Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b₅ with kinetic parameters of K_M = 11 ± 1 ??M and k_cat = 6.1 ± 0.4 per min for permethrin (1:1 cis-trans) and K_M = 2.0 ± 0.3 ??M and k_cat = 1.2 ± 0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4′-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.     

Humoral response to the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to Afrotropical malaria vectors

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.     

Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae

Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.     

Human antibody responses to the Anopheles salivary gSG6-P1 peptide: a novel tool for evaluating the efficacy of ITNs in malaria vector control

To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.     

The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae

The D7-related (D7r) proteins of the malaria vector Anopheles gambiae have been shown to bind the biogenic amines serotonin, norepinephrine, and histamine with high affinity. One member of the group (D7r1 or hamadarin) has also been shown to have an anticoagulant/antikinin activity. To understand the mechanistic details of its antihemostatic/anti-inflammatory effects, we have determined the crystal structure of one member of this group, D7r4, along with the structures of ligand complexes with serotonin, tryptamine, histamine, and norepinephrine. The D7 fold consists of an arrangement of eight alpha-helices stabilized by three disulfide bonds. The structure is similar to those of the arthropod odorant-binding proteins, a relationship that had been predicted based on sequence comparisons. Although odorant-binding proteins commonly have six alpha-helices, D7r4 has eight, resulting in significantly different positioning and structure of the ligand binding pocket. The pocket itself is lined by hydrophobic side chains along with polar and charged groups oriented to form hydrogen bonds with the aliphatic amino group and with groups on the aromatic portions of the ligands. These structures, along with accompanying mutagenesis studies, have allowed us to identify critical residues for biogenic amine binding and to predict which members of the large D7 protein family found in blood-feeding nematocerous Diptera will function as biogenic amine-binding proteins.     

The role of the United States military in the development of vector control products,including insect repellents,insecticides, and bed nets

Arthropod‐borne diseases such as malaria, dengue, scrub typhus, and leishmaniasis continue to pose a significant threat to U.S. military forces deployed in support of operational and humanitarian missions. These diseases are transmitted by a variety of arthropods, including mosquitoes, ticks, chiggers, sand flies, and biting midges. In addition to disease threats, biting arthropods can cause dermatitis, allergic reactions, and sleep loss; therefore, monitoring of vector impact and integrated use of personal protective measures (PPM) and methods to reduce the vector populations are needed to protect service members. The U.S. military has played a vital role in vector identification tools and the development and testing of many of the most effective PPM and vector control products available today, including the topical repellent DEET and the repellent/insecticide permethrin, which is applied to clothing and bed nets. Efforts to develop superior products are ongoing. Although the U.S. military often needs vector control products with rather specific properties (e.g., undetectable, long‐lasting in multiple climates) in order to protect its service members, many Department of Defense vector control products have had global impacts on endemic disease control.     

The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.     

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito,Anopheles stephensi

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.     

The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.     

Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa

Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction.Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the ??-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique.The distribution of the Rdl^R mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions.     

Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae

Odor-detection in the malaria mosquito Anopheles gambiae involves large families of diverse proteins, including multiple odorant binding proteins (AgOBPs) and olfactory receptors (AgORs). The receptors AgOR1 and AgOR2, as well as the binding protein AgOBP1, have been implicated in the recognition of human host odors. In this study, we have explored the expression of these olfactory proteins, as well as the ubiquitous odorant receptor heteromerization partner AgOR7, in the thirteen flagellomeres (segments) of female and male antenna. Expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. In female mosquitoes, AgOR1-expressing olfactory receptor neurons (ORNs) were almost exclusively segregated in segments 3 to 9, whereas AgOR2-expressing ORNs were distributed over flagellomeres 2 to 13. Different individuals comprised a similar number of cells expressing a distinct AgOR type, although their antennal topography and number per flagellomere varied. AgOBP1-expressing support cells were present in segments 3 to 13 of the female antenna, with increasing numbers towards the distal end. In male mosquitoes, total numbers of AgOR- and AgOBP1-expressing cells were much lower. While AgOR2-expressing cells were found on both terminal flagellomeres, AgOR1 cells were restricted to the most distal segment. High densities of AgOBP1-expressing cells were identified in segment 13, whereas segment 12 comprised very few. Altogether, the results demonstrate that both sexes express the two olfactory receptor types as well as the binding protein AgOBP1 but there is a significant sexual dimorphism concerning the number and distribution of these cells. This may suggest gender-specific differences in the ability to detect distinct odorants, specifically human host-derived volatiles.     

Barrier spraying to control the malaria vector Anopheles albimanus: laboratory and field evaluation in the Dominican Republic

Abstract. To develop a new strategy for control of exophilic/exophagic malaria vectors which rest on peridomestic foliage, Anopheles albimanus was used for laboratory bioassays of mosquito adulticides applied to various vegetation types. Of the three classes of insecticide tested, the pyrethroid (permethrin at 112 g/ha) showed greater foliar persistence than either the carbamate (bendiocarb at 340g/ha) or the organophosphorus compound (malathion at 277g/ha). Field evaluation of barrier spraying against An.albimanus was evaluated in rural villages of southwest Dominican Republic during 1989. The pyrethroid deltamethrin was sprayed aerially for ultra-low volume application at treatment rates of 17-19ga.i./ha in a radius of 500m around two villages. An.albimanus were monitored by light-traps and human bait collections at both treated villages, compared with two similar untreated villages, up to 9 nights post-treatment. Densities of female An.albimanus were significantly reduced in the sprayed villages for at least 8 or 9 nights. Further evaluation of barrier spraying is recommended to determine optimal pyrethroid formulations and applications rates, their impact on non-target fauna and efficacy against malaria transmission.     

Identification of Glossina palpalis gambiensis specific salivary antigens: towards the development of a serologic biomarker of human exposure to tsetse flies in West Africa

The saliva of blood sucking arthropods contains a number of pharmacologically active compounds that induce an antibody response in exposed human individuals. The objectives of the present study were (i) to assess the human IgG response directed against salivary antigens of Glossina palpalis gambiensis, the main vector of Trypanosoma brucei gambiense in West Africa, as a biomarker of human–tsetse contacts; and (ii) to identify specific salivary antigens. Immune reactivity of human plasma collected within active human African trypanosomiasis (HAT) foci (coastal Guinea), historical foci where tsetse flies are still present (South-West Burkina Faso) and a tsetse free area (Bobo-Dioulasso, Burkina Faso), was measured by ELISA against whole saliva extracts. In the active HAT foci and areas where tsetse flies were present in high densities, specific IgG responses were significantly higher (p < 0.0001) to those in Bobo-Dioulasso or in Loropeni, where tsetse flies were either absent or only present at low densities. Furthermore, 2D-electrophoresis combined with mass spectrometry enabled to reveal that several antigens were specifically recognized by plasma from exposed individuals. Among them, four salivary proteins were successfully identified (Ada, 5′Nuc, Ag5 and Tsgf1). These results represent a first attempt to identify Glossina salivary proteins or synthetic peptides to develop a standardized and specific biomarker of tsetse exposure in West Africa.     

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.     

Genetic differentiation at the 3'UTR of PROS-Ag25: a cDNA homologue of Drosophila melanogaster proteasome subunit PROS-Dm25 in the malaria vector Anopheles gambiae

The Anopheles gambiae cDNA encoding the homologue of the Drosophila melanogaster proteasome PROS-Dm25 was identified and analysed in terms of nucleotide sequence, and chromosomal localisation. In the 3 untranslated region, a GA-rich sequence was mapped which was found to be widely polymorphic among taxa belonging to the A. gambiae complex.     

The impact of long-lasting microbial larvicides in reducing malaria transmission and clinical malaria incidence: study protocol for a cluster randomized controlled trial

BackgroundThe massive scale-up of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) has led to a substantial increase in malaria vector insecticide resistance as well as in increased outdoor transmission, both of which hamper the effectiveness and efficiency of ITN and IRS. Long-lasting microbial larvicide can be a cost-effective new supplemental intervention tool for malaria control.Methods/designWe will implement the long-lasting microbial larvicide intervention in 28 clusters in two counties in western Kenya. We will test FourStar controlled release larvicide (6 % by weight Bacillus thuringiensis israelensis and 1 % Bacillus sphaerius) by applying FourStar controlled release granule formulation, 90-day briquettes, and 180-day briquettes in different habitat types. The primary endpoint is clinical malaria incidence rate and the secondary endpoint is malaria vector abundance and transmission intensity. The intervention will be conducted as a two-step approach. First, we will conduct a four-cluster trial (two clusters per county, with one of the two clusters randomly assigned to the intervention arm) to optimize the larvicide application scheme. Second, we will conduct an open-label, cluster-randomized trial to evaluate the effectiveness and cost-effectiveness of the larvicide. Fourteen clusters in each county will be assigned to intervention (treatment) or no intervention (control) by a block randomization on the basis of clinical malaria incidence, vector density, and human population size per site. We will treat each treatment cluster with larvicide for three rounds at 4-month intervals, followed by no treatment for the following 8 months. Next, we will switch the control and treatment sites. The former control sites will receive three rounds of larvicide treatment at appropriate time intervals, and former treatment sites will receive no larvicide. We will monitor indoor and outdoor vector abundance using CO₂-baited CDC light traps equipped with collection bottle rotators. Clinical malaria data will be aggregated from government-run malaria treatment centers.DiscussionSince current first-line vector intervention methods do not target outdoor transmission and will select for higher insecticide resistance, new methods beyond bed nets and IRS should be considered. Long-lasting microbial larviciding represents a promising new tool that can target both indoor and outdoor transmission and alleviate the problem of pyrethroid resistance. It also has the potential to diminish costs by reducing larvicide reapplications. If successful, it could revolutionize malaria vector control in Africa, just as long-lasting bed nets have done.

Trial registration

U.S. National Institute of Health, study ID {"type":"clinical-trial","attrs":{"text":"NCT02392832","term_id":"NCT02392832"}}NCT02392832. Registered on 3 February 2015.     

The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

Background  

Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.     

Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.