Development of gene-edited Zeugodacus tau and Bactrocera zonata white pupae lines in support of sterile insect technique applications

Development of gene-edited Zeugodacus tau and Bactrocera zonata white pupae lines in support of sterile insect technique applications Zhi Gong1,**, Chrysanthi Ioannidou2,3,**, Marc F. Schetelig4,5, Elena Drosopoulou3, Kostas Bourtzis2,* 1 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Hainan, China 2 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, P.O. Box 100, 1400 Vienna, Austria 3 Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 24 124 Thessaloniki, Greece 4 Department of Insect Biotechnology in Plant Protection, Institute for Insect Biotechnology, Justus-Liebig-University Gießen, Winchesterstr. 2, 35394 Gießen, Germany 5 Liebig Centre for Agroecology and Climate Impact Research, Justus-Liebig-University Gießen; 35394 Gießen, Germany. * Corresponding author: [email protected] ** These authors contributed equally to this work. With 2 figures Abstract: Zeugodacus tau and Bactrocera zonata, native to South and Southeast Asia, are invasive quarantine pests causing significant economic damage to over 50 crops. The sterile insect technique (SIT), a proven method for controlling tephritid pests such as the Mediterranean fruit fly (Ceratitis capitata), could offer a sustainable control solution for these species. However, the absence of genetic sexing strains (GSS), essential for efficient and cost-effective SIT programs, hampers their operational use. The white pupae (wp) gene is a promising selectable marker for GSS development, but its applicability in these species has not been demonstrated. In this study, we identified and disrupted the orthologous wp gene in both species using CRISPR/Cas9 gene editing, targeting exon 3 as a first step in a proposed “neoclassical approach” to GSS development. Embryo microinjection resulted in high survival rates, and several homozygous mutant lines were established and validated through sequencing and protein structure modeling. Four wp mutant lines were generated in each species. Zeugodacus tau mutants exhibited reduced apical wing spots, while B. zonata mutants displayed partial loss of pigmentation in the thorax and abdominal tergites. These results confirm the wp gene as a functional and transferable GSS marker and demonstrate the potential of CRISPR/Cas9 for rapid GSS development in non-model tephritids. This work lays the foundation for future SIT applications against Z. tau and B. zonata, illustrating the transformative potential of genome editing in insect pest management. Keywords: CRISPR/Cas9 mutagenesis; white pupae gene; genetic sexing strain (GSS); Tephritidae; genome editing; pumpkin fruit fly; Phenotype characterization; Pigmentation 1 Introduction Zeugodacus tau (Walker 1849) and Bactrocera zonata (Saunders 1841) (Diptera: Tephritidae) are significant quarantine pests across South and Southeast Asia (Ullah et al. 2023; Liu & Ji 2024). Bactrocera zonata, the peach fruit fly, has expanded beyond its native range to established populations in parts of the Middle East and extensive regions of Africa, including Egypt, Sudan, and Libya (Ullah et al. 2023). Both species inflict substantial agricultural and economic impacts, infesting more than 50 plant hosts with a preference to fleshy fruits, particularly bananas for B. zonata and cucurbits for Z. tau (Liu & Ji 2024; EFSA et al. 2025). Their polyphagous nature, rapid reproduction, and long-distance flight ability, contribute to their rapid spread in climatically suitable regions, posing severe threats to global trade Entomologia Generalis, Vol. 45 (2025), Issue 6, 1689–1697 Open Access Published online December 4, 2025 © 2025 The authors DOI: 10.1127/entomologia/3833 E. Schweizerbart’sche Verlagsbuchhandlung, 70176 Stuttgart, Germany, www.schweizerbart.de Article and food security (Liu & Ji 2024; Malod et al. 2023). Various control methods are employed against these flies including male annihilation (EFSA et al. 2025; Liu & Ji 2024), insecticides (El-Maghraby et al. 2023; Liu & Ji 2024), entomopathogenic agents (Liu & Ji 2024; Murtaza et al. 2022), and parasitoids (Liu & Ji 2024). The sterile insect technique (SIT) offers an environmentally friendly and species-specific pest control strategy ideally implemented on an area-wide basis (Dyck et al. 2021; Hendrichs et al. 2021; Knipling 1955). This approach is based on the release of radiation-sterilized males in an area-wide manner to control insect populations and has been applied against diverse pest and disease vector species globally (Dyck et al. 2021; Hendrichs et al. 2021). Although SIT has been successfully implemented by releasing both sterile males and sterile females, with the most remarkable result being the eradication of Cochliomyia hominivorax from Southern US, Mexico and whole Central America with a permanent barrier established at the Panama Canal, it has been documented that the release of only males significantly improves its efficiency and cost-effectiveness (Rendón et al. 2004). Sex separation remains a key bottleneck for SIT scalability and the genetic sexing strains (GSS) can offer an efficient and precise way for separating males from females (Franz et al. 2021). The construction of a GSS is based on the availability of a selectable marker and the linkage of the wild-type allele of this marker to the male determining region. The selectable markers which have mainly been used for the construction of classical (non-transgenic) GSS are insecticide resistance, coloration/pigmentation or conditional lethal genes (Franz et al. 2021). Among them, the white pupae marker has been used for the construction of GSS in three tephritid species: the Mediterranean fruit fly Ceratitis capitata, the Oriental fruit fly Bactrocera dorsalis, and the melon fruit fly Zeugodacus cucurbitae. The practical application of these white pupae GSS has been demonstrated in successful operational SIT programs (Calla et al. 2014; McInnis et al. 2007; Kongratarpon et al. 2025). The recent discovery of the white pupae gene in combination with the development of gene-editing approaches paved the way for the induction of novel white pupae mutations in diverse SIT target tephritid species (Ward et al. 2021). Such mutations have now been developed for several Bactrocera species including Z. cucurbitae (Ward et al. 2021; Paulo et al. 2022; Zhang et al. 2023). The recently proposed neoclassical approach accelerates GSS development using genome editing instead of classical mutagenesis and screening (Petrucci et al. 2025). Here, we apply the first part of the strategy to generate and characterize wp knockout lines in Z. tau and B. zonata. We assess the feasibility and transferability of the neoclassical approach and the wp marker across SIT-targeted tephritid pest species. 2 Materials and methods 2.1 Insect strains and rearing All experiments were carried out at the Insect Pest Control Laboratory (IPCL) of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria. Laboratory colonies of Zeugodacus tau (Palampur, India) and Bactrocera zonata (Pakistan) had been maintained for 41 and 115 generations, respectively. Both strains were reared under constant laboratory conditions at 25 ± 2 ℃, 65 ± 5% RH, and a photoperiod of L12:D12. The adult fly diet consisted of a sugar and yeast extract mixture (3:1), with water ad libitum. Eggs of Z. tau were collected using zucchini slices and transferred to an artificial diet (Sollazzo et al. 2022), with the pH adjusted to 4.7. Bactrocera zonata eggs were collected in guava juicecoated artificial devices with peripheral holes and transferred to water-filled Petri dishes on a sponge and tissue paper to prevent desiccation. Upon hatching, first instar larvae were moved to Petri dishes containing the same diet. For pupation, the dishes were placed in containers with a layer of sand. Pupae were collected by sieving the sand and kept in Petri dishes until adult emergence. 2.2 Identifying the white pupae gene and designing primers and single-guide RNAs The wp gene was identified in Z. tau and B. zonata through BLAST homology searches against their respective genome assemblies, Z. tau (GCA_031772095.1) and B. zonata (GCA_043005645.1), available in the NCBI database (www.ncbi.nlm.nih.gov/datasets/genome) (Wang et al. 2023; Deschepper et al. 2024). The queries under review for the homology searches were the previously identified wp genes from Z. cucurbitae (LOC105216239) and B. dorsalis (LOC105232189) (Ward et al. 2021). The resulting sequences were used to design speciesspecific primer pairs (Table S1, Fig. S1) for amplification and sequencing of the target gene in the laboratory strains. Alignment of the obtained wp gene for Z. tau with its homolog from Z. cucurbitae (LOC105216239) and the corresponding mRNA sequence obtained from NCBI assisted in identifying the exon–intron boundaries of the wp gene. Similarly, the exon–intron structure of the B. zonata wp gene was determined by alignment with the B. dorsalis homolog (LOC105232189) and its corresponding mRNA sequence, also obtained from NCBI. All sequence alignments and comparative analyses were performed using Geneious Prime 2022.2.2. (https://www.geneious.com). The single-guide RNAs, gRNA_GMB_27 and gRNA_ GMB_28 (Table S1) targeting exon 3 of the wp gene in B. zonata and Z. tau, respectively, were designed using Geneious Prime 2022.2.2 (https://www.geneious.com) and CHOPCHOP v3 (CHOPCHOP). The crRNA and tracrRNA components were synthesized by MERCK-Sigma Aldrich 1690 Zhi Gong et al. (Germany) and resuspended in nuclease-free water before they were used for injections. 2.3 Detection and characterization of the white pupae gene Genomic DNA of Z. tau and B. zonata were extracted from six individual virgin adult flies (three females and three males) using the NucleoSpin® Tissue kit (MACHEREY‐NAGEL, Germany) according to the manufacturer’s instructions. The extracted DNA was used as a template for PCR gene amplification using InvitrogenTM PlatinumTM Hot-start Green PCR Master Mix (Invitrogen, Waltham, Massachusetts, USA), the primers listed in Table S1 with cycling conditions described in Supplementary material. The PCR products were purified using the DNA Clean & Concentrator-25 kit (ZYMO Research, Irvine, CA, USA), sequenced via Sanger sequencing and analyzed using Geneious Prime 2022.2.2. Following the sequence analysis, full-length nucleotide sequences of the wp gene from six tephritid species were aligned in MEGA 12.0.10 using CLUSTALW to assess sequence similarity, and pairwise comparisons were conducted using the *p*-distance model. To construct the phylogenetic tree, multiple sequence alignment was performed in MEGA12.0.10 using CLUSTALW, based on the amino acid sequences encoded by the wp genes of Z. tau and B. zonata, along with orthologs from selected tephritid species retrieved from GenBank. A neighbor-joining phylogenetic tree was constructed in MEGA 12.0.10 with 500 bootstrap replicates. Jalview was also used for aligning the wp genes of Z. tau and B. zonata to identify molecular structures and domains (https://www.jalview.org/). 2.4 Zeugodacus tau and Bactrocera zonata embryonic microinjections Embryo microinjections were carried out at 22 °C according to standard procedures with minor modifications. Zeugodacus tau embryos were collected within 20 min of oviposition, while for B. zonata, collection occurred within 30 min. The embryos were transferred into a mesh basket placed inside a Petri dish containing a layer of distilled water to prevent desiccation. A treatment with 1% sodium hypochlorite for 45 sec in Z. tau and for 120 sec in B. zonata was used for embryo dechorionation. To terminate the treatment, the embryos were rinsed thoroughly with distilled water to remove residual chemicals. The embryos were aligned in a uniform orientation on a glass slide using a double-sided adhesive tape (Scotch 3M, 665). After a 5–10 min air desiccation period, they were covered with Halocarbon oil 700 (Merck-Sigma Aldrich, Darmstadt, Germany) to prevent dehydration during the injection process. Detailed description of the instruments used for the microinjection prosses can be found in the supplementary material. 2.5 Establishment of white pupae mutant lines G0 adults produced from the microinjections were mated with virgin wild-type individuals of the opposite sex at a 1:3 sex ratio (Fig. S2). G1 progeny were intercrossed, resulting in the G2 generation (Fig. S2). Non-lethal genotyping was applied to all white pupae G2 individuals to detect the CRISPR/Cas9 causal mutation. Insects carrying the same mutation were inbred to establish a homozygous mutant line. Insect color at different developmental stages during the first generations (until a mutant line was established) was monitored under a Leica M205 FA stereo microscope and photographed using a Leica DFC 450C camera (Leica Microsystems) and the LAS software 4.13.0. For the non-lethal genotyping, DNA was extracted from a single leg using the Platinum Direct PCR Universal Master Mix kit (Invitrogen, Waltham, Massachusetts, USA). The extracted DNA was used as a template for PCR amplification using the primer pair GMB 668 – GMB 669 (Table S1) and the polymerase provided in the kit, with cycling conditions described in the supplementary material. The amplified products were purified using DNA Clean & Concentrator-25 (ZYMO Research, Irvine, CA, USA) and under review to Sanger sequencing. 2.6 Protein analysis The AlphaFold Protein Structure Database (https://www. alphafoldserver.com) was used to predict the 3D structures of the wild-type and mutant Wp proteins from Z. tau and B. zonata. This database uses deep learning algorithms to model protein structures from sequence data. Structural quality, including domain arrangement and confidence scores (pLDDT and PAE), were assessed using the AlphaFold online viewer. Additionally, membrane topology was analyzed using the DeepTMHMM web server (https://dtu.biolib.com/DeepTMHMM), which predicts transmembrane helices and their orientation using a neural network-based method. Default parameters were applied for all analyses. 3 Results 3.1 Sequence conservation of the wp gene in Zeugodacus tau and Bactrocera zonata The nucleotide sequences of the wp gene identified in the genome assemblies of Zeugodacus tau and Bactrocera zonata were compared to the ortholog sequences of the Tephritidae species in which wp mutations, either naturally occurring or induced, were known at the time of this writing, and of Drosophila melanogaster. At the nucleotide level, Z. tau exhibited the highest identity with Zeugodacus cucurbitae (98.75%), followed by Bactrocera tryoni (91.35%), Bactrocera dorsalis (91.29%), B. zonata (91.23%) and Ceratitis capitata (84.96%); the lowest identity was observed with D. melanogaster (71.13%) (Fig. S3 A). Bactrocera zonata shared the highest nucleotide identity with B. dorsalis (97.68%), followed by B. tryoni (97.62%), Z. tau (91.23%), Z. cucurbitae (91.1%), and C. capitata (84.65%); its lowest Zeugodacus tau and Bactrocera zonata white pupae lines 1691 identity was also with D. melanogaster (71.51%) (Fig. S3 A). Maximum Likelihood phylogenetic analysis using the Wp protein sequences of the above species (Fig. S3 B) grouped the Z. tau wp sequence with that of Z. cucurbitae, while the B. zonata sequence was placed as a sister clade to B. dorsalis and B. tryoni. 3.2 Establishment and characterization of white pupae mutant strains The wp gene was knocked out in Z. tau and B. zonata by targeting its third exon using the CRISPR/Cas9 system via nonhomologous end joining (NHEJ). For Z. tau, 1,318 embryos were injected. Of these, 906 embryos hatched into larvae (68.74%), 151 larvae developed into pupae (11.45%), and 139 pupae eclosed as adults. The overall egg-to-adult survival rate was 10.55%. The G₀ adults comprised 58 females and 81 males (Table S2). For B. zonata, 105 embryos were injected. Twenty-five of them hatched into larvae (23.80%), 22 of them pupated successfully (20.95%), and 15 adults emerged. The overall eggto-adult survival rate was 14.27%. The G₀ adults included six females and nine males (Table S2). Zeugodacus tau and B. zonata G0 adults were individually backcrossed to their respective wild-type parental strains (Fig. S2). Due to the recessive nature of the wp phenotype, G1 progeny were inbred to generate G2 progeny. Genotyping of virgin G2 individuals displaying the white pupae phenotype, via sequencing of the third exon of the wp gene, revealed distinct mutations in both species. An analysis of the wp gene with DeepTMHMM predicted twelve transmembrane helices in the wild-type protein of both species (Fig. S4). AlphaFold-predicted structural models of wildtype and mutant proteins revealed truncations and domain mislocalization in the mutants (Fig. S5, Fig. S6). Among the 139 crosses established in Z. tau, fourteen produced progeny exhibiting the white pupae phenotype, corresponding to seven distinct mutation events (ZtI_wp – CRISPR 1–7) (Fig. 1A). Homozygous lines were established for four of these events (ZtI_wp-CRISPR 1–4). The specific mutations identified in these lines comprised a 4-bp deletion (ZtI_wp – CRISPR 1), a 22-bp deletion with an 8-bp insertion (ZtI_wp – CRISPR 2), an 8-bp deletion with a 10-bp insertion (ZtI_wp – CRISPR 3), and a 9-bp deletion with a 7-bp insertion (ZtI_wp – CRISPR 4). All four mutations resulted in frameshifts and introduced premature stop codons, truncating the Wp protein to 344, 314, 346, and 318 amino acids, respectively. All mutations occurred within the fourth extracellular domain. Structural prediction indicated that these truncated proteins lack the ability to reinsert into the membrane and form the eighth transmembrane helix (Fig. S5). Three additional mutation events were identified. The ZtI_wp – CRISPR 5 mutants carried an 18-bp deletion along with a 5-bp insertion, while ZtI_wp – CRISPR 6 involved a 5-bp deletion. Both mutations caused frameshifts and introduced premature stop codons, resulting in truncated Wp proteins of 341 and 317 amino acids, respectively. The mutant ZtI_wp – CRISPR 7 was characterized by a 6-bp in-frame deletion, leading to the deletion of Asp304 and Val305 from the fourth extracellular domain of the Wp protein. This mutation did not cause a frameshift and resulted in a protein of 529 amino acids (compared to 531 amino acids in the wild-type). All Z. tau wp mutants exhibited the characteristic white pupae coloration. Interestingly, the adults also carried an additional morphological change as the size of the apical wing spot was significantly reduced (Fig. 1B, Fig. S7). Among the 15 crosses established in B. zonata, two crosses produced individuals exhibiting the white pupae phenotype. Six distinct mutational events in the wp gene were identified (Fig. 2A). Homozygous lines were established for four events (BzP_wp – CRISPR1–4). The remaining two events (BzP_wp – CRISPR 5, BzP_wp – CRISPR 6) were detected only in single individuals that died prior to producing progeny. The BzP_wp – CRISPR1 line harbored a homozygous 3-bp in-frame deletion, resulting in the loss of Arg283 within the seventh transmembrane helix of the Wp protein (Fig. 2A and S6). The BzP_wp – CRISPR 2, BzP_wp – CRISPR 3, and BzP_wp – CRISPR 4 lines carried homozygous deletions of 10-bp, 23-bp, and 7-bp, respectively. These deletions caused frameshifts, introducing premature stop codons and producing truncated Wp proteins of 284 amino acids (BzP_wp – CRISPR 2, BzP_wp – CRISPR 3) or 285 amino acids (BzP_wp – CRISPR 4) (Fig. S6). The mutations in BzP_wp – CRISPR 5 and BzP_wp – CRISPR 6 comprised an 18-bp in-frame deletion (resulting in the loss of Ile282, Arg283, His284, Ser285, Gly286, Gly287) and a 27-bp deletion with a 1-bp insertion (causing a frameshift and truncation to 283 amino acids), respectively. All identified mutations disrupted the coding sequence, preventing formation of the full twelve-helix membrane topology (Fig. S6). All B. zonata wp mutant lines presented the expected white pupae coloration. In addition, the adults showed loss of pigmentation in the thorax and abdominal tergite banding patterns, except for the post-ocular head spots. Wing pigmentation showed no observable differences (Fig. 2B). 4 Discussion Zeugodacus tau and Bactrocera zonata are among the most destructive tephritid pests in South and Southeast Asia, with invasive populations threatening fruit production and international trade (Liu & Ji 2024; EFSA et al. 2025). The SIT, applied as a part of area-wide pest management programs, offers a sustainable and species-specific solution for their control (Dyck et al. 2021). However, the absence of GSS in both species limits practical SIT implementation. Here, we report the first successful use of CRISPR/Cas9mediated knockout of the white pupae (wp) gene in Z. tau 1692 Zhi Gong et al. Fig. 1. CRISPR/Cas9-induced knockout mutations in Zeugodacus tau. A) Nucleotide and amino acid sequence comparisons of mutant wp alleles in G2 individuals versus wild-type reference sequence. Amino acids are numbered based on the wild-type sequence (area 303–322). Deletions are represented by dashes, insertions are shown in bold, while alterations leading to premature stop codons are marked by asterisks. The mutations induced in each line include: a 4-bp deletion (ZtI_wp – CRISPR 1), a 22-bp deletion with an 8-bp insertion (ZtI_wp – CRISPR 2), an 8-bp deletion with a 10-bp insertion (ZtI_wp – CRISPR 3), a 9-bp deletion with a 7-bp insertion (ZtI_wp – CRISPR 4), an 18-bp deletion with a 5-bp insertion (ZtI_wp – CRISPR 5), a 5-bp deletion (ZtI_wp – CRISPR 6), and a 6-bp in-frame deletion (ZtI_wp – CRISPR 7). Mutations in ZtI_wp – CRISPR 1–6 resulted in frameshifts and introduced premature stop codons, in the positions 345 (not shown here), 315, 347 (not shown here), 317, 341 (not shown here) and 317, respectively. The mutation ZtI_wp – CRISPR 7 consisted of a 6-bp in-frame deletion, leading to the deletion of Asp304 and Val305. B) Comparative phenotypic analysis of wild-type and CRISPR/Cas9-induced homozygous wp knockout mutants at the pupal and adult stage. Arrows indicate the apical wing spots which are smaller in the wp mutants. Generated using BioRender. Zeugodacus tau and Bactrocera zonata white pupae lines 1693 Fig. 2. CRISPR/Cas9-induced knockout mutations in Bactrocera zonata. A) Nucleotide and amino acid sequence comparisons of mutant wp alleles in G2 individuals versus wild-type reference sequences. Amino acids are numbered based on the wild-type sequence (area 273–293). Deletions are represented by dashes, insertions are shown in bold, while alterations leading to premature stop codons are marked by asterisks. The BzP_wp – CRISPR 1 carries a 3-bp in-frame deletion, resulting in the loss of Arg283. The BzP_wp – CRISPR 2, BzP_wp – CRISPR 3 and BzP_wp – CRISPR 4 lines carry deletions of 10-bp, 23-bp, and 7-bp, respectively. These deletions caused frameshifts, introducing premature stop codons in positions 285 (BzP_wp – CRISPR 2, BzP_wp – CRISPR 3) and 286 (BzP_wp – CRISPR 4). The mutation BzP_wp – CRISPR 5 has an 18-bp in-frame deletion resulting in the loss of Ile282, Arg283, His284, Ser285, Gly286 and Gly287. BzP_wp – CRISPR 6 mutation is a 27-bp deletion with a 1-bp insertion causing a frameshift and an induction of a premature stop codon in position 284. B) Comparative phenotypic analysis of wild-type and CRISPR/Cas9-induced homozygous wp knockout mutants at the pupal and adult stage. Arrows indicate the thorax and the abdominal tergite bands where loss of pigmentation is observed in the wp mutants. Generated with BioRender. 1694 Zhi Gong et al. and B. zonata, establishing homozygous mutant lines with consistent selectable phenotypes and morphological traits. Survival and editing efficiency (Table S2) were high for both species, comparing favorably to published CRISPR/ Cas9 studies in other tephritids such as C. capitata (Ward et al. 2021), B. tryoni (Choo et al. 2018), B. dorsalis (Bai et al. 2019), and B. oleae (Koidou et al. 2020). The relatively high survival in Z. tau may be attributed to the precise optimization of microinjection parameters and needle design, minimizing embryonic damage. Conversely, in B. zonata, survival was lower than in the recent work by Nazarov et al. (2025), likely due to our longer dechorionation time (1% NaOCl for 2 min vs. 0.5% for 30 s), which may increase early mortality. However, our earlier injection timing (<1 h post-oviposition) may have enhanced larval-to-adult survival (60% vs. 41%). This underscores the species-specific balance between embryo viability and editing success. The generated wp mutations were predominantly frameshift-inducing indels, introducing premature stop codons and truncating the Wp protein (Fig. 1, 2, S5 and S6), thereby abolishing function. In Z. tau, six of the seven characterized alleles caused premature truncation, removing multiple predicted extracellular and transmembrane domains. One allele (ZtI_wp – CRISPR 7) involved an in-frame deletion (ΔAsp304– Val305) within an extracellular loop. Despite preserving overall protein length, this line still displayed a white pupae phenotype similar to an observation in medfly (Ward et al. 2021). Structural modeling suggests that Asp304 may stabilize local folding via salt-bridge formation (Nick Pace et al. 2014; Ban et al. 2019), and Val305 may contribute to hydrophobic core integrity (Strub et al. 2004). Their deletion likely destabilizes domain conformation, impairing function. Similar functional consequences were observed in B. zonata. Notably, BzP_wp – CRISPR 1 carried a single amino acid deletion (Arg283) within the seventh transmembrane helix. This residue is likely involved in helix–helix packing or electrostatic stabilization, as previously shown for arginines in membrane-spanning regions (Ulmschneider et al. 2017; Armstrong et al. 2016). The resulting phenotype, white pupae with reduced adult pigmentation, indicates lossof-function despite minimal structural alteration. The other mutations (BzP_wp – CRISPR 2–6) caused frameshifts or deletions disrupting transmembrane topology. To our knowledge, this study provides the first clear evidence that wp mutations in tephritids can affect not only pupal but also adult morphology, as seen in reduced wing spot pigmentation (Z. tau) and thoracic/abdominal pigment loss (B. zonata). Previously, wp mutants were known only for their pupal phenotype (Ward et al. 2021; Paulo et al. 2022). The identification of the wp gene as a conserved gene encoding a Major Facilitator Superfamily (MSF) transporter protein (Ward et al. 2021) supports earlier biochemical studies showing that normal sclerotization and pigmentation require the transport of hemolymph pigment precursorscatecholaminesto the pupal cuticle (Wappner et al. 1995). The altered adult traits could reflect broader roles of wp in transporter-mediated movement of pigment precursors during development, possibly through shared pathways with eye and cuticle pigmentation. Interestingly, all white pupae mutant insects also displayed adult pigmentation defects, suggesting residual or stage-specific function of Wp isoforms. The role of the mucK gene, the orthologue of wp in Bombyx mori, in the pigmentation process of insect larvae (Zhao et al. 2012), as well as the higher expression levels of Bdorwp in both male and female adults (Zhang et al. 2023) support the physiological significance of the wp gene in different developmental stages. The development of wp lines in Z. tau and B. zonata shows how CRISPR/Cas9 can overcome the critical challenge of absent naturally occurring markers in non-model pest species. This study provides the first validated selectable marker for Z. tau and B. zonata, which could allow sex separation at the pupal stage, establishing essential tools for their genetic control. Our results support the “neoclassical approach” as a reproducible pipeline that can be rapidly adapted to other high-priority tephritid pests (Petrucci et al. 2025). Next steps to improve operational SIT programs should: (i) evaluate rearing efficiency parameters of wp mutants, (ii) combine this marker with conditional lethality systems (e.g., the recently identified tsl gene (Aumann et al. 2025), mirroring the success of VIENNA 8 in C. capitata, and (iii) develop sex-linkage through either classical translocation or CRISPR/Cas9 approaches. Combined, these steps would enable the development of robust, cost-effective GSS in both Z. tau and B. zonata, paving the way for their inclusion in SIT programs in Asia and Africa. Given their polyphagy, dispersal potential, and invasiveness, effective suppression of these species would have substantial agronomic and biosecurity impact. Acknowledgments: This study benefited from discussions at meetings for the Coordinated Research Project D44003, ‘Generic approach for the development of genetic sexing strains for SIT applications’, funded by the International Atomic Energy Agency (IAEA). This study was financially supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, and the Technical Cooperation Department of the IAEA through a fellowship to Z.G. Additional funding was provided by the European Union’s Horizon Europe Research and Innovation Program REACT (Grant Agreement number 101059523 to MFS). We also thank Georgia Gouvi and Germano Sollazzo for their contribution in designing the sgRNAs targeting the wp gene for both species. References Armstrong, C. T., Mason, P. E., Anderson, J. L. R., & Dempsey, C. E. (2016). Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage-sensitive ion channels. 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Journal of Insect Physiology, 58(11), 1397–1405. https://doi.org/10.1016/j. jinsphys.2012.08.002 Manuscript received: July 17, 2025 Revisions requested: August 12, 2025 Revised version received: September 15, 2025 Manuscript accepted: October 6, 2025 The pdf version (Adobe JavaScript must be enabled) of this paper includes an electronic supplement: Supplementary methods, Fig. S1–S7, Table S1, S2. Zeugodacus tau and Bactrocera zonata white pupae lines 1697