The impact of yeast-encapsulated orange oil in Aedes aegypti oviposition.

The yeast-encapsulated orange oil (YEOO) is a novel larvicide under development against vector mosquitoes. Despite its efficiency against Aedes aegypti (L.) in small scale experiments, its applicability in vector control can be influenced by other effects on mosquito behaviour or physiology. For this reason, the impact of YEOO particles in mosquito oviposition was evaluated in laboratory and semi-field conditions. Oviposition assays with one gravid Aedes aegypti female were carried under laboratory and semi-field conditions with natural light and temperature fluctuation. For all ovitraps, the number of eggs was manually counted in the wooden paddle and in the solution of each ovitrap. The proportion of eggs between substrates (wooden paddle and solution) varied between conditions, with females in laboratory presenting a lower preference to lay eggs in paddles when compared with studies in semi-field. This behaviour shifts in laboratory can create challenges to extrapolate results from laboratory to the field. Here, studies in both conditions indicate a similar impact of YEOO particles in Aedes aegypti oviposition. The potential treatment concentration of YEOO particles presents a strong repellent/deterrent effect (-0.559 > OAI > -0.760) within the initial 72h of application when compared with water, and weak repellent/deterrent signal (OAI = -0.220) when compared against inactivated yeast. Control ovitraps with water were more positive for egg presence than treated ovitraps, while ovitraps with YEOO particles and inactivated yeast present similar number of positive ovitraps. It is possible that the repellent/deterrent action is partially driven by the delivery system, since most times Citrus sinensis EO oviposition repellent/deterrent signal is weak, and it seem influenced by solvent/delivery used. However, it is unclear how the yeast wall that protect/surrounds the orange oil will negatively affect oviposition since live yeast are normally consider an attractant for mosquito oviposition.

Evaluation of Insect Growth Regulators (IGRs) as biological pesticides for control of Aedes aegypti mosquitoes.

BACKGROUND OBJECTIVES: Insect growth regulators (IGRs) are biological hormone analogue or mimics used as pesticides to inhibit the growth of larva during their molting and skin shedding. This study aimed to test the effect of IGRs on the eggs hatching and post-hatching inhibition of Aedes mosquitoes and understanding its effect in the mosquito breeding habitats for reduction in adult emergence. METHODS: Experiments on the evaluation of three insect growth regulators (IGRs) for the control of different stages of Aedes aegypti was carried out during 2020-21. Each experiment consisted of four treatments viz., Pyriproxyfen, Novaluron, and Larvicol at 1.0 ppm and distilled water as a control. All experiments were carried out in completely randomized design (CRD) except eggs which were carried out in factorial design each with three replications. RESULTS: All tested IGRs performed better in affecting eggs, larval and pupal stages of Ae. aegypti. Highest eggs hatching inhibition (80%) of fresh eggs occurred in Pyriproxyfen followed by Novaluron (66%) and lowest in Larvicol (62%). Eggs hatch inhibition of embryonated eggs was lower than fresh eggs. Pyriproxyfen caused 69%, Novaluron 59% and Larvicol 39% eggs hatch inhibition of embryonated eggs. Both Pyriproxyfen and Novaluron performed better in causing 98-100% larval mortality followed by Larvicol (39%). Larval development to pupal stage was completely prevented by both Pyriproxyfen and Novaluron. Although Larvicol resulted in lowest eggs hatch and larval inhibition but prevented pupae to emerge as adults. Results further showed 70-89% mortality of 3rd instar larvae of Ae. aegypti when exposed to Pyriproxyfen and Novaluron solutions after 30 days storage at lab. temperature (27±2°C), RH 70±5. INTERPRETATION CONCLUSION: None of the IGRs was more effective at the pupal stage but showed carry-on activity of growth inhibition and mortality of the successive stages of development when used against eggs stages. Therefore, we recommend early application of IGRs at mosquito habitats during the beginning and onset of the season when very early stages of mosquitoes are available in the field.

Evaluation of the DN-Mini (miniaturized double net) trap for sampling host-seeking Anopheles mosquitoes in malaria-endemic villages of southern Tanzania.

BACKGROUND: Surveillance of malaria vectors is crucial for assessing the transmission risk and impact of control measures. Human landing catches (HLC) directly estimate the biting rates but raise ethical concerns due to the exposure of volunteers to mosquito-borne pathogens. A common alternative is the CDC-light trap, which is effective for catching host-seeking mosquitoes indoors but not outdoors. New, exposure-free methods are needed for sampling mosquitoes indoors and outdoors in ways that reflect their natural risk profiles. The aim of this study was therefore to evaluate the efficacy of the miniaturized double net trap (DN-Mini) for sampling host-seeking mosquitoes in south-eastern Tanzania, where malaria transmission is dominated by Anopheles funestus. METHODS: Adult mosquitoes were collected from 222 randomly selected houses across three villages (74 per village) in Ulanga district, south-eastern Tanzania, using the DN-Mini traps, CDC-Light traps, and Prokopack aspirators. First, we compared CDC-light and DN-Mini traps for collecting indoor host-seeking mosquitoes, while Prokopack aspirators were used for indoor-resting mosquitoes. Second, we deployed the DN-Mini and Prokopack aspirators to collect host-seeking and resting mosquitoes indoors and outdoors. Generalized linear mixed models (GLMM) with a negative binomial distribution were used to compare the effectiveness of the traps for catching different mosquito species. RESULTS: The DN-Mini was 1.53 times more efficient in collecting An. funestus indoors (RR = 1.53, 95% CI: 1.190-1.98) compared to the CDC-Light trap. However, for Anopheles arabiensis, the DN-Mini caught only 0.32 times as many mosquitoes indoors as the CDC-Light traps (RR = 0.32, 95% CI: 0.183-0.567). Both An. funestus and An. arabiensis were found to be more abundant indoors than outdoors when collected using the DN-Mini trap. Similarly, the Prokopack aspirator was greater indoors than outdoors for both An. funestus and An. arabiensis. CONCLUSION: The DN-Mini outperformed the CDC-light trap in sampling the dominant malaria vector, An. funestus species, but was less effective in capturing An. arabiensis, and for both vector species, the biting risk was greater indoors than outdoors when measured using the DN-Mini trap. These findings highlight the importance of selecting appropriate trapping methods based on mosquito species and behaviors.

Biological effects of Lippia alba essential oil against Anopheles gambiae and Aedes aegypti.

The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods. The use of plants is increasingly considered as a sustainable biological solution for vector control. The aim of this study was to evaluate the biological effects of the essential oil (EO) of Lippia alba harvested in Abidjan (Côte d'Ivoire) against Anopheles gambiae and Aedes aegypti mosquitoes. Phytochemical compounds were identified by GC-MS. Knockdown and mortality were determined according to the WHO test tube protocol. Contact irritancy was assessed by observing the movement of mosquitoes from a treated WHO tube to a second untreated tube. Non-contact repellency was assessed using a standardised high-throughput screening system (HITSS). Blood meal inhibition was assessed using a membrane feeding assay treated with EO. The EO was identified as the citral chemotype. The EO gave 100% KD60 in both species at a concentration of 1%. Mortalities of 100% were recorded with An. gambiae and Ae. aegypti at concentrations of 1% and 5% respectively. The highest proportions of females escaping during the contact irritancy test were 100% for An. gambiae at 1% concentration and 94% for Ae. aegypti at 2.5% concentration. The 1% concentration produced the highest proportions of repelled mosquitoes in the non-contact repellency tests: 76.8% (An. gambiae) and 68.5% (Ae. aegypti). The blood meal inhibition rate at a dose of 10% was 98.4% in Ae. aegypti but only 15.5% in An. gambiae. The citral chemotype of L. alba EO has promising biological effects in both species that make it a potentially good candidate for its use in mosquito control. The results obtained in this study encourage the further evaluation of L. alba EOs from other localities and of different chemotypes, under laboratory and field conditions.

Adenosine triphosphate overrides the aversive effect of antifeedants and toxicants: a model alternative phagostimulant for sugar-based vector control tools.

BACKGROUND: Sugar, when used as the phagostimulant in attractive toxic bait control tools, limits the efficacy and selectivity of this technology. Thus, more potent and selective phagostimulants than sugar are required to improve this technology. The potency of adenosine triphosphate (ATP) as an alternative model phagostimulant was assessed to determine its capacity to override the aversive effects of select antifeedants and toxicants. How ATP and sucrose modulate the rate of toxicity in the yellow fever mosquito Aedes aegypti was also examined. METHODS: A no-choice feeding assay was used to investigate the phagostimulatory ability of ATP to override the aversive effects of structurally divergent antifeedant and toxicant compounds, and to modulate the rate of toxicity over 24 h. Binary combinations of antifeedant and toxicant compounds, at various concentrations, were similarly assessed for enhanced lethal potency. In comparison, no-choice open access and cotton wick feeding assays were used to determine the phagostimulatory role of sucrose in the ingestion of boric acid-laced diets. Dissections of the guts were performed to determine the diet destination as dependant on the phagostimulant. RESULTS: ATP is a potent phagostimulant that dose dependently overrides aversion to antifeedant and toxicant tastants. Feeding on antifeedant- or toxicant-laced diets that was induced by ATP selectively resulted in rapid knockdown (nicotine, lobeline and caffeine) or death (boric acid and propylene glycol), with a combination of the two lethal compounds inducing a synergistic effect at lower concentrations. ATP- and sucrose-induced feeding predominantly directed the antifeedant- or toxicant-laced meals to the midgut and the crop, respectively. CONCLUSIONS: ATP is an efficacious alternative model phagostimulant to sucrose that overrides the aversive effects of antifeedants and toxicants, resulting in rapid toxic effects. Furthermore, this study demonstrates that variation in the rate of toxicity between ATP- and sugar-induced feeding is at least partly regulated by the differential feeding response, volume imbibed and the destination of the meals. Additional research is needed to identify structurally related, stable analogues of ATP due to the ephemeral nature of this molecule. For future applications, the workflow presented in this study may be used to evaluate such analogues for their suitability for use in attractive bait stations designed to target a broad range of haematophagous arthropods and prevent off-target species' feeding.

Vegetable oil-based surfactants are adjuvants that enhance the efficacy of neonicotinoid insecticides and can bias susceptibility testing in adult mosquitoes.

BACKGROUND: The standard operating procedure for testing the susceptibility of adult mosquitoes to neonicotinoid or butenolide insecticides recommends using a vegetable oil ester (Mero) as a surfactant. However, there is growing evidence that this adjuvant contains surfactants that can enhance insecticide activity, mask resistance and bias the bioassay. METHODOLOGY/PRINCIPAL FINDINGS: Using standard bioassays, we tested the effects of commercial formulations of vegetable oil-based surfactants similar to Mero on the activity of a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). We found that three different brands of linseed oil soap used as cleaning products drastically enhanced neonicotinoid activity in Anopheles mosquitoes. At 1% (v/v), the surfactant reduced the median lethal concentration, LC50, of clothianidin more than 10-fold both in susceptible and in resistant populations of Anopheles gambiae. At 1% or 0.5% (v/v), linseed oil soap restored the susceptibility of adult mosquitoes fully to clothianidin, thiamethoxam and imidacloprid and partially to acetamiprid. By contrast, adding soap to the active ingredient did not significantly affect the level of resistance to permethrin or deltamethrin suggesting that vegetable oil-based surfactants specifically enhance the potency of some classes of insecticides. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that surfactants are not inert ingredients, and their use in susceptibility testing may jeopardize the ability to detect resistance. Further research is needed to evaluate the potential, the limitations and the challenges of using some surfactants as adjuvants to enhance the potency of some chemicals applied in mosquito control.

Laboratory evaluation of the efficacy of deltamethrin-laced attractive toxic sugar bait formulation on Anopheles stephensi.

BACKGROUND: Attractive toxic sugar bait (ATSB) is a promising "attract and kill"-based approach for mosquito control. It is a combination of flower nectar/fruit juice to attract the mosquitoes, sugar solution to stimulate feeding, and a toxin to kill them. Selecting an effective attractant and optimizing concentration of toxicant is significant in the formulation of ATSB. METHODS: Current study formulated an ATSB using fruit juice, sugar and deltamethrin, a synthetic pyrethroid. It was evaluated against two laboratory strains of Anopheles stephensi. Initial studies evaluated comparative attractiveness of nine different fruit juices to An. stephensi adults. Nine ASBs were prepared by adding fermented juices of plum, guava, sweet lemon, orange, mango, pineapple, muskmelon, papaya, and watermelon with 10% sucrose solution (w/v) in 1:1 ratio. Cage bioassays were conducted to assess relative attraction potential of ASBs based on the number of mosquito landings on each and the most effective ASB was identified. Ten ATSBs were prepared by adding the identified ASB with different deltamethrin concentrations (0.015625-8.0 mg/10 mL) in 1:9 ratio. Each ATSB was assessed for the toxic potential against both the strains of An. stephensi. The data was statistically analysed using PASW (SPSS) software 19.0 program. RESULTS: The cage bioassays with nine ASBs revealed higher efficacy (p < 0.05) of Guava juice-ASB > Plum juice-ASB > Mango juice-ASB in comparison to rest of the six ASB's. The bioassay with these three ASB's ascertained the highest attractancy potential of guava juice-ASB against both the strains of An. stephensi. The ATSB formulations resulted in 5.1-97.9% mortality in Sonepat (NIMR strain) with calculated LC30, LC50, and LC90 values of 0.17 mg deltamethrin/10 mL, 0.61 mg deltamethrin/10 mL, and 13.84 mg deltamethrin/10 mL ATSB, respectively. Whereas, 6.12-86.12% mortality was recorded in the GVD-Delhi (AND strain) with calculated LC30, LC50, and LC90 values of 0.25 mg deltamethrin/10 mL, 0.73 mg deltamethrin/10 mL and 10.22 mg deltamethrin/10 mL ATSB, respectively. CONCLUSION: The ATSB formulated with guava juice-ASB and deltamethrin (0.0015625-0.8%) in 9:1 ratio showed promising results against two laboratory strains of An. stephensi. Field assessment of these formulations is being conducted to estimate their feasibility for use in mosquito control.

Radiation dose-fractionation in adult Aedes aegypti mosquitoes.

Balancing process efficiency and adult sterile male biological quality is one of the challenges in the success of the sterile insect technique (SIT) against insect pest populations. For the SIT against mosquitoes, many stress factors need to be taken into consideration when producing sterile males that require high biological quality to remain competitive once released in the field. Pressures of mass rearing, sex sorting, irradiation treatments, packing, transport and release including handling procedures for each step, add to the overall stress budget of the sterile male post-release. Optimizing the irradiation step to achieve maximum sterility while keeping off-target somatic damage to a minimum can significantly improve male mating competitiveness. It is therefore worth examining various protocols that have been found to be effective in other insect species, such as dose fractionation. A fully sterilizing dose of 70 Gy was administered to Aedes aegypti males as one acute dose or fractionated into either two equal doses of 35 Gy, or one low dose of 10 Gy followed by a second dose of 60 Gy. The two doses were separated by either 1- or 2-day intervals. Longevity, flight ability, and mating competitiveness tests were performed to identify beneficial effects of the various treatments. Positive effects of fractionating dose were seen in terms of male longevity and mating competitiveness. Although applying split doses generally improved male quality parameters, the benefits may not outweigh the added labor in SIT programmes for the management of mosquito vectors.

Title: Fractionnement de la dose d'irradiation chez les moustiques Aedes aegypti adultes. Abstract: Équilibrer l'efficacité du processus et la qualité biologique des mâles adultes stériles est l'un des défis du succès de la technique des insectes stériles (TIS) contre les populations d'insectes nuisibles. Pour la TIS contre les moustiques, de nombreux facteurs de stress sont à prendre en compte lors de la production de mâles stériles qui nécessitent une haute qualité biologique pour rester compétitifs une fois relâchés au champ. Les pressions de l'élevage en masse, du triage par sexe, des traitements d'irradiation, de l'emballage, du transport et de la libération, y compris les procédures de manipulation pour chaque étape, s'ajoutent au budget de stress global du mâle stérile après la libération. L'optimisation de l'étape d'irradiation pour atteindre une stérilité maximale tout en minimisant les dommages somatiques hors cible peut améliorer considérablement la compétitivité de l'accouplement des mâles et il est donc important d'examiner divers protocoles qui se sont révélés efficaces chez d'autres espèces d'insectes, comme le fractionnement de dose. Une dose entièrement stérilisante de 70 Gy a été administrée aux mâles Aedes aegypti en une dose unique ou fractionnée en deux doses égales de 35 Gy, ou une faible dose de 10 Gy suivie d'une seconde dose de 60 Gy. Les deux doses étaient séparées par des intervalles de 1 ou 2 jours. Des tests de longévité, d'aptitude au vol et de compétitivité à l'accouplement ont été réalisés pour identifier les effets bénéfiques des différents traitements. Des effets positifs de la dose de fractionnement ont été observés en termes de longévité des mâles et de compétitivité à l'accouplement. Bien que l'application de doses fractionnées améliore généralement les paramètres de qualité des mâles, les avantages peuvent ne pas compenser le travail supplémentaire dans les programmes TIS pour la gestion des moustiques vecteurs.

Developing the radiation-based sterile insect technique (SIT) for controlling Aedes aegypti: identification of a sterilizing dose.

BACKGROUND: The sterile insect technique (SIT) is emerging as a tool to supplement traditional pesticide-based control of Aedes aegypti, a prominent mosquito vector of microbes that has increased the global burden of human morbidity and mortality over the past 50 years. SIT relies on rearing, sterilizing and releasing large numbers of male mosquitoes that will mate with fertile wild females, thus reducing production of offspring from the target population. In this study, we investigated the effects of ionizing radiation (gamma) on male and female survival, longevity, mating behavior, and sterility of Ae. aegypti in a dose-response design. This work is a first step towards developing an operational SIT field suppression program against Ae. aegypti in St. Augustine, Florida, USA. RESULTS: Exposing late-stage pupae to 50 Gy of radiation yielded 99% male sterility while maintaining similar survival of pupae to adult emergence, adult longevity and male mating competitiveness compared to unirradiated males. Females were completely sterilized at 30 Gy, and when females were dosed with 50 Gy, they had a lower incidence of blood-feeding than unirradiated females. CONCLUSION: Our work suggests that an ionizing radiation dose of 50 Gy should be used for future development of operational SIT in our program area because at this dose males are 99% sterile while maintaining mating competitiveness against unirradiated males. Furthermore, females that might be accidentally released with sterile males as a result of errors in sex sorting also are sterile and less likely to blood-feed than unirradiated females at our 50 Gy dose. © 2022 Society of Chemical Industry.

Mosquitocidal activities of Chenopodium botrys whole plant n-hexane extract against Culex quinquefasciatus / Atividades mosquitocidas do extrato de n-hexano de planta inteira de Chenopodium botrys contra Culex quinquefasciatus

This research aimed to investigate various mosquitocidal activities of Chenopodium botrys whole- plant n-hexane extract against Culex quinquefasciatus. The extract showed remarkable larvicidal, pupicidal, adulticidal, oviposition deterrent and adult emergence inhibitory activities against Cx. quinquefasciatus. During the larvicidal and pupicidal activities, the 24-hour lethal concentration (LC50) of extract against 2nd instar larvae, 4th instar larvae and pupae were 324.6, 495.6 and 950.8 ppm, respectively. During the CDC (Centers for Disease Control and Prevention) bottle bioassay for adulticidal activity, the median knockdown times (KDT50) at 1.25% concentration was 123.4 minutes. During the filter paper impregnation bioassay for adulticidal activity, the KDT50 value at 0.138 mg/cm2 concentration was 48.6 minutes. The extract was fractionated into 14 fractions through silica gel column chromatography which were then combined into six fractions on the basis of similar retention factor (Rf) value. These fractions were screened for adulticidal activity by applying CDC bottle bioassay. The fraction obtained through 60:40 to 50:50% n-hexanes-chloroform mobile phase with 0.5 Rf value showed 100% adulticidal activity at 0.2% concentration. During oviposition deterrent activity, the highest concentration (1000 ppm) showed 71.3 ± 4.4% effective repellence and 0.6 ± 0.1 oviposition activity index. During adult emergence inhibition activity, the median emergence inhibition (EI50) value was 312.3 ppm. From the outcome of the present investigation, it is concluded that the n-hexane extract of C. botrys whole- plant possesses strong larvicidal, pupicidal, adulticidal, oviposition deterrent and adult emergence inhibitory activities against Cx. quinquefasciatus.

Esta pesquisa teve como objetivo investigar várias atividades mosquitocidas do extrato n-hexano de planta inteira de Chenopodium botrys contra Culex quinquefasciatus. O extrato mostrou atividades larvicida, pupicida, adulticida, dissuasora de oviposição e inibidora da emergência de adultos contra a Cx. quinquefasciatus. Durante as atividades larvicida e pupicida, a concentração letal de 24 horas (CL50) do extrato contra larvas de 2º estádio, larvas de 4º estádio e pupa foi de 324,6, 495,6 e 950,8 ppm, respectivamente. Durante o bioensaio com frasco do CDC (Centros para Controle e Prevenção de Doenças) para adulticida, o tempo médio de desativação (KDT50) na concentração de 1,25% foi de 123,4 minutos. Durante o bioensaio de impregnação com papel de filtro para a atividade adulticida do extrato, o valor KDT50 na concentração de 0,138 mg / cm2 foi de 48,6 minutos. O extrato foi fracionado em 14 frações através de cromatografia em coluna de gel de sílica que foram então combinadas em seis frações com base em um valor de fator de retenção (Rf) semelhante. Essas frações foram selecionadas quanto à atividade adulticida por meio da aplicação do bioensaio com garrafa do CDC. A fração obtida através da fase móvel de n-hexanos-clorofórmio 60:40% a 50:50% com valor de 0,5 Rf apresentou atividade adulticida de 100% na concentração de 0,2%. Durante a atividade de dissuasão da oviposição, a maior concentração de extrato (1000 ppm) apresentou repelência efetiva de 71,3 ± 4,4% e índice de atividade de oviposição de 0,6 ± 0,1. Durante a atividade de inibição da emergência de adultos, o valor médio de inibição da emergência (EI50) foi de 312,3 ppm. A partir do resultado da presente investigação, conclui-se que o extrato de n-hexano da planta inteira de C. botrys possui fortes atividades larvicida, pupicida, adulticida, dissuasora da oviposição e inibidora da emergência de adultos contra a Cx. quinquefasciatus.

A standalone incompatible insect technique enables mosquito suppression in the urban subtropics.

The strong suppression of Aedes albopictus on two Guangzhou islands in China has been successfully achieved by releasing males with an artificial triple-Wolbachia infection. However, it requires the use of radiation to sterilize residual females to prevent population replacement. To develop a highly effective tool for dengue control, we tested a standalone incompatible insect technique (IIT) to control A. albopictus in the urban area of Changsha, an inland city where dengue recently emerged. Male mosquitoes were produced in a mass rearing facility in Guangzhou and transported over 670 km under low temperature to the release site. After a once-per-week release with high numbers of males (phase I) and a subsequent twice-per-week release with low numbers of males (phase II), the average numbers of hatched eggs and female adults collected weekly per trap were reduced by 97% and 85%, respectively. The population suppression caused a 94% decrease in mosquito biting at the release site compared to the control site. Remarkably, this strong suppression was achieved using only 28% of the number of males released in a previous trial. Despite the lack of irradiation to sterilize residual females, no triple-infected mosquitoes were detected in the field post release based on the monitoring of adult and larval A. albopictus populations for two years, indicating that population replacement was prevented. Our results support the feasibility of implementing a standalone IIT for dengue control in urban areas.

A high-throughput HPLC method for simultaneous quantification of pyrethroid and pyriproxyfen in long-lasting insecticide-treated nets.

Long-lasting insecticide-treated nets (LLINs) play a crucial role in preventing malaria transmission. LLINs should remain effective for at least three years, even after repeated washings. Currently, monitoring insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. Our aim was to develop a simple, high-throughput and low-resource method for measuring insecticides in LLINs. To extract insecticides, polyethylene-LLIN samples were heated at 85 °C for 45 min in a non-hazardous solvent mix containing dicyclohexylphthalate as an internal standard. The extraction solvent was reduced from 50 to 5 ml using a 0.2 g sample, 90% smaller than the recommended sample size. By optimizing HPLC chromatography, we simultaneously detected pyrethroid and pyriproxyfen insecticides with high sensitivity in LLIN's extract. The method can quantify levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing for insecticide tracking before and after the use of LLINs. This method can be used to assess LLINs with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset® Net), 1% pyriproxyfen and 2% permethrin (Olyset® Duo), or 0.55% pyriproxyfen and 0.55% alpha-cypermethrin (Royal Gaurd®). One can run 120 samples (40 nets) simultaneously with high precision and accuracy, improving throughput and reducing labour, costs, and environmental impact.

Effects of agricultural pesticides on the susceptibility and fitness of malaria vectors in rural south-eastern Tanzania.

BACKGROUND: Agricultural pesticides may exert strong selection pressures on malaria vectors during the aquatic life stages and may contribute to resistance in adult mosquitoes. This could reduce the performance of key vector control interventions such as indoor-residual spraying and insecticide-treated nets. The aim of this study was to investigate effects of agrochemicals on susceptibility and fitness of the malaria vectors across farming areas in Tanzania. METHODS: An exploratory mixed-methods study was conducted to assess pesticide use in four villages (V1-V4) in south-eastern Tanzania. Anopheles gambiae (s.l.) larvae were collected from agricultural fields in the same villages and their emergent adults examined for insecticide susceptibility, egg-laying and wing lengths (as proxy for body size). These tests were repeated using two groups of laboratory-reared An. arabiensis, one of which was pre-exposed for 48 h to sub-lethal aquatic doses of agricultural pesticides found in the villages. RESULTS: Farmers lacked awareness about the linkages between the public health and agriculture sectors but were interested in being more informed. Agrochemical usage was reported as extensive in V1, V2 and V3 but minimal in V4. Similarly, mosquitoes from V1 to V3 but not V4 were resistant to pyrethroids and either pirimiphos-methyl or bendiocarb, or both. Adding the synergist piperonyl butoxide restored potency of the pyrethroids. Pre-exposure of laboratory-reared mosquitoes to pesticides during aquatic stages did not affect insecticide susceptibility in emergent adults of the same filial generation. There was also no effect on fecundity, except after pre-exposure to organophosphates, which were associated with fewer eggs and smaller mosquitoes. Wild mosquitoes were smaller than laboratory-reared ones, but fecundity was similar. CONCLUSIONS: Safeguarding the potential of insecticide-based interventions requires improved understanding of how agricultural pesticides influence important life cycle processes and transmission potential of mosquito vectors. In this study, susceptibility of mosquitoes to public health insecticides was lower in villages reporting frequent use of pesticides compared to villages with little or no pesticide use. Variations in the fitness parameters, fecundity and wing length marginally reflected the differences in exposure to agrochemicals and should be investigated further. Pesticide use may exert additional life cycle constraints on mosquito vectors, but this likely occurs after multi-generational exposures.

Development of the Sterile Insect Technique to control the dengue vector Aedes aegypti (Linnaeus) in Sri Lanka.

BACKGROUND: The Sterile Insect Technique (SIT) is presently being tested to control dengue in several countries. SIT aims to cause the decline of the target insect population through the release of a sufficient number of sterilized male insects. This induces sterility in the female population, as females that mate with sterilized males produce no offspring. Male insects are sterilized through the use of ionizing irradiation. This study aimed to evaluate variable parameters that may affect irradiation in mosquito pupae. METHODS: An Ae. aegypti colony was maintained under standard laboratory conditions. Male and female Ae. aegypti pupae were separated using a Fay and Morlan glass sorter and exposed to different doses of gamma radiation (40, 50, 60, 70 and 80 Gy) using a Co60 source. The effects of radiation on survival, flight ability and the reproductive capacity of Ae. aegypti were evaluated under laboratory conditions. In addition, mating competitiveness was evaluated for irradiated male Ae. aegypti mosquitoes to be used for future SIT programmes in Sri Lanka. RESULTS: Survival of irradiated pupae was reduced by irradiation in a dose-dependent manner but it was invariably greater than 90% in control, 40, 50, 60, 70 Gy in both male and female Ae. aegypti. Irradiation didn't show any significant adverse effects on flight ability of male and female mosquitoes, which consistently exceeded 90%. A similar number of eggs per female was observed between the non-irradiated groups and the irradiated groups for both irradiated males and females. Egg hatch rates were significantly lower when an irradiation dose above 50 Gy was used as compared to 40 Gy in both males and females. Irradiation at higher doses significantly reduced male and female survival when compared to the non-irradiated Ae. aegypti mosquitoes. Competitiveness index (C) scores of sterile and non-sterile males compared with non-irradiated male mosquitoes under laboratory and semi-field conditions were 0.56 and 0.51 respectively at 50 Gy. SIGNIFICATION: Based on the results obtained from the current study, a 50 Gy dose was selected as the optimal radiation dose for the production of sterile Ae. aegypti males for future SIT-based dengue control programmes aiming at the suppression of Ae. aegypti populations in Sri Lanka.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation.

Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries. The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place. Insect neuropeptidergic system offers potential targets to control the insect vectors. The essential roles of the neuropeptide ecdysis triggering hormone (ETH) in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae. One of two ETH peptides of the African malaria mosquito (AgETH1), a small peptide hormone with 17 amino acid residues (SESPGFFIKLSKSVPRI-NH2 ), was studied to elucidate its molecular structure. N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation. The solution structure of AgETH1 using 2D 1 H-1 H nuclear magnetic resonance (NMR) spectroscopy and nuclear overhauser effect (NOE) derived constraints revealed a short alpha helix between residues 3S and 11S. The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes.

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect's avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85-97% escape) and Anopheles minimus Theobald (97-99%) at high concentrations (2.5-5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90-100% escape) and An. minimus (83-100%). Whereas moderate contact irritancy was observed against An. dirus (40-50% escape) and Ae. aegypti (51-59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Aula invertida en el desarrollo de competencias para el control domicilario de mosquitos de importancia para la salud pública / Flipped classroom in the development of skills for home control of mosquitoes of importance to public health

El método de enseñanza-aprendizaje de aula invertida propicia actividades participativas, de esfuerzo y colaboración antes, durante y fuera del salón de clases. Por tal razón, que se propuso la aplicación del aula invertida en el desarrollo de competencias para el control domicilario de mosquitos de importancia para la salud pública en la Universidad Nacional Autónoma de Chota, Camarja, Perú. El estudio fue experimental con una muestra de 72 estudiantes. Para la recolección de datos se aplicó la estrategia didáctica del Modelo Flipped Classroom en dos tiempos (pre y post test). Se usó el software SPSS para medianas y prueba de Wilcoxon considerando un alpha del 5%. Como resultado, en el post-test sobre los factores de riesgo identificados en el domicilio se evidenciaron cambios significativos, donde 72,22% identificaron los recipientes de almacenamiento de agua expuestos, 51,39% Frascos/botellas/envases con agua estancada, 45,83% piletas sin liempieza, 29,17% Llantas/desechos al aire libre con agua estancada, 26,39% Floreros con agua vieja, 20,83% analetas/grietas con agua estancada, 12,50% otros y 8,33% no identifico ningún factor, la comparación de la puntuación promedio en competencias para el control domicilario de mosquitos, antes y después de la aplicación del aula invertida en la Prueba de Wilcoxon mostró una z de -4,81 en la dimesión de identificar, -4,860 proponer y -4862 actuar, todos con p 0,000% (diferencia altamente significativa). El método de aula invertida a través de métodos prácticos interactivos contribuye de manera significativa al desarrollo de competencias para el control domicilario de mosquitos de importancia de salud pública(AU)

The flipped classroom teaching-learning method encourages participatory, effortful, and collaborative activities before, during, and outside the classroom. For this reason, the application of the flipped classroom was proposed in the development of skills for the home control of mosquitoes of importance for public health at the National Autonomous University of Chota, Camarja, Peru. The study was experimental with a sample of 72 students. For data collection, the didactic strategy of the Flipped Classroom Model was applied in two stages (pre and post test). The SPSS software was used for medians and the Wilcoxon test considering an alpha of 5%. As a result, in the post-test on the risk factors identified at home, significant changes were evidenced, where 72.22% identified exposed water storage containers, 51.39% Jars/bottles/containers with stagnant water, 45 83% pools without cleaning, 29.17% tires/outdoor debris with stagnant water, 26.39% vases with old water, 20.83% holes/cracks with stagnant water, 12.50% others and 8.33 % did not identify any factor, the comparison of the average score in skills for home control of mosquitoes, before and after the application of the flipped classroom in the Wilcoxon Test showed a z of -4.81 in the dimension of identifying, - 4,860 propose and -4,862 act, all with p 0.000% (highly significant difference). The flipped classroom method through interactive practical methods contributes significantly to the development of skills for home control of mosquitoes of public health importance(AU)

Microbiota and transcriptome changes of Culex pipiens pallens larvae exposed to Bacillus thuringiensis israelensis.

Culex pipiens pallens is an important vector of lymphatic filariasis and epidemic encephalitis. Mosquito control is the main strategy used for the prevention of mosquito-borne diseases. Bacillus thuringiensis israelensis (Bti) is an entomopathogenic bacterium widely used in mosquito control. In this study, we profiled the microbiota and transcriptional response of the larvae of Cx. pipiens pallens exposed to different concentrations of Bti. The results demonstrated that Bti induced a significant effect on both the microbiota and gene expression of Cx. pipiens pallens. Compared to the control group, the predominant bacteria changed from Actinobacteria to Firmicutes, and with increase in the concentration of Bti, the abundance of Actinobacteria was gradually reduced. Similar changes were also detected at the genus level, where Bacillus replaced Microbacterium, becoming the predominant genus in Bti-exposed groups. Furthermore, alpha diversity analysis indicated that Bti exposure changed the diversity of the microbota, possibly because the dysbiosis caused by the Bti infection inhibits some bacteria and provides opportunities to other opportunistic taxa. Pathway analysis revealed significant enhancement for processes associated with sphingolipid metabolism, glutathione metabolism and glycerophospholipid metabolism between all Bti-exposed groups and control group. Additionally, genes associated with the Toll and Imd signaling pathway were found to be notably upregulated. Bti infection significantly changed the bacterial community of larvae of Cx. pipiens pallens.

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

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

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.