Bioactive components in Psidium guajava extracts elicit biotoxic attributes and distinct antioxidant enzyme modulation in the larvae of vectors of lymphatic filariasis and dengue.

Control of mosquito vectors, which have caused a global disease burden, has employed various methods. However, the challenges posed by current physical and chemical methods have raised concerns about vector control programs, leading to the search for alternative methods that are less toxic, eco-friendly, and cost-effective. This study investigated the larvicidal potential of aqueous, methanol, and ethylacetate extracts of Guava (Psidium guajava) against Aedes aegypti and Culex quinquefasciatus larvae. Functional group and phytochemical characterization were performed using Fourier-Transform Infrared Spectroscopy (FTIR) and GC-MS analysis to identify the bioactive compounds in the extracts. Larval bioassays were conducted using WHO standard procedures at concentrations of 12.5, 25, 50, 125, and 250 mg/L, and mortality was recorded after 24, 48, and 72 h. Additionally, antioxidant enzyme profiles in the larvae were studied. All of the solvent extracts showed larvicidal activity, with the methanol extract exhibiting the highest mortality against Ae. aegypti and Cx. quinquefasciatus larvae, followed by aqueous and ethylacetate extracts. FTIR spectroscopic analysis revealed the presence of OH, C-H of methyl and methylene, CO and CC. The GC-MS analysis indicated that the methanol, aqueous, and ethylacetate extracts all had 27, 34, and 43 phytoactive compounds that were effective at causing larvicidal effects, respectively. Different concentrations of each extract significantly modulated the levels of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione in larvae. This study's findings indicate the potential for developing environmentally friendly vector control products using the bioactive components of extracts from P. guajava leaves.

Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle.

Dengue virus (DENV) subdues cell membranes for its cellular cycle by reconfiguring phospholipids in humans and mosquitoes. Here, we determined how and why DENV reconfigures phospholipids in the mosquito vector. By inhibiting and activating the de novo phospholipid biosynthesis, we demonstrated the antiviral impact of de novo-produced phospholipids. In line with the virus hijacking lipids for its benefit, metabolomics analyses indicated that DENV actively inhibited the de novo phospholipid pathway and instead triggered phospholipid remodeling. We demonstrated the early induction of remodeling during infection by using isotope tracing in mosquito cells. We then confirmed in mosquitoes the antiviral impact of de novo phospholipids by supplementing infectious blood meals with a de novo phospholipid precursor. Eventually, we determined that phospholipid reconfiguration was required for viral genome replication but not for the other steps of the virus cellular cycle. Overall, we now propose that DENV reconfigures phospholipids through the remodeling cycle to modify the endomembrane and facilitate formation of the replication complex. Furthermore, our study identified de novo phospholipid precursor as a blood determinant of DENV human-to-mosquito transmission.

Biochemical profiling of functionally expressed CYP6P9 variants of the malaria vector Anopheles funestus with special reference to cytochrome b5 and its role in pyrethroid and coumarin substrate metabolism.

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.

Cytochrome P450 metabolic resistance (CYP6P9a) to pyrethroids imposes a fitness cost in the major African malaria vector Anopheles funestus.

Metabolic resistance threatens the sustainability of pyrethroid-based malaria control interventions. Elucidating the fitness cost and potential reversal of metabolic resistance is crucial to design suitable resistance management strategies. Here, we deciphered the fitness cost associated with the CYP6P9a (P450-mediated metabolic resistance) in the major African malaria vector Anopheles funestus. Reciprocal crosses were performed between a pyrethroid susceptible (FANG) and resistant (FUMOZ-R) laboratory strains and the hybrid strains showed intermediate resistance. Genotyping the CYP6P9a-R resistance allele in oviposited females revealed that CYP6P9a negatively impacts the fecundity as homozygote susceptible mosquitoes (CYP6P9a-SS) lay more eggs than heterozygote (OR = 2.04: P = 0.01) and homozygote resistant mosquitoes. CYP6P9a also imposes a significant fitness cost on the larval development as homozygote resistant larvae (CYP6P9a-RR) developed significantly slower than heterozygote and homozygote susceptible mosquitoes (χ2 = 11.2; P = 0.0008). This fitness cost was further supported by the late pupation of homozygote resistant than susceptible mosquitoes (OR = 2.50; P < 0.01). However, CYP6P9a does not impact the longevity as no difference was observed in the life span of mosquitoes with different genotypes (χ2 = 1.6; P = 0.9). In this hybrid strain, a significant decrease of the resistant CYP6P9a-RR genotype was observed after ten generations (χ2 = 6.6; P = 0.01) suggesting a reversal of P450-based resistance in the absence of selection. This study shows that the P450-mediated metabolic resistance imposes a high fitness cost in malaria vectors supporting that a resistance management strategy based on rotation could help mitigate the impact of such resistance.

A survey of Anopheles species composition and insecticide resistance on the island of Bubaque, Bijagos Archipelago, Guinea-Bissau.

BACKGROUND: Bubaque is the most populous island of the Bijagos archipelago, a group of malaria-endemic islands situated off the coast of Guinea-Bissau, West Africa. Malaria vector control on Bubaque relies almost exclusively on the use of long-lasting insecticidal nets (LLINs). However, there is little information on local vector bionomics and insecticide resistance. METHODS: A survey of mosquito species composition was performed at the onset of the wet season (June/July) and the beginning of the dry season (November/December). Sampling was performed using indoor adult light-traps and larval dipping. Anopheles mosquitoes were identified to species level and assessed for kdr allele frequency by TaqMan PCR. Females were analysed for sporozoite positivity by CSP-ELISA. Resistance to permethrin and α-cypermethrin was measured using the CDC-bottle bioassay incorporating the synergist piperonyl-butoxide. RESULTS: Several Anopheles species were found on the island, all belonging to the Anopheles gambiae sensu lato (s.l.) complex, including An. gambiae sensu stricto, Anopheles coluzzii, Anopheles melas, and An. gambiae/An. coluzzii hybrids. Endophagic Anopheles species composition and abundance showed strong seasonal variation, with a majority of An. gambiae (50% of adults collected) caught in June/July, while An. melas was dominant in November/December (83.9% of adults collected). Anopheles gambiae had the highest sporozoite rate in both seasons, with infection rates of 13.9% and 20% in June/July and November/December, respectively. Moderate frequencies of the West African kdr allele were found in An. gambiae (36%), An. coluzzii (35%), An. gambiae/An. coluzzii hybrids (42%). Bioassays suggest moderate resistance to α-cypermethrin, but full susceptibility to permethrin. CONCLUSIONS: The island of Bubaque maintained an An. gambiae s.l. population in both June/July and November/December. Anopheles gambiae was the primary vector at the onset of the wet season, while An. melas is likely to be responsible for most dry season transmission. There was moderate kdr allele frequency and synergist assays suggest likely metabolic resistance, which could reduce the efficacy of LLINs. Future control of malaria on the islands should consider the seasonal shift in mosquito species, and should employ continuous monitoring for insecticide resistance.

[Insecticide resistance in Aedes aegypti and Ae. albopictus (Diptera: Culicidae) populations from Tapachula, Chiapas, Mexico]. / Resistencia a insecticidas en Aedes aegypti y Aedes albopictus (Diptera: Culicidae) de Tapachula, Chiapas, México.

OBJECTIVE: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México. MATERIALS AND METHODS: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively. RESULTS: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species. CONCLUSIONS: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur.

OBJETIVO: Determinar la resistencia a insecticidas en Ae. aegypti y Ae. albopictus de Tapachula, Chiapas, México. MATERIAL Y MÉTODOS: Se utilizaron ovitrampas para obtener huevos de mosquitos Aedes y se realizaron pruebas de susceptibilidad (CDC) y ensayos enzimáticos con la primera generación. RESULTADOS: Aedes aegypti mostró resistencia a deltametrina, permetrina, malatión, clorpirifos, temefos y a bendiocarb (CARB), mientras que Aedes albopictus a malatión y en menor grado a cloripirifos, temefos, permetrina y deltametrina. Ambas especies mostraron altos niveles de enzimas como citocomo P450 y glutatión S-tranferasa, mientras que los niveles de esterasas variaron por especie y sitio muestreado. Se detectó acetilcolinesterasa insensible a insecticidas en ambas especies. CONCLUSIONES: En un hábitat urbano de Tapachula, Chiapas, México donde se aplica control con insecticidas Ae. aegypti y Ae. albopictus sólo son susceptibles al propoxur.

Biocontrol and non-target effect of fractions and compound isolated from Streptomyces rimosus on the immature stages of filarial vector Culex quinquefasciatus Say (Diptera: Culicidae) and the compound interaction with Acetylcholinesterase (AChE1).

The present study was aimed to check the mosquitocidal activity of intracellular methanol extract fractions and the compound di (2-ethylhexyl) phthalate isolated from Streptomyces rimosus. The isolated compound was also analyzed for its interaction with Acetylcholinesterase (AChE1). The larvae and eggs of Culex quinquefasciatus were exposed to four different concentrations such as 2.5, 5.0, 7.5 and 10 ppm for fractions and 0.5, 1.0, 1.5 and 2.0 ppm for compound. After 24 and 120 h post treatment, the larval mortality and ovicidal activity were recorded. Fractions collected from the intracellular methanol extract were tested for larvicidal activity; among them Fraction 4 was found to be the active fraction. Fraction 4 showed 74% larvicidal activity with LC50 and LC90 values of 6.9 and 17.2 ppm, respectively, in 24 h against the larvae of Cx. quinquefasciatus. Fraction 4 showed 95% ovicidal activity at 10 ppm concentration after 120 h post treatment. The eluted compound di(2-ethylhexyl) phthalate was highly toxic and exhibited promising activity against the eggs of Cx. quinquefasciatus. The compound presented 94% ovicidal activity at 2.0 ppm concentration after 120 h post treatment. The larvae of Cx. quinquefasciatus were exposed to di(2-ethylhexyl) phthalate which showed good activity in a concentration-dependent manner. The compound showed 76% larvicidal activity against the larvae of Cx. quinquefasciatus with LC50 and LC90 values of 1.22 and 3.28 ppm, respectively, at 2 ppm concentration in 24 h. Fraction 4 and the compound were subjected to toxicity study against non-target organism and were found to be nontoxic. The present studies revealed that the treated larvae showed serious damage in the midgut cells. Growth disruption and larval deformities were observed in compound-treated larvae. The compound was highly active and inhibited AChE in a concentration-dependent manner. Computational analysis of the compound had strong interaction with AChE1 of Cx. quinquefasciatus. These results clearly showed that Fraction 4 and the compound isolated from S. rimosus can be used to control the life stages of Cx. quinquefasciatus; it will be a good alternative to synthetic insecticides.

Potential mode of action of a novel plumbagin as a mosquito repellent against the malarial vector Anopheles stephensi, (Culicidae: Diptera).

Plumbagin was isolated and characterized from the roots of Plumbago zeylanica using chromatography: TLC, Column chromatogram, HPLC, FTIR and 1H NMR. The isolated pure compounds were assayed for potency as inhibitors of: acetylcholine esterase (AchE), glutathione S-transferases (GST), superoxide dismutase (SOD), cytochrome P450 and α, ß-esterase, and for repellency with Anopheles stephensi at four different concentrations (25, 50, 75 and 100ppm). The enzyme assay against the pure compound reveals that the level of esterase and SOD was decreased significantly in contrast the level of GST and cytochrome P450 was increased significantly. Our results suggests that novel Plumbagin has significantly alters the level of enzyme comparable to the control. Evaluations resulted in Plumbagin producing maximum repellency scores against An. stephensi mosquitoes in dose dependent manner with highest repellence was observed in the 100ppm. Histological examination showed that the midgut, hindgut and muscles are the most affected tissues. These tissues affected with major changes including separation and collapse of epithelial layer and cellular vacuolization. The results support the utility of plant compound Plumbagin for vector control as an alternative to synthetic insecticides, however, more vigorous field trials are needed to determine viability under natural conditions.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are associated with insecticide resistance in the major malaria vectors Anopheles gambiae s.l. and Anopheles funestus.

Malaria remains one of the highest causes of morbidity and mortality, with 249 million cases and over 608,000 deaths in 2022. Insecticides, which target the Anopheles mosquito vector, are the primary method to control malaria. The widespread nature of resistance to the most important insecticide class, the pyrethroids, threatens the control of this disease. To reverse the stall in malaria control there is urgent need for new vector control tools, which necessitates understanding the molecular basis of pyrethroid resistance. In this study we utilised multi-omics data to identify uridine-diphosphate (UDP)-glycosyltransferases (UGTs) potentially involved in resistance across multiple Anopheles species. Phylogenetic analysis identifies sequence similarities between Anopheline UGTs and those involved in agricultural pesticide resistance to pyrethroids, pyrroles and spinosyns. Expression of five UGTs was characterised in An. gambiae and An. coluzzii to determine constitutive over-expression, induction, and tissue specificity. Furthermore, a UGT inhibitor, sulfinpyrazone, restored susceptibility to pyrethroids and DDT in An. gambiae, An. coluzzii, An. arabiensis and An. funestus, the major African malaria vectors. Taken together, this study provides clear association of UGTs with pyrethroid resistance as well as highlighting the potential use of sulfinpyrazone as a novel synergist for vector control.

Mosquito vector proteins homologous to α1-3 galactosyl transferases of tick vectors in the context of protective immunity against malaria and hypersensitivity to vector bites.

BACKGROUND: An epitope, Galα1-3Galß1-4GlcNAc-R, termed α-gal, is present in glycoconjugates of New World monkeys (platyrrhines) and other mammals but not in hominoids and Old World monkeys (catarrhines). The difference is due to the inactivation of α1-3 galactosyl transferase (α1-3 GT) genes in catarrhines. Natural antibodies to α-gal are therefore developed in catarrhines but not platyrrhines and other mammals. Hypersensitivity reactions are commonly elicited by mosquito and tick vector bites. IgE antibodies against α-gal cause food allergy to red meat in persons who have been exposed to tick bites. Three enzymes synthesising the terminal α1-3-linked galactose in α-gal, that are homologous to mammalian α and ß1-4 GTs but not mammalian α1-3 GTs, were recently identified in the tick vector Ixodes scapularis. IgG and IgM antibodies to α-gal are reported to protect against malaria because mosquito-derived sporozoites of malaria parasites express α-gal on their surface. This article explores the possibility that the α-gal in sporozoites are acquired from glycoconjugates synthesised by mosquitoes rather than through de novo synthesis by sporozoites. METHODS: The presence of proteins homologous to the three identified tick α1-3 GTs and mammalian α1-3 GTs in two important mosquito vectors, Aedes aegypti and Anopheles gambiae, as well as Plasmodium malaria parasites, was investigated by BLASTp analysis to help clarify the source of the α-gal on sporozoite surfaces. RESULTS: Anopheles gambiae and Ae. aegypti possessed several different proteins homologous to the three I. scapularis proteins with α1-3 GT activity, but not mammalian α1-3 GTs. The putative mosquito α1-3 GTs possessed conserved protein domains characteristic of glycosyl transferases. However, the genus Plasmodium lacked proteins homologous to the three I. scapularis proteins with α1-3 GT activity and mammalian α1-3 GTs. CONCLUSIONS: The putative α1-3 GTs identified in the two mosquito vectors may synthesise glycoconjugates containing α-gal that can be transferred to sporozoite surfaces before they are inoculated into skin during blood feeding. The findings merit further investigation because of their implications for immunity against malaria, hypersensitivity to mosquito bites, primate evolution, and proposals for immunisation against α-gal.

Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations.

BACKGROUND: It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach. METHODS: Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. RESULTS: We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations. CONCLUSION: Importantly, 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.

Overexpression of Activated AMPK in the Anopheles stephensi Midgut Impacts Mosquito Metabolism, Reproduction and Plasmodium Resistance.

Mitochondrial integrity and homeostasis in the midgut are key factors controlling mosquito fitness and anti-pathogen resistance. Targeting genes that regulate mitochondrial dynamics represents a potential strategy for limiting mosquito-borne diseases. AMP-activated protein kinase (AMPK) is a key cellular energy sensor found in nearly all eukaryotic cells. When activated, AMPK inhibits anabolic pathways that consume ATP and activates catabolic processes that synthesize ATP. In this study, we overexpressed a truncated and constitutively active α-subunit of AMPK under the control of the midgut-specific carboxypeptidase promotor in the midgut of female Anopheles stephensi. As expected, AMPK overexpression in homozygous transgenic mosquitoes was associated with changes in nutrient storage and metabolism, decreasing glycogen levels at 24 h post-blood feeding when transgene expression was maximal, and concurrently increasing circulating trehalose at the same time point. When transgenic lines were challenged with Plasmodium falciparum, we observed a significant decrease in the prevalence and intensity of infection relative to wild type controls. Surprisingly, we did not observe a significant difference in the survival of adult mosquitoes fed either sugar only or both sugar and bloodmeals throughout adult life. This may be due to the limited period that the transgene was activated before homeostasis was restored. However, we did observe a significant decrease in egg production, suggesting that manipulation of AMPK activity in the mosquito midgut resulted in the re-allocation of resources away from egg production. In summary, this work identifies midgut AMPK activity as an important regulator of metabolism, reproduction, and innate immunity in An. stephensi, a highly invasive and important malaria vector species.

Drug targeting of aminoacyl-tRNA synthetases in Anopheles species and Aedes aegypti that cause malaria and dengue.

BACKGROUND: Mosquito-borne diseases have a devastating impact on human civilization. A few species of Anopheles mosquitoes are responsible for malaria transmission, and while there has been a reduction in malaria-related deaths worldwide, growing insecticide resistance is a cause for concern. Aedes mosquitoes are known vectors of viral infections, including dengue, yellow fever, chikungunya, and Zika. Aminoacyl-tRNA synthetases (aaRSs) are key players in protein synthesis and are potent anti-infective drug targets. The structure-function activity relationship of aaRSs in mosquitoes (in particular, Anopheles and Aedes spp.) remains unexplored. METHODS: We employed computational techniques to identify aaRSs from five different mosquito species (Anopheles culicifacies, Anopheles stephensi, Anopheles gambiae, Anopheles minimus, and Aedes aegypti). The VectorBase database ( https://vectorbase.org/vectorbase/app ) and web-based tools were utilized to predict the subcellular localizations (TargetP-2.0, UniProt, DeepLoc-1.0), physicochemical characteristics (ProtParam), and domain arrangements (PfAM, InterPro) of the aaRSs. Structural models for prolyl (PRS)-, and phenylalanyl (FRS)-tRNA synthetases-were generated using the I-TASSER and Phyre protein modeling servers. RESULTS: Among the vector species, a total of 37 (An. gambiae), 37 (An. culicifacies), 37 (An. stephensi), 37 (An. minimus), and 35 (Ae. aegypti) different aaRSs were characterized within their respective mosquito genomes. Sequence identity amongst the aaRSs from the four Anopheles spp. was > 80% and in Ae. aegypti was > 50%. CONCLUSIONS: Structural analysis of two important aminoacyl-tRNA synthetases [prolyl (PRS) and phenylanalyl (FRS)] of Anopheles spp. suggests structural and sequence similarity with potential antimalarial inhibitor [halofuginone (HF) and bicyclic azetidine (BRD1369)] binding sites. This suggests the potential for repurposing of these inhibitors against the studied Anopheles spp. and Ae. aegypti.

The Effect of Entomopathogenic Fungi on Enzymatic Activity in Chlorpyrifos-Resistant Mosquitoes, Culex quinquefasciatus (Diptera: Culicidae).

Culex quinquefasciatus Say is an important pest species and a vector of multiple pathogens. Insecticide applications are necessary for the effective control of mosquitoes. In the current study, a laboratory population of Cx. quinquefasciatus was exposed to chlorpyrifos for 15 consecutive generations and then assessed for the changes in detoxification enzyme activities before and after exposure to Metarhizium anisopliae (Metschn.) Sorokin and Beauveria bassiana (Bals.) Vuill. during 14th-15th generations. Activities of acetylcholinesterase (AChE), glutathione S-transferase (GST), esterase (EST), acid phosphatases (ACP), and alkaline phosphatases (ALP) were increased in the chlorpyrifos-selected (Chlor-SEL) population in relation to an unselected (Un-SEL) population. The resistance ratio of Chlor-SEL 15th generation (G15) was increased 3,583-fold against first generation (G1) and 6,026-fold against the Un-SEL population. The results depicted maximum activities of ACP (83.48), ALP (65.54), GST (13.047), EST (10.42), and AChE (4.86) µmol/min of mg/ml protein at G15 after consecutive chlorpyrifos applications. The Chlor-SEL populations at G14-G15 were treated with different concentrations of M. anisopliae and B. bassiana for possible suppression of enzymatic activities. Activities of ACP were suppressed to 24.22 µmol/min of mg/ml protein at G15 when exposed to B. bassiana and 22.40 µmol/min of mg/ml protein at G14 after exposure to M. anisopliae. The suppression of detoxification enzymes by application of fungi in resistant population of Cx. quinquefasciatus will aid in the mosquito's management programs.

Acetylcholinesterase (ace-1R) target site mutation G119S and resistance to carbamates in Anopheles gambiae (sensu lato) populations from Mali.

BACKGROUND: The long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are major malaria vector control strategies in Mali. The success of control strategies depends on a better understanding of the status of malaria vectors with respect to the insecticides used. In this study we evaluate the level of resistance of Anopheles gambiae (sensu lato) to bendiocarb and the molecular mechanism that underlies it. METHODS: Larvae of An. gambiae (s.l.) were collected from breeding habitats encountered in the three study sites and bioassayed with bendiocarb. The ace-1 target site substitution G119S was genotyped using a TaqMan assay. RESULTS: The three species of the An. gambiae complex in Mali, i.e. An. arabiensis, An. coluzzii and An. gambiae (s.s.) were found in sympatry in the three surveyed localities with different frequencies. We observed a resistance and suspicious resistance of the three species to bendiocarb with a mortality rate ranging from 37% to 86%. The allelic frequency of the G119S mutation was higher in An. gambiae (s.s.) compared to the other two species; 42.86%, 25.61% and 16.67% respectively in Dangassa, Koula, and Karadié. The allelic frequency of G119S in An. coluzzii ranged from 4.5% to 8.33% and from 1.43% to 21.15% for An. arabiensis. After exposure to bendiocarb, the G119S mutation was found only in survivors. The survival of Anopheles gambiae (s.l) populations from the three surveyed localities was associated with the presence of the mutation. CONCLUSIONS: The study highlights the implication of G119S mutation in bendiocarb resistance in An. gambiae (s.s.), An. arabiensis and An. coluzzii populations from the three surveyed localities.

Comparative Molecular Description of a Novel GST Gene in Culex pipiens (Diptera: Culicidae).

Repeated exposure to insecticides, particularly pyrethroids and organophosphates, has resulted in the development of insecticide resistance in the mosquito Culex pipiens, a primary disease vector. Glutathione S-transferase (GST) is involved in the phase II detoxification of numerous xenobiotics, including insecticides. In this study, a GST gene (CPIJ002678) was amplified, sequenced, and used in comprehensive molecular analyses ending up in development of a rapid assay to distinguish more tolerant individuals from susceptible Culex pipiens using the Restriction Fragment Length Polymorphism (RFLP) technique. Field collected Culex pipiens strains from untreated areas, organophosphates-treated areas and a lab strain reared for many generations, all were used in CDC bottle bioassays to evaluate the susceptibility status of the studied individuals to malathion insecticide. Interestingly, both field sites collected groups showed high levels of resistance at the malathion diagnostic time. Gene amplification, and bidirectional direct sequencing results were analyzed. Compared with the reference genome sequence, the pairwise alignment of the amplified sequences showed 96.6% similarity to the reference sequence in the GenBank database. The confirmed gene sequences were assembled and aligned using various bioinformatic softwares. The assembled contigs were used in NEBcutter V2.0 for constructing restriction maps and checked for the availability of differences (if present) between susceptible and more tolerant strains. Specific molecular RFLP markers were successfully recognized to differentiate the more tolerant from the susceptible Culex pipiens phenotypes.

Insecticide Resistance Status of Aedes aegypti (Diptera: Culicidae) in California by Biochemical Assays.

Insecticide resistance in Aedes aegypti mosquitoes poses a major threat to public health worldwide. There are two primary biological mechanisms that can lead to insecticide resistance, target site and metabolic resistance, both of which confer resistance to specific classes of insecticides. Due to the limited number of chemical compounds available for mosquito control, it is important to determine current enzymatic profiles among mosquito populations. This study assessed resistance profiles for three metabolic pathways, α-esterases, ß-esterases, and mixed-function oxidases (MFOs), as well as insensitivity of the acetylcholinesterase (iAChE) enzyme in the presence of propoxur, among Ae. aegypti from the Central Valley and southern California. All field-collected Ae. aegypti demonstrated elevated MFOs and iAChE activity, indicating potential development of pyrethroid and organophosphate resistance, respectively. Although regional variations were found among α-esterase and ß-esterase activity, levels were generally elevated, further suggesting additional mechanisms for developing organophosphate resistance. Furthermore, mosquito samples from southern California exhibited a higher expression level to all three metabolic enzymes and iAChE activity in comparison to mosquitoes from the central region. These results could help guide future mosquito control efforts, directing the effective use of insecticides while limiting the spread of resistance.

Identification, characterization and expression analysis of Anopheles stephensi double peroxidase.

Peroxidases catalyze the reduction of peroxides and that, in turn, oxidize various substrates. They have been widely reported to play an important role in mosquito innate immunity against various pathogens. Here, we have characterized double heme peroxidase (AsDBLOX) gene from the Indian malaria vector Anopheles stephensi. It is a true ortholog of An. gambiae DBLOX. This 4209 bp AsDBLOX gene encodes for a protein of 1402 amino acids that has two duplicated peroxidase domains, domain I (from amino acid 61 to 527) and domain II (from amino acid 714 to 1252). The first domain has only substrate binding sites and lacks all other motifs of a functional heme peroxidase (e.g. heme binding site, calcium binding site and homodimer interface). Instead, it has two integrin binding motifs-LDV (Leu-Asp-Val) and RGD (Arg-Gly-Asp). The second peroxidase domain, however, has all the features of a complete heme peroxidase along with an integrin binding motif LDI (Leu-Asp-Ile). Thus, AsDBLOX gene is a unique type of peroxinectin as these groups of proteins are characterized by integrin binding motifs along with a heme peroxidase domain. We also observed that the AsDBLOX gene is expressed in all the life cycle stages of mosquito and is highly induced in the pupal stage of development which indicates its possible role in development.

Malaria load affects the activity of mosquito salivary apyrase.

Mosquitoes infected by sporozoites, the infectious stage of malaria, bite more frequently than uninfected mosquitoes. One of the mechanisms underlying this behavioural change appears to be that the sporozoites decrease the activity of apyrase, an ADP-degrading enzyme that helps the mosquitoes to locate blood. Using the parasite Plasmodium berghei and the mosquito Anopheles gambiae, we confirmed that sporozoite infection alters the host-seeking behaviour of mosquitoes by making them more likely to refeed after a first blood meal, and that apyrase activity is one of the mechanisms of the increased biting persistence and motivation of infectious mosquitoes. We further showed that apyrase activity decreases as the sporozoite load increases, and that mosquitoes with lower apyrase activity take up less blood, making it more likely that they would return to top up their blood meal. Finally, by comparing full-sib families of mosquitoes, we showed that there was genetic variation for apyrase activity, but not for the resistance of parasites to be manipulated. Our results give new insights in understanding how malaria parasites change their hosts to affect their own transmission.

Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

BACKGROUND: Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor. METHODS: Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations. RESULTS: Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids. CONCLUSIONS: In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.