Stop the biting: targeting a mosquito's sense of smell.

Mosquitoes are a great threat to human health. Fortunately, they have a weakness: they utilize their sense of smell to target a human host. Recent studies examine the effectiveness of protecting humans from attack by ablating or odorant targeting mosquito olfactory receptors. The results are both promising and alarming.

A perspective on the impacts of microplastics on mosquito biology and their vectorial capacity.

Microplastics (plastic particles <5 mm) permeate aquatic and terrestrial ecosystems and constitute a hazard to animal life. Although much research has been conducted on the effects of microplastics on marine and benthic organisms, less consideration has been given to insects, especially those adapted to urban environments. Here, we provide a perspective on the potential consequences of exposure to microplastics within typical larval habitat on mosquito biology. Mosquitoes represent an ideal organism in which to explore the biological effects of microplastics on terrestrial insects, not least because of their importance as an infectious disease vector. Drawing on evidence from other organisms and knowledge of the mosquito life cycle, we summarise some of the more plausible impacts of microplastics including physiological, ecotoxicological and immunological responses. We conclude that although there remains little experimental evidence demonstrating any adverse effect on mosquito biology or pathogen transmission, significant knowledge gaps remain, and there is now a need to quantify the effects that microplastic pollution could have on such an important disease vector.

Comparative evaluation of two lures to attract female mosquitoes in an urban natural reserve during daytime.

The most widely used attractant to capture adult female mosquitoes is CO2. However, there are also baits available on the market that emit a scent resembling human skin. These baits were specifically designed to attract highly anthropophilic species such as Aedes albopictus and Aedes aegypti. In this study, we compare the effectiveness of CDC traps baited either with CO2 or with a commercial blend simulating skin odor, BG-Sweetscent, for trapping female mosquitoes during daylight hours in an urban reserve in the City of Buenos Aires. We employed a hurdle generalized linear mixed model to analyze trap capture probability and the number of mosquitoes captured per hour, considering the effects of attractant, mosquito species, and their interaction. Traps baited with CO2 captured ten mosquito species, while those baited with BG-Sweetscent captured six in overall significantly lower abundance. The odds of capturing mosquitoes were 292% higher for the CO2-baited traps than for those baited with BG-Sweetscent. No evidence of a combined effect of attractant type and species on female mosquito captures per hour was found. Results indicated that CDC traps baited with CO2 were more effective than those baited with BG-Sweetscent in capturing more mosquito species and a higher number of mosquitoes within each species, even if the species captured with CO2 exhibited a certain level of anthropophilia. This result has practical implications for mosquito surveillance and control in urban natural reserves.

Chemical Composition, Repellent, and Oviposition Deterrent Potential of Wild Plant Essential Oils against Three Mosquito Species.

In this study, the chemical composition, repellent, and oviposition deterrent effects of five plant essential oils (EOs) extracted from Lantana camara (Verbenaceae), Schinus terebinthifolia (Anacardiaceae), Callistemon viminalis (Myrtaceae), Helichrysum odoratissimum (Asteraceae), and Hyptis suaveolens (Lamiaceae) were evaluated against Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus. When tested at 33.3 µg/cm2, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum were effective repellents against Ae. aegypti (89%, 91%, 90%, and 51% repellency, respectively), but they were less repellent against An. gambiae (66%, 86%, 59%, and 49% repellency, respectively). Interestingly, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum exhibited 100% repellency against Cx. quinquefasciatus at 33.3 µg/cm2. In time-span bioassays performed at 333 µg/cm2, the EO of L. camara exhibited 100% repellence against Ae. aegypti and An. gambiae for up to 15 min and against Cx. quinquefasciatus for 75 min. The oviposition bioassays revealed that L. camara exhibited the highest activity, showing 85%, 59%, and 89% oviposition deterrence against Ae. aegypti, An. gambiae, and Cx. quinquefasciatus, respectively. The major compounds of L. camara, S. terebinthifolia, and C. viminalis were trans-ß-caryophyllene (16.7%), α-pinene (15.5%), and 1,8-cineole (38.1%), respectively. In conclusion, the L. camara and S. terebinthifolia EOs have the potential to be natural mosquito repellents.

A First Report on the Isolation and Characterization of a Highly Potential Indigenous Mosquitocidal Bacterium (Bacillus thuringiensis Subspecies Israelensis VCRC B647) from Red Soil, India.

BACKGROUND: To control mosquito vectors causing human diseases, bacterial biopesticides are currently in use. Indeed, the recent development of resistance to these bacterial agents has impeded its applications. Under these circumstances, the search for novel bacterial agents with mosquitocidal activity is unavoidable. In this study, a novel mosquitocidal bacterium was isolated from red soils of agricultural field. OBJECTIVES: The objective of this study was to isolate and identify new mosquitocidal bacteria from the natural environment. MATERIALS AND METHODS: Soil samples were collected during 2021-2022 from Tirupathur district of Tamil Nadu, South India. The samples were bioprocessed for culturing the bacterial colony in a suitable culture medium (Nutrient Yeast Salt Medium), and after 72 h, the bacterial cell mass was removed and lyophilized. Bioassays (mosquito toxicity assays) were carried out to screen the bacterial colonies for mosquitocidal effect. The potential colony was further analyzed, and identified for its application in mosquito control. RESULTS: The new isolate screened from red soil was identified as Bacillus thuringiensis subspecies israelensis (VCRC B647) as per the ilvD gene sequence analysis. The strain was found to be potentially effective in controlling mosquito larvae, and further biochemical analyses, bacterial growth, biomass, and protein content were investigated. The new isolate did not show any toxic effect on nontarget aquatic organisms. CONCLUSION: It is significant to depict that the mosquitocidal action of this new isolate (Bti) is highly significant than the reference strain of Bti-H14. It is concluded that this is the first report that an indigenous strain of Bti VCRC B647 is very effective in mosquito control.

Small-scale field evaluation of the entomological efficacy and the residual activity of Fludora® Fusion WP-SB indoor residual spraying against Anopheles culicifacies s.l. in Gujarat, India.

OBJECTIVES: To evaluate the entomological efficacy and the residual activity of indoor residual spraying with Fludora® Fusion 562.5 WP-SB, a combination formulation containing clothianidin, a neonicotinoid and deltamethrin, a pyrethroid, against the main rural malaria vector, Anopheles culicifacies s.l., in India in a small-scale trial. METHODS: In three study villages, suitable households were randomly allocated to five treatments: Fludora® Fusion 562.5 WP-SB (target dose 225 mg active ingredient AI/m2 ); clothianidin 70 WG (target dose 200 mg AI/m2 ); K-Othrine 250 WG (deltamethrin, target dose 25 mg AI/m2 ); Ficam VC 80 WP-SB (bendiocarb, target dose 400 mg AI/m2 ) and unsprayed control. Insecticides were sprayed by hand compression sprayers with control flow valves and 8002E nozzles. Post-spray cone bioassays were done on insecticide-treated walls using a colonised, deltamethrin-resistant strain of An. culicifacies. Mosquitoes were collected from treated rooms by different methods. The insecticide content on filter papers collected from the sprayed walls was determined by chemical assay to assess the spray quality. RESULTS: The ratios of applied to target doses of insecticides were within 0.84 to 1.4, showing a good spray quality. The cone bioassays revealed residual action lasting 7 months for all insecticides without significant differences in mortality between different surfaces treated nor between the four treatment arms (P > 0.05). Considering all entomological parameters such as indoor resting density, excito-repellency, blood-feeding inhibition and delayed mortality, the overall efficacy of Fludora® Fusion WG-SB was equal or better compared with other insecticides. CONCLUSIONS: Fludora® Fusion showed overall equal or better efficacy than deltamethrin and bendiocarb alone against a pyrethroid-resistant malaria vector population and can be considered as an alternative product for management of pyrethroid resistance in malaria vectors.

Evaluation of malaria preventive measures among adult patients attending the Bamendjou and Foumbot district hospitals of the West Region of Cameroon.

BACKGROUND: Although a significant decrease in entomological and epidemiological indicators was reported in Cameroon since the introduction of insecticide-treated bed nets, malaria prevalence remains high also in some parts of the West Region of Cameroon. This study was designed to evaluate malaria preventive measures among patients attending the Bamendjou and Foumbot District hospitals of the West Region of Cameroon. METHODS: This was a cross-sectional study carried out within a period of 3 months, from January to March 2020. Data was obtained using a structured questionnaire and laboratory analysis. The CareStart™ Pf Malaria HRP2 qualitative rapid diagnostic test was used for malaria diagnosis. The questionnaire was designed to collect information on respondent's socio-demographic characteristics, and the use of malaria preventive measures. Data were analysed using descriptive statistics, regression analysis, and Chi-square (and Fisher's exact) test. RESULTS: A total of 170 study participants were recruited in Foumbot and 197 in Bamendjou. Malaria was significantly (P < 0.0001) more prevalent in Foumbot (47.06%) than in Bamendjou (19.8%). In Foumbot, non-use of insect repellent spray (P = 0.0214), insect repellent body cream (P = 0.0009), mosquito spray (P = 0.0001) and not draining stagnant water (P = 0.0004) predisposed to higher risk of malaria. In Bamendjou, non-use of insect repellent spray (P = 0.0012), long-lasting insecticidal bed nets (P = 0.0001), window and door nets (P = 0.0286), predisposed to a higher risk of malaria. CONCLUSIONS: Malaria prevalence was high among the study participants especially in Foumbot. An adequate follow-up to ensure effective execution of the recently launched third phase of LLINs distribution campaign in Cameroon is recommended. Additionally, integrated vector management is required to ensure effective control of malaria transmission in Foumbot and Bamendjou.

Antifungal and Larvicidal Activities of Phlorotannins from Brown Seaweeds.

Phlorotannins are secondary metabolites produced by brown seaweeds with antiviral, antibacterial, antifungal, and larvicidal activities. Phlorotannins' structures are formed by dibenzodioxin, ether and phenyl, ether, or phenyl linkages. The polymerization of phlorotannins is used to classify and characterize. The structural diversity of phlorotannins grows as polymerization increases. They have been characterized extensively with respect to chemical properties and functionality. However, review papers of the biological activities of phlorotannins have focused on their antibacterial and antiviral effects, and reviews of their broad antifungal and larvicidal effects are lacking. Accordingly, evidence for the effectiveness of phlorotannins as antifungal and larvicidal agents is discussed in this review. Online databases (ScienceDirect, PubMed, MEDLINE, and Web of Science) were used to identify relevant articles. In total, 11 articles were retrieved after duplicates were removed and exclusion criteria were applied. Phlorotannins from brown seaweeds show antifungal activity against dermal and plant fungi, and larvicidal activity against mosquitos and marine invertebrate larvae. However, further studies of the biological activity of phlorotannins against fungal and parasitic infections in aquaculture fish, livestock, and companion animals are needed for systematic analyses of their effectiveness. The research described in this review emphasizes the potential applications of phlorotannins as pharmaceutical, functional food, pesticide, and antifouling agents.

Associated patterns of insecticide resistance in field populations of malaria vectors across Africa.

The development of insecticide resistance in African malaria vectors threatens the continued efficacy of important vector control methods that rely on a limited set of insecticides. To understand the operational significance of resistance we require quantitative information about levels of resistance in field populations to the suite of vector control insecticides. Estimation of resistance is complicated by the sparsity of observations in field populations, variation in resistance over time and space at local and regional scales, and cross-resistance between different insecticide types. Using observations of the prevalence of resistance in mosquito species from the Anopheles gambiae complex sampled from 1,183 locations throughout Africa, we applied Bayesian geostatistical models to quantify patterns of covariation in resistance phenotypes across different insecticides. For resistance to the three pyrethroids tested, deltamethrin, permethrin, and λ-cyhalothrin, we found consistent forms of covariation across sub-Saharan Africa and covariation between resistance to these pyrethroids and resistance to DDT. We found no evidence of resistance interactions between carbamate and organophosphate insecticides or between these insecticides and those from other classes. For pyrethroids and DDT we found significant associations between predicted mean resistance and the observed frequency of kdr mutations in the Vgsc gene in field mosquito samples, with DDT showing the strongest association. These results improve our capacity to understand and predict resistance patterns throughout Africa and can guide the development of monitoring strategies.

Properties of Mosquito Repellent-Plasticized Poly(lactic acid) Strands.

Poly(lactic acid) (PLA) is an attractive candidate for replacing petrochemical polymers because it is fully biodegradable. This study investigated the potential of PLA as a sustainable and environmentally friendly alternative material that can be developed into commercially viable wearable mosquito repellent devices with desirable characteristics. PLA strands containing DEET and IR3535 were prepared by twin screw extrusion compounding and simultaneously functioned as plasticizers for the polymer. The plasticizing effect was investigated by thermal and rheological studies. DSC studies showed that the addition of DEET and IR3535 into PLA strands reduced the glass transition temperature consistent with predictions of the Fox equation, thus proving their efficiency as plasticizers. The rheology of molten samples of neat PLA and PLA/repellents blends, evaluated at 200 °C, was consistent with shear-thinning pseudoplastic behaviour. Raman studies revealed a nonlinear concentration gradient for DEET in the PLA strand, indicating non-Fickian Type II transport controlling the desorption process. Release data obtained at 50 °C showed initial rapid release followed by a slower, near constant rate at longer times. The release rate data were fitted to a novel modification of the Peppas-Sahlin desorption model.

Chemical Composition, Insecticidal and Mosquito Larvicidal Activities of Allspice (Pimenta dioica) Essential Oil.

Essential oils are biologically and environmentally safe pesticidal compounds yielded from aromatic plants. Spices are important sources of essential oils, and they are widely used in the medicine, food, and various other industries. Among the different spices, Allspice (Pimenta dioica) is underexplored in terms of its biological efficacy and a limited number of studies are available on the chemical composition of Allspice essential oil (AEO); thus, the present study evaluated the larvicidal property, the repellency, and the fumigant toxicity against common pests of stored products of AEO. AEO was found to inhibit the survival of larvae of such vectors as Aedis, Culex, and Armigeres species. Further, AEO was found to exert repellant effects against the pests of such stored products as Sitophilus, Callosobruchus, and Tribolium. Similarly, the fumigant toxicity was found to be high for AEO against these species. The contact toxicity of AEO was high against Sitophilus and Callosobruchus. Apart from that, the essential oil was found to be safe against a non-target organism (guppy fishes) and was found to be non-genotoxic in an Allium cepa model. Overall, the results of the present study indicate that the essential oil from Allspice could be used as an environmentally safe larvicidal and biopesticidal compound.

Effects of Kathon, a Chemical Used Widely as a Microbicide, on the Survival of Two Species of Mosquitoes.

In recent decades, demands for novel insecticides against mosquitoes are soaring, yet candidate chemicals with desirable properties are limited. Kathon is a broad-spectrum isothiazolinone microbicide, but other applications remain uncharacterized. First, we treated larvae of Culex quinquefasciatus and Aedes albopictus, two major mosquito vectors of human viral diseases, with Kathon at 15 mg/L (a concentration considered safe in cosmetic and body care products), and at lower concentrations, and found that Kathon treatment resulted in high mortality of larvae. Second, sublethal concentration of Kathon can cause significantly prolonged larval development of C. quinquefasciatus. Third, we explored the effects of two constituents of Kathon, chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT), on the survival of larvae, and found that CMIT was the major toxic component. Further, we explored the mechanisms of action of Kathon against insect cells and found that Kathon reduces cell viability and adenosine triphosphate production but promotes the release of lactate dehydrogenase in Drosophila melanogaster S2 cells. Our results indicate that Kathon is highly toxic to mosquito larvae, and we highlight its potential in the development of new larvicides for mosquito control.

Comparative functional survival and equivalent annual cost of 3 long-lasting insecticidal net (LLIN) products in Tanzania: A randomised trial with 3-year follow up.

BACKGROUND: Two billion long-lasting insecticidal nets (LLINs) have been procured for malaria control. A functional LLIN is one that is present, is in good physical condition, and remains insecticidal, thereby providing protection against vector-borne diseases through preventing bites and killing disease vectors. The World Health Organization (WHO) prequalifies LLINs that remain adequately insecticidal 3 years after deployment. Therefore, institutional buyers often assume that prequalified LLINs are functionally identical with a 3-year lifespan. We measured the lifespans of 3 LLIN products, and calculated their cost per year of functional life, to demonstrate the economic and public health importance of procuring the most cost-effective LLIN product based on its lifespan. METHODS AND FINDINGS: A randomised double-blinded trial of 3 pyrethroid LLIN products (10,571 nets in total) was conducted at 3 follow-up points: 10 months (August-October 2014), 22 months (August-October 2015), and 36 months (October-December 2016) among 3,393 households in Tanzania using WHO-recommended methods. Primary outcome was LLIN functional survival (LLIN present and in serviceable condition). Secondary outcomes were (1) bioefficacy and chemical content (residual insecticidal activity) and (2) protective efficacy for volunteers sleeping under the LLINs (bite reduction and mosquitoes killed). Median LLIN functional survival was significantly different between the 3 net products (p = 0.001): 2.0 years (95% CI 1.7-2.3) for Olyset, 2.5 years (95% CI 2.2-2.8) for PermaNet 2.0 (hazard ratio [HR] 0.73 [95% CI 0.64-0.85], p = 0.001), and 2.6 years (95% CI 2.3-2.8) for NetProtect (HR = 0.70 [95% CI 0.62-0.77], p < 0.001). Functional survival was affected by accumulation of holes, leading to users discarding nets. Protective efficacy also significantly differed between products as they aged. Equivalent annual cost varied between US$1.2 (95% CI $1.1-$1.4) and US$1.5 (95% CI $1.3-$1.7), assuming that each net was priced identically at US$3. The 2 longer-lived nets (PermaNet and NetProtect) were 20% cheaper than the shorter-lived product (Olyset). The trial was limited to only the most widely sold LLINs in Tanzania. Functional survival varies by country, so the single country setting is a limitation. CONCLUSIONS: These results suggest that LLIN functional survival is less than 3 years and differs substantially between products, and these differences strongly influence LLIN value for money. LLIN tendering processes should consider local expectations of cost per year of functional life and not unit price. As new LLIN products come on the market, especially those with new insecticides, it will be imperative to monitor their comparative durability to ensure that the most cost-effective products are procured for malaria control.

A Biomimetic Nanodecoy Traps Zika Virus To Prevent Viral Infection and Fetal Microcephaly Development.

Zika virus (ZIKV) has emerged as a global health threat due to its unexpected causal link to devastating neurological disorders such as fetal microcephaly; however, to date, no approved vaccine or specific treatment is available for ZIKV infection. Here we develop a biomimetic nanodecoy (ND) that can trap ZIKV, divert ZIKV away from its intended targets, and inhibit ZIKV infection. The ND, which is composed of a gelatin nanoparticle core camouflaged by mosquito medium host cell membranes, effectively adsorbs ZIKV and inhibits ZIKV replication in ZIKV-susceptible cells. Using a mouse model, we demonstrate that NDs significantly attenuate the ZIKV-induced inflammatory responses and degenerative changes and thus improve the survival rate of ZIKV-challenged mice. Moreover, by trapping ZIKV, NDs successfully prevent ZIKV from passing through physiologic barriers into the fetal brain and thereby mitigate ZIKV-induced fetal microcephaly in pregnant mice. We anticipate that this study will provide new insights into the development of safe and effective protection against ZIKV and various other viruses that threaten public health.

Mosquitocidal and Anti-Inflammatory Properties of The Essential Oils Obtained from Monoecious, Male, and Female Inflorescences of Hemp (Cannabis sativa L.) and Their Encapsulation in Nanoemulsions.

Among the various innovative products obtainable from hemp (Cannabis sativa L.) waste biomass originating from different industrial processes, the essential oil (EO) deserves special attention in order to understand its possible application in different fields, such as cosmetics, pharmaceuticals, and botanical insecticides. For the purpose, in the present work, we studied the chemical composition of EOs obtained from different hemp varieties, namely Felina 32 and Carmagnola Selezionata (CS) using monoecious, male, and female inflorescences, and we evaluated their mosquitocidal activities on larvae and pupae of two main malaria vectors, Anopheles gambiae and An. stephensi. Then, in order to evaluate the safe use of hemp EOs for operators, the potential pro- or anti-inflammatory effect of hemp EOs together with their toxicological profile were determined on dermal fibroblasts and keratinocytes. Given the promising results obtained by insecticidal and anti-inflammatory studies, a preliminary evaluation of EOs encapsulation into nanoemulsions (NEs) has been performed with the aim to develop a formulation able to improve their poor physicochemical stability. Felina 32 and CS inflorescences provided EOs with an interesting chemical profile, with monoterpene and sesquiterpene hydrocarbons as the major components. This study highlighted the potential application of male inflorescences, which are usually discharged during hemp product processing. These EOs could be exploited as potential sustainable and eco-friendly insecticides, given their capability to be toxic against mosquitoes and the possibility to use them to prepare stable and safe formulations. The LC50 values found in this study (<80 ppm) are lower, on average, than those of many plant EOs, with the advantage of using an industrial waste product. From MTT assay and gene and protein expression analysis, EOs showed no cytotoxicity at the appropriate doses and exerted an anti-inflammatory effect on the human cell lines tested. These findings encourage further applied research on hemp EOs in order support their industrial exploitation.

Chemical Compositions, Mosquito Larvicidal and Antimicrobial Activities of Essential Oils from Five Species of Cinnamomum Growing Wild in North Central Vietnam.

Members of the genus Cinnamomum (Lauraceae) have aromatic volatiles in their leaves and bark and some species are commercially important herbs and spices. In this work, the essential oils from five species of Cinnamomum (C. damhaensis, C. longipetiolatum, C. ovatum, C. polyadelphum and C. tonkinense) growing wild in north central Vietnam were obtained by hydrodistillation, analyzed by gas chromatography and screened for antimicrobial and mosquito larvicidal activity. The leaf essential oil of C. tonkinense, rich in ß-phellandrene (23.1%) and linalool (32.2%), showed excellent antimicrobial activity (MIC of 32 µg/mL against Enterococcus faecalis and Candida albicans) and larvicidal activity (24 h LC50 of 17.4 µg/mL on Aedes aegypti and 14.1 µg/mL against Culex quinquefasciatus). Cinnamomum polyadelphum leaf essential oil also showed notable antimicrobial activity against Gram-positive bacteria and mosquito larvicidal activity, attributable to relatively high concentrations of neral (11.7%) and geranial (16.6%). Thus, members of the genus Cinnamomum from Vietnam have shown promise as antimicrobial agents and as potential vector control agents for mosquitoes.

Ammi Visnaga L., a Potential Medicinal Plant: A Review.

Ammi visnaga L. (Visnaga daucoides Gaertn., Family Apiaceae), also known as Khella Baldi or toothpick weed, is an annual or biennial herb indigenous to the Mediterranean region of North Africa, Asia, and Europe. The plant is known to have been used in traditional medicine a long time ago. Nowadays, it is used in modern medicine to treat many aliments such as renal colic and coronary insufficiency, and is used as an antioxidant, antifungal, and antibacterial, with a larvicidal effect on mosquito larvae. Peer-reviewed studies show that these pharmacological activities are due its valuable chemical constituents that include mainly essential oil, polyphenolic compounds including flavonoids, as well as γ-pyrones, represented mainly by khellin and visnagin. Its essential oil is reported to have antiviral, antibacterial, and larvicidal effects, while its flavonoid content is responsible for its antioxidant activity. Its γ-pyrone content has a powerful effect on facilitating the passage of kidney stones and relieving renal colic, in addition to having a relaxant effect on smooth muscle including that of the coronary arteries. The current review represents the progress in research on A. visnaga in terms of either its chemistry or biological activities. This review represents scientific support material for the use of the plant by the pharmaceutical industry.

Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial).

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria, due to its ability to kill mosquitoes feeding on recently treated individuals. In a recent trial, 3-day courses of 300 and 600 mcg/kg/day were shown to kill Anopheles mosquitoes for at least 28 days post-treatment when fed patients' venous blood using membrane feeding assays. Direct skin feeding on humans may lead to higher mosquito mortality, as ivermectin capillary concentrations are higher. We compared mosquito mortality following direct skin and membrane feeding. METHODS: We conducted a mosquito feeding study, nested within a randomized, double-blind, placebo-controlled trial of 141 adults with uncomplicated malaria in Kenya, comparing 3 days of ivermectin 300 mcg/kg/day, ivermectin 600 mcg/kg/day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. On post-treatment day 7, direct skin and membrane feeding assays were conducted using laboratory-reared Anopheles gambiae sensu stricto. Mosquito survival was assessed daily for 28 days post-feeding. RESULTS: Between July 20, 2015, and May 7, 2016, 69 of 141 patients participated in both direct skin and membrane feeding (placebo, n = 23; 300 mcg/kg/day, n = 24; 600 mcg/kg/day, n = 22). The 14-day post-feeding mortality for mosquitoes fed 7 days post-treatment on blood from pooled patients in both ivermectin arms was similar with direct skin feeding (mosquitoes observed, n = 2941) versus membrane feeding (mosquitoes observed, n = 7380): cumulative mortality (risk ratio 0.99, 95% confidence interval [CI] 0.95-1.03, P = .69) and survival time (hazard ratio 0.96, 95% CI 0.91-1.02, P = .19). Results were consistent by sex, by body mass index, and across the range of ivermectin capillary concentrations studied (0.72-73.9 ng/mL). CONCLUSIONS: Direct skin feeding and membrane feeding on day 7 resulted in similar mosquitocidal effects of ivermectin across a wide range of drug concentrations, suggesting that the mosquitocidal effects seen with membrane feeding accurately reflect those of natural biting. Membrane feeding, which is more patient friendly and ethically acceptable, can likely reliably be used to assess ivermectin's mosquitocidal efficacy. CLINICAL TRIALS REGISTRATION: NCT02511353.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.