Generating Cisgenic Sexing Strains in Insect Pests

Generating Cisgenic Sexing Strains in Insect Pests Angela Meccariello Imperial College London https://orcid.org/0000-0001-9706-5764 Article Keywords: CRISPR/Cas9, Sex Specic Alternative Splicing, Genetic Sexing Strains, Medy Posted Date: April 21st, 2025 DOI: https://doi.org/10.21203/rs.3.rs-6449302/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License Additional Declarations: Yes there is potential Competing Interest. A patent has been led on this technology. O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. N.P.K is a founder of 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 conict-of-interest policies. All other authors declare no competing interests. Table 1 is available in the Supplementary Files section. 1 Generating Cisgenic Sexing Strains in Insect Pests 1 2 Serafima Davydova1$, Junru Liu2$, Nikolay P. Kandul2, Igor Antoshechkin3, Jonathan Mann1, W. Evan 3 Braswell4, Omar S. Akbari2*, Angela Meccariello1* 4 1. Department of Life Sciences, Imperial College London, London, SW7 2AZ, United Kingdom 5 2. School of Biological Sciences, Department of Cell and Developmental Biology, University of California, San 6 Diego, La Jolla, CA 92093, United States of America 7 3. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, 8 United States of America 9 4. USDA APHIS PPQ Science & Technology Insect Management and Molecular Diagnostic Laboratory, 22675 10 North Moorefield Road, Edinburg, Texas, 78541, United States of America 11 12 * Corresponding authors: Angela Meccariello, Ph.D. Email: [email protected] 13 Omar S. Akbari, Ph.D. Email: [email protected] 14 $ These authors contributed equally: Serafima Davydova and Junru Liu 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 2 Abstract 36 Insect pest population control via sterile insect technique severely benefits from separation by sex 37 prior to release. To simplify this process, traditional genetics has been deployed to develop 38 genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic 39 significance, although very few are applied in the field due to associated fitness costs and 40 instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We 41 use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific 42 alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain 43 that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and 44 relied on the sex-specifically spliced intron of the endogenous transformer gene, which we 45 seamlessly inserted into the pupal colouration white pupae gene. This minimal modification 46 resulted in the generation of a homozygous strain we term IMPERIAL that was phenotypically 47 stable where all female pupae are brown while male pupae are white with overall good fitness. 48 By minimally editing the genome, our CGSS approach can be applied to other pests that may aid 49 more efficient and economically suitable pest control. 50 51 Key words: CRISPR/Cas9; Sex Specific Alternative Splicing; Genetic Sexing Strains, Medfly 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 3 Main text 70 Genetic sexing strains (GSSs) have been developed in multiple insect species of economic 71 significance to allow easier male and female separation necessary for efficient population control. 72 Specifically, GSSs are used within sterile insect technique (SIT) programmes which work via 73 frequent releases of sterilised insects into the wild for temporary population control (1-3). Male-74 only releases, aided by GSS implementation, strongly succour in released fly dispersal and their 75 mating frequency with wild females, thus enhancing SIT success (4-6). Vast efforts have focused 76 on GSS generation in mosquito disease vectors and agricultural fruit fly pests to avoid labour-77 intensive sex-sorting by eliminating the females from the released population early in the life cycle 78 (7-12). 79 80 The traditional GSS approach requires two key attributes: a selectable marker and a Y-81 chromosome or male-determining locus linkage from which it needs to be expressed (4). A 82 primary example of a traditional GSS is the VIENNA 8 strain developed in the tephritid fruit fly 83 pest Ceratitis capitata (Mediterranean fruit fly or medfly) possessing an increasing threat to the 84 agricultural industry with its expanding global distribution and vast host range (13-14). VIENNA 8, 85 similarly to its predecessor VIENNA 7, relies on a radiation-induced simultaneous translocation 86 of wp gene and temperature sensitive lethal (tsl) genes onto the Y-chromosome involved in pupal 87 colouration and heat tolerance respectively (15-16). Whilst the former, wp, has been 88 characterised in multiple tephritids, the latter, tsl, remains to be identified in C. capitata and its 89 relatives. As the VIENNA 8 strain has a wp and tsl double-mutant background, the white-pupaed 90 females die upon embryonic heat exposure, whilst the brown-pupaed males persevere into 91 adulthood (4; 17). Across the Tephritidae family, such traditional GSSs have been successfully 92 developed in multiple species, although only those developed in C. capitata and Anastrepha 93 ludens have been implemented on a SIT facility scale (18-19). Other traditional examples tested 94 on a larger scale include the eye colour phenotype-dependent Aedes aegypti GSSs (20). 95 96 In parallel to similar GSSs in other tephritid species, the VIENNA 8 strain is infrequently 97 susceptible to phenotype loss via recombination requiring an extra filtering step at SIT facilities 98 (4, 21). Furthermore, they have notable reductions in fertility, thus creating obstacles for mass 99 rearing (18, 22). To elevate GSS fitness, multiple transgenic approaches have been engineered 100 for female exclusion either through a selectable phenotypic marker or female-specific lethality in 101 the medfly and other fruit fly species (23-33). Abundantly, a sex-specific intron from the 102 transformer (tra) gene has been implemented in the medfly for female-specific transgene 103 4 expression from the autosomes (25-26, 30). Most recently this was completed for female-specific 104 fluorescence marker expression in a Sexing Element Produced by Alternative RNA-splicing of 105 Transgenic Observable reporter (SEPARATOR) system whereby both the C. capitata and newly 106 the A. ludens tra introns were used (25). While these transgene-based approaches provide 107 exciting alternatives, they still require regulatory authorisation which can be a slow and 108 cumbersome process. 109 110 Herein, we wanted to develop a universal method in insects to engineer GSSs that use modern 111 engineering techniques to generate minimal genetic modifications wherein both donor and 112 recipient are derived from the same species which we call Cisgenic GSS (CGSS). We used the 113 medfly as our model system to engineer a non-transgenic CGSS without exogenous elements. 114 Sex-specific expression of the wp selectable marker was achieved through sex-specific splicing 115 of the tra intron. This was attained through a homology-directed repair (HDR)-dependent knock-116 in of the tra intron seamlessly into the white pupae (wp) locus of wild-type Benakeion medfly 117 embryos, feasible due to recent success of CRISPR/Cas9-mediated HDR in the species (34-36). 118 119 Collectively, using the intron of tra, a master gene of tephritid female sex determination (37-39), 120 results in extremely robust desired phenotypes, rendering it suitable for further use in CGSSs, yet 121 to be developed. The knock-in construct (1167A) was engineered using the endogenous 122 transformer (tra) intron, which was placed between the homology arms of each approximately 123 700 bp in length. The anticipated outcome was female-specific wp gene expression, whereby 124 males harbouring two copies of the tra-containing wp gene would have a white pupae phenotype 125 and females harbouring two copies of the tra-containing wp gene would have a brown pupae 126 phenotype, which can be distinguished by eye (Fig. 1A). As the wp mutation is recessive in nature, 127 the knock-in strain, hereupon named IMPERIAL, was isolated using backcrosses to the 128 irradiation-generated white pupae knock-out (wp-/-) strain at G0 and G1 (Fig. 1B). We confirmed 129 successful knock-in of the tra intron (wpKI+/-) in all obtained brown-pupaed G2 females using 130 amplicon sequencing of the integration site. A homozygous wpKI+/+ line was established at G7 (F0) 131 after crossing sibling white-pupaed males with brown-pupaed females, genotyping all parents and 132 screening the whole progeny at every intermediate generation. To verify the integration in the 133 wpKI+/+ IMPERIAL strain, genomic DNA from CGSS females was sequenced. The sequencing 134 revealed reads with the expected 1345 bp CRISPR-HDR insertion indicating a seamless 135 intragenic insertion of the tra intron into the wp gene (fig. S1). 136 137 5 To verify the sex-sorting suitability of the strain, phenotypic pupae colour stability and sex ratios 138 of the IMPERIAL strain were examined alongside the existing VIENNA 8 GSS, where males and 139 females emerge from brown and white pupae accordingly. For five consecutive generations (F2-140 F6), pupae and adult phenotypes were recorded for IMPERIAL (pupae n = 4147; adult n = 4074) 141 and VIENNA 8 (pupae n = 1386; adult n = 1227) strains in parallel, maintained under the same 142 conditions. As anticipated, in the wpKI+/+ IMPERIAL strain all females emerged from brown pupae 143 and all males emerged from white pupae (Fig. 2A). The reverse phenotypes were universally 144 observed amongst individuals from the VIENNA 8 strain. Altogether, the proportion of adult 145 females in the IMPERIAL strain (mean = 49.8%) exceeded that of the VIENNA 8 strain (mean = 146 37.4%). Whilst populations within every generation of the IMPERIAL strain adhered to the 147 expected 1:1 male: female sex ratio (chi-square goodness of fit tests), the VIENNA 8 did not at 148 F2, F4 and F5 (Fig. 2A). In sum these results confirm the phenotypic stability of the IMPERIAL 149 strain that, dissimilarly to VIENNA 8, consistently confines to the expected 1:1 male: female sex 150 ratio. 151 152 To better understand differences in the tra intron-dependent wp splicing in males and females of 153 the wpKI+/+ IMPERIAL strain, we performed reverse-transcription PCR (RT-PCR) on genomic and 154 complementary DNA (cDNA) templates from adult flies (fig. S2). A single female band was 155 amplified from the cDNA template, corresponding to a transcript with the fully spliced-out tra intron 156 (37), confirmed by sequencing thereafter. Male cDNA banding entirely consisted of larger 157 fragments from which two unique male isoforms with premature stop codons were isolated via 158 clonal sequencing, both containing sequences of the two male-specific exons. These results are 159 suggestive of functional White pupae protein production in females and ablation of its translation 160 in males. Further investigation into wp splicing of the IMPERIAL strain was conducted using 161 RNAseq. As expected, all three female libraries had multiple reads that spanned the junction 162 splicing the tra intron. In contrast, the three male libraries had no such reads indicating that the 163 intron is spliced in females and not in males (fig. S3). As expected, the clustering and principal 164 component analyses indicated a close relationship of samples by sex (fig. S4). 165 166 The IMPERIAL sexing strain was further compared to its parental wild-type Benakeion and 167 VIENNA 8 strains in terms of general fitness, and thus suitability for larger-scale employment. 168 First, a standard egg-adult survival assay utilising triplicate sibling crosses of 10 males with 20 169 females was conducted, whereby rates of egg laying, egg hatching, hatched larvae-pupae 170 recovery, pupae-adult recovery, and total egg-adult recovery were assessed (Fig. 2B). Kruskal-171 6 Wallis and sequential Dunn’s tests were performed as means of statistical analysis. Notably, egg 172 production within the measured time period was significantly elevated in the IMPERIAL strain 173 compared to both wild-type and VIENNA 8 strains (p < 0.05*). Hatching rate in VIENNA 8 was 174 significantly lower than in wild-type (p = 0.0036**), although insignificant reductions in the 175 IMPERIAL strain were also observed (p = 0.0899). We recorded the highest larval-pupal survival 176 in the IMPERIAL strain, which was significantly raised compared to VIENNA 8 (p = 0.0036**). In 177 line with the earlier phenotypic stability experiments, the pupal-adult recovery rates were 178 significantly lower in the VIENNA 8 strain, compared to both wild-type and IMPERIAL strains (p < 179 0.05*). Overall egg-adult survival was significantly reduced in VIENNA 8 when assessed against 180 wild-type (p = 0.0127*) and IMPERIAL (p = 0.0368*) strains alike. Egg-adult survival in wild-type 181 and IMPERIAL strains, however, was statistically similar (p = 0.3274), indicative of good fitness 182 in the IMPERIAL strain. 183 184 We also explored adult longevity with virgin males and females restricted to separate husbandry 185 under regular lab conditions (Fig. 2C). Highest survival was observed in wild-type females, whilst 186 the shortest longevity belonged to VIENNA 8 females. Pairwise comparisons via log-rank tests 187 were performed between strains, and the combinations of stain and sex (tables S1 and S2). 188 Altogether, VIENNA 8 had significant reductions in longevity compared to both wild-type (p = 189 0.0022**) and IMPERIAL (p = 0.0048**) strains. Wild-type Benakeion and IMPERIAL strains with 190 the same genetic background, on the other hand, did not have a significant difference between 191 one another (p = 0.3920). Amongst females, significant differences were recorded for VIENNA 8 192 against both wild-type (p < 0.0001****) and IMPERIAL (p < 0.0001****) strains, while male 193 comparisons between strains were statistically insignificant. These results are suggestive of 194 comparable longevity of the males and importantly females from the IMPERIAL strain with wild-195 type. 196 197 A delay in white-pupaed female development has been documented in the VIENNA 8 strain (40). 198 To determine whether similar issues occur in the males of the IMPERIAL strain because of their 199 white-pupaed phenotype, we compared pupal eclosion times from age-matched egg collections 200 by sex. The development times were significantly different by strain and sex (nested ANOVA, F 201 = 178.6, p < 0.0001****) (Fig. 2D). The time for wild-type males (mean = 18.60 days) and females 202 (mean = 18.75 days) to reach adulthood were statistically similar to IMPERIAL males (mean = 203 18.51 days) and females (mean = 18.59 days) (table S3). Flies from the VIENNA 8 strain were 204 significantly slower to eclose, with means of 20.11 and 24.00 days for males and females 205 7 accordingly. Whilst there may be differences in acclimation of the VIENNA 8 to a newer laboratory 206 environment in comparison to the other two strains, there was a significant difference between 207 VIENNA 8 males and females (p < 0.0001****), which is absent in both wild-type (p = 0.94854) 208 and IMPERIAL (p = 0.99781) strains. The observed trends strongly indicate that the IMPERIAL 209 strain does not experience a developmental discrepancy between sexes which is present in the 210 current VIENNA 8 GSS. 211 212 The mating preferences of females towards the IMPERIAL, Benakeion and VIENNA 8 strains 213 were assessed through simultaneous allowing of mating between males from all three strains with 214 females from the irradiation-generated wp-/- strain. Due to the recessive nature of the wp 215 mutation, the father(s) were easily ‘revealed’ through pupal colour and corresponding adult 216 phenotype screening (Table 1). The most common parentage belonged to wild-type male(s) 217 (36.93%), followed by equal parentage by IMPERIAL male(s) (28.41%) and VIENNA 8 male(s) 218 (28.41%). We also observed offspring cohorts from single mothers with mixed strain paternity at 219 a low frequency (6.25%). This data indicates that the pupal colour of the IMPERIAL males does 220 not disadvantage their mating success in respect to brown-pupaed VIENNA 8 males. 221 222 Here, we describe an entirely novel CRISPR/Cas9-generated CGSS method with proof of concept 223 for the major agricultural pest C. capitata using the wp gene involved in pupal pigmentation (16). 224 Although multiple robust next-generation GSS approaches have been established in the medfly 225 to date (24-26, 33), the herein-established IMPERIAL strain is the first to exclusively encompass 226 endogenous elements. Our approach uses the sex-specific intron of tra, a gene responsible for 227 female-specific fate induction in the Tephritidae fruit fly family and beyond, making it an appealing 228 target for cross-species application (41-42). Importantly, the Y-chromosome-independent GSS 229 approach highlighted herein is time-effective as it only requires comprehensible cross completion 230 for line establishment. Via CRISPR/Cas9-mediated HDR, we integrated the tra intron into the wp 231 gene to achieve its expression in females exclusively, resulting in a brown-pupae phenotype. Due 232 to sex-specific splicing (fig. S2), males in the strain do not translate wp transcripts successfully. 233 Specifically, this is caused by the inclusion of early termination codons upon transcription leading 234 to a white-pupae phenotype. Given that no foreign DNA is inserted into the genome, our approach 235 may be easier to gain regulatory approvals for release, but this remains to be determined. 236 237 Characterisation of the IMPERIAL strain revealed that its fitness is comparative to its ancestral 238 wild-type strain from which it was generated. This included survival during development and upon 239 8 adulthood, as well as eclosion time comparison between males and females (Fig. 2). Despite 240 statistically similar egg-adult survival, however, the IMPERIAL strain egg hatching rates was 241 insignificantly reduced. In all above-mentioned assessments the VIENNA 8 strain performance 242 indicated greater fitness costs than in the herein generated strain, although mating 243 competitiveness was similar between the two GSSs (Table 1). To explore the fitness of the 244 IMPERIAL strain in further detail, and hence its suitability for SIT implementation, larger scale 245 trials need to be performed. Among all males and females screened in the process of line 246 characterisation in this study, no reverse phenotypes were observed, suggestive of vast line 247 stability. Furthermore, similarly to wild-type flies, the males and females are consistently 248 distributed in equal proportions in the IMPERIAL strain (Fig. 2A). This highlights the limited effect 249 of the white-pupae phenotype on the male survival to adulthood in our GSS, opening a possibility 250 for its efficient facility-based rearing. In theory, the sex-sorting of this strain can be performed at 251 pupal developmental stage using automated machinery, already employed, and optimised at SIT 252 C. capitata facilities. Thus, with its stability and cisgenic nature, our system could be an 253 advantageous and cost-effective alternative for currently used strategies in SIT-mediated 254 population control. 255 256 The latest reiteration of the traditional GSSs, VIENNA 8, possesses a heat sensitivity component 257 via the tsl gene (15). As its wild-type copy is expressed from the Y-chromosome in an 258 autosomally-mutant tsl (tsl-/-) background, only males are tolerant to heat (4). To improve our 259 system further, a heat-inducible component can be added to the IMPERIAL strain. This will 260 additionally allow for a thorough investigation of the fitness costs in VIENNA 8 observed in this 261 work. Once tsl locus is characterised in full, alternative iterations of the tra intron for functional 262 male-specific splicing, or entirely different sex-specific introns (37, 43) can be used for its 263 expression. 264 265 Data availability 266 Complete plasmid sequence is available at Addgene.org (#218233). The raw data used for figure 267 generation is provided in the Source data files. The sequencing data are available at NCBI under 268 BioProject PRJNA1189200 (Reviewer link 269 https://dataview.ncbi.nlm.nih.gov/object/PRJNA1189200?reviewer=8am21kg9tm2eskhv6ls27knsd)270 The herein-generated knock-in strain is available upon request from A.M. 271 272 Disclaimer 273 15 469 470 471 472 473 474 Supplementary materials 475 Generating Cisgenic Sexing Strains in Insect Pests 476 477 Serafima Davydova1$, Junru Liu2$, Nikolay P. Kandul2, Igor Antoshechkin3, Jonathan Mann1, W. 478 Evan Braswell4, Omar S. Akbari2*, Angela Meccariello1* 479 5. Department of Life Sciences, Imperial College London, London, SW7 2AZ, United Kingdom 480 6. School of Biological Sciences, Department of Cell and Developmental Biology, University of California, San 481 Diego, La Jolla, CA 92093, United States of America 482 7. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, 483 United States of America 484 8. USDA APHIS PPQ Science & Technology Insect Management and Molecular Diagnostic Laboratory, 22675 485 North Moorefield Road, Edinburg, Texas, 78541, United States of America 486 487 * Corresponding authors: Angela Meccariello, Ph.D. Email: [email protected] 488 Omar S. Akbari, Ph.D. Email: [email protected] 489 $ These authors contributed equally: Serafima Davydova and Junru Liu 490 491 492 493 494 495 496 497 498 499 500 16 501 Materials & Methods 502 Plasmid design and construction 503 We used Gibson enzymatic assembly to build the 1167A plasmid, which contains C. capitata MFS 504 transporter Exon 3 (LOC101451947), the tra intron and Opie2-DsRed outside the homology arms. 505 A pre-existing plasmid containing piggyBac flanks, with an Opie2 promoter regulating DsRed, was 506 linearised by NdeI and KpnI to clone 1167A. The MFS transporter Exon3 was amplified into two 507 fragments from C. capitata genomic DNA using primer pairs 1167A.c1F and c2R, as well as 508 1167A.c5F and c6R (table S4). The tra intron was amplified from 795H1 (Addgene #205482) 509 using primer pair 1167A.c3F and c4R, then inserted inside of the MFS exon 3 coding sequence 510 (table S4). 511 512 C. capitata maintenance 513 All fly stains were reared under standard lab conditions described previously (1). A carrot-based 514 diet was provided for larval development (2) and a 1:1 yeast: glucose mix was given to adult flies. 515 The Benakeion wild-type strain was supplied by the Saccone Lab (University of Naples “Federico 516 II”), whilst irradiation-generated white pupae -/- (wp-/-) (3) and VIENNA 8 D53strains were 517 obtained from the FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture (Seibersdorf, 518 Austria). 519 520 C. capitata germline transformation 521 Microinjections of the 1167A plasmid were performed into embryos of the wild-type Benakeion 522 strain. The plasmid (250 ng/μl) was injected alongside a pre-assembled ribonucleoprotein (RNP) 523 complex of Cas9 protein (200 ng/μl) (PNA Bio) and pre-synthesized gRNA-wp (100 ng/μl) 524 (Synthego) (4). 525 526 wpKI+/+ line establishment 527 The cross scheme for G0-G3 line generation is summarized in Fig. 1. The injected G0s were 528 reciprocally crossed to the characterised irradiation-generated wp-/- strain (3). The resulting G1 529 progeny was separated by pupal colour, and all males emerging from white pupae were 530 backcrossed in pools to the females from the irradiation-generated wp-/- strain. At G2, the brown 531 pupae were isolated, and all 10 female adults from the same parental G1 cross were collectively 532 crossed to five sibling white-pupaed males. After mating, genomic DNA (gDNA) was individually 533 extracted from G2 brown-pupaed females using an altered phenol-chloroform protocol (5). The 534 17 knock-in site was amplified using Phusion High-Fidelity PCR Master Mix with HF Buffer (New 535 England Biolabs®) with genome-specific 1167A_F and 1167A_R primers (table S4) designed in 536 Geneious Prime 2023.1.2. To confirm the initial integration, the PCR products were purified via 537 Monarch® PCR & DNA Cleanup Kit (New England Biolabs®) and analysed via Oxford Nanopore 538 sequencing (Full Circle Labs). 539 540 Over the course of the following 4 generations (G3-G6) multiple sibling crosses were performed 541 in parallel and the parental genotypes were verified. Hereby three possible alleles were 542 differentiated: 1) the irradiation-generated wp-/-; 2) the intron-less wp-/- with indels and 3) the tra 543 intron knock-in (fig. S5). For this, a multiplex 3-primer PCR was designed using a forward 544 (1167A_F) binding upstream of the integration site, a reverse (1167A_R) downstream of the 545 integration site and a second reverse (8kb_B_R) binding to the irradiation-generated wp-/- 546 inserted sequence (fig. S5). 547 548 Verification of genome integration site in the homozygous strain 549 To verify the insertion site in the wpKI+/+ IMPERIAL strain, we conducted Oxford Nanopore 550 genomic DNA sequencing. Genomic DNA was extracted from four knock-in adult males and four 551 knock-in adult females using the Blood & Cell Culture DNA Midi Kit (Qiagen). 552 553 PCR tra intron splicing confirmation 554 In parallel, single males and females were separately collected for gDNA and RNA extractions. 555 gDNA was extracted as detailed above. RNA was extracted via an adapted TRIzol® (Ambion)-556 chloroform-based protocol (6). cDNA was synthesised from total RNA using Maxima H Minus 557 First Strand cDNA Synthesis Kit with dsDNase (ThermoFisher) according to the instructions 558 provided by the manufacturer. Phusion High-Fidelity PCR Master Mix with HF Buffer (New 559 England Biolabs®) was used for PCR amplification from gDNA and cDNA templates using the 560 1167_F_V1 splicing and 1167A_R primer pair (table S4). Bands amplified from cDNA of the 561 IMPERIAL strain were purified using a Monarch® DNA Gel Extraction Kit (New England Biolabs®) 562 and Sanger sequenced (Genewiz Inc.). Male cDNA PCR reaction was additionally subjected to 563 Sanger sequencing post-PCR product cloning using the StrataClone PCR Cloning Kit (Agilent) 564 whereby different isoforms were isolated. 565 566 RNA sequencing 567 18 To confirm the sex-specific expression of the wp gene, we performed Illumina RNA sequencing. 568 Total RNA was extracted from mature wpKI+/+ adult males and females from the IMPERIAL strain 569 in three biological replicates (six samples) using the miRNeasy Tissue/Cells Advanced Mini Kit 570 (Qiagen), following the manufacturer’s protocol. Genomic DNA was removed using the gDNA 571 eliminator column included with the kit. RNA integrity was tested with the RNA 6000 Pico Kit for 572 Bioanalyzer (Agilent Technologies). 573 574 Male (ID# 27026-27028) and female (ID# 27023-27025) libraries with three replicates each were 575 sequenced to approximate depth of 20M paired end reads (table S5). The reads were aligned 576 with STAR (https://github.com/alexdobin/STAR) to the EGII-3.2.1 genome assembly 577 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_905071925.1/), into which traF intron was 578 inserted at the white pupae locus (GCA_905071925.1_EGII-3.2.1_genomic.traF-intron.fna). To 579 generate a more complete annotation file we transferred the Ccap_2.1 annotations 580 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_000347755.4/) to the EGII-3.2.1 genome 581 by aligning Ccap_2.1 transcript sequences with BLAT and parsing the alignments to generate a 582 GTF file (Supplemental File 1), which was used for all subsequent analysis steps. Gene 583 abundances were quantified with featureCounts 584 (https://subread.sourceforge.net/featureCounts.html), count data (table S6, source data) were 585 converted to TPM (table S7, source data) and FPKM (table S8, source data) values and combined 586 using Perl scripts. Gene annotations were downloaded from EnsemblMetazoa using the BioMart 587 tool (https://metazoa.ensembl.org/Ceratitis_capitata_gca000347755v4/Info/Index) and added to 588 the quantification data (tables S6-S8, source data). TPM values were used to perform PCA and 589 clustering analyses in R to identify possible sample outliers. Replicates for each sex clustered 590 together as expected and displayed high correlations between each other without obvious outliers 591 (Fig. S4, table S9, source data). To visualize splicing of the traF intron within the white pupae 592 locus (LOC101451947), BAM files produced by STAR were imported into IGV 593 (https://igv.org/doc/desktop/). 594 595 Stability assay 596 From G3 until G7 all pupae were separated by colour and corresponding adults were scored by 597 sex. When the homozygosity of the IMPERIAL strain was verified at G7 (F0), alongside the 598 VIENNA 8 D53strain, for 5 consecutive generations (F2-F6) eggs were collected five days after 599 eclosion and raised under regular conditions until pupal stage of development. Brown and white 600 pupae were then separated, and the sex of all adults from each pool was recorded. 601 19 602 Egg-adult survival assay 603 Sibling crosses of 10 males and 20 females were set up in triplicates simultaneously for the 604 IMPERIAL, VIENNA 8 D53-, and wild-type Benakeion strains. Egg numbers and their hatching 605 rates were determined as described previously (7). Specifically, five days after eclosion, all eggs 606 laid within a 5-hour period were collected and unhatched eggs were counted twice four days apart 607 using Fiji (8). Pupal and adult recovery rates were determined thereafter. 608 609 Adult longevity assay 610 Age-matched adults from the wild-type Benakeion, IMPERIAL and VIENNA 8 D53strains were 611 separated by sex upon eclosion and placed into cages of 10 individuals. Three male and three 612 female replicates were set up simultaneously for each strain; and maintained under standard 613 conditions thereafter. Daily, dead flies were counted and removed from the cages for 30 614 consecutive days. 615 616 Mating-preference assay 617 90 females from the irradiation-generated wp-/- strain (3) were simultaneously placed together 618 with 15 males from each of the Benakeion, IMPERIAL and VIENNA 8 D53strains for a total of a 619 1:2 male: female ratio. The experiment was repeated 3 times. The flies were left to mate for 4 full 620 days, after which females were separated into individual small cages. Upon oviposition, eggs from 621 all females which oviposited were separately collected and reared normally until pupation. In the 622 cases where white and brown pupae were present, they were separated by colour. Adults 623 eclosing from both mixed and brown pupae-only collections were screened by sex. 624 625 Eclosion assay 626 24-hour egg collections were made from sibling crosses of 10 males and 20 females of the 627 IMPERIAL, VIENNA 8 D53-, and wild-type Benakeion strains, set up in parallel triplicates. The 628 offspring were reared under normal conditions until pupation, whereby IMPERIAL and VIENNA 8 629 pupae were sorted by colour. The eclosing adults were therein scored by sex every day. 630 631 Figure generation and statistical analyses 632 All statistical analysis and plot generation was performed in RStudio. Chi-squared tests were used 633 for sex ratio and pupal colour analysis. Egg-adult survival was assessed using Kruskal-Wallis and 634 Dunn’s tests. Longevity data was plotted and analysed using survival and survminer packages. 635 20 Eclosion rates were assessed using nested ANOVA by strain, with sex added in as an extra factor. 636 Pairwise comparisons were conducted via a post hoc Tukey HSD test thereafter. Microsoft 637 PowerPoint and Inkscape 1.3.2 (9) were used to create construct and fly-centred diagrams. 638 639 Supplementary references 640 1. Meccariello, A., Krsticevic, F., Colonna, R., Del Corsano, G., Fasulo, B., Papathanos, P. 641 A. & Windbichler, N. (2021) Engineered sex ratio distortion by X-shredding in the global 642 agricultural pest Ceratitis capitata. BMC Biology. 19 (1), 1–14. 643 2. Sollazzo, G., Gouvi, G., Nikolouli, K., Martinez, E. I. C., Schetelig, M. F. & Bourtzis, K. 644 (2022) Temperature sensitivity of wild-type, mutant and genetic sexing strains of Ceratitis 645 capitata. Insects. 13 (10), 943. 646 3. Ward, C. M., Aumann, R. A., Whitehead, M. A., Nikolouli, K., Leveque, G., Gouvi, G., 647 Fung, E., Reiling, S. J., Djambazian, H., Hughes, M. A., Whiteford, S., Caceres-Barrios, 648 C., Nguyen, T. N. M., Choo, A., Crisp, P., Sim, S. B., Geib, S. M., Marec, F., Häcker, I., 649 Ragoussis, J., Darby, A. C., Bourtzis, K., Baxter, S. W. & Schetelig, M. F. (2021) White 650 pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter. Nature 651 Communications. 12 (1), 491. 652 4. Meccariello, A., Hou, S., Davydova, S., Fawcett, J. D., Siddall, A., Leftwich, P. T., 653 Krsticevic, F., Papathanos, P. A. & Windbichler, N. (2024) Gene drive and genetic sex 654 conversion in the global agricultural pest Ceratitis capitata. Nature Communications. 15 655 (1), 372. 656 5. Holmes, D. S. & Bonner, J. (1973) Preparation, molecular weight, base composition, and 657 secondary structure of giant nuclear ribonucleic acid. Biochemistry. 12 (12), 2330–2338. 658 6. Chomczynski, P. & Mackey, K. (1995) Substitution of chloroform by bromo-chloropropane 659 in the single-step method of RNA isolation. Analytical Biochemistry. 225 (1), 163–164. 660 7. Davydova, S., Liu, J., Kandul, N. P., Braswell, W. E., Akbari, O. S. & Meccariello, A. (2023) 661 Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis 662 capitata. Scientific Reports. 13 (1), 19866. 663 8. Abràmoff, M. D., Magalhães, P. J. & Ram, S. J. (2004) Image processing with ImageJ. 664 Biophotonics International. 11 (7), 36–42. 665 9. Harrington, B. & Engelen, J. (2004) Inkscape. URL: Http://Www.Inkscape.Org. 666 667 Supplementary Figure legends 668 21 Fig. S1 Oxford Nanopore genome sequencing of the white pupae genomic site. 669 Diagrams depicting the nanopore sequencing reads (top grey) aligning to the white pupae 670 genomic location (blue) with the tra intron inserted in the IMPERIAL strain. 671 672 Fig. S2 white pupae is spliced sex-specifically in the IMPERIAL strain. 673 (A) The diagram showing the white pupae gene in the genome of the transformer (tra) intron 674 IMPERIAL knock-in (KI) strain with forward and reverse primers labelled as F and R, accordingly 675 (table S4). (B) The annotated electrophoresis gel of the PCR products using primers from (A). 676 The PCR was performed on genomic and complementary DNA templates from wild-type and 677 IMPERIAL adults. The DNA ladder and the negative control were run in the first and tenth wells, 678 respectively. 679 680 Fig. S3 RNA sequencing alignments to the wp genomic locus. 681 A zoomed-out genome browser view of the white pupae genomic locus and gene structure (blue 682 bars on bottom). RNAseq reads were aligned (light grey bars) for the female samples (27023-683 27025) and the male samples (27026-27028). The inserted tra intron is indicated in dotted red 684 box. 685 686 Fig. S4 Medfly RNAseq Clustering and Principal Component Analysis. 687 688 Fig. S5 white pupae genotyping for homozygous IMPERIAL strain generation. 689 Diagrams depicting the genotyping strategy to distinguish the three possible alleles, used at every 690 generation from G2 till G7. 691 692 Supplementary Table legends 693 Table S1 Statistical analysis of adult longevity by strain. 694 22 P-values for strain-by-strain pairwise log-rank test comparisons for adult longevity with Bonferroni 695 corrections. 696 697 Table S2 Statistical analysis of adult longevity by strain and sex. 698 P-values for pairwise log-rank test comparisons by sex and strain for adult longevity with 699 Bonferroni corrections. 700 701 Table S3 Statistical analysis of pupal eclosion times by strain and sex. 702 P-values for pairwise comparisons of post hoc Tukey HSD test after nested ANOVA by strain and 703 sex. 704 705 Table S4 Primer summary. 706 Sequences of primers used for construct cloning and cisgenic strain characterisation. 707 Figures Figure 1 Design behind the cisgenic IMPERIAL strain. (A) A simplied diagram showcasing the IMPERIAL strain generation and its underlying mechanism. The knock-in, mediated via homology-directed repair was performed into the Benakeion wild-type strain. Due to the presence of premature stop codons in the male-specic exons, the males are phenotypically whitepupaed, whilst in females, gene rescue occurs resulting in a brown-pupae phenotype (E1-E4, Exons 1-4; LHA, left homology arm; MFS, Major Facilitator Superfamily; RHA, right homology arm; tra, transformer; wp, white pupae). (B) Graphic summary of IMPERIAL strain establishment via outcrosses to the irradiationgenerated homozygous recessive white pupae mutant (wp-/-) strain (KI, knock-in).