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Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti [1]

['Shih-Che Weng', 'School Of Biological Sciences', 'Department Of Cell', 'Developmental Biology', 'University Of California', 'San Diego', 'La Jolla', 'California', 'United States Of America', 'Igor Antoshechkin']

Date: 2023-12

Abstract Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.

Author summary Female mosquitoes, exclusive blood-feeders and carriers of dangerous human diseases, necessitate elimination before implementing genetic biocontrol methods. Enter SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter), a robust sex-sorting approach with key attributes: (i) harnessing male-specific expression through sex-specific alternative splicing (SSAS) of a safe, bright fluorescent marker; (ii) early larval sex-sorting and beyond; (iii) adaptable high-throughput sorting with COPAS; (iv) independence from sex-chromosome linkage; (v) genetic stability, resistant to meiotic recombination or chromosomal rearrangements; (vi) allowing positive selection of males for added reliability; (vii) transferable to various species through ubiquitous sex-specific alternative splicing, cross-species compatible promoters, and markers; and (viii) enabling early-stage mosquito separation for efficient male mosquito production. SEPARATOR reliably separates sexes during Aedes aegypti larval and pupal stages, and facilitates transcriptome sequencing of early larval males and females, revealing sex-specific mosquito genes. This method streamlines male production for release programs, offering cross-species adaptability to support genetic biocontrol initiatives.

Citation: Weng S-C, Antoshechkin I, Marois E, Akbari OS (2023) Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti. PLoS Genet 19(11): e1011065. https://doi.org/10.1371/journal.pgen.1011065 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, UNITED STATES Received: August 14, 2023; Accepted: November 14, 2023; Published: November 27, 2023 Copyright: © 2023 Weng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Complete sequence maps and plasmids are deposited at Addgene.org (#200012). All Illumina and Nanopore sequencing data has been deposited to the NCBI-SRA (PRJNA985064). All data used to generate figures are provided in the Supplementary Materials/Tables. Funding: This work was supported by funding from an NIH awards (R01AI151004, R01GM132825, RO1AI148300, RO1AI175152, DP2AI152071), EPA STAR award (RD84020401), and an Open Philanthropy award (309937-0001) awarded to O.S.A. E.M. received funding from Agence Nationale de la Recherche through grant #ANR-19-CE35-0007 GDaMO. The views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the U.S. government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: O.S.A. and S.W. have filed a patent on this technology. O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. All other authors declare no competing interests.

Introduction The mosquito is the most deadly animal on the planet, estimated to have killed roughly 50% of humans since the dawn of humanity [1]. This year, roughly 700 million cases of mosquito transmitted diseases worldwide shall occur resulting in over a million deaths [2]. Insecticides and larvicides have traditionally been used to control mosquitoes, however mosquitoes have evolved resistance [3–7], and harmful chemicals are unfortunately poisoning our environment [8–10] and killing non-target species such as pollinators [11–13]. Moreover, a warming global climate is expanding habitable ranges of mosquitoes and their pathogens resulting in an increased number of people at risk of contracting mosquito transmitted pathogens [14–16]. Given these issues, innovative technologies are critical to combat this expanding global threat. To genetically suppress mosquito populations, several biocontrol strategies have been developed including the classical sterile insect technique (SIT) [17], release of insects carrying a dominant lethal (RIDL) [18], female specific RIDL (fsRIDL) [19–23], Wolbachia-based incompatible insect technique (IIT) [24], the precision guided sterile insect technique (pgSIT)[25], Ifegenia (inherited female elimination by genetically encoded nucleases to interrupt alleles)[26], and homing-based suppression gene drives [27]. The ultimate goal for each of these approaches is to scalably release modified mosquitoes into the environment to safely achieve sustained species-specific population suppression. Given that only female mosquitoes consume blood and transmit pathogens, exclusive release of male-only cohorts is critical. While some of these approaches have built-in self-sexing mechanisms (e.g. gene drives [27], fsRIDL [23], Ifegenia [26], pgSIT [25]), others require laborious manual separation of females prior to final release (e.g. classical SIT, Wolbachia-based IIT [24]) significantly reducing scalability. Moreover, for Wolbachia-based IIT any accidental female releases can undermine the intervention [28,29], underscoring the imminent need for efficient and reliable sexing. Sex-sorting mosquitoes can be done manually, however this is laborious, error-prone, and not scalable for genetic biocontrol interventions. In Aedes and Culex mosquitoes, sexual dimorphism of pupa size can aid sex separation [30,31], however pupal size is density-dependent requiring optimized rearing conditions. To overcome this limitation, AI-assisted optical adult sex-sorting systems have been developed that can differentiate adults based upon sexual dimorphic features with impressive accuracy [32]. However, these have only been developed in Aedes aegypti and require sorting at the fragile adult stage which is not ideal, slow, and difficult to scale. Alternatively, selectable markers have been genetically linked to sex chromosomes in Anopheles [33–37], or to sex-determining loci in Aedes [38–40]. However, genetic linkage is often broken by meiotic recombination, or translocations, making these strains less stable when scaled [17,41]. Strains have also been developed using sex-specifically expressed promoters in mosquitoes [22,42–44] to express fluorescent proteins in the gonads, however given the small size of the gonads the expression is often weak and stage-specific [45]. There has also been efforts to exploit sex-specific alternative splicing (SSAS) to enable red fluorescent protein (RFP) expression in the flight muscles in Aedes [20], or to bias green fluorescent protein (EGFP) expression in larvae in Anopheles [46,47]. However, these sexing strains are engineered using species-specific components making them less portable across species. Additionally, the expression patterns are often not strong, or early enough, to allow for reliable sorting in initial larval stages which would be ideal for COPAS sorting and for genetic biocontrol interventions. Taken together, the development of simple, stable, and reliable sex-sorting technologies that can be facilely engineered in multiple mosquito species and enable early larval sex sorting remains to be developed. Here we exploit the sex specifically alternative spliced intron within Ae. aegypti doublesex (AaeDsx), which is a highly conserved transcription factor involved in sex determination of insects, to enable robust sex-specific alternative splicing of EGFP. We call our approach SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter). SEPARATOR exploits SSAS of a widely expressed fluorescent reporter to provide positive selection of males that can be integrated into genetic biocontrol interventions. To engineer SEPARATOR, any intron that is sex-specifically spliced could be utilized. SEPARATOR has many desirable features including: (i) it exploits male specific expression via sex-specific alternative splicing (SSAS) of an innocuous bright fluorescent marker; (ii) it enables sex-sorting during early larval development and beyond; (iii) is adaptable for high-throughput sorting using COPAS; (iv) does not rely on sex-chromosome linkage; (v) is genetically stable and not prone to breakage by meiotic recombination or chromosomal rearrangements; (vi) it enables positive selection of males to further increase robustness; and (vii) is facilely portable to alternate species as it utilizes the process of sex-specific alternative splicing which is used throughout the insect kingdom in addition to transposable elements, promoters, and markers that are cross-species compatible; and (viii) it enables separating mosquitoes at early stages such as L1, which allows the breeding facility to use space and food more efficiently to raise male mosquitoes only. Using SEPARATOR, we demonstrate positive identification of male larvae in Aedes mosquitoes, as early as mature embryos and beyond. By harnessing the capabilities of COPAS, we demonstrate sorting of over 100,000 EGFP+ males with speeds of up to 740 larvae/minute, achieving a EGFP-negative female contamination rate between 0.01% to 0.03%. Taken together, this method provides a much needed tool to scale and reliably release males for genetic biocontrol interventions.

Discussion Efficient and scalable sex separation is critical in the process of utilizing insects for genetic biocontrol [17]. To make this approach more sustainable and cost-effective, it is important to improve methods for sex separation. In this study, we present a highly efficient method for selecting male larvae/pupae in Aedes mosquitoes called SEPARATOR. This approach utilizes a COPAS instrument to positively select male L1 larvae that express a dominant male-specific reporter gene (EGFP). Importantly, this was designed as a positive-selection system to ensure that only those larvae expressing the dominant male-specific EGFP are precision sorted. Therefore, if EGFP becomes mutated or breaks, those individuals will not be selected making this system incredibly robust. Moreover, the use of a male spliced intron further increases the robustness since females will not have the ability to properly splice this intron to generate EGFP. In comparison to existing techniques, SEPARATOR has several advantages, including its exceptional efficiency, precision, and potential for portability across multiple species. In fact SEPARATOR only relies on a few simple components that have been shown to function in many insects. These include a transposable element for genetic transformation—here we use piggybac but there are many other choices that could also work including mariner, minos, Hermes, p-element, CRISPR-HDR etc. Another component is a strong ubiquitous promoter that expresses early in development that can drive expression of the dsx-EGFP cassette. Here we use a baculovirus promoter Hr5ie1 that has been shown to work in many insects—but this could be swapped out for an alternative species-specific promoter if desired. The most important component is the sex-spliced intron and here we use a DSX derived intron from Ae. aegypti. Given that DSX is a highly conserved gene in insects and known to undergo sex-speific alternative splicing indicates that introns from this gene derived from species of interest will likely work for SEPARATOR. Notwithstanding, any intron that is sex-spliced in the species of interest could also work—it will just need to be carefully designed and tested. Finally, EGFP could also be swapped out for other dominant markers or even desirable genes that could benefit from our sex-splicing module. Taken together, SEPARATOR encodes a few components that should make it readily adaptable to other species. In Aedes, there is a distinct size differential between female and male pupae, with females being larger. This characteristic allows for pupae to be size-sorted through a sieve, which serves as the first step in sex-sorting. While this method is effective in separating the sexes, up to 2–5% of female pupae can still remain in the male-enriched batch. To further reduce the number of female mosquitoes, a secondary sex-sorting step is employed using an AI-assisted optical system [31,32]. This system utilizes visual cues from sexual dimorphic differences in adult mosquito morphology to identify and remove any remaining female mosquitoes. This second step significantly reduces female contamination rates to an incredibly low level of 0.02% to 1.13⨯10−7% [62]. However, it should be noted that this step is done at the adult stage, which is not ideal for sorting as adults are very fragile. As a comparison, sorting SEPARATOR expressing larvae using a single COPAS instrument can sort up to 36K larvae per hour which really makes this a scalable approach. There were a few unexpected results associated with SEPARATOR. During the 15 generations reported, no DsRed-expressed mosquitoes were observed. Previous research has shown that in D. melanogaster and Ae. aegypti, the presence of the fruitless (Fru) protein is not detected in females, despite the expression of the corresponding transcripts [63–65]. Furthermore, a study conducted in D. melanogaster has revealed that the translation of the fru female splicing product is suppressed by the Tra protein [64]. This finding suggests that perhaps the transcripts specific to females are not translated. In D. melanogaster, both Dsx and Fru function as sex-determining factors and are regulated by the Tra protein. Nevertheless, proving the inability of a gene to undergo translation presents significant challenges. As a result, uncertainty persists regarding whether Dsx follows a comparable mechanism of translation inhibition, emphasizing the necessity for additional investigation. Fortunately, the male-specific product of AaeDsx demonstrates successful functionality (Fig 1C–1E). Additionally, our objective is to generate a positively selected population of males to enhance reliability and ensure accurate sex sorting. Hence, considering the next generation of SEPARATOR, utilizing it for positive male selection and incorporating a ubiquitous expression system for a transgenic marker could be a viable approach. SEPARATOR is robust and can be easily adapted for use in both classical Sterile Insect Technique (SIT) and Wolbachia-based Insecticide-Treated Insect Technique (IIT) [24,28,29] (Fig 4). The current method for breeding male mosquitoes involves culturing both sexes to the pupal / adult stage for sex sorting, after which the female mosquitoes are removed. In the context of Wolbachia-based IIT, the precise release of only males is crucial. The inadvertent release of females can compromise the effectiveness of the intervention [28,29], highlighting the urgent requirement for accurate sexing methods. However, current methods are not as efficient as SEPARATOR. SEPARATOR utilizes less time for male-sorting at the L1 larvae stage, making it a more efficient use of breeding space and food to raise male mosquitoes. The rapidity of COPAS sorting allows for multiple iterations of screening, effectively minimizing the risk of female contamination. With this system, researchers and breeders can save time and resources by identifying and separating male mosquitoes at an early stage of development. This allows for a more efficient and cost-effective method of breeding and raising male mosquitoes for research and other purposes. Taken together, SEPARATOR provides a valuable tool to help revolutionize the way we sort sexes, making the process more efficient, cost-effective, and reliable and may prove to be a valuable addition to genetic biocontrol programs. PPT PowerPoint slide

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TIFF original image Download: Fig 4. SEPARATOR provides a more simplified and economical sex-sorting of Ae. aegypti mosquitoes than the currently available two-step sex-sorting approaches. (A) The process of generating radiation-induced sterile male mosquitoes begins with the cultivation of mosquitoes. The larger female pupae are removed using a sieve. However, it should be noted that smaller female pupae may still remain after the first sex sorting. Once the mosquitoes have been irradiated, emerging adult mosquitoes are screened out by an image recognition AI that has been trained to discriminate between female and male adult morphologies during the second sex sorting. The SEPARATOR approach utilizes a male-specific reporter (GFP) to positively select male L1 larvae. This can be done using the COPAS instrument, which is capable of high-throughput selection at a speed of up to 10 larvae per second. By removing female larvae early in the development process, SEPARATOR supports a more efficient production of males for SIT application. Furthermore, SEPARATOR allows for the transportation and release of irradiated sex-sorted pupae. This means that adult male mosquitoes can emerge directly into the environment without incurring additional fitness costs from handling and transportation. (B) The Wolbachia-based incompatible insect technique (IIT) utilizes a two-step sex-sorting approach for sorting the Wolbachia-infected male mosquitoes currently in use. The process of generating Wolbachia-infected SEPARATOR mosquitoes is relatively simple, as it involves crossing Wolbachia-infected female mosquitoes with SEPARATOR male mosquitoes. The resulting Wolbachia-infected male L1 larvae that express a male-specific reporter (GFP) are then positively selected using the COPAS instrument. Figures were created using BioRender. https://doi.org/10.1371/journal.pgen.1011065.g004

Materials and methods Molecular cloning and transgenesis To create the endogenous AaeDsx splicing module construct, we amplified the fragment of endogenous exons and introns from the genomic DNA of Ae. aegypti using PCR. We then linearized the previous mCherry and EGFP containing plasmid, 1122I, using the restriction enzyme PacI. The linearized 1122I plasmid and the fragment of endogenous exons and introns were used in a Gibson enzymatic assembly method to build the 1174CX plasmid. To open the reading frame of the female-specific product and allow for in-frame expression of the mCherry coding sequence, we substituted the endogenous exon 5b with an engineered exon 5b that had the stop codons removed. The sequence of the engineered exon 5b was synthesized using the gBlocks Gene Fragment service. We removed the endogenous exon 5b by cutting it with the restriction enzymes PmlI and SnaBI, and then used Gibson assembly to incorporate the engineered exon 5b containing fragment into the cut 1174CX plasmid, resulting in the 1174C plasmid. To generate a plasmid for sex-specific expression of DsRed and EGFP, we amplified the fragment of the engineered exon 5b and DsRed coding sequences from the 1174C plasmid and the previous 874Y plasmid. We then fused these two fragments together using overlapping PCR. Finally, we used omega PCR to substitute the sequence from the mCherry coding sequence to the 3xP3 promoter in the 1174C plasmid with the DsRed coding sequence, resulting in the 1174D plasmid. During each cloning step, we selected single colonies and cultured them in LB medium with ampicillin. We then extracted the plasmids (using the Zymo Research Zyppy plasmid miniprep kit) and performed Sanger sequencing. The final plasmids were maxi-prepped (using the Zymo Research ZymoPURE II Plasmid Maxiprep kit) and fully sequenced by Primordium. All primers are listed in S18 Table. The complete annotated plasmid sequences and plasmid DNA are available at Addgene (ID: 200012). Transgenic lines were created by microinjecting preblastoderm stage embryos with a mixture of the piggyBac plasmid and a transposase helper plasmid. Four days after microinjection, the G0 embryos were hatched and the surviving pupae were separated and sexed. The pupae were placed in separate cages for males and females, along with wild-type male pupae in the female cages and wild-type female pupae in the male cages, in a 5:1 ratio. After several days to allow for development and mating, a blood meal was provided and eggs were collected, aged, and hatched. The larvae with positive fluorescent markers were isolated using a fluorescent stereomicroscope. To isolate separate insertion events, male transformants with fluorescent markers were crossed with female transformants without fluorescent markers, and separate lines were established. The individual genetic sexing lines (1174D) were maintained as mixtures of homozygotes and heterozygotes, with periodic elimination of wild-type individuals. The genetic sexing line (1174D) was homozygosed through approximately ten generations of single-pair sibling matings, selecting individuals with the brightest marker expression each generation. Mosquito rearing and maintenance Ae. aegypti mosquitoes were obtained from the Liverpool strain, which was previously used to generate the reference genome [60]. These mosquitoes were raised in incubators at 30°C with 20–40% humidity and a 12-hour light/dark cycle in cages (Bugdorm, 24.5 x 24.5 x 24.5 cm). Adults were given 10% (m/V) aqueous sucrose ad libitum, and females were given a blood meal by feeding on anesthetized mice for approximately 15 minutes. Oviposition substrates were provided about 3 days after the blood meal. Eggs were collected, aged for about 4 days to allow for embryonic development, and then hatched in deionized water in a vacuum chamber. Approximately 400 larvae were reared in plastic containers (Sterilite, 34.6 x 21 x 12.4 cm, USA) with about 3 liters of deionized water and fed fish food (TetraMin Tropical Flakes, Tetra Werke, Melle, Germany). For genetic crosses, female virginity was ensured by separating and sexing the pupae under the microscope based on sex-specific morphological differences in the genital lobe shape (at the end of the pupal abdominal segments just below the paddles) before releasing them to eclose in cages. These general rearing procedures were followed unless otherwise noted. In order to increase the number of homozygotes in the 1174D transgenic line, we transferred both the high-intensity EGFP pupae and female EGFP-negative pupae to a cage and allowed them to mate after eclosion. Female mosquitoes were fed a blood meal, and five adult females were individually transferred to egg tubes for colonization and egg collection. The eggs from each colony were hatched and reared. The colonies with a higher proportion of female EGFP negatives and male EGFP positives were selected for colonization in the next generation. After a few rounds of colonization, the colonies with 100% of males in the strong EGFP positive group and 100% of females in the EGFP negative group were selected and propagated for expansion. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as recommended by the National Institutes of Health and approved by the University of California San Diego Institutional Animal Care and Use Committee (IACUC, Animal Use Protocol #S17187) and University of California San Diego Biological Use Authorization (BUA #R2401). COPAS fluorescent sorting, sexing and imaging To determine the precise number of larvae in COPAS clusters, the COPAS raw data was filtered based on the optical density and size measurements of the individuals, "log(EXT)" and "log(TOF)," to remove outliers such as egg debris and dust. Then, a filter was applied based on the individuals’ fluorescence measurements, "log(first fluorescence)" and "log(second fluorescence)", to further refine the data. Finally, the fluorescence measurements were automatically clustered and denoised using Density-Based Spatial Clustering of Applications with Noise (DBSCAN) [66,67]. The analysis of COPAS data was conducted using the web-based app, Floreada.io (https://floreada.io/). COPAS sorting was performed largely as described for Anopheles larvae [47]. Aedes eggs stuck to their egg laying paper were briefly rinsed to eliminate dust and debris, immersed in deionized water in a small container, and their hatching was stimulated under partial vacuum (25% of atmospheric pressure) in a vacuum chamber for 30–60 minutes. They were then incubated overnight at 28°C to maximize larval hatching. On the next day, resulting unfed neonate larvae were transferred to the reservoir of a large particle flow cytometry COPAS SELECT instrument (Union Biometrica, Holliston, MA, USA) equipped with a multiline argon laser (488, 514 nm) and a diode laser (670 nm). Larvae were analyzed and sorted with the Biosort5281 software using a 488 nm filter and the following acquisition parameters: Green PMT 500, Red PMT 600, Delay 8; Width 6, pure mode with superdrops. Flow rate was kept between 20 and 70 objects per second through adjusting of the concentration of larvae in the sample. Larvae identified as males (EGFP positive) were dispensed in a Petri dish. In these conditions, sorting speed ranged from 4000 to 7400 larvae in 10 minutes (+ 6 minutes for system initialization and 6 minutes for system cleaning and shutdown), the total number of sorted larvae being limited by the number of available larvae. Sorted larval counts provided by the COPAS software were recorded on sorting. For quality control of sorted larvae, the reservoir and fluidics of the instrument were carefully rinsed and the sorted larvae analyzed by passing them once more in the machine. In some experiments, objects falling outside the EGFP positive gate were collected in “Enrich” mode to remove EGFP negative contaminants from the pool of EGFP positive larvae, and verified by microscopy. Mosquitoes were examined, scored, and imaged using the Leica M165FC fluorescent stereomicroscope equipped with the Leica DMC2900 camera. For higher-resolution images, we used a Leica DM4B upright microscope equipped with a VIEW4K camera. To distinguish between male and female pupae in mosquitoes, we used a microscope to observe the sex-specific morphological differences in the genital lobe shape located at the end of the pupal abdominal segments just below the paddles. This allowed us to ensure that we were selecting both male and female pupae for our experiments. Determination of genome integration sites To determine the transgene insertion sites, we performed Oxford Nanopore genome DNA sequencing. We extracted genomic DNA using the Blood & Cell Culture DNA Midi Kit (Qiagen, Cat. No. / ID: 13343) from 5 adult males and 5 adult females of SEPARATOR, following the manufacturer’s protocol. The sequencing library was prepared using the Oxford Nanopore SQK-LSK110 genomic library kit and sequenced on a single MinION flowcell (R9.4.1) for 72 hrs. Basecalling was performed with ONT Guppy base calling software version 6.4.6 using dna_r9.4.1_450bps_sup model generating 3.03 million reads above the quality threshold of Q≧10 with N50 of 7941 bp and total yield of 11.08 Gb. To identify transgene insertion sites, nanopore reads were mapped to plasmids carrying SEPARATOR (1174D, Addgene as plasmid #200012) using minimap2 [68] and further aligned them to the AaegL5.0 genome (GCF_002204515.2). Subsequently, we calculated the average depth of coverage for the three autosomes and the transgene using samtools and visualized the results in R. The coverage depths for chr1, chr2, and chr3 were determined to be 6.31, 6.30, and 6.08, respectively. Interestingly, the coverage depth for SEPARATOR transgene was notably higher at 16.14. Based on the coverage analysis, it appears that the SEPARATOR transgene is present in three copies (S3 Fig). By examining the read alignments using Interactive Genomics Viewer (IGV), we were able to determine the exact insertion sites. Notably, we identified three copies of the SEPARATOR constructs sequence. The three integration sites are NC_035109.1:92046983, NC_035108.1:444508475 and NC_035107.1:299022928. This finding aligns with the results of the depth of coverage analysis, further supporting the presence of three insertion sites. The second integration site on NC_035108.1 overlaps with the AAEL005024 gene, which is currently classified as an uncharacterized protein. However, the other two integration sites do not overlap with any known genes. The nanopore sequencing data has been deposited to the NCBI SRA (PRJNA985064). RNA sequencing (RNA-seq) analysis To quantify target gene reduction and expression from transgenes as well as to assess global expression patterns, we performed Illumina RNA sequencing. We extracted total RNA using miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, Cat. No. / ID: 217604) from 50 EGFP-positive male mosquitoes and 50 EGFP-negative female mosquitoes at L1 larva stage in biological triplicate (6 samples total), following the manufacturer’s protocol. Genomic DNA was depleted using the gDNA eliminator column provided by the kit. RNA integrity was assessed using the RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies, Cat. No. / ID: #5067–1513), and mRNA was isolated from ∼1 μg of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Cat. No. / ID: E7490). RNA-seq libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, Cat. No. / ID: E7770) following the manufacturer’s protocols. Briefly, mRNA was fragmented to an average size of 200 nt by incubating at 94°C for 15 min in the first strand buffer. cDNA was then synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed, and ligated to NEBNext hairpin adaptors (NEB, Cat. No. / ID: E7335). Following ligation, adaptors were converted to the “Y” shape by treating with USER enzyme, and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250–350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified using a Qubit dsDNA HS Kit (ThermoFisher Scientific, Cat. No. / ID: Q32854), and the size distribution was confirmed using a High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, Cat. No. / ID: 5067–4626). Libraries were sequenced on an Illumina NextSeq2000 in paired end mode with the read length of 50 nt and sequencing depth of 25 million reads per library. Base calls and FASTQ generation were performed with DRAGEN 3.8.4. The reads were mapped to the AaegL5.0 genome (GCF_002204515.2) supplemented with SEPARATOR transgene sequences using STAR. On average, ∼97.4% of the reads were mapped. The analysis of RNA-Seq data was performed using an integrated web application called iDEP [69]. TPM values were calculated from counts produced by feature counts and combined (S4 Table). Hierarchical clustering of the data shows that for each genotype, all replicates cluster together, as expected (S5A and S5B Fig). DESeq2 was then used to perform differential expression analyses between male (EGFP-positive) and female (EGFP-negative) at L1 larvae stage (S5C Fig). For each DESeq2 comparison, gene ontology enrichments were performed on significantly differentially expressed genes. (S5D and S5E Fig and S5 Table). Illumina RNA sequencing data has been deposited to the NCBI-SRA (PRJNA985064). For transcriptome comparing analysis, we acquired 47 files consisting of six developmental stages (L3 larvae, L4 larvae, early pupae, mid pupae, late pupae, and adult carcass) from SRA (S6 Table). These files were then aligned to the AaegL5 genome (GCF_002204515.2) using STAR. The analysis of RNA-Seq data was performed using an integrated web application called iDEP [69]. TPM values were calculated from counts produced by feature counts and combined (S8 Table). Statistical analysis Statistical analysis was performed in Prism9 for macOS by GraphPad Software, LLC. At least three biological replicates were used to generate statistical means for comparisons.

Acknowledgments We thank Ming Li, Judy Ishikawa, Sammy Lee, and Dylan Turksoy for helping with mosquito husbandry and microinjections.The views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the U.S. government. Figures were created using www.BioRender.com.

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