Mosquito Control Strategies Research Articles

Boosting the efficacy of fungal biocontrol: miRNA339-5p-mediated mosquito immunity regulation.

Aedes mosquitoes are vectors for numerous viral diseases, including dengue, zika, chikungunya, and yellow fever. Therefore, underscoring the urgent need for eco-friendly alternatives to combat insecticide resistance and the scarcity of effective vaccines. Entomopathogenic fungi present a sustainable alternative to chemical insecticides; however, their widespread application is limited by their relatively low virulence. Here, we investigated the immunological interactions between Metarhizium anisopliae and Aedes albopictus, demonstrating that fungal infection significantly up-regulated immune-related genes in the Toll and melanization pathways, thereby enhancing antifungal and antibacterial defenses at 48 h post-infection (hpi). Small RNA sequencing identified miR339-5p as a crucial modulator, targeting the immune genes Gram-Negative Binding Protein 1 (GNBP1) and CLIP-domain Serine Protease B15 (CLIPB15), which are critical for Toll and phenoloxidase (PO) pathway activation. The administration of a synthetic miR339-5p mimic increased fungal virulence, resulting in a higher mortality rate among adult mosquitoes and a significant increase in the mortality rate of mosquito larvae within 24 hpi. GNBP1 was found to regulate both Toll and PO pathways, while CLIPB15 specifically modulated the PO system by cleaving prophenoloxidase (PPO). This research highlights the potential of leveraging Ae. albopictus-encoded miR339-5p through advanced genetic engineering techniques to bolster the efficacy of existing fungal-based mosquito control strategies, providing a promising approach in the fight against mosquito-borne diseases. © 2024 Society of Chemical Industry.

Wolbachia-Based Approaches to Controlling Mosquito-Borne Viral Threats: Innovations, AI Integration, and Future Directions in the Context of Climate Change

Wolbachia-based mosquito control strategies have gained significant attention as a sustainable approach to reduce the transmission of vector-borne diseases such as dengue, Zika, and chikungunya. These endosymbiotic bacteria can limit the ability of mosquitoes to transmit pathogens, offering a promising alternative to traditional chemical-based interventions. With the growing impact of climate change on mosquito population dynamics and disease transmission, Wolbachia interventions represent an adaptable and resilient strategy for mitigating the public health burden of vector-borne diseases. Changes in temperature, humidity, and rainfall patterns can alter mosquito breeding habitats and extend the geographical range of disease vectors, increasing the urgency for effective control measures. This review highlights innovations in Wolbachia-based mosquito control and explores future directions in the context of climate change. It emphasizes the integration of Wolbachia with other biological approaches and the need for multidisciplinary efforts to address climate-amplified disease risks. As ecosystems shift, Wolbachia interventions could be crucial in reducing mosquito-borne diseases, especially in vulnerable regions. AI integration in Wolbachia research presents opportunities to enhance mosquito control strategies by modeling ecological data, predicting mosquito dynamics, and optimizing intervention outcomes. Key areas include refining release strategies, real-time monitoring, and scaling interventions. Future opportunities lie in advancing AI-driven approaches for integrating Wolbachia with other vector control measures, promoting adaptive, data-driven responses to climate-amplified disease transmission.

Larvicidal and enzyme inhibition effects of Phoenix pusilla derived Methyl oleate and malathion on Aedes aegypti strains

This study explores the larvicidal potential of methanolic flower extracts from Phoenix pusilla (Pp-Fe), its major compound, and malathion (MLT), against laboratory strain (LS) and field strain (FS) of Aedes aegypti, the dengue mosquito vector. We identified thirty-one derivatives, with methyl oleate (MO) comprising 28.5% of Pp-Fe. Comparative efficacy evaluations were performed using peak dosages of Pp-Fe (500 ppm), MO (5 ppm), and MLT (5 ppm) on LS and FS larvae. Both LS and FS second instars showed higher susceptibility to Pp-Fe (95% and 93%, respectively) and MO (85% and 83%, respectively). MLT resulted in significant mortality rates among LS larvae (98%) and notable reductions among FS larvae (71%). The expression levels of key biomarker enzymes (carboxylesterase, GST, and CYP450) exhibited a consistent decrease and subsequent upregulation in LS and FS larvae following exposure to Pp-Fe and MO, contrasting with the significant expression variations observed in LS and FS larvae exposed to MLT. LS larvae demonstrated heightened susceptibility and evident midgut cell damage following all treatments, suggesting potential disparities in susceptibility and adaptive responses between LS and FS strains towards MLT. These observations underscore the promising larvicidal attributes of Pp-Fe and MO, emphasizing the need for further exploration of their mechanisms of action in the development of environmentally sustainable mosquito control strategies and resistance management.

Mosquito diversity, abundance and phenology in Mexico City: A heavily urbanized environment

We need to understand how zoonotic diseases make their way in urbanized areas, and one way is via gathering well-structured temporal and spatial accounts of zoonotic disease vectors. Due to its large biotic and abiotic heterogeneity, Mexico City provides favorable environmental conditions for the proliferation of mosquitoes including vector species of medical importance. Hereby we present the results of one year sampling (May 2023-April 2024) in eight contrasting locations in Mexico City. In total, 4,861 mosquitoes belonging to 11 species were captured, with Culex stigmatosoma and Culex restuans emerging as the most dominant, while Anopheles aztecus, Culex erythrothorax and Aedes trivittatus were the least abundant. Monthly fluctuations in the composition and abundance of species were observed at each site, highlighting cemeteries as the places with the highest mosquito density, making them key areas for monitoring. This research should serve local health authorities to guide mosquito control strategies, and two key candidates are Culex quinquefasciatus and Cx. restuans, particularly from June to November which is when their abundance reaches a peak.

Yeast encapsulation of photosensitive insecticides increases toxicity against mosquito larvae while protecting microorganisms

An important defense against the deadly diseases that mosquitoes transmit is the application of insecticides that reduce mosquito populations. Unfortunately, the evolution and subsequent spread of insecticide resistance has decreased their efficacy. Therefore, new mosquito control strategies are needed. One class of larvicides, known as photosensitive insecticides, or PSIs, kills larvae via light-activated oxidative damage. PSIs are promising larvicides because of their high larvicidal efficacy, rapid photodegradation, inexpensive cost, and mechanism that is dissimilar to other insecticide classes. We explored a novel delivery strategy for increasing both the larvicidal efficiency and environmental biocompatibility of PSIs, known as yeast encapsulation. Using the PSIs, curcumin and methylene blue, we measured the survival of Anopheles gambiae larvae and Escherichia coli following exposure to either non-encapsulated or yeast-encapsulated PSIs and a photoperiod. Yeast encapsulation increased the phototoxicity of both curcumin and methylene blue against mosquito larvae, likely by increasing ingestion. Furthermore, yeast encapsulation protected E. coli from the phototoxicity of yeast-encapsulated curcumin, but not yeast-encapsulated methylene blue. Yeast encapsulation increases the larvicidal efficacy of a PSI while also increasing biocompatibility. Therefore, yeast encapsulation of PSIs is a promising insecticide delivery strategy for mosquito control.

Synthesis and insecticidal assessment of nitrogenous heterocycles derived from 2-pyridone derivative against Culex pipiens L. Larvae

This study reports the synthesis of a novel 1,6-diamino-4-(2,4-dichlorophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (1) and its subsequent functionalization to yield a library of diverse heterocycles (2–10). The nucleophilicity of compound 1 was exploited to react with various electrophiles to afford a range of structurally enriched derivatives. The structures of all synthesized heterocycles were confirmed by elemental analysis and spectroscopic techniques as (FT-IR, 1H NMR, 13C NMR, and mass spectroscopy). Bioassays revealed significant larvicidal activity against mosquito larvae (Culex pipiens L.) for compounds 1–4, 6, 7 and 9. In silico docking studies suggest a potential mode of action through acetylcholinesterase (AChE) inhibition, potentially explaining the observed larvicidal effect. Additionally, interactions with other neuroreceptors were predicted. Finally, analyzing of the structure activity relationship (SAR) of the novel constructed heterocycles gave us a lot of interesting promising results for developing novel and sustainable mosquito control strategies.

Genome resequencing and genome-wide polymorphisms in mosquito vectors Aedes aegypti and Aedes albopictus from south India

Aedes aegypti and Aedes albopictus mosquitoes spread major vector-borne viral diseases in tropical and sub-tropical regions of the globe. In this study, we sequenced the genome of Indian Ae. aegypti and Ae. albopictus and mapped to their reference genomes. Comparative genomics were performed between our strain and the reference strains. A total of 14,416,484 single nucleotide polymorphisms (SNPs) and 156,487 insertions and deletions (InDels) were found in Ae. aegypti, and 28,940,433 SNPs and 188,987 InDels in Ae. albopictus. Particular emphasis was given to gene families involved in mosquito digestion, development, and innate immunity, which could be putative candidates for vector control. Serine protease cascades and their inhibitors called serpins, play a central role in these processes. We extracted high-impact variants in genes associated with serine proteases and serpins. This study reports for the first time a high coverage genome sequence data of an Indian Ae. albopictus mosquito. The results from this study will provide insights into Indian Aedes specific polymorphisms and the evolution of immune related genes in mosquitoes, which can serve as a resource for future comparative genomics and those pursuing the development of targeted biopesticides for effective mosquito control strategies.

Gene Drive and Symbiont Technologies for Control of Mosquito-Borne Diseases.

Mosquito-borne diseases, such as dengue and malaria, pose a significant burden to global health. Current control strategies with insecticides are only moderately effective. Scalable solutions are needed to reduce the transmission risk of these diseases. Symbionts and genome engineering-based mosquito control strategies have been proposed to address these problems. Bacterial, fungal, and viral symbionts affect mosquito reproduction, reduce mosquito lifespan, and block pathogen transmission. Field tests of endosymbiont Wolbachia-based methods have yielded promising results, but there are hurdles to overcome due to the large-scale rearing and accurate sex sorting required for Wolbachia-based suppression approaches and the ecological impediments to Wolbachia invasion in replacement approaches. Genome engineering-based methods, in which mosquitoes are genetically altered for the modification or suppression of wild populations, offer an additional approach for control of mosquito-borne diseases. In particular, the use of gene drive alleles that bias inheritance in their favor is a potentially powerful approach. Several drives are frequency dependent, potentially giving them broadly similar population dynamics to Wolbachia. However, public acceptance and the behavior of released drives in natural mosquito populations remain challenges. We summarize the latest developments and discuss the knowledge gaps in both symbiont- and gene drive-based methods.

High-Throughput Screening System Evaluation of Andrographis paniculata (Burm.f.) Extracts and Their Fractions against Mosquito Vectors.

Infectious diseases that cause illness and/or death in humans can be contracted from mosquito bites. A viable and alternate method of personal protection that can lower the danger of human exposure to mosquito-borne diseases is the use of plant-based repellents. Using a high-throughput screening system, the current work examined the toxicity, contact irritancy, and spatial repellency of Andrographis paniculata crude extract and its fractions against Aedes aegypti, Anopheles minimus, and An. dirus. Five fractions (i.e., F1, F2, F3, F4, and F5) were separated from the crude extract by column and thin layer chromatography and analyzed using high-performance liquid chromatography and mass spectrometry. The major active compounds identified from F3 and F5 were 4-deoxy-11,12-didehydroandrographolide and andrographolide. Three concentrations (1.0, 2.5, and 5.0%) for each of the crude extracts and the five fractions were individually impregnated on nylon netting strips and evaluated against the three mosquito species. Results showed that the highest contact irritancy was elicited by the crude extract at 5% concentration against Ae. aegypti (43.70% escaped). Results of the spatial activity index (SAI) showed that fractions F3 and F5 at 2.5% demonstrated the strongest repellency against Ae. aegypti (SAI = 0.84) and An. minimus (SAI = 0.83), respectively. Both the crude extract and its components did not cause any knockdown or mortality. These findings suggest that fractionation of A. paniculata extracts is valuable in assessing their spatial repellent efficacy against mosquitoes. Fractions F3 and F5 hold promise as natural mosquito repellents and could contribute to developing effective mosquito control strategies.

Larvicidal potential of Trachyspermum ammi essential oil and Delphinium speciosum extract against malaria, dengue, and filariasis mosquito vectors

Mosquito-borne diseases, such as malaria, dengue, and Zika, pose major public health challenges globally, affecting millions of people. The growing resistance of mosquito populations to synthetic insecticides underscores the critical need for effective and environmentally friendly larvicides. Although chemical pesticides can initially be effective, they often lead to negative environmental consequences and health hazards for non-target species, including humans. This study aimed to evaluate the larvicidal effects of Trachyspermum ammi essential oil and Delphinium speciosum extract on the larvae of three major mosquito species: Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Mosquito larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were reared under controlled laboratory conditions. The larvicidal activity of T. ammi essential oil and D. speciosum extract was evaluated through standard bioassays, using various concentrations of essential oils (10, 20, 40, 80, and 160 ppm) and extracts (160, 320, 640, 1280, and 2560 ppm) to determine the lethal concentration (LC50) values after 24 h of exposure. Fresh plant materials were collected, with the essential oil extracted via hydro-distillation, and the extract prepared using methanol solvent extraction. The chemical composition of T. ammi essential oil was examined using gas chromatography-mass spectrometry (GC-MS). Additionally, the preliminary analysis of the chemical compounds in D. speciosum extract was carried out using thin layer chromatography (TLC) and nuclear magnetic resonance spectroscopy (NMR) techniques. The results indicated that the essential oil of T. ammi exhibited more effective larvicidal activity compared to the D. speciosum extract. Specifically, the essential oil demonstrated LC50 values of 18 ppm for Cx. quinquefasciatus and 19 ppm for Ae. aegypti. In contrast, the D. speciosum extract showed the strongest larvicidal effect against An. stephensi, with an LC50 of 517 ppm. Concentrations of 40 ppm of the essential oil and 1280 ppm of the extract resulted in 100% mortality across all three species. Both the essential oil of T. ammi and the D. speciosum extract exhibited concentration-dependent larvicidal activity, and these results were statistically significant (p < 0.001) compared to the no-treatment group. GC-MS analysis revealed thymol (88.95%), o-cymen-5-ol (4.11%), and γ-terpinene (2.10%) as the major constituents of the T. ammi essential oil. Additionally, TLC verified the presence of alkaloids in both chloroform and methanolic extracts. Proton NMR identified a diterpene structure for these alkaloids. These findings suggest that T. ammi essential oil is a promising candidate for natural mosquito control strategies. Given its efficacy, further research is warranted to explore its potential in integrated vector management programs.

Characterization of the microbiome of Aedes albopictus populations in different habitats from Spain and São Tomé

The mosquito microbiome significantly influences vector competence, including in Aedes albopictus, a globally invasive vector. Describing the microbiome and Wolbachia strains of Ae. albopictus from different regions can guide area-specific control strategies. Mosquito samples from Spain and São Tomé were analyzed using 16S rRNA gene sequencing and metagenomic sequencing. Wolbachia infection patterns were observed by sex and population. Female mosquitoes were blood-fed, a factor considered in analyzing their microbiota. Results revealed a dominance of dual Wolbachia infections, strains A and B, in the microbiome of both populations of Ae. albopictus, especially among females. Both populations shared a core microbiome, although 5 and 9 other genera were only present in Spain and São Tomé populations, respectively. Genera like Pelomonas and Nevskia were identified for the first time in Aedes mosquitoes. This study is the first to describe the Ae. albopictus bacteriome in Spain and São Tomé, offering insights for the development of targeted mosquito control strategies. Understanding the specific microbiome composition can help in designing more effective interventions, such as microbiome manipulation and Wolbachia-based approaches, to reduce vector competence and transmission potential of these mosquitoes.

The complexities of blood-feeding patterns in mosquitoes and sandflies and the burden of disease: A minireview.

Mosquitoes and sandflies exhibit a wide range of blood feeding patterns, targeting a wide range of vertebrate species, including birds, mammals, reptiles, and amphibians, for proteins vital for egg development. This broad host range increases the opportunity for them to acquire pathogens of numerous debilitating-and-fatal diseases from various animal reservoirs, playing a significant role in disease crossover between animals and humans, also known as zoonotic transmission. This review focuses on the intricate blood-feeding habits of these dipteran vectors, their sensory systems and the complex dance between host and pathogen during disease transmission. We delve into the influence of blood sources on pathogen spread by examining the insect immune response and its intricate interplay with pathogens. The remarkable sense of smell guiding them towards food sources and hosts is explored, highlighting the interplay of multiple sensory cues in their navigation. Finally, we examine the challenges in mosquito control strategies and explore innovations in this field, emphasizing the need for sustainable solutions to combat this global health threat. By understanding the biology and behaviour of these insects, we can develop more effective strategies to protect ourselves and mitigate the burden of vector-borne diseases.

The invasive Aedes albopictus in the Doñana World Heritage Site

BackgroundAedes albopictus is catalogued as one of the 100 most dangerous species worldwide. Native to Asia, the species has drastically increased its distribution range, reaching all continents except Antarctica. The presence of Ae. albopictus in Spain was first reported in 2004 in Cataluña (NE Spain), and it is spreading in the country.MethodsWe conducted an extensive mosquito monitoring study in the natural protected area of the Doñana National Park (SW Spain) in 2023. After identifying the presence of Ae. albopictus, a mosquito control strategy was developed and implemented to eradicate the species in the area.ResultsOverall, 12,652 mosquito females of 14 different species were captured at nine sites within the park. For the first time, the presence of Ae. albopictus was recorded in the area, despite intensive trapping performed at some localities since 2003. The presence of this invasive species in the park is most likely linked to human activities, potentially facilitated by daily car trips of personnel. Although larvae of Culex, Anopheles, and Culiseta mosquitoes were identified in these containers, the presence of Ae. albopictus larvae was not recorded in those locations. In spite of that, the biological larvicide Bacillus thuringiensis israelensis (Bti) was applied to artificial containers potentially used by Ae. albopictus as breeding sites.ConclusionsThis work evidences the high capacity of Ae. albopictus to reach highly conserved natural areas far from urban foci. We discuss the implications of the presence of Ae. albopictus in this endangered ecosystem and the potential control measures necessary to prevent its reintroduction.Graphical

A Review of the Latest Control Strategies for Mosquito-Borne Diseases.

Mosquito-borne diseases are persistent and potentially severe posing a threat to global pandemic preparedness. The risk of mosquito-borne virus transmission is rapidly increasing due to the unprecedented spread of viruses such as dengue and chikungunya, the disruption of global mosquito-borne disease control efforts following the emergence of coronavirus diseases 2019 (COVID-19) in 2019, global warming, and human activities. To address this global challenge, various innovative mosquito control technologies are being developed worldwide. This paper summarizes the latest advances in mosquito vector control, focusing on China's latest mosquito control strategies, to provide insights into implementing novel mosquito-borne disease control measures.

The odorant-binding protein genes obp67 and obp56d-like encode products that guide oviposition site selection in the Asian tiger mosquito, Aedes albopictus.

Aedes albopictus is an important vector of arboviruses and prefers small containers of stagnant water as oviposition sites. One of the mechanisms mosquitoes use to search for suitable oviposition sites is relying on odor cues from prospective sites and their surroundings. The genetic and molecular bases of this behavior are not known for Ae.albopictus. Oviposition site-searching behavior can be separated into 2 stages: container location and water detection. We applied a glue compound to the antennae and the maxillary palps of adult females to mask their ability to detect molecules that may guide them to preferred oviposition sites. Treatment of the antennae significantly reduces the location index (P<0.001), indicating a decreased ability to find oviposition sites, whereas no significant difference was observed in mosquitoes with maxillary palps treated with the same glue compound (P>0.05). The detection time, measured as the duration from contact with the water surface to the deposition of the first egg, was extended in mosquitoes with treated antennae or maxillary palps, supporting the conclusion that olfaction is involved in the detection of oviposition site. Transcriptomic analysis identified differentially expressed olfactory-related genes, including obp67, obp56d-like, obp19d-like and obp67-like. RNA interference (RNAi)-mediated knockdown of obp67 and obp56d-like significantly affected the location index and detection time, respectively. Cas9/guide RNA-mediated knockout of obp56d-like resulted in a prolonged detection time, compared with the wild type (P<0.05). These findings help to elucidate aspects of the olfactory mechanisms involved in Ae.albopictus oviposition site selection, and provide a basis for the development of mosquito surveillance and control strategies.

Rhodamine-B for the mark, release, and recapture experiments in gamma-irradiated male Aedes aegypti (Diptera: Culicidae): Persistence, dispersal, and its effect on survival.

Rhodamine-B (Rh-B) marking shows a great potential for use in mark-release-recapture (MRR) studies for rear-and-release mosquito control strategies, including the radiation-based sterile insect technique. However, its applicability and evaluation in body-stain-irradiated males of Aedes aegypti have received little attention. The present study evaluated the use of Rh-B to mark gamma-irradiated male A. aegypti. Male A. aegypti were irradiated at the pupal stage at a dose of 70 Gy. After emergence, males were fed 0.1, 0.2, 0.3, or 0.4% Rh-B in 10% glucose solution for 4 days. Groups of unirradiated males that received the same feeding treatments were used as control groups. We evaluated the persistence of Rh-B and the longevity of males after Rh-B feeding. Furthermore, the use of Rh-B in irradiated A. aegypti for MRR experiments was evaluated at an urban site. No difference was observed in the Rh-B persistence among all concentrations at the 24-h postmarking period ranging from 91.25 ± 1.61% to 96.25 ± 1.61% and from 90.00 ± 2.28% to 93.13 ± 2.77% for the unirradiated and irradiated groups, respectively. Rh-B persistence significantly decreased over time, and persistence was significantly longer with increased concentrations in both the unirradiated and irradiated groups. Longevity was considerably decreased by Rh-B feeding and irradiation. However, no significant difference in longevity was found among males fed various concentrations of Rh-B. Through MRR experiments, irradiated-Rh-B marked males were mostly detected within a radius of 20 m and 40 m from the center-release point. The mean distance traveled of the released males from the three MRR events was calculated to be 42.6 m. This study confirms that Rh-B body marking through sugar feeding is applicable for irradiated male A. aegypti, with only a slight effect on longevity. Furthermore, considering the significant reduction in persistence over time, further study is needed to assess the impact of this reduction on the calculation of field biological parameters resulting from MRR experiments.

DENGUE MANAGEMENT THROUGH MOSQUITO CONTROL: ASPHYXIATION TECHNIQUES AND HOMEMADE REPELLENTS FOR SKIN PROTECTION

Mosquito-borne diseases pose significant health challenges globally, particularly in regions such as Bangladesh where chikungunya and dengue have been prevalent in recent years. These diseases are transmitted through the bites of infected mosquitoes, which breed in stagnant water. Common mosquito-borne diseases include chikungunya, dengue, malaria, and Zika virus, among others. Efforts to control mosquito populations have included the destruction of breeding sites, the application of larvicides, and more recently, the use of Bacillus thuringiensis israelensis (BTI) in Dhaka. While these measures have helped manage chikungunya, dengue remains a persistent issue despite ongoing control efforts. This paper explores innovative mosquito control strategies, focusing on asphyxiation techniques to reduce mosquito populations and the use of homemade repellents for personal protection against mosquito bites. The effectiveness of these methods in the context of dengue management is critically examined.

Synthesis, insecticidal Activity, and molecular docking analysis of some benzo[h]quinoline derivatives against Culex pipiens L. Larvae

Some heterocycles bearing a benzo[h]quinoline moiety were synthesized through treating a 3-((2-chlorobenzo[h]quinolin-3-yl)methylene)-5-(p-tolyl)furan-2(3H)-one with four nitrogen nucleophiles comprising ammonium acetate, benzylamine, dodecan-1-amine, and 1,2-diaminoethane. Also, thiation reactions of furanone and pyrrolinone derivatives were investigated. The insecticidal activity of these compounds against mosquito larvae (Culex pipiens L.) was evaluated. All tested compounds exhibited significant larvicidal activity, surpassing that of the conventional insecticide chlorpyrifos. In silico docking analysis revealed that these compounds may act as acetyl cholinesterase (AChE) inhibitors, potentially explaining their larvicidal effect. Additionally, interactions with other neuroreceptors, such as nicotinic acetylcholine receptor and sodium channel voltage-gated alpha subunit were also predicted. The results obtained from this study reflected the potential of benzo[h]quinoline derivatives as promising candidates for developing more effective and sustainable mosquito control strategies. The ADME (absorption, distribution, metabolism, and excretion) analyses displayed their desirable drug-likeness and oral bioavailability properties.

Assessment of toxicity of pyriproxyfen, Bacillus thuringiensis, and malathion and their mixtures used for mosquito control on embryo-larval development and behavior of zebrafish.

Pyriproxyfen (PPF), Bacillus thuringiensis israelensis (BTI), and malathion (MLT) are widely used worldwide to control the population of mosquitos that transmit arboviruses. The current work aimed to evaluate the toxicity of these single pesticides and their binary mixtures of PPF + BTI, PPF + MLT, and MLT + BTI on the embryo-larval stage of zebrafish (Danio rerio) as an animal model. Epiboly, mortality, apical endpoints, affected animals, heart rate, morphometric, thigmotaxis, touch sensitivity, and optomotor response tests were evaluated. PPF and MLT and all mixtures reduced the epiboly percentage. Mortality increased significantly in all exposed groups, except BTI, with MLT being the most toxic. The observed apical endpoints were pericardial and yolk sac edemas, and tail and spine deformation. Exposure to MLT showed a higher percentage of affected animals. A reduction in heart rate was also observed in MLT- and PPF + MLT-exposed groups. The PPF + MLT mixture decreased head measurements. Behavioral alterations were observed, with a decrease in thigmotaxis and touch sensitivity responses in PPF + MLT and MLT + BTI groups. Finally, optomotor responses were affected in all groups. The above data obtained suggest that the MLT + PFF mixture has the greatest toxicity effects. This mixture affected embryo-larval development and behavior and is close to the reality in several cities that use both pesticides for mosquito control rather than single pesticides, leading to a reevaluation of the strategy for mosquito control.

Impact of Ae-GRD on Ivermectin Resistance and Its Regulation by miR-71-5p in Aedes aegypti.

iGABAR, a member of the Cys-loop ligand-gated ion channel superfamily, is a significant target of the insecticide ivermectin (IVM). GRD is the potential subunit of the insect iGABAR. However, little information about GRD in Ae. aegypti has been reported. In this study, we involved cloning and characterizing the iGABAR subunit GRD of Ae. aegypti (Ae-GRD). Sequence analysis indicated that Ae-GRD, as part of the cysteine-loop ligand-gated ion channel family, is similar to other insect GRD. RNA interference (RNAi) was employed to explore IVM resistance in Ae. aegypti, resulting in a significant reduction in Ae-GRD expression (p < 0.05), and the mortality of Ae. aegypti adults with Ae-GRD knockdown was significantly decreased after exposure to ivermectin. Bioinformatics prediction identified miR-71-5p as a potential regulator of Ae-GRD. In vitro, dual-luciferase reporter assays confirmed that Ae-GRD expression was regulated by miR-71-5p. Microinjection of miR-71-5p mimics upregulated miR-71-5p expression and downregulated Ae-GRD gene expression, reducing mortality by 34.52% following IVM treatment. Conversely, microinjection of a miR-71-5p inhibitor decreased miR-71-5p expression but did not affect the susceptibility to IVM despite increased Ae-GRD expression (p < 0.05). In conclusion, Ae-GRD, as one of the iGABA receptor subunits, is a potential target of ivermectin. It may influence ivermectin resistance by modulating the GABA signaling pathway. The inhibition of Ae-GRD expression by miR-71-5p decreased ivermectin resistance and consequently lowered the mortality rate of Ae. aegypti mosquitoes. This finding provides empirical evidence of the relationship between Ae-GRD and its miRNA in modulating insecticide resistance, offering novel perspectives for mosquito control strategies.