Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes.

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.

Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

An Optimized/Scale Up-Ready Protocol for Extraction of Bacterially Produced dsRNA at Good Yield and Low Costs.

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates.

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Evaluation of Hydrogen Peroxide Fumigation and Heat Treatment for Standard Emergency Arthropod Inactivation in BSL-3 Insectaries.

Climate change and global movements of people and goods have accelerated the spread of invasive species, including insects that vector infectious diseases, which threaten the health of more than half of the world's population. Increasing research efforts to control these diseases include the study of vector - pathogen interactions, involving the handling of pathogen-infected vector insects under biosafety level (BSL) 2 and 3 conditions. Like microbiology BSL-3 laboratories, BSL-3 insectaries are usually subjected to fixed-term or emergency room decontamination using recognized methods such as hydrogen peroxide (H2O2) or formaldehyde fumigation. While these procedures have been standardized and approved for the inactivation of diverse pathogens on surfaces, to date, there are no current standards for effective room-wide inactivation of insects in BSL-3 facilities in case of an emergency such as the accidental release of a large number of infected vectors. As H2O2 is often used for standard room decontamination in BSL-3 facilities, we evaluated H2O2 fumigation as a potential standard method for the safe, room-wide deactivation of insects in BSL-3 insectaries in comparison to heat treatment. To account for physiological diversity in vector insect species, six species from three different orders were tested. For the H2O2 fumigation we observed a strong but also varying resilience across all species. Lethal exposure time for the tested dipterans ranged from nine to more than 24 h. Furthermore, the coleopteran, Tribolium castaneum, did not respond to continuous H2O2 exposure for 48 h under standard room decontamination conditions. In contrast, temperatures of 50°C effectively killed all the tested species within 2 to 10 min. The response to lower temperatures (40-48°C) again showed a strong variation between species. In summary, results suggest that H2O2 fumigation, especially in cases where a gas generator is part of the laboratory equipment, may be used for the inactivation of selected species but is not suitable as a general emergency insect inactivation method under normal room decontamination conditions. In contrast, heat treatment at 48 to 50°C has the potential to be developed as an approved standard procedure for the effective inactivation of insects in BSL-3 facilities.

Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2.

The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

Molecular tools to create new strains for mosquito sexing and vector control.

Vector control programs based on population reduction by matings with mass-released sterile insects require the release of only male mosquitoes, as the release of females, even if sterile, would increase the number of biting and potentially disease-transmitting individuals. While small-scale releases demonstrated the applicability of sterile males releases to control the yellow fever mosquito Aedes aegypti, large-scale programs for mosquitoes are currently prevented by the lack of efficient sexing systems in any of the vector species.Different approaches of sexing are pursued, including classical genetic and mechanical methods of sex separation. Another strategy is the development of transgenic sexing systems. Such systems already exist in other insect pests. Genome modification tools could be used to apply similar strategies to mosquitoes. Three major tools to modify mosquito genomes are currently used: transposable elements, site-specific recombination systems, and genome editing via TALEN or CRISPR/Cas. All three can serve the purpose of developing sexing systems and vector control strains in mosquitoes in two ways: first, via their use in basic research. A better understanding of mosquito biology, including the sex-determining pathways and the involved genes can greatly facilitate the development of sexing strains. Moreover, basic research can help to identify other regulatory elements and genes potentially useful for the construction of transgenic sexing systems. Second, these genome modification tools can be used to apply the gained knowledge to build and test mosquito sexing strains for vector control.

Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing enormous economic damage in horticultural systems. A successful and environment-friendly control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. However, the SIT is not readily applicable to each pest species. While transgenic approaches hold great promise to improve critical aspects of the SIT to transfer it to new species, they are suspect to strict or even prohibitive legislation regarding the release of genetically modified (GM) organisms. In contrast, specific mutations created via CRISPR-Cas genome editing are not regulated as GM in the US, and might thus allow creating optimal strains for SIT. Here, we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring. Based on the achieved efficiency, this approach could also be used to introduce mutations which do not produce a screenable phenotype and identify positive mutants with a reasonable workload. Furthermore, CRISPR-Cas HDR would allow to recreate mutations formerly identified in classical mutagenesis screens and to transfer them to related species to establish new (SIT-like) pest control systems. Considering the potential that CRISPR-induced alterations in organisms could be classified as non-GM in additional countries, such new strains could potentially be used for pest control applications without the need to struggle with GMO directives.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

Localization of Prp8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy.

The U4/U6-U5 tri-small nuclear ribonucleoprotein (snRNP) is a major, evolutionarily highly conserved spliceosome subunit. Unwinding of its U4/U6 snRNA duplex is a central event of spliceosome activation that requires several components of the U5 portion of the tri-snRNP, including the RNA helicase Brr2, Prp8 and the GTPase Snu114. Here we report the EM projection structure of the Saccharomyces cerevisiae tri-snRNP. It shows a modular organization comprising three extruding domains that contact one another in its central portion. We have visualized genetically tagged tri-snRNP proteins by EM and show here that U4/U6 snRNP forms a domain termed the arm. Conversely, a separate head domain adjacent to the arm harbors Brr2, whereas Prp8 and the GTPase Snu114 are located centrally. The head and arm adopt variable relative positions. This molecular organization and dynamics suggest possible scenarios for structural events during catalytic activation.