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1. A regional One Health approach to the risk of invasion by Anopheles stephensi in Mauritius

   https://doi.org/10.1371/journal.pntd.0011827  

   Iyaloo, Diana P; Zohdy, Sarah; Carney, Ryan M; Mosawa, Varina Ramdonee; Elahee, Khouaildi B; Munglee, Nabiihah; Latchooman, Nilesh; Puryag, Surendra; Bheecarry, Ambicadutt; Bhoobun, Hemant; et al (September 2024, PLOS Neglected Tropical Diseases)

   Christofferson, Rebecca C (Ed.)

   BackgroundAnopheles stephensiis an invasive malaria vector in Africa that threatens to put an additional 126 million people at risk of malaria if it continues to spread. The island nation of Mauritius is highly connected to Asia and Africa and is at risk of introduction due to this connectivity. For early detection ofAn.stephensi, the Vector Biology and Control Division under the Ministry of Health in Mauritius, leveraged a well-establishedAedesprogram, asAn.stephensiis known to shareAedeshabitats. These efforts triggered multisectoral coordination and cascading benefits of integrated vector and One Health approaches. MethodsBeginning June 2021, entomological surveys were conducted at points of entry (seaport, airport) and on ships transporting livestock in collaboration with the Civil Aviation Department, the Mauritian Port Authority and National Veterinary Services.A total of 18, 39, 723 mosquito larval surveys were respectively conducted in the airport, seaport, and other localities in Mauritius while two, 20, and 26 adult mosquito surveys were respectively conducted in the airport, seaport, and twenty-six animal assembly points. Alongside adult mosquito surveys, surveillance of vectors of veterinary importance (e.g.-Culicoidesspp.) was also carried out in collaboration with National Parks and Conservation Service and land owners. ResultsA total of 8,428 adult mosquitoes were collected and 1,844 larval habitats were positive for mosquitoes. All collected mosquitoes were morphologically identified and 151Anophelesand 339Aedesmosquitoes were also molecularly characterized. Mosquito species detected wereAedes albopictus,Anopheles arabiensis,An.coustani,An.merus,Culex quinquefasciatus,Cx.thalassiusandLutzia tigripes.Anopheles stephensiwas not detected. The One Health approach was shared with the French Agricultural Research Centre for International Development (CIRAD), strengthening collaboration between Mauritius and Réunion Island on vector surveillance at entry points and insecticide resistance monitoring. The Indian Ocean Commission (IOC) was also alerted to the risk ofAn.stephensi, leading to regional efforts supporting trainings and development of a response strategy toAn.stephensibringing together stakeholders from Comoros, Madagascar, Mauritius, Réunion Island and Seychelles. ConclusionsMauritius is a model system showing how existing public health entomology capabilities can be used to enhance vector surveillance and control and create multisectoral networks to respond to any emerging public and veterinary health vector-borne disease threat. 

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2. Assessing Temperature-Dependent Deltamethrin Toxicity in Various kdr Genotypes of Aedes aegypti Mosquitoes

   https://doi.org/10.3390/insects16030254  

   Kalmouni, Joshua; Jensen, Brook M; Ain, Joshua; Paaijmans, Krijn P; Huijben, Silvie (March 2025, Insects)

   Insecticide resistance surveillance systems for vector-borne diseases are crucial for early detection of resistance and the implementation of evidence-based resistance management strategies. While insecticide susceptibility bioassays are typically conducted under controlled laboratory conditions, mosquitoes in the field experience varying environmental conditions, with temperature being a key determinant. Understanding the relationship between temperature and insecticide toxicity is essential for interpreting and extrapolating assay results across different climate zones or more locally across days with different weather conditions. In this study, we examined Aedes aegypti mosquitoes with different genetic backgrounds of insecticide resistance. Mosquitoes were homozygous for the knockdown resistance (kdr) F1534C mutation, plus either (1) homozygous for the kdr 1016V wildtype allele, (2) homozygous for the kdr V1016I mutant allele, or (3) heterozygous genetic crosses. These three genotypes were exposed to deltamethrin using WHO tube tests at three temperatures (22 °C, 27 °C, and 32 °C) and varying dosages. LC50 values were determined for each genotype and temperature combination. A negative temperature coefficient was observed exclusively in female mosquitoes homozygous for the 1016V wildtype allele, indicating reduced pyrethroid toxicity at higher temperatures. No temperature–toxicity relationship was found in males of this genotype or in other genotypes of either sex. These findings suggest that temperature may interact with kdr mutations and possibly even sex, highlighting the complex interactions between genetic mutations and environmental factors, such as temperature, in determining the insecticide resistance phenotype. Given the wide distribution of Ae. aegypti, understanding how local climate conditions influence insecticide performance will help improve control strategies and slow resistance evolution, protecting public health efforts against mosquito-borne diseases 

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3. Expression of endogenous Anopheles gambiae microRNAs using an Anopheles gambiae densovirus (AgDNV) intronic expression system

   https://doi.org/10.1186/s13071-025-06994-7  

   Johnson, Rebecca_M; Metz, Hillery_C; Suzuki, Yasutsugu; McLean, Kyle_J; Rasgon, Jason_L (August 2025, Parasites & Vectors)

   Abstract BackgroundAnopheles gambiaedensovirus (AgDNV) is a highly species-specific parvovirus that reaches high titers in adultAnopheles gambiaemosquitoes with few transcriptomic effects and minimal significant fitness effects. Given these characteristics, AgDNV has been proposed as a viral vector for basic research and mosquito control. Previous work created an AgDNV co-expression system with a wild-type AgDNV helper plasmid and a transducing plasmid expressing enhanced green fluorescent protein (EGFP) that can be used to co-transfect cells to generate infectious recombinant transducing AgDNV virions. Generated virions infect theAn. gambiaemidgut, fat body, and ovaries, yet this viral vector system is limited in the size of transgenes that can be expressed due to capsid packaging limitations. MethodsConsidering these size constraints, we created an artificial intron within the EGFP gene of the transducing construct that can express small pieces of genetic material such as microRNAs (miRNAs), microRNA sponges, or other small sequences. Placement of this intron in EGFP created a fluorescent reporter such that incorrect splicing produces a frameshift mutation in EGFP and an early stop codon, whereas correct splicing results in normal EGFP expression and co-transcription of the intronic genetic cargo. A selection of miRNAs with predicted or demonstrated importance in mosquito immunity and reproduction with expression localized to the fat body or ovaries were chosen as intronic cargo. Construct expression and splicing was evaluated, and the impact of miRNA expression on putative miRNA targets was measuredin vitroandin vivo. ResultsThe created intron was correctly spliced in cells and mosquitoes; however, miRNA delivery resulted in inconsistent changes to miRNA and predicted target gene transcript levels—possibly due to organ-specific miRNA expression or inaccurate putative target predictions leading to miRNA–target gene sequence mismatch. ConclusionsAlthough our results on target gene expression were inconsistent, with optimization this viral vector and developed intron have potential as an expression tool withinAn. gambiaemosquitoes or cell lines. Graphical Abstract 

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4. Comparison of the variability in mortality data generated by CDC bottle bioassay, WHO tube test, and topical application bioassay using Aedes aegypti mosquitoes

   https://doi.org/10.1186/s13071-022-05583-2  

   Althoff, Rachel A.; Huijben, Silvie (December 2022, Parasites & Vectors)

   Abstract BackgroundInsecticide resistance remains a major public health problem. Resistance surveillance is critical for effective vector control and resistance management planning. Commonly used insecticide susceptibility bioassays for mosquitoes are the CDC bottle bioassay and the WHO tube test. Less commonly used in the field but considered the gold standard for assessing insecticide susceptibility in the development of novel insecticides is the topical application bioassay. Each of these bioassays has critical differences in how they assess insecticide susceptibility that impacts their ability to differentiate between resistant and susceptible populations or determine different levels of resistance intensity. MethodsWe compared the CDC bottle bioassay, the WHO tube test, and the topical application bioassay in establishing the dose–response against deltamethrin (DM) using the DM-resistantAedes aegyptistrain MC1. Mosquitoes were exposed to a range of insecticide concentrations to establish a dose–response curve and assess variation around model predictions. In addition, 10 replicates of 20–25 mosquitoes were exposed to a fixed dose with intermediate mortality to assess the degree of variation in mortality. ResultsThe topical application bioassay exhibited the lowest amount of variation in the dose–response data, followed by the WHO tube test. The CDC bottle bioassay had the highest level of variation. In the fixed-dose experiment, a higher variance was similarly found for the CDC bottle bioassay compared with the WHO tube test and topical application bioassay. ConclusionThese data suggest that the CDC bottle bioassay has the lowest power and the topical application bioassay the highest power to differentiate between resistant and susceptible populations and assess changes over time and between populations. This observation has significant implications for the interpretation of surveillance results from different assays. Ultimately, it will be important to discuss optimal insecticide resistance surveillance tools in terms of the surveillance objective, practicality in the field, and accuracy of the tool to reach that objective. Graphical Abstract 

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5. Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control

   https://doi.org/10.1534/g3.120.401133  

   Macias, Vanessa M.; McKeand, Sage; Chaverra-Rodriguez, Duverney; Hughes, Grant L.; Fazekas, Aniko; Pujhari, Sujit; Jasinskiene, Nijole; James, Anthony A.; Rasgon, Jason L. (April 2020, G3: Genes|Genomes|Genetics)

   Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles ) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control ( Re ceptor- m ediated O vary T ransduction of C argo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles , ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species. 

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