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1. ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae)

   https://doi.org/10.1093/jme/tjab016  

   Li, Xixi; Xu, Yang; Zhang, Hongbo; Yin, Haitao; Zhou, Dan; Sun, Yan; Ma, Lei; Shen, Bo; Zhu, Changliang (February 2021, Journal of Medical Entomology)

   Slotman, Michel (Ed.)

   Abstract The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes—14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control. 

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2. Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR‐Cas9 ribonucleoprotein

   https://doi.org/10.1111/imb.12663  

   Chaverra‐Rodriguez, D.; Dalla Benetta, E.; Heu, C. C.; Rasgon, J. L.; Ferree, P. M.; Akbari, O. S. (August 2020, Insect Molecular Biology)

   Abstract CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel waspNasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adultN. vitripennisfemales using either ReMOT control (Receptor‐Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used theDrosophila melanogaster‐derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of thecinnabargene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR‐applied genetic manipulation in this and other rising model organisms. 

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3. The Development and Expansion of in vivo Germline Editing Technologies in Arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and Beyond

   https://doi.org/10.1093/icb/icad123  

   Terradas, Gerard; Macias, Vanessa_M; Peterson, Hillary; McKeand, Sage; Krawczyk, Grzegorz; Rasgon, Jason_L (September 2023, Integrative And Comparative Biology)

   Synopsis In the past 20 years, sequencing technologies have led to easy access to genomic data from nonmodel organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene-editing vectors into early stage embryos have been used for arthropod transgenesis and the discovery of Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR–Cas) technologies allowed for targeted mutagenesis and the creation of knockouts or knock-ins in arthropods. Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which require expensive equipment and extensive hands-on training. ReMOT Control’s main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges in the application across species after 5 years since its development, including novel extensions of the technique such as direct parental (DIPA)-CRISPR. 

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4. Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

   https://doi.org/10.1371/journal.ppat.1009131  

   Shaw, W. Robert; Holmdahl, Inga E.; Itoe, Maurice A.; Werling, Kristine; Marquette, Meghan; Paton, Douglas G.; Singh, Naresh; Buckee, Caroline O.; Childs, Lauren M.; Catteruccia, Flaminia (December 2020, PLOS Pathogens)

   Billker, Oliver (Ed.)

   Many mosquito species, including the major malaria vector Anopheles gambiae , naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P . falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An . gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R 0 , and find the average R 0 is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions. 

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5. 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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