mosquitoes with minimal supervision from a research team at considerably lower cost than a research team for lymphatic filariasis xenomonitoring. However, evaluation of the selection and motivation of CVCs, acceptability of CVCs strategy and its epidemiological relevance for lymphatic filariasis xenomonitoring programmes need to be assessed in greater detail.

Molecular xenomonitoring for Wuchereria bancrofti in Culex quinquefasciatus in two districts in Bangladesh supports transmission assessment survey findings.

BACKGROUND: Careful monitoring for recrudescence of Wuchereria bancrofti infection is necessary in communities where mass drug administration (MDA) for the elimination of lymphatic filariasis (LF) as a public health problem has been stopped. During the post-MDA period, transmission assessment surveys (TAS) are recommended by the World Health Organization to monitor the presence of the parasite in humans. Molecular xenomonitoring (MX), a method by which parasite infection in the mosquito population is monitored, has also been proposed as a sensitive method to determine whether the parasite is still present in the human population. The aim of this study was to conduct an MX evaluation in two areas of Bangladesh, one previously endemic district that had stopped MDA (Panchagarh), and part of a non-endemic district (Gaibandha) that borders the district where transmission was most recently recorded. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were systematically collected from 180 trap sites per district and mosquito pools were tested for W. bancrofti using real-time PCR. A total of 23,436 intact mosquitoes, representing 31 species, were collected from the two districts, of which 10,344 (41%) were Culex quinquefasciatus, the vector of W. bancrofti in Bangladesh. All of the 594 pools of Cx. quinquefasciatus tested by real-time PCR were negative for the presence of W. bancrofti DNA. CONCLUSIONS/SIGNIFICANCE: This study suggested the absence of W. bancrofti in these districts. MX could be a sensitive tool to confirm interruption of LF transmission in areas considered at higher risk of recrudescence, particularly in countries like Bangladesh where entomological and laboratory capacity to perform MX is available.