RESUMEN
BACKGROUND: Resistance to major public health insecticides in Côte d'Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. METHODS: This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from Southeast Côte d'Ivoire in 2019. RESULTS: Phenotypic resistance was intense: >25% of mosquitoes survived exposure to 10 times the doses of pyrethroids required to kill susceptible populations. Similarly, the 24-hour mortality rate with deltamethrin-only LLINs was very low and not significantly different from that with an untreated net. Sublethal pyrethroid exposure did not induce significant delayed vector mortality effects 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide, or new insecticides clothianidin and chlorfenapyr, were highly toxic to A. coluzzii. Pyrethroid-susceptible A. coluzzii were significantly more likely to be infected with malaria, compared with those that survived insecticidal exposure. Pyrethroid resistance was associated with significant overexpression of CYP6P4, CYP6P3, and CYP6Z1. CONCLUSIONS: Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating piperonyl butoxide, chlorfenapyr, or clothianidin in areas of high resistance intensity in Côte d'Ivoire.
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Anopheles , Mosquiteros Tratados con Insecticida , Insecticidas , Malaria , Piretrinas , Animales , Côte d'Ivoire , Resistencia a los Insecticidas , Insecticidas/farmacología , Malaria/prevención & control , Control de Mosquitos , Mosquitos Vectores , Butóxido de Piperonilo/farmacología , Piretrinas/farmacologíaRESUMEN
BACKGROUND: In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance. METHODS: Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3). RESULTS: In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy-Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah. CONCLUSIONS: Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Programme (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region.
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Anopheles/efectos de los fármacos , Resistencia a los Insecticidas/fisiología , Insecticidas/farmacología , Mosquitos Vectores/efectos de los fármacos , Animales , Anopheles/genética , Anopheles/fisiología , Femenino , Guinea , Resistencia a los Insecticidas/genética , Malaria/prevención & control , Mosquitos Vectores/genética , Mosquitos Vectores/fisiologíaRESUMEN
BACKGROUND: The proportion of mosquito blood-meals that are of human origin, referred to as the 'human blood index' or HBI, is a key determinant of malaria transmission. METHODS: A systematic review was conducted followed by meta-regression of the HBI for the major African malaria vectors. RESULTS: Evidence is presented for higher HBI among Anopheles gambiae (M/S forms and Anopheles coluzzii/An. gambiae sensu stricto are not distinguished for most studies and, therefore, combined) as well as Anopheles funestus when compared with Anopheles arabiensis (prevalence odds ratio adjusted for collection location [i.e. indoor or outdoor]: 1.62; 95% CI 1.09-2.42; 1.84; 95% CI 1.35-2.52, respectively). This finding is in keeping with the entomological literature which describes An. arabiensis to be more zoophagic than the other major African vectors. However, analysis also revealed that HBI was more associated with location of mosquito captures (R2 = 0.29) than with mosquito (sibling) species (R2 = 0.11). CONCLUSIONS: These findings call into question the appropriateness of current methods of assessing host preferences among disease vectors and have important implications for strategizing vector control.
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Anopheles/fisiología , Análisis Químico de la Sangre/métodos , Control de Mosquitos/métodos , Mosquitos Vectores/fisiología , África , Animales , Conducta Alimentaria , Humanos , MalariaRESUMEN
BACKGROUND: Vector control based on indoor residual spraying (IRS) is one of the main components of the visceral leishmaniasis (VL) elimination programme in India. Dichlorodiphenyltrichloroethane (DDT) was used for IRS until 2015 and was later replaced by the synthetic pyrethroid alpha-cypermethrin. Both classes of insecticides share the same target site, the voltage-gated sodium channel (Vgsc). As high levels of resistance to DDT have been documented in the local sand fly vector, Phlebotomus argentipes, it is possible that mutations in the Vgsc gene could provide resistance to alpha-cypermethrin, affecting current IRS pyrethroid-based vector control. METHODS: This study aimed to compare frequencies of knockdown resistance (kdr) mutations in Vgsc between two sprayed and two unsprayed villages in Bihar state, India, which had the highest VL burden of the four endemic states. Across four villages, 350 female P. argentipes were collected as part of a 2019 molecular xenomonitoring study. DNA was extracted and used for sequence analysis of the IIS6 fragment of the Vgsc gene to assess the presence of kdr mutations. RESULTS: Mutations were identified at various positions, most frequently at codon 1014, a common site known to be associated with insecticide resistance in mosquitoes and sand flies. Significant inter-village variation was observed, with sand flies from Dharampur, an unsprayed village, showing a significantly higher proportion of wild-type alleles (55.8%) compared with the three other villages (8.5-14.3%). The allele differences observed across the four villages may result from selection pressure caused by previous exposure to DDT. CONCLUSIONS: While DDT resistance has been reported in Bihar, P. argentipes is still susceptible to pyrethroids. However, the presence of kdr mutations in sand flies could present a threat to IRS used for VL control in endemic villages in India. Continuous surveillance of vector bionomics and insecticide resistance, using bioassays and target genotyping, is required to inform India's vector control strategies and to ensure the VL elimination target is reached and sustained.
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Resistencia a los Insecticidas , Insecticidas , Leishmaniasis Visceral , Mutación , Phlebotomus , Piretrinas , Animales , India , Phlebotomus/genética , Phlebotomus/efectos de los fármacos , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Piretrinas/farmacología , Femenino , Leishmaniasis Visceral/transmisión , Leishmaniasis Visceral/parasitología , Canales de Sodio Activados por Voltaje/genética , Insectos Vectores/genética , Insectos Vectores/efectos de los fármacos , DDT/farmacología , Proteínas de Insectos/genéticaRESUMEN
Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA-LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10-100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.
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Anopheles , Malaria , Mosquitos Vectores , Técnicas de Amplificación de Ácido Nucleico , Plasmodium , Humanos , Animales , Anopheles/parasitología , Plasmodium/genética , Plasmodium/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Malaria/diagnóstico , Malaria/parasitología , Mosquitos Vectores/parasitología , Recombinasas/metabolismo , Recombinasas/genética , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificaciónRESUMEN
With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.
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Acetamidas , Antimaláricos , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Acetamidas/farmacología , Acetamidas/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Antimaláricos/química , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Mutación , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Malaria Falciparum/tratamiento farmacológico , Humanos , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacosRESUMEN
BACKGROUND: As the control of malaria remains heavily dependent on vector management interventions, it is important to understand the impact of these on mosquito populations. Age-grading is a valuable tool for this; however, logistical challenges in remote, resource-poor areas make current methodologies difficult to incorporate into clinical trials and routine surveillance. Our aim was to validate a methodology that could be easily implemented in such settings. Using dried mosquito specimens instead of freshly killed ones, we validated the commonly used ovarian tracheation technique for assessing population age structure. METHODS: Laboratory-reared Anopheles coluzzii mosquitoes with known parity status were dry preserved in silica gel for up to 12 weeks and rehydrated prior to parity assessment. The results were compared to parity results for freshly killed mosquitoes from the same colony. Preserved, field-caught Anopheles gambiae sensu lato (s.l.) from Guinea-Bissau were assessed by three different assessors blinded to each other's scores. An overall index of agreement was calculated using inter-rater reliability of all assessor pairings. The impact of preservation time was investigated using a one-way ANOVA to look for differences in assessor agreement over three time periods. RESULTS: The parity status was correctly identified for 90% of dry preserved and rehydrated insectary-reared An. coluzzii and for 98% of freshly killed insectary-reared An. coluzzii. The inter-rater reliability was highest (0.94) for freshly killed An. coluzzii. The results for all time points showed excellent strength of agreement between assessors. For field-caught An. gambiae s.l., the overall index of agreement between all three assessors was 0.86 (95% confidence interval 0.78-0.93), indicating almost perfect agreement. There was no significant difference between assessor agreement between time frames. CONCLUSIONS: Dry preserving and rehydrating Anopheles mosquitoes provides an alternative to using freshly killed mosquitoes to assess the efficacy of a control intervention in remote settings where it is logistically difficult to dissect fresh specimens. This method also provides the flexibility required for parity assessment to be done on larger scales over bigger areas.
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Anopheles , Animales , Fluidoterapia , Mosquitos Vectores , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The kala-azar elimination programme has resulted in a significant reduction in visceral leishmaniasis (VL) cases across the Indian Subcontinent. To detect any resurgence of transmission, a sensitive cost-effective surveillance system is required. Molecular xenomonitoring (MX), detection of pathogen DNA/RNA in vectors, provides a proxy of human infection in the lymphatic filariasis elimination programme. To determine whether MX can be used for VL surveillance in a low transmission setting, large numbers of the sand fly vector Phlebotomus argentipes are required. This study will determine the best method for capturing P. argentipes females for MX. METHODOLOGY/PRINCIPAL FINDINGS: The field study was performed in two programmatic and two non-programmatic villages in Bihar, India. A total of 48 households (12/village) were recruited. Centers for Disease Control and Prevention light traps (CDC-LTs) were compared with Improved Prokopack (PKP) and mechanical vacuum aspirators (MVA) using standardised methods. Four 12x12 Latin squares, 576 collections, were attempted (12/house, 144/village,192/method). Molecular analyses of collections were conducted to confirm identification of P. argentipes and to detect human and Leishmania DNA. Operational factors, such as time burden, acceptance to householders and RNA preservation, were also considered. A total of 562 collections (97.7%) were completed with 6,809 sand flies captured. Females comprised 49.0% of captures, of which 1,934 (57.9%) were identified as P. argentipes. CDC-LTs collected 4.04 times more P. argentipes females than MVA and 3.62 times more than PKP (p<0.0001 for each). Of 21,735 mosquitoes in the same collections, no significant differences between collection methods were observed. CDC-LTs took less time to install and collect than to perform aspirations and their greater yield compensated for increased sorting time. No significant differences in Leishmania RNA detection and quantitation between methods were observed in experimentally infected sand flies maintained in conditions simulating field conditions. CDC-LTs were favoured by householders. CONCLUSIONS/SIGNIFICANCE: CDC-LTs are the most useful collection tool of those tested for MX surveillance since they collected higher numbers of P. argentipes females without compromising mosquito captures or the preservation of RNA. However, capture rates are still low.
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Culicidae , Leishmaniasis Visceral , Phlebotomus , Psychodidae , Estados Unidos , Femenino , Humanos , Animales , Masculino , Leishmaniasis Visceral/epidemiología , Mosquitos Vectores , ARNRESUMEN
The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16-32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.
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Aedes , Anopheles , Insecticidas , Animales , Anopheles/genética , Aedes/genética , Larva/genética , Mosquitos Vectores/genética , FitomejoramientoRESUMEN
Since its first detection in 2012 in Djibouti, Anopheles stephensi has invaded and established in the Horn of Africa, and more recently Nigeria. The expansion of this vector poses a significant threat to malaria control and elimination efforts. Integrated vector management is the primary strategy used to interrupt disease transmission; however, growing insecticide resistance is threatening to reverse gains in global malaria control. We present a next-generation amplicon-sequencing approach, for high-throughput monitoring of insecticide resistance genes (ace1, GSTe2, vgsc and rdl), species identification and characterization of genetic diversity (its2 and cox1) in An. stephensi. Ninety-five An. stephensi mosquitoes, collected in Ethiopia, were screened, identifying 104 SNPs, including the knock-down mutation L958F (L1014F in Musca domestica), and for the first time in this vector species, the A296S substitution (A301S in Drosophila melanogaster) in the rdl locus. Two other amino acid substitutions (ace1-N177D, GSTe2-V189L) were also identified but have not been previously implicated in insecticide resistance. Genetic diversity in the mitochondrial cox1 gene revealed shared haplotypes between Ethiopian An. stephensi with samples from Pakistan, Sudan, and Djibouti. Overall, we present a reliable, cost-effective strategy using amplicon-sequencing to monitor known insecticide resistance mutations, with the potential to identify new genetic variants, to assist in the high-throughput surveillance of insecticide resistance in An. stephensi populations.
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Anopheles , Insecticidas , Malaria , Animales , Resistencia a los Insecticidas/genética , Anopheles/genética , Drosophila melanogaster , Mosquitos Vectores/genética , Insecticidas/farmacología , EtiopíaRESUMEN
BACKGROUND: Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. METHODOLOGY AND PRINCIPAL FINDINGS: Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. CONCLUSION: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.
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Leishmaniasis Visceral , Humanos , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/epidemiología , Antígenos de Protozoos , Técnicas y Procedimientos Diagnósticos , Sensibilidad y Especificidad , Anticuerpos Antiprotozoarios , ADN , Pruebas con Sangre Seca/métodosRESUMEN
Chagas disease vector control relies on prompt, accurate identification of houses infested with triatomine bugs for targeted insecticide spraying. However, most current detection methods are laborious, lack standardization, have substantial operational costs and limited sensitivity, especially when triatomine bug densities are low or highly focal. We evaluated the use of FTA cards or cotton-tipped swabs to develop a low-technology, non-invasive method of detecting environmental DNA (eDNA) from both triatomine bugs and Trypanosoma cruzi for use in household surveillance in eastern Colombia, an endemic region for Chagas disease. Study findings demonstrated that Rhodnius prolixus eDNA, collected on FTA cards, can be detected at temperatures between 21 and 32 °C, when deposited by individual, recently blood-fed nymphs. Additionally, cotton-tipped swabs are a feasible tool for field sampling of both T. cruzi and R. prolixus eDNA in infested households and may be preferable due to their lower cost. eDNA detection should not yet replace current surveillance tools, but instead be evaluated in parallel as a more sensitive, higher-throughput, lower cost alternative. eDNA collection requires virtually no skills or resources in situ and therefore has the potential to be implemented in endemic communities as part of citizen science initiatives to control Chagas disease transmission.
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Enfermedad de Chagas , Insecticidas , Rhodnius , Trypanosoma cruzi , Animales , Insectos Vectores , Rhodnius/genética , Trypanosoma cruzi/genéticaRESUMEN
Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126-54.00 µg/ml or LC95 × 3 = 0.7437-17.82 µg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.
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Anopheles , Insecticidas , Malaria , Piretrinas , Animales , Insecticidas/farmacología , Anopheles/genética , Dieldrín/farmacología , Estudios Prospectivos , Salud Pública , Resistencia a los Insecticidas/genética , Mosquitos Vectores , Malaria/prevención & control , Piretrinas/farmacología , Control de MosquitosRESUMEN
Surveillance of malaria vector species and the monitoring of insecticide resistance are essential to inform malaria control strategies and support the reduction of infections and disease. Genetic barcoding of mosquitoes is a useful tool to assist the high-throughput surveillance of insecticide resistance, discriminate between sibling species and to detect the presence of Plasmodium infections. In this study, we combined multiplex PCR, custom designed dual indexing, and Illumina next generation sequencing for high throughput single nucleotide polymorphism (SNP)-profiling of four species from the Anopheles (An.) gambiae complex (An. gambiae sensu stricto, An. coluzzii, An. arabiensis and An. melas). By amplifying and sequencing only 14 genetic fragments (500 bp each), we were able to simultaneously detect Plasmodium infection; insecticide resistance-conferring SNPs in ace1, gste2, vgsc and rdl genes; the partial sequences of nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and intergenic spacers (IGS), Short INterspersed Elements (SINE), as well as mitochondrial genes (cox1 and nd4) for species identification and genetic diversity. Using this amplicon sequencing approach with the four selected An. gambiae complex species, we identified a total of 15 non-synonymous mutations in the insecticide target genes, including previously described mutations associated with resistance and two new mutations (F1525L in vgsc and D148E in gste2). Overall, we present a reliable and cost-effective high-throughput panel for surveillance of An. gambiae complex mosquitoes in malaria endemic regions.
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Anopheles , Insecticidas , Malaria , Animales , Anopheles/genética , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Mosquitos Vectores/genéticaRESUMEN
Insecticide resistance among mosquito species is now a pervasive phenomenon that threatens to jeopardize global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterized and compared the microbiota of Anopheles coluzzii in relation to their deltamethrin resistance and exposure profiles. Comparisons between 2- and 3-day-old deltamethrin-resistant and -susceptible mosquitoes demonstrated significant differences in microbiota diversity. Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera, each of which comprised insecticide-degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals, with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin-exposed and -unexposed 5- to 6-day-old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5- to 6-day-old mosquitoes, which had reduced microbial diversity compared to 2- to 3-day-old mosquitoes. Our findings revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population-level field screening of bacterial microbiota may be feasibly integrated into wider resistance monitoring, if reliable and reproducible markers associated with phenotype can be identified. IMPORTANCE Control of insecticide-resistant vector populations remains a significant challenge to global malaria control and while substantial progress has been made elucidating key target site mutations, overexpressed detoxification enzymes and alternate gene families, the contribution of the mosquito microbiota to phenotypic insecticide resistance has been largely overlooked. We focused on determining the effects of deltamethrin resistance intensity on Anopheles coluzzii microbiota and identifying any microbial taxa associated with phenotype. We demonstrated a significant reduction in microbial diversity between deltamethrin-resistant and -susceptible mosquitoes. Insecticide degrading bacterial species belonging to Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera were significantly enriched in resistant mosquitoes, while Asaia and Serratia dominated microbial profiles of susceptible individuals. Our results revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for surveillance and opportunities for designing innovative control techniques to prevent the further evolution and spread of insecticide resistance.
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Acetobacteraceae/metabolismo , Anopheles/efectos de los fármacos , Anopheles/microbiología , Resistencia a los Insecticidas/fisiología , Insecticidas/farmacología , Nitrilos/farmacología , Piretrinas/farmacología , Serratia/metabolismo , Animales , Côte d'Ivoire , Malaria/prevención & control , Microbiota/genética , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/microbiología , ARN Ribosómico 16S/genéticaRESUMEN
Wolbachia, a widespread bacterium which can influence mosquito-borne pathogen transmission, has recently been detected within Anopheles (An.) species that are malaria vectors in Sub-Saharan Africa. Although studies have reported Wolbachia strains in the An. gambiae complex, apparent low density and prevalence rates require confirmation. In this study, wild Anopheles mosquitoes collected from two regions of Guinea were investigated. In contrast with previous studies, RNA was extracted from adult females (n = 516) to increase the chances for the detection of actively expressed Wolbachia genes, determine Wolbachia prevalence rates and estimate relative strain densities. Molecular confirmation of mosquito species and Wolbachia multilocus sequence typing (MLST) were carried out to analyse phylogenetic relationships of mosquito hosts and newly discovered Wolbachia strains. Strains were detected in An. melas (prevalence rate of 11.6%-16/138) and hybrids between An. melas and An. gambiae sensu stricto (prevalence rate of 40.0%-6/15) from Senguelen in the Maferinyah region. Furthermore, a novel high-density strain, termed wAnsX, was found in an unclassified Anopheles species. The discovery of novel Wolbachia strains (particularly in members, and hybrids, of the An. gambiae complex) provides further candidate strains that could be used for future Wolbachia-based malaria biocontrol strategies.
RESUMEN
Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria.
Asunto(s)
Anopheles , Malaria , Wolbachia , Animales , Anopheles/genética , Hibridación Fluorescente in Situ , Herencia Materna , Mosquitos Vectores , Wolbachia/genéticaRESUMEN
Land-use practices such as agriculture can impact mosquito vector breeding ecology, resulting in changes in disease transmission. The typical breeding habitats of Africa's second most important malaria vector Anopheles funestus are large, semipermanent water bodies, which make them potential candidates for targeted larval source management. This is a technical workflow for the integration of drone surveys and mosquito larval sampling, designed for a case study aiming to characterise An. funestus breeding sites near two villages in an agricultural setting in Côte d'Ivoire. Using satellite remote sensing data, we developed an environmentally and spatially representative sampling frame and conducted paired mosquito larvae and drone mapping surveys from June to August 2021. To categorise the drone imagery, we also developed a land cover classification scheme with classes relative to An. funestus breeding ecology. We sampled 189 potential breeding habitats, of which 119 (63%) were positive for the Anopheles genus and nine (4.8%) were positive for An. funestus. We mapped 30.42 km2 of the region of interest including all water bodies which were sampled for larvae. These data can be used to inform targeted vector control efforts, although its generalisability over a large region is limited by the fine-scale nature of this study area. This paper develops protocols for integrating drone surveys and statistically rigorous entomological sampling, which can be adjusted to collect data on vector breeding habitats in other ecological contexts. Further research using data collected in this study can enable the development of deep-learning algorithms for identifying An. funestus breeding habitats across rural agricultural landscapes in Côte d'Ivoire and the analysis of risk factors for these sites.
Asunto(s)
Anopheles , Malaria , Agricultura , Animales , Côte d'Ivoire , Ecosistema , Larva , Mosquitos Vectores , Estaciones del Año , Flujo de TrabajoRESUMEN
One of the key determinants of a haematophagous vector's capacity to transmit pathogens is its selection of which host to secure a blood meal from. This choice is influenced by both intrinsic (genetic) and extrinsic (environmental) factors, but little is known of their relative contributions. Blood fed Anopheles mosquitoes were collected from a malaria endemic village in Ghana. Collections were conducted across a range of different host availabilities and from both indoor and outdoor locations. These environmental factors were shown to impact dramatically the host choice of caught malaria vectors: mosquitoes caught indoors were ten-fold more likely to have sourced their blood meal from humans; and a halving in odds of being human-fed was found for mosquitoes caught only 25 m from the centre of the village. For the first time, we demonstrate that anthropophagy was better explained by extrinsic factors (namely, local host availability and indoor/outdoor trapping location) than intrinsic factors (namely, the (sibling) species of the mosquito caught) (respective Akaike information criterion estimates: 243.0 versus 359.8). Instead of characterizing biting behaviour on a taxonomic level, we illustrate the importance of assessing local entomology. Accounting for this behavioural plasticity is important, both in terms of measuring effectiveness of control programmes and in informing optimal disease control strategies.
Asunto(s)
Anopheles/fisiología , Sangre , Entomología/métodos , Ambiente , Conducta Alimentaria , Preferencias Alimentarias , Interacciones Huésped-Parásitos , Mordeduras y Picaduras de Insectos , Malaria/transmisión , Control de Mosquitos/métodos , Mosquitos Vectores , Animales , Ghana , HumanosRESUMEN
BACKGROUND: Several mosquito collection methods are routinely used in vector control programmes. However, they target different behaviours causing bias in estimation of species diversity and abundance. Given the paucity of mosquito trap data in West Africa, we compared the performance of five trap-lure combinations and Human Landing Catches (HLCs) in Guinea. METHODS: CDC light traps (LT), BG sentinel 2 traps (BG2T), gravid traps (GT) and Stealth traps (ST) were compared in a 5 × 5 Latin Square design in three villages in Guinea between June and July 2018. The ST, a portable trap which performs similarly to a LT but incorporates LEDs and incandescent light, was included since it has not been widely tested. BG2T were used with BG and MB5 lures instead of CO2 to test the efficacy of these attractants. HLCs were performed for 5 nights, but not as part of the Latin Square. A Generalised Linear Mixed Model was applied to compare the effect of the traps, sites and collection times on mosquito abundance. Species identification was confirmed using PCR-based analysis and Sanger sequencing. RESULTS: A total of 10,610 mosquitoes were captured across five traps. ST collected significantly more mosquitoes (7096) than the rest of the traps, but resulted in a higher number of damaged specimens. ST and BG2T collected the highest numbers of Anopheles gambiae (s.l.) and Aedes aegypti mosquitoes, respectively. HLCs captured predominantly An. coluzzii (41%) and hybrids of An. gambiae and An. coluzzii (36%) in contrast to the five traps, which captured predominantly An. melas (83%). The rural site (Senguelen) presented the highest abundance of mosquitoes and overall diversity in comparison with Fandie (semi-rural) and Maferinyah Centre I (semi-urban). Our results confirm the presence of four species for the first time in Guinea. CONCLUSIONS: ST collected the highest number of mosquitoes suggesting this trap may play an important role for mosquito surveillance in Guinea and similar sites in West Africa. We recommend the incorporation of molecular tools in entomological studies since they have helped to identify 25 mosquito species in this area.