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.

Vectorial competence, insecticide resistance in Anopheles funestus and operational implications for malaria vector control strategies in Benin Republic.

The primary reason for the failure of malaria vector control across endemic regions is the widespread insecticide resistance observed in Anopheles vectors. The most dominant African vectors of malaria parasites are Anopheles gambiae and Anopheles funestus mosquitoes. These species often exhibit divergent behaviours and adaptive changes underscoring the importance of deploying active and effective measures in their control. Unlike An. gambiae, An. funestus mosquitoes are poorly studied in Benin Republic. However, recent reports indicated that An. funestus can adapt and colonize various ecological niches owing to its resistance against insecticides and adaptation to changing breeding habitats. Unfortunately, scientific investigations on the contribution of An. funestus to malaria transmission, their susceptibility to insecticide and resistance mechanism developed are currently insufficient for the design of better control strategies. In an attempt to gather valuable information on An. funestus, the present review examines the progress made on this malaria vector species in Benin Republic and highlights future research perspectives on insecticide resistance profiles and related mechanisms, as well as new potential control strategies against An. funestus. Literature analysis revealed that An. funestus is distributed all over the country, although present in low density compared to other dominant malaria vectors. Interestingly, An. funestus is being found in abundance during the dry seasons, suggesting an adaptation to desiccation. Among the An. funestus group, only An. funestus sensu stricto (s.s.) and Anopheles leesoni were found in the country with An. funestus s.s. being the most abundant species. Furthermore, An. funestus s.s. is the only one species in the group contributing to malaria transmission and have adapted biting times that allow them to bite at dawn. In addition, across the country, An. funestus were found resistant to pyrethroid insecticides used for bed nets impregnation and also resistant to bendiocarb which is currently being introduced in indoor residual spraying formulation in malaria endemic regions. All these findings highlight the challenges faced in controlling this malaria vector. Therefore, advancing the knowledge of vectorial competence of An. funestus, understanding the dynamics of insecticide resistance in this malaria vector, and exploring alternative vector control measures, are critical for sustainable malaria control efforts in Benin Republic.

Relative expression of key genes involved in nucleic acids methylation in Anopheles gambiae sensu stricto.

In vertebrates, enzymes responsible for DNA methylation, one of the epigenetic mechanisms, are encoded by genes falling into the cytosine methyltransferases genes family (Dnmt1, Dnmt3a,b and Dnmt3L). However, in Diptera, only the methyltransferase Dnmt2 was found, suggesting that DNA methylation might act differently for species in this order. Moreover, genes involved in epigenetic dynamics, such as Ten-eleven Translocation dioxygenases (TET) and Methyl-CpG-binding domain (MBDs), present in vertebrates, might play a role in insects. This work aimed at investigating nucleic acids methylation in the malaria vector Anopheles gambiae (Diptera: Culicidae) by analysing the expression of Dnmt2, TET2 and MBDs genes using quantitative real-time polymerase chain reaction (qRT-PCR) at pre-immature stages and in reproductive tissues of adult mosquitoes. In addition, the effect of two DNA methylation inhibitors on larval survival was evaluated. The qPCR results showed an overall low expression of Dnmt2 at all developmental stages and in adult reproductive tissues. In contrast, MBD and TET2 showed an overall higher expression. In adult mosquito reproductive tissues, the expression level of the three genes in males' testes was significantly higher than that in females' ovaries. The chemical treatments did not affect larval survival. The findings suggest that mechanisms other than DNA methylation underlie epigenetic regulation in An. gambiae.

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.

P. falciparum msp1 and msp2 genetic diversity in P. falciparum single and mixed infection with P. malariae among the asymptomatic population in Southern Benin.

Plasmodium falciparum and Plasmodium malariae infections are prevalent in malaria-endemic countries. However, very little is known about their interactions especially the effect of P. malariae on P. falciparum genetic diversity. This study aimed to assess P. falciparum genetic diversity in P. falciparum and mixed infection P. falciparum/P. malariae isolates among the asymptomatic populations in Southern Benin. Two hundred and fifty blood samples (125 of P. falciparum and 125 P. falciparum/P. malariae isolates) were analysed by a nested PCR amplification of msp1 and msp2 genes. The R033 allelic family was the most represented for the msp1 gene in mono and mixed infection isolates (99.2% vs 86.4%), while the K1 family had the lowest frequency (38.3% vs 20.4%). However, with the msp2 gene, the two allelic families displayed similar frequencies in P. falciparum isolates while the 3D7 allelic family was more represented in P. falciparum/P. malariae isolates (88.7%). Polyclonal infections were also lower (62.9%) in P. falciparum/P. malariae isolates (p < 0.05). Overall, 96 individual alleles were identified (47 for msp1 and 49 for msp2) in P. falciparum isolates while a total of 50 individual alleles were identified (23 for msp1 and 27 for msp2) in P. falciparum/P. malariae isolates. The Multiplicity of Infection (MOI) was lower in P. falciparum/P. malariae isolates (p < 0.05). This study revealed a lower genetic diversity of P. falciparum in P. falciparum/P. malariae isolates using msp1 and msp2 genes among the asymptomatic population in Southern Benin.

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.

Single nucleotide polymorphism (SNP) in the doublesex ( dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae.

Background: Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex ( dsx) gene, involved in mosquitos' somatic sexually dimorphic traits determination. However, the pathways that trigger the signal of dsx gene exon skipping alternative splicing mechanism in anopheline mosquitoes are not well characterized. This study aims to screen the An. gambiae dsx gene splice site sequences for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene ( Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs.   Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

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.

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

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

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.