scholarly journals Overexpression of Two Members of D7 Salivary Genes Family is Associated with Pyrethroid Resistance in the Malaria Vector Anopheles Funestus s.s. but Not in Anopheles Gambiae in Cameroon

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 211 ◽  
Author(s):  
Emmanuel Elanga-Ndille ◽  
Lynda Nouage ◽  
Achille Binyang ◽  
Tatiane Assatse ◽  
Billy Tene-Fossog ◽  
...  

D7 family proteins are among the most expressed salivary proteins in mosquitoes. They facilitate blood meal intake of the mosquito by scavenging host amines that induce vasoconstriction, platelet aggregation and pain. Despite this important role, little information is available on the impact of insecticide resistance on the regulation of D7 proteins and consequently on the blood feeding success. In this study, real-time quantitative polymerase chain reaction (qPCR) analyses were performed to investigate how pyrethroid resistance could influence the expression of genes encoding D7 family proteins in Anopheles gambiae and Anopheles funestus s.s. mosquitoes from Elon in the Central Cameroon. Out of 328 collected mosquitoes, 256 were identified as An. funestus sl and 64 as An. gambiae sl. Within the An. funestus group, An. funestus s.s. was the most abundant species (95.95%) with An. rivulorum, An. parensis and An. rivulorum-like also detected. All An. gambiae s.l mosquitoes were identified as An. gambiae. High levels of pyrethroid resistance were observed in both An. gambiae and An. funestus mosquitoes. RT-qPCR analyses revealed a significant overexpression of two genes encoding D7 proteins, D7r3 and D7r4, in pyrethroids resistant An. funestus. However, no association was observed between the polymorphism of these genes and their overexpression. In contrast, overall D7 salivary genes were under-expressed in pyrethroid resistant An. gambiae. This study provides preliminary evidences that pyrethroid resistance could influence blood meal intake through over-expression of D7 proteins although future studies will help establishing potential impact on vectorial capacity.

2021 ◽  
Author(s):  
Priscille Barreaux ◽  
Jacob C. Koella ◽  
Raphael N’Guessan ◽  
Matthew B. Thomas

Abstract Background: There is a pressing need to improve understanding of how insecticide resistance affects the functional performance of Insecticide Treated Nets (ITNs). Standard WHO insecticide resistance monitoring assays are designed for resistance surveillance and do not necessarily provide insight into how different frequencies, mechanisms or intensities of resistance affect the ability of ITNs to reduce malaria transmission. Methods: The current study presents some novel laboratory-based assays that attempt to better simulate realistic exposure of mosquitoes to ITNs and to quantify impact of exposure not only on instantaneous mortality, but also blood feeding and longevity, two traits that are central to transmission. The assays evaluated the performance of a standard ITN (Permanet® 2.0), a ‘next generation’ combination ITN that includes a resistance breaking synergist (Permanet® 3.0), and an untreated net (UTN), against field-derived Anopheles gambiae s.l. mosquitoes from Côte d’Ivoire exhibiting 1500-fold pyrethroid resistance. Results: The study revealed that a standard ITN induced negligible instantaneous mortality against the resistant mosquitoes, whereas the resistance breaking net caused high mortality and a reduction in blood feeding. However, the ITNs still impacted long term survival relative to the UTN. The impact on longevity depended on feeding status, with blood-fed mosquitoes living longer than unfed mosquitoes following ITN exposure. The ITNs also reduced the blood feeding success, the time spent on the net, and blood-feeding duration, relative to the untreated net. Conclusion: Thus, while the standard ITN did not have as substantial instantaneous impact as the resistance breaking net, it still had significant impacts on traits important for transmission. These results highlight the benefit of improved bioefficacy assays that allow for realistic exposure and consider sub- or pre-lethal effects to help assess the functional significance of insecticide resistance.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Patrick K. Tungu ◽  
Elisante Michael ◽  
Wema Sudi ◽  
William W. Kisinza ◽  
Mark Rowland

Abstract Background The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. Methods Experimental hut trials tested IG2 efficacy against two positive controls—a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)—consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. Results In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13–4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83–1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74–1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44–1.11 vs IG1: 0.61, CI 0.39–0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus. Conclusions The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector.


Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 143 ◽  
Author(s):  
Benjamin D. Menze ◽  
Mersimine F. Kouamo ◽  
Murielle J. Wondji ◽  
Williams Tchapga ◽  
Micareme Tchoupo ◽  
...  

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.


mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Rebekah A. Reynolds ◽  
Hyeogsun Kwon ◽  
Ryan C. Smith

ABSTRACT Blood feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood feeding promotes production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E plays an important role in priming the immune response for pathogen challenge. Here, we examine the immunological effects of priming Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. Transcriptome sequencing (RNA-seq) analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in antipathogen defense. Together, these data demonstrate that 20E influences cellular immune function and antipathogen immunity following mosquito blood feeding, arguing the importance of hormones in the regulation of mosquito innate immune function. IMPORTANCE Blood feeding is required to provide nutrients for mosquito egg production and serves as a mechanism to acquire and transmit pathogens. Shortly after a blood meal is taken, there is a peak in the production of 20-hydroxyecdysone (20E), a mosquito hormone that initiates physiological changes, including yolk protein production and mating refractoriness. Here, we examine additional roles of 20E in the regulation of mosquito immunity, demonstrating that priming the immune system with 20E increases mosquito resistance to pathogens. We identify differentially expressed genes in response to 20E treatment, including several involved in innate immune function as well as lipid metabolism and transport. Together, these data argue that 20E stimulates mosquito cellular immune function and innate immunity shortly after blood feeding.


mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Hyeogsun Kwon ◽  
Maria L. Simões ◽  
Rebekah A. Reynolds ◽  
George Dimopoulos ◽  
Ryan C. Smith

ABSTRACT Mosquitoes may feed multiple times during their life span in addition to those times needed to acquire and transmit malaria. To determine the impact of subsequent blood feeding on parasite development in Anopheles gambiae, we examined Plasmodium parasite infection with or without an additional noninfected blood meal. We found that an additional blood meal significantly reduced Plasmodium berghei immature oocyst numbers, yet had no effect on the human parasite Plasmodium falciparum. These observations were reproduced when mosquitoes were fed an artificial protein meal, suggesting that parasite losses are independent of blood ingestion. We found that feeding with either a blood or protein meal compromises midgut basal lamina integrity as a result of the physical distention of the midgut, enabling the recognition and lysis of immature P. berghei oocysts by mosquito complement. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood feeding to accelerate their growth. This is in contrast to experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate distinct differences in Plasmodium species in evading immune detection and utilizing host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for the transmission of malaria. IMPORTANCE Mosquitoes must blood feed multiple times to acquire and transmit malaria. However, the impact of an additional mosquito blood meal following malaria parasite infection has not been closely examined. Here, we demonstrate that additional feeding affects mosquito vector competence; namely, additional feeding significantly limits Plasmodium berghei infection, yet has no effect on infection of the human parasite P. falciparum. Our experiments support that these killing responses are mediated by the physical distension of the midgut and by temporary damage to the midgut basal lamina that exposes immature P. berghei oocysts to mosquito complement, while human malaria parasites are able to evade these killing mechanisms. In addition, we provide evidence that additional feeding promotes P. falciparum oocyst growth. This is in contrast to P. berghei, where oocyst size is independent of an additional blood meal. This suggests that human malaria parasites are able to exploit host resources provided by an additional feeding to accelerate their growth. In summary, our data highlight distinct differences in malaria parasite species in evading immune recognition and adapting to mosquito blood feeding. These observations have important, yet previously unexplored, implications for the impact of multiple blood meals on the transmission of malaria.


2021 ◽  
Author(s):  
Patrick Tungu ◽  
Elisante Michael ◽  
Wema Sudi ◽  
William Kisinza ◽  
Mark Rowland

Abstract BackgroundThe effectiveness of long-lasting insecticidal nets (LLINs), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. Our objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheline funestus mosquitoes. MethodsExperimental hut trials tested IG2 efficacy against two positive controls - a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1) - consistent with World Health Organisation (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated twice and analysed using multivariate and meta-analysis. ResultsIn the two trials held in NE Tanzania, A. funestus mortality was 2.27 (risk ratio 95% CI 1.13-4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p=0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83-1.30, p=0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74-1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44-1.11 vs IG1: 0.61, CI 0.39-0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q=36, P=0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in A. funestus. ConclusionsThe high mortality recorded by IG2 against pyrethroid-resistant A. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector.


2019 ◽  
Author(s):  
Hyeogsun Kwon ◽  
Rebekah A. Reynolds ◽  
Maria L. Simões ◽  
George Dimopoulos ◽  
Ryan C. Smith

AbstractA minimum of two blood meals are required for a mosquito to acquire and transmit malaria, yet Anopheles mosquitoes frequently obtain additional blood meals during their adult lifespan. To determine the impact of subsequent blood-feeding on parasite development in Anopheles gambiae, we examined rodent and human Plasmodium parasite infection with or without an additional non-infected blood meal. We find that an additional blood meal significantly reduces P. berghei immature oocyst numbers, yet does not influence mature oocysts that have already begun sporogony. This is in contrast to experiments performed with the human parasite, P. falciparum, where an additional blood meal does not affect oocyst numbers. These observations are reproduced when mosquitoes were similarly challenged with an artificial protein meal, suggesting that parasite losses are due to the physical distension of the mosquito midgut. We provide evidence that feeding compromises the integrity of the midgut basal lamina, enabling the recognition and lysis of immature P. berghei oocysts by the mosquito complement system. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood-feeding to accelerate their growth. This contrasts experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate differences in the ability of Plasmodium species to evade immune detection and adapt to utilize host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for transmission of malaria.


2020 ◽  
Author(s):  
Catherine L. Moyes ◽  
Rosemary S. Lees ◽  
Cristina Yunta ◽  
Kyle J. Walker ◽  
Kay Hemmings ◽  
...  

Abstract The primary malaria control intervention in high burden countries is the deployment of long-lasting insecticide-treated nets (LLINs) treated with pyrethroids, alone or in combination with a second active ingredient or synergist. It is essential to understand whether the impact of pyrethroid resistance can be mitigated by switching between different pyrethroids or whether cross-resistance precludes this. Structural diversity within the pyrethroids could mean some compounds are better able to counteract the resistance mechanisms that have evolved in malaria vectors. Here we consider variation in vulnerability to the P450 enzymes that confer metabolic pyrethroid resistance in Anopheles gambiae s.l. and Anopheles funestus. We assess the relationships among pyrethroids in terms of their binding affinity to key P450s and the percent dep­letion by these P450s, in order to identify which pyrethroids diverge from the others. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. We found that etofenprox, which lacks the common structural moiety of other pyrethroids, potentially diverges from the commonly deployed pyrethroids in terms of P450 binding affinity and resistance in malaria vector populations, but not depletion by the P450s tested. These results are supplemented by an analysis of resistance to the same pyrethroids in Aedes aegypti populations, which also found etofenprox diverges from the other pyrethroids in terms of resistance in wild populations. In addition, we found that bifenthrin, which also lacks the common structural moiety of most pyrethroids, diverges from the commonly deployed pyrethroids in terms of P450 binding affinity and depletion by P450s. However, resistance to bifenthrin in vector populations is largely untested. The prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin, and permethrin was correlated across malaria vector populations and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.


2020 ◽  
Author(s):  
David A. Larsen ◽  
Rachael L. Church

AbstractBackgroundPyrethroid resistance is a major concern for malaria vector control programs that predominantly rely on insecticide-treated mosquito nets (ITN). Contradictory results of the impact of resistance have been observed in field studies.MethodsWe combined continent-wide estimates of pyrethroid resistance in Anopheles gambiae from 2006-2017 with continent-wide survey data to assess the effect of increasing pyrethroid resistance on the effectiveness of ITNs to prevent malaria infections in sub-Saharan Africa. We utilized both a pooled-data approach and meta-regression of survey regions to assess how pyrethroid resistance affects the association between ITN ownership and malaria outcomes in children aged 6-59 months.FindingsITN ownership reduced the risk of malaria outcomes in both pooled and meta-regression approaches. In the pooled analysis, there was no observed interaction between ITN ownership and estimated level of pyrethroid resistance (Likelihood ratio [LR] test = 1.127 for the outcome of rapid diagnostic test confirmed malaria infection, p = 0.2885; LR test = 0.161 for the outcome of microscopy confirmed malaria infection, p = 0.161; LR test = 0.646 for the outcome of moderate or severe anemia, p = 0.4215). In the meta-regression approach the level of pyrethroid resistance did not explain any of the variance in subnational estimates of ITN effectiveness for any of the outcomes.InterpretationITNs decreased risk of malaria outcomes independent of the levels of pyrethroid resistance in the malaria vector populations.FundingDAL did not receive funding and RC received a SOURCE grant from Syracuse University for this project.


2020 ◽  
Author(s):  
Lynda Nouage ◽  
Emmanuel Elanga-Ndille ◽  
Achille Binyang ◽  
Magellan Tchouakui ◽  
Tatiane Atsatse ◽  
...  

AbstractInsecticide resistance genes are often associated with pleiotropic effects on various mosquito life-history traits. However, very little information is available on the impact of insecticide resistance, especially metabolic resistance, on blood feeding process in mosquitoes. Here, using two recently detected DNA-based metabolic markers in the major malaria vector, An. funestus, we investigated how metabolic resistance genes could affect blood meal intake.After allowing both field F1 and lab F8 Anopheles funestus strains to feed on human arm for 30 minutes, we assessed the association between key parameters of blood meal process including, probing time, feeding duration, blood feeding success and blood meal size, and markers of glutathione S-transferase (L119F-GSTe2) and cytochrome P450 (CYP6P9a_R) - mediated metabolic resistance. None of the parameters of blood meal process was associated with L119F-GSTe2 genotypes. In contrast, for CYP6P9a_R, homozygote resistant mosquitoes were significantly more able to blood-feed than homozygote susceptible (OR = 3.3; CI 95%: 1.4-7.7; P =0.01) mosquitoes. Moreover, the volume of blood meal ingested by CYP6P9a-SS mosquitoes was lower than that of CYP6P9a-RS (P<0.004) and of CYP6P9a-RR (P<0.006). This suggests that CYP6P9a gene affects the feeding success and blood meal size of An. funestus. However, no correlation was found in the expression of CYP6P9a and that of genes encoding for salivary proteins involved in blood meal process.This study suggests that P450-based metabolic resistance may increase the blood feeding ability of malaria vectors and potential impacting their vectorial capacity.


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