Beninese Plant Extracts with Antiplasmodial Activity Select New Allele Variants Msp1 and Msp2 in Plasmodium falciparum.

Background: Natural medicinal products are commonly used as a remedy against malaria infections in African populations and have become a major source of information for the screening of new and more effective antiplasmodial molecules. Therefore, in vitro studies are needed to validate the efficacy of these medicinal products and to explore the potential effects of such drugs on the genetic diversity of Plasmodium falciparum. The current study has investigated the impact of some Beninese plant extracts with antiplasmodial activity on the genetic diversity of P. falciparum. Method: Five (5) ethanolic plant extracts (Dissotis rotundifolia, Ehretia cymosa Thonn, Hibiscus surattensis L., Cola millenii K. Shum, and Costus afer Ker Gawl) and a compound extracted from Ehretia cymosa Thonn (encoded CpE2) were tested against asexual stage parasites of a culture-adapted strain of P. falciparum. Subsequently, the P. falciparum Msp1 and Msp2 markers were genotyped, and the number of allelic variants and the multiplicity of infection (MOI) were compared between drug-exposed and unexposed parasites. Results: All plant extracts have shown inhibitory activity against asexual P. falciparum and selected new allelic variants of the Msp1 and Msp2 genes compared to unexposed parasites. The newly selected allelic variants were K1_100bp and RO33_300bp of the Msp1 gene and FC27_150bp, FC27_300bp, FC27_400bp, and FC27_600bp of the Msp2 gene. However, there was no significant difference in MOI between drug-exposed and unexposed parasites. Conclusion: Our study highlights a source for the selection of new Msp1 and Msp2 alleles after exposure to antimalarial drugs. These findings pave the way for further studies investigating the true roles of these newly selected alleles in P. falciparum.

Asymptomatic Plasmodium infection among primary schoolchildren and Anopheles-mediated malaria transmission: A cross-sectional study in Ouidah; south-western Benin.

Understanding the contribution of asymptomatic Plasmodium carriers in malaria transmission might be helpful to design and implement new control measures. The present study explored the prevalence of asymptomatic and symptomatic Plasmodium infections (asexual and sexual stages) and the contribution of asymptomatic P. falciparum carriers to Anopheles-mediated malaria transmission in Ouidah (Benin). Thick and thin blood smears were examined from finger-prick blood specimens using light microscopy, and the density of both asexual and sexual stages of Plasmodium species was calculated. Infectivity of gametocyte-infected blood samples to Anopheles gambiae was assessed through direct membrane feeding assays. The prevalence of asymptomatic Plasmodium infections was 28.73% (289/1006). All the asymptomatic gametocyte-carriers (19/19), with gametocytaemia ranging from 10 - 1200 gametocytes/µL of blood, were infectious to An. gambiae mosquitoes. The mean oocyst prevalences varied significantly (χ 2  = 16.42, df = 7, p = 0.02) among laboratory mosquito strains (6.9 - 39.4%) and near-field mosquitoes (4.9 - 27.2%). Likewise, significant variation (χ 2  = 56.85, df = 7, p = 6.39 × 10-10) was observed in oocyst intensity. Our findings indicate that asymptomatic Plasmodium carriers could significantly contribute to malaria transmission. Overall, this study highlights the importance of diagnosing and treating asymptomatic and symptomatic infection carriers during malaria control programmes.

Euclasta condylotricha flowers essential oils: A new source of juvenile hormones and its larvicidal activity against Anopheles gambiae s.s. (Diptera: Culicidae).

The essential oil (EO) of plants of the Poaceae family has diverse chemical constituents with several biological properties. But, data on the chemical constituents and toxicity are still unavailable for some species belonging to this family, such as Euclasta condylotricha Steud (Eu. condylotricha). In this study, the chemical composition of the EOs of Eu. condylotricha flowers was evaluated by gas chromatography coupled with mass spectrometry (GC-MS). The EOs larvicidal property was assessed against third instar larvae of three Anopheles gambiae laboratory strains (Kisumu, Acerkis and Kiskdr) according to the WHO standard protocol. The percentage yields of the EOs obtained from hydro distillation of Eu. condylotricha flowers varied 0.070 to 0.097%. Gas Chromatography-Mass Spectrometry (GC-MS) applied to the EOs revealed fifty-five (55) chemical constituents, representing 94.95% to 97.78% of the total essential oils. Although different chemical profiles of the dominant terpenes were observed for each sample, EOs were generally dominated by sesquiterpenoids with juvenile hormones as the major compounds. The primary compounds were juvenile hormone C16 (JH III) (35.97-48.72%), Methyl farnesoate 10,11-diol (18.56-28.73%), tau-Cadinol (18.54%), and ß-Eudesmene (12.75-13.46%). Eu. condylotricha EOs showed a strong larvicidal activity with LC50 values ranging from 35.21 to 52.34 ppm after 24 hours of exposition. This study showed that Eu. Condylotricha flowers essential oils are potent sources of juvenile hormones that could be a promising tool for developing an eco-friendly malaria vector control strategy.

Malaria-Transmitting Vectors Microbiota: Overview and Interactions With Anopheles Mosquito Biology.

Malaria remains a vector-borne infectious disease that is still a major public health concern worldwide, especially in tropical regions. Malaria is caused by a protozoan parasite of the genus Plasmodium and transmitted through the bite of infected female Anopheles mosquitoes. The control interventions targeting mosquito vectors have achieved significant success during the last two decades and rely mainly on the use of chemical insecticides through the insecticide-treated nets (ITNs) and indoor residual spraying (IRS). Unfortunately, resistance to conventional insecticides currently being used in public health is spreading in the natural mosquito populations, hampering the long-term success of the current vector control strategies. Thus, to achieve the goal of malaria elimination, it appears necessary to improve vector control approaches through the development of novel environment-friendly tools. Mosquito microbiota has by now given rise to the expansion of innovative control tools, such as the use of endosymbionts to target insect vectors, known as "symbiotic control." In this review, we will present the viral, fungal and bacterial diversity of Anopheles mosquitoes, including the bacteriophages. This review discusses the likely interactions between the vector microbiota and its fitness and resistance to insecticides.

Plasmodium falciparum msp1 and msp2 genetic diversity in parasites isolated from symptomatic and asymptomatic malaria subjects in the South of Benin.

Symptomatic and asymptomatic malaria patients are considered as the reservoirs of human Plasmodium. In the present study, we have evaluated the Plasmodium falciparum merozoite surface protein-1 (Pfmsp1) and protein-2 (Pfmsp2) genetic diversity among the symptomatic and asymptomatic malaria infection from health facilities in Cotonou, Benin Republic. A cross-sectional study recruited 158 individuals, including 77 from the asymptomatic and 81 from the symptomatic groups. The parasites were genotyped using Nested Polymerase Chain Reaction. Samples identified as Plasmodium falciparum were genotyped for their genetic diversity. No significant difference was observed in the overall multiplicity of infection (MOI) between the asymptomatic and symptomatic groups. In the symptomatic group, the overall frequency of K1, MAD20, and RO33 allelic family was more predominant (98.5%) followed by 3D7 (87.3%) and FC27 (83.1%). However, in asymptomatic group, the K1 alleles were the most prevalent (100%) followed by FC27 (89.9%), 3D7 (76.8%), MAD20 (60.5%), and RO33 (35.5%). The frequency of multiple allelic types (K1+MAD20+RO33) at the Pfmsp1 loci in the symptomatic infections was significantly higher when compared to that of the asymptomatic ones (97% vs. 34%, p < 0.05), whereas no difference was observed in the frequency of multiple allelic types (3D7 and FC27) at the Pfmsp2 loci between the two groups. The high presence of msp1 multiple infections in the symptomatic group compared to asymptomatic ones suggests an association between the genetic diversity and the onset of malaria symptoms. These data can provide valuable information in the development of a vaccine that could reduce the symptomatic disease.

Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae.

BACKGROUND: Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. METHODS: The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. RESULTS: The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. CONCLUSIONS: The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Interplay Between Oxytetracycline and the Homozygote kdr (L1014F) Resistance Genotype on Fecundity in Anopheles gambiae (Diptera: Culicidae) Mosquitoes.

The insecticide resistance in Anopheles gambiae mosquitoes has remained the major threat for vector control programs but the fitness effects conferred by these mechanisms are poorly understood. To fill this knowledge gap, the present study aimed at testing the hypothesis that antibiotic oxytetracycline could have an interaction with insecticide resistance genotypes and consequently inhibit the fecundity in An. gambiae. Four strains of An. gambiae: Kisumu (susceptible), KisKdr (kdr (L1014F) resistant), AcerKis (ace-1 (G119S) resistant) and AcerKdrKis (both kdr (L1014F) and ace-1 (G119S) resistant) were used in this study. The different strains were allowed to bloodfeed on a rabbit previously treated with antibiotic oxytetracycline at a concentration of 39·10-5 M. Three days later, ovarian follicles were dissected from individual mosquito ovaries into physiological saline solution (0.9% NaCl) under a stereomicroscope and the eggs were counted. Fecundity was substantially lower in oxytetracycline-exposed KisKdr females when compared to that of the untreated individuals and oxytetracycline-exposed Kisumu females. The exposed AcerKis females displayed an increased fecundity compared to their nontreated counterparts whereas they had reduced fecundity compared to that of oxytetracycline-exposed Kisumu females. There was no substantial difference between the fecundity in the treated and untreated AcerKdrKis females. The oxytetracycline-exposed AcerKdrKis mosquitoes had an increased fecundity compared to that of the exposed Kisumu females. Our data indicate an indirect effect of oxytetracycline in reducing fecundity of An. gambiae mosquitoes carrying kdrR (L1014F) genotype. These findings could be useful for designing new integrated approaches for malaria vector control in endemic countries.

Phenotypic Insecticide Resistance in Anopheles gambiae (Diptera: Culicidae): Specific Characterization of Underlying Resistance Mechanisms Still Matters.

An effective control of malaria vectors requires an extensive knowledge of mechanisms underlying the resistance-phenotypes developed by these vectors against insecticides. We investigated Anopheles gambiae mosquitoes from Benin and Togo for their intensity of insecticide resistance and we discussed the involvement of genotyped mechanisms in the resistance-phenotypes observed. Three- to five-day-old adult mosquitoes emerged from field and laboratory An. gambiae larvae were assayed using WHO tube intensity tests against various doses of deltamethrin: 1× (0.05%); 2× (0.1%); 5× (0.25%); 7.5× (0.375%) and those of pirimiphos-methyl: 0.5× (0.125%); 1× (0.25%). Members of An. gambiae complex were screened in field populations using polymerase chain reaction (PCR) assays. The presence of kdrR(1014F/1014S) and ace-1R(119S) mutations was also investigated using TaqMan and PCR-RFLP techniques, respectively. Anopheles gambiae from field were very resistant to deltamethrin, whereas KisKdr and AcerKdrKis strains displayed 100% mortality rates at 2× the diagnostic dose. In contrast, the field mosquitoes displayed a low resistance-intensity against 1× the diagnostic dose of pirimiphos-methyl, whereas AcerKis and AcerKdrKis strains showed susceptibility at 0.5× the diagnostic dose. Anopheles gambiae s.s., Anopheles coluzzii, and Anopheles arabiensis were identified. Allelic frequencies of kdrR (1014F) and ace-1R (119S) mutations in the field populations varied from 0.65 to 1 and 0 to 0.84, respectively. The field An. gambiae displayed high-resistance levels against deltamethrin and pirimiphos-methyl when compared with those of the laboratory An. gambiae-resistant strains. These results exhibit the complexity of underlying insecticide resistance mechanisms in these field malaria vectors.

Phenotypic effects of concomitant insensitive acetylcholinesterase (ace-1(R)) and knockdown resistance (kdr(R)) in Anopheles gambiae: a hindrance for insecticide resistance management for malaria vector control.

BACKGROUND: Malaria is endemic in sub-Saharan Africa with considerable burden for human health. Major insecticide resistance mechanisms such as kdr(R) and ace-1(R) alleles constitute a hindrance to malaria vector control programs. Anopheles gambiae bearing both kdr and ace-1 resistant alleles are increasingly recorded in wild populations. In order to maintain the efficacy of vector control strategies, the characterization of concomitant kdr and ace-1 resistance, and their pleiotropic effects on malaria vector phenotype on insecticide efficacy are important. METHODS: Larval and adult bioassays were performed with different insecticide classes used in public health following WHO standard guidelines on four laboratory Anopheles gambiae strains, sharing the same genetic background but harboring distinct resistance status: KISUMU with no resistance allele; ACERKIS with ace-1(R) allele; KISKDR with kdr(R) allele and ACERKDRKIS with both resistance alleles' ace-1(R) and kdr(R) . RESULTS: Larval bioassays indicate that the homozygote resistant strain harboring both alleles (ACERKDRKIS) displayed slightly but significantly higher resistance level to various insecticides like carbamates (bendiocarb, p < 0.001; propoxur, p = 0.02) and organophosphates (chlorpyriphos-methyl, p = 0.002; fenitrothion, p < 0.001) when compared to ACERKIS strain. However, no differences were recorded between ACERKDRKIS and KISKDR resistance level against permethrin (Pyrethroid, p = 0.7) and DDT (Organochlorine, p = 0.24). For adult bioassays, the percentages of mosquitoes knocked down were significantly lower for ACERKDRKIS than for KISKDR with permethrin (p = 0.003) but not with deltamethrin. The percentage of mortality from adult bioassays was similar between ACERKDRKIS and ACERKIS with carbamates and organophosphates, or between ACERKDRKIS and KISKDR with pyrethroid and DDT. Concerning acetylcholinesterase enzyme, ACERKDRKIS strain showed similarAChE1 activity than that of ACERKIS. CONCLUSION: The presence of both kdr(R) and ace-1(R) alleles seems to increase the resistance levels to both carbamate and organophosphate insecticides and at operational level, may represent an important threat to malaria vector control programs in West Africa.