Genome assembly of five Tephritid species for the enhancement of the Sterile Insect Technique

Tephritidae insect pests account for extensive crop damage and yield losses globally. Modern, sustainable pest management approaches are species-specific and, therefore, high- quality genome assemblies are required for their application. Here, we present chromosome- level assemblies for five members of the Tephritidae family: Anastrepha fraterculus , Anastrepha ludens, Bactrocera dorsalis, Bactrocera zonata and Zeugodacus cucurbitae. The assemblies used long read sequencing polished with short read sequencing and scaffolded using Hi-C (chromatin conformation capture) sequencing. Prior to scaffolding the assembly deduplication was performed to separate a primary assembly and an alternate assembly, and each was then scaffolded independently. The scaffolded assemblies reached N50 length in the range of 60Mb to 120Mb. The scaffolded assemblies were verified with BUSCO and completeness was in the range 97% to 98.5% and had very low duplicated, fragmented and missing orthologs.

chromosome -level genome assembly, long read sequencing, Hi -C scaffolding, invasive species

Members of the Tephritidae family that cause significant yield and economic losses are worldwide distributed in areas that include: tropical areas of Southeast Asia and in the sub-Saharan region in Africa ( Bactrocera dorsalis ), the Southern and South -East Asian countries and the Arabian Peninsula ( Bactrocera zonata ), Asian and Australian -Oceanian Regions (Z eugodacus cucurbitae ), Central America ( Anastrepha ludens and Anastrepha fraterculus) and in the Southern United States, Caribbean Islands, and South Am erica (Anastrepha fraterculus) (EPPO 2024). The spread of these species can occur either through natural movement of the flies or by human -assisted means of transport (imports of fruit commodities or fruit in passenger luggage) thus elevating the risk of invasion in new areas. Additionally, the ongoing climate crisis is expected to provoke the spread of invasive insects into new regions (Papadopoulos et al. 2024; Qin et al. 2019; Ullah et al. 2023). Bactrocera dorsalis (Hendel), the oriental fruit fly, belongs to the “dorsalis complex” which includes pest species that cause devastating fruit losses in global fruit production. Due to its extensive host range (more than 270 plant species) (Vargas et al. 2015), its high reproductive rate and the continuously rising global temperatures, it is estimated that previously unsuitable areas are or will soon be available for the introduction and establishment of B. dorsalis (Jaffar et al. 2023; Zhao et al. 2024). Bactrocera zonata (Saunders), the peach fruit fly, mainly attacks peach and guava cultivars but mango and citrus fruits are also among its long list of cultivated hosts (Delrio & Cocco 2012; White & Elsonharris 1994). Bactrocera zonata’s polyphagous habits and the absence of dormancy allow the pest to be continuously active and complete several generations throughout the year (El-Mahdy et al. 2009; Khan & Naveed 2017). Zeugodacus cucurbitae (Coquillett), the melon fruit fly, is considered native to India but it has been introduced in several Asian and African countries, as well as in regions of Oceania .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint (De Meyer et al. 2015). Cucurbit crops are the main hosts of Z. cucurbitae, although non - cucurbit crops have also been reported (Dhillon et al. 2005). Zeugodacus cucurbitae has been shown to withstand a wide range of temperatures at all developmental stages, which can enhance its geographical distribution potential (Ahn et al. 2022). The Mexican fruit fly, Anastrepha ludens (Loew), is a major pest of citrus and mango in tropical and subtropical areas (CABI 2019). It is considered one of the most abundant fruit flies in these areas with substantial economic impacts that range from fruit losses to increased production costs and trade restrictions (Hernández et al. 2017; Zapata 2022). Another member of the Anastrepha spp., the South American fruit fly, Anastrepha fraterculus (Wiedemann), is a highly destructive pest of a wide range of fruits including citrus, apples and stone fruits (Ovruski et al. 2003; Segura et al. 2006). Due to its extent morphological and genetic variability, A. fraterculus is considered a cryptic species complex, a factor that has contributed to enhanced quarantine regulations and international trade regulations (Hendrichs et al. 2015). The need to develop efficient pest management techniques against the above fruit flies along with the global shift away from chemical applications have advanced the sterile insect technique (SIT) into a prime approach. The SIT is based on the release of ir radiated sterile males in the wild that mate with wild females resulting in a decrease of the population in the field (Dyck et al. 2021). It is an environmentally friendly and sustainable control method that has been applied for the suppression, eradication, prevention or containment of several insect pests that affect economically important crops, livestock, and human health (Dyck et al. 2021; Klassen et al. 2021). SIT application efficiency and cost -effectiveness is improved when genetic sexing strains (GSS) of the target pest are employed. Male -only releases have been previously proven to increase the SIT effectiveness and decrease the overall cost of the technique (Hendrichs et al. 1995; Rendón et al. 2004). Genetic sexing strains of fruit flies like Ceratitis capitata have been developed through irradiation and classical genetic approaches and are used worldwide with a proven success record (Franz et al. 2021; Aug ustinos et al. 2017). These GSSs are based not only on visible markers (white pupae - wp phenotype), but also on conditionally lethal traits (temperature -sensitive lethal - tsl phenotype), which allow for the removal of females early in the developmental process (Franz et al. 2021; Caceres 2002). The identification of selectable markers for the current GSS, like VIENNA 7 and VIENNA 8 of Ceratitis capitata, was achieved by classical genetic approaches and lasted over two decades (Franz et al. 2021; Augustinos et al. 2017). The continuous introduction and establishment of insect pests (Zingore et al. 2020) have urged scientific efforts to find faster and transferable approaches for the identification of new selectable markers across different species (FAO/IAEA 2019, 2021; Ward et al. 2021; Sollazzo et al. 2024). Recent efforts have been focused on a generic (neo-classical) approach that is based on the construction of non-transgenic GSS for SIT applications (FAO/IAEA 2019, 2021). The generic approach involves the identification of genes and their specific mutations responsible for desirable traits in a species that can function as selectable markers. Induction of mutations in the orthologous genes of other species and the linkage of the wild-type allele of the gene to .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint the male sex can lead to the construction of a new GSS for an SIT target species (Ward et al. 2021; Sollazzo et al. 2024). A high -quality genome sequencing and assembly can facilitate the identification of genes and their unique polymorphisms and advance the application of the generic approach in several species. This will subsequently allow for efficient genome editing and prevention of off-target changes through the implementation of genome editing approaches, such as the CRISPR/Cas9 technology (Bai et al. 2019; Komal et al. 2023; Paulo et al. 2022; Sollazzo et al. 2024; Ward et al. 2021). The CRISPR/Cas9 technology targets predetermined locations in the genome and therefore genomic data of high accuracy are required for efficient editing and mutation induction. In addition, a highly precise Y-chromosome assembly of the species used for GSS development can aid in the identification of suitable regions for knock-ins that will link the wild type allele of the selectable marker to males (FAO/IAEA 2019, 2021). In this study we sequenced and assembled at the contig level, male samples of five Tephritid fruit flies. Using state -of-the-art genome sequencing technologies (Illumina NovaSeq; Oxford Nanopore and PromethION) a valuable resource of genomic data with improved quality and accuracy was created. These new genome assemblies can be used for the identification of selectable markers and the induction of mutations in these five fruit fly species, while the assembly of the Y-chromosome further supports the development of GSS. Since the samples used for sequencing were in all cases males, valuable data for the Y - chromosome are also offered that will assist in the identification of suitable locations for gene insertions in this chromosome. Although all our assemblies are at the contig rather than the chromosome level, they bear value for population structure and global invasion route studies and offer essential data for studying the evolutionary history of the species (Zhang et al. 2023). These contig level assemblies will serve as an index for pinpointing the best targets for genetic manipulation and development of tephritid fruit flies GSS. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint

The assembly workflow produced high quality contig -level genomes using PacBio (continuous long reads) and Nanopore long reads based on BUSCO results. The Hi -C sequencing produced chromosome -scale scaffolds for all the genomes (See Table 1 and Figure 1). The assembly workflow also yielded highly complete haploid principal assemblies. The long read polished assemblies have a >97% complete BUSCO d etected (Table 2). The duplicate purging process also successfully removed duplicated sequences present in the initial long read assembly. The Hi-C maps shown in Figure 2 show a very clean picture of the scaffolding with no apparent mis -assemblies. The rep eat annotation (Table 3) shows the repeat types and numbers detected in the assemblies. The number of base repeats masked in each of the genomes ranges from 3.17% to 3.7%. Statistics Anastrepha fraterculus Anastrepha ludens Bactrocera dorsalis Bactrocera zonata Zeugodacus cucurbitae # contigs 4,973 1,238 588 660 5,140 # contigs (>= 50000 bp) 67 403 58 162 239 Largest scaffold 182,068,020 125,331,998 102,926,020 103,247,910 70,658,899 Total length 793,193,562 720,811,084 537,310,284 600,442,075 374,778,270 Total length (>= 50000 bp) 766,717,392 706,688,108 531,140,571 593,885,298 349,303,203 N50 121,046,886 118,249,840 76,567,395 65,592,266 62,275,020 L50 3 3 4 4 3 GC (%) 37 37 37 36 35 # N's 225,200 231,400 105,800 118,600 134,300 NG50 109,980,515 114,208,257 40,758,100 45,696,414 - LG50 4 5 8 7 - Table 1. Genome statistics for different, final principal (haploid) assemblies are shown. The scaffolded assemblies all reach an N50 length ranging from 62 Mb to 121 Mb with three to four scaffolds, which is indicative of a successful scaffolding process. Only Zeugodacus cucurbitae scaffolded to a smaller than expected genome size. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Figure 1. Plot of the percentage of the genome length against the sum of the scaffold sizes adding scaffold from longest to shortest using principal assemblies. To compare the species, we used a common genome size of 1Gb. Anastrepha fraterculus (AF), Anastrepha ludens (AL), Bactrocera dorsalis (BD), Bactrocera zonata (BZ) and Zeugodacus cucurbitae (ZC). Anastrepha fraterculus and Anastrepha ludens have their largest contigs sum up to 70% and 65% of the 1 Gb reference size, respectively. Bactrocera dorsalis and Bactrocera zonata both have their largest contigs sum to 50%- 55% of the 1 Gb reference size. Zeugodacus cucurbitae assembled to the smallest size at 30% of the 1 Gb reference size. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Complete Single Duplicated Fragmented Missing Anastrepha fraterculus Assembly error corrected (long and short reads) 98.0% 64.7% 33.3% 1.1% 0.9% Assembly error corrected (Hi-C reads) 97.9% 64.7% 33.2% 1.2% 0.9% Assembly duplicate purged 96.7% 94.9% 1.8% 1.5% 1.8% Assembly duplicate purged rescued 97.0% 95.2% 1.8% 1.5% 1.5% Final Hi-C scaffolded 98.0% 96.6% 1.4% 0.9% 1.1% Anastrepha ludens Assembly error corrected (long and short reads) 98.1% 80.0% 18.1% 0.5% 1.4% Assembly error corrected (Hi-C reads) 98.0% 79.9% 18.1% 0.6% 1.4% Assembly duplicate purged 95.7% 95.2% 0.5% 1.2% 3.1% Assembly duplicate purged rescued 96.9% 96.0% 0.9% 1.2% 1.9% Final Hi-C scaffolded 97.1% 96.6% 0.5% 1.1% 1.8% Bactrocera dorsalis Assembly error corrected (long and short reads) 99.2% 35.4% 63.8% 0.4% 0.4% Assembly error corrected (Hi-C reads) 99.1% 35.4% 63.7% 0.4% 0.5% Assembly duplicate purged 95.2% 93.5% 1.7% 0.8% 4.0% Assembly duplicate purged rescued 98.0% 95.5% 2.5% 0.9% 1.1% Final Hi-C scaffolded 98.3% 97.3% 1.0% 0.6% 1.1% Bactrocera zonata Assembly error corrected (long and short reads) 99.3% 47.3% 52.0% 0.2% 0.5% Assembly error corrected (Hi-C reads) 99.3% 47.4% 51.9% 0.2% 0.5% Assembly duplicate purged 97.8% 95.4% 2.4% 0.7% 1.5% Assembly duplicate purged rescued 98.3% 95.2% 3.1% 0.8% 0.9% Final Hi-C scaffolded 98.5% 96.3% 2.2% 0.7% 0.8% Zeugodacus cucurbitae Assembly error corrected (long and short reads) 98.2% 97.2% 1.0% 0.8% 1.0% Assembly error corrected (Hi-C reads) 98.3% 97.3% 1.0% 0.8% 0.9% Assembly duplicate purged 97.7% 97.2% 0.5% 0.8% 1.5% Assembly duplicate purged rescued 98.1% 97.6% 0.5% 0.8% 1.1% Final Hi-C scaffolded 98.4% 98.0% 0.4% 0.6% 1.0% Table 2. BUSCO statistics for the principal assemblies at the different stages of the assembly process (using diptera_odb10). .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint a) b) c) d) e) f) Figure 2. Hi-C maps generated with PretextGraph of the principal assembly for the following species: Anastrepha fraterculus (a), Anastrepha ludens (b), Bactrocera dorsalis (c), Bactrocera zonata (d) and Zeugodacus cucurbitae (e). In each plot the x- axis and y axis represents the scaffolds of the principal assembly with grid lines separating the scaffolds on both axis. Inside each square, using a heatmap we show the contact map of the interactions in the genome for inter and intra scaffold interactions measured from the HiC read alignments. In panel (f) we show a closeup of the largest primary scaffold for Zeugodacus cucurbitae mapped against itself showing the heat map in more detail. As expected, for all the species we mostly see intra-scaffold interactions near the diagonal and no inter-scaffold interactions. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Anastrepha fraterculus Anastrepha ludens Bactrocera dorsalis Bactrocera zonata Zeugodacus cucurbitae sequences: 5,502 7,879 7,027 7,749 9,971 total length: 682,452,311 721,912,558 539,979,902 594,029,458 374,234,511 GC level: 37% 37% 36% 36% 35% bases masked: 22,626,981 (3.2%) 24,285,344 (3.3%) 17,129,886 (3.1%) 19,389,731 (3.1%) 13,844,259 (3.5%) number of elements number of elements number of elements number of elements number of elements Retroelements 11,922 12,534 6,950 7,529 4,606 SINEs: 508 440 127 157 95 Penelope: 188 199 136 134 71 LINEs: 8,821 9,338 5,445 6,034 3,818 L2/CR1/Rex 4,285 4,260 1,871 2,166 725 R2/R4/NeSL 466 490 0 2 0 RTE/Bov-B 3,707 4,153 3,377 3,664 2,972 L1/CIN4 346 406 180 169 111 LTR elements: 2,593 2,756 1,378 1,338 693 Gypsy/DIRS1 2,478 2,643 1,308 1,255 642 Retroviral 110 109 66 72 47 DNA transposons 17,928 23,209 12,455 17,838 3,703 hobo-Activator 1,161 1,345 1,502 1,849 394 Tc1-IS630-Pogo 16,059 21,154 10,740 15,725 3,193 MULE-MuDR 22 17 6 27 0 PiggyBac 346 356 111 109 18 Tourist/Harbinger 1 1 3 3 1 Rolling-circles 1,202 1,539 822 524 486 Unclassified: 2,747 3,016 2,315 2,320 830 Small RNA: 1,756 1,544 774 940 635 Satellites: 229 218 124 139 60 Simple repeats: 295,682 311,488 241,586 262,464 226,297 Low complexity: 51,191 53,640 33,921 39,229 31,227 Table 3. Repeat annotation using Repeat Masker with Dfam database, including Diptera family. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint

In this work we present assemblies for Anastrepha fraterculus, Anastrepha ludens , Bactrocera dorsalis, Bactrocera zonata and Zeugodacus cucurbitae. The assemblies were constructed using state-of-the-art technologies, combining both long read sequencing and Hi- C chromatin proximity ligation. Critical to a good initial assembly is the molecular weight and amount of reads used. A large enough contig N50 is important for the Hi-C scaffolding to be successful. A second factor that can impact the Hi-C scaffolding is the presence of duplicated contigs in the assembly as these will cause Hi -C reads to be removed because of duplicate mapping. An optimal duplicate purging step is critical to separate the initial assembly into haploid assemblies and ensure the success of the scaffold sizes into the chromosome scale. Existing assemblies are deposited in NCBI for Anastrepha ludens, Bactrocera dorsalis, and Zeugodacus cucurbitae. Although the strains differ and there is merit in each of these assemblies on its own, we compared the BUSCO scores (Table 4) across the assemblies to show that the assemblies presented here are in the same range as in previous studies. Assembly BUSCO Comple te BUSCO Duplicat ed BUSCO Frag. BUSCO Missing # contigs Total length N50 L50 BD (this paper) 98.5% 1.8% 0.6% 0.9% 588 537,310,284 76,567,395 4 BD_GCA_020283865.1 92.6% 0.2% 0.8% 6.6% 6 468,671,466 90,458,965 3 BD_GCA_023373825.1 98.1% 0.2% 0.8% 1.1% 586 530,310,706 93,297,747 3 BD_GCA_029030885.1 97.3% 2.9% 1.0% 1.7% 952 524,751,100 1,217,930 109 BD_GCA_030710565.1 95.1% 1.3% 0.9% 4.0% 1,038 566,605,033 103,769,376 3 ZC (this paper) 98.5% 0.5% 0.6% 0.9% 5,140 374,778,270 62,275,020 3 ZC_GCA_000806345.2 98.2% 0.2% 0.7% 1.1% 5,545 374,633,570 1,415,739 65 ZC_GCF_028554725.1 98.8% 0.3% 0.4% 0.8% 56 439,259,567 75,513,634 3 AL (this paper) 97.5% 0.7% 0.8% 1.7% 1,238 720,811,084 118,249,840 3 AL_GCA_029783585.1 77.0% 9.2% 6.6% 16.4% 155,863 988,756,442 11,016 26,222 AL_GCA_028408465.1 99.0% 0.3% 0.3% 0.7% 141 820,783,141 131,385,309 3 Table 4. BUSCO scores across different assembly versions published for Anastrepha ludens (AL), Bactrocera dorsalis (BD) and Zeugodacus cucurbitae (ZC). .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint

Sample collection and DNA extraction Virgin freshly emerged adult males were collected from long -established laboratory colonies of the five fruit fly species at the Insect Pest Control Laboratory of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture (Seibersdorf, Austria). The geographic origin and colony generation used for sampling the five species are shown in Supplementary Table S1. High-Molecular-Weight (HMW) genomic DNA was extracted from the male flies (2 -3 flies per extraction) with different kits. The Nanobind tissue kit (Pacific Biosciences) was used for Anastrepha fraterculus and Zeugodacus cucurbitae . The New England Biolabs (NEB) Monarch DNA extraction kit was used for Bactrocera dorsalis and Bactrocera zonata. The QIAGEN Genomic tip 20/G kit (Qiagen, Germany) was used for Anastrepha ludens. The same extracted DNA was used for long-read sequencing and short-read sequencing. Short-read and long-read library construction The short-read sequencing libraries for all species except Anastrepha ludens were constructed using an ultrasonicator (Covaris) for the shearing and the NxSeq AmpFREE Low DNA ligation kit with xGen dual index adapters from Integrated DNA Technologies (IDT). For Anastrepha ludens the short read libraries were constructed using TruSeq PCR-free (Illlumina) The Oxford Nanopore libraries were constructed with unsheared DNA using the Pacific Biosciences short read elimination kit (SRE <25 kb) first and the Oxford Nanopore kit SQK - LSK-109 kit for the library for R9 pore chemistry following the manufacturer’s recommendations. For the Anastrepha ludens PacBio sequencing the sample was purified with AMPure beads (Beckman Coulter, UK) (0.6 volumes) and QC checked for concentration, size, integrity, and purity using Qubit (Qiagen, UK), Fragment Analyser (Agilent Technologies) and Nanodrop (Thermo Fisher) ma chines. The samples were then processed without shearing using the PacBio Express kit 1 for library construction and an input of 4 µg DNA following the manufacturer’s protocol. The final library was size-selected using the Sage Blue Pippin (Sage Sciences) 0.75% cassette U1 marker in the range of 25 –80 kb. The final library size and concentrations were obtained on the Fragment Analyser before being sequenced using the Sequel 1 2.1 chemistry with V4 primers at a loading on plate conc entration of 6 pM and 10 h movie times. Hi-C proximity ligation library construction Each proximity ligation library was prepared from several frozen whole flies which were first ground with an SP Bel -Art liquid nitrogen cooled mini mortar (Fisher Scientific). The ground material was then fixed, digested and proximity ligated using the Arima High Coverage HiC+ kit. The final ready -to-sequence library was constructed using a Covaris ultrasonicator for the shearing and the NxSeq AmpFREE Low DNA ligation kit and xGen dual index adapters from Integrated DNA Technologies (IDT). .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Sequencing The sequencing of the whole genome sequencing libraries was performed on Illumina NovaSeq 6000 S4 (PE150), on Illumina HiSeq 4000 (PE150), and on MGI tech. DNB -G400 (PE150). The Hi-C libraries were sequenced on Illumina NovaSeq 6000 S4 (PE150) and MGI tech. DNB-G400. For MGI sequencing the Illumina-compatible libraries were first circularised for MGI to allow the DNA nanoballs to be generated. The long read sequencing was performed using both the Pacific Biosciences (PacBio) Sequel and the Oxford Nanopore PromethION P48. See supplementary table S2 for sequencing instruments and sequencing yields. The goat database (goat.genomehubs.org) reports the genome size to be 949Mb for all five species. This genome size was used to calculate the raw read coverage for each type of sequencing. Genome assembly and scaffolding The Anastrepha ludens PacBio continuous long reads (CLR) were assembled with the Canu assembler. The other four species ( Anastrepha fraterculus , Bactrocera dorsalis , Bactrocera zonata , and Zeugodacus cucurbitae ) sequenced with Oxford Nanopore were assembled with the Flye assembler. All the assemblies were polished for four rounds using Pilon. The short reads were trimmed with Trimmomatic before being used for polishing. Tigmint was then used with the long reads to further error correct the assemblies. A second correction is performed using the Hi-C data and YaHS by converting the inital_break agp file to a fasta file. Following the short read, long read, and Hi -C error correction of the assemblies, a haploid version of the assembly is created using Purge_Dups to remove duplicate contigs. This step results in a first version of the principal haploid assembly and an alternate assembly containing the second allele and contig duplicates. To avoid overpurging contigs from the principal haplotype a contig rescue step is performed. The rescue is done by aligning transcripts from the species or closely related species to the principal and alternate assemblies and moving contigs from the alternate to the primary in the case where transcripts only map to the alternate assembly. In the case of Anastrepha fraterculus transcripts from Anastrepha ludens were used. In the case of Bactrocera zonata transcripts from Bactrocera dorsalis were used with the addition of the Maleness-on-the-Y (MoY) gene. At this stage, the main scaffolding is performed on both the principal assembly and on the alternate assembly using chromap for the trimming/mapping and YaHS for the scaffolding. Before scaffolding the alternate assembly, Purge_Dups is used to remove any d uplications still present in the alternate assembly. All tool versions used are listed in supplementary table S4. The assembly workflow is illustrated in Figure S1. The version of the tools is listed in supplementary Table S4. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Additional assembly processing steps for NCBI submission Before NCBI submission we removed mitochondrial contigs using the following mitochondrial (MT) genome sequences: NC_027725.1 for Bactrocera zonata, NC_008748.1 for Bactrocera dorsalis , NC_034912.1 for Anastrepha fraterculus , MH900082.1 for Zeugodacus cucurbitae and MT121222.1 for Anastrepha ludens . Scaffolds from the assemblies were aligned to the MT sequences with minimap2 and were removed if more than 80% of the scaffold was aligned. Finally at the end of the workflow the alternative assemblies are missing homozygote scaffolds that were not duplicated in the initial assembly. For NCBI these missing scaffolds were copied back into the alternative assembly to form a more complete alternative assembly. Repeat sequence annotation The primary assemblies were annotated for repeats using RepeatMasker with the Dfam database (RepeatMasker version 4.1.6, rmblastn version 2.14.1+, Dfam 3.8 with Diptera family). .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Data availability All sequencing datasets and assemblies have been deposited to National Center for Biotechnology Information (NCBI) in separate Bioprojects for each species. Raw Read Data The raw sequencing data for the five species have been deposited to NCBI under the sequence read archive (SRA) section. The short read whole genome sequencing (WGS) and Hi-C proximity ligation datasets are provided as untrimmed fastq files. The Oxford Nano pore and PacBio long reads are both provided in fastq format. All the raw read datasets have been submitted under the Bioproject of the corresponding principal assembly (see Table 4). Table 4. Raw sequencing reads are submitted to the NCBI sequence read archive. Species NCBI Bioproject Dataset type NCBI SRA accessions Anastrepha fraterculus PRJNA1065016 Long read WGS SRR29293661, SRR29293662, SRR29293663, SRR29293664 Short read WGS SRR29319412, SRR29319413 Hi-C SRR29319415, SRR29319414 Anastrepha ludens PRJNA1076526 Long read WGS SRR29307005, SRR29307006, SRR29307007 Short read WGS SRR29323014, SRR29323013 Hi-C SRR29323016, SRR29323015 Bactrocera dorsalis PRJNA1076946 Long read WGS SRR29299427 Short read WGS SRR29323025, SRR29323026 Hi-C SRR29323028, SRR29323027 Bactrocera zonata PRJNA1082643 Long read WGS SRR29299502, SRR29299503 Short read WGS SRR29319677, SRR29319676 Hi-C SRR29319678 Zeugodacus cucurbitae PRJNA1070629 Long read WGS SRR29294704, SRR29294702, SRR29294703 Short read WGS SRR29322956, SRR29322955 Hi-C SRR29322957, SRR29322958 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint NCBI Assemblies The contig level principal and alternate assemblies for the five species have been deposited to the NCBI genomes section under separate Bioprojects linked by the same species Biosample (see Table 5). The scaffolded versions are submitted to NCBI. Table 5. Bioprojects and GeneBank assembly IDs in NCBI. 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). The authors also wish to thank Elena Isabel Cancio Martinez and Gülizar Pillwax for insect rearing. Funding This study was financially supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, the German Research Foundation through the Middle East Cooperation project 491548882, the Canada Foundation for Innovation grants 40104 and 3544 and the BBSRC (Biotechnology and Biological Sciences Research Council) under the research grants BB/P000843/1 and BB/W00304X/1. Species Biosample Bioproject (principal) Bioproject (alternate) GenBank assembly (principal) GenBank assembly (alternate) Anastrepha fraterculus SAMN39449916 PRJNA1065016 PRJNA1065015 GCA_037575425.2 GCA_037575645.2 Anastrepha ludens SAMN39944021 PRJNA1076526 PRJNA1076525 GCA_037783455.2 GCA_037783485.2 Bactrocera dorsalis SAMN39957830 PRJNA1076946 PRJNA1076945 GCA_037783525.2 GCA_037783465.2 Bactrocera zonata SAMN40214030 PRJNA1082643 PRJNA1082642 GCA_037783105.2 GCA_037783125.2 Zeugodacus cucurbitae SAMN39655635 PRJNA1070629 PRJNA1070628 GCA_037783285.2 GCA_037783305.2 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Supplementary Information Supplementary Figure S1. Assembly workflow Species Generation Geographic origin (strain) Anastrepha fraterculus G63 Brazil (Vacaria) Anastrepha ludens G117 Mexico Bactrocera dorsalis G113 Thailand (Saraburi) Bactrocera zonata G17 Israel Zeugodacus cucurbitae G127 Mauritius (Sookar 5/a) Supplementary Table S1. Description of fly strains and generations used for the DNA extraction. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 16, 2025. ; https://doi.org/10.1101/2025.09.15.670340doi: bioRxiv preprint Species DataSet Type Sequencer Total reads Total bases Taxon ID Genome SIze in Mb Genome fold Coverage Anastrepha fraterculus Long read WGS PrometION R9 18,054,709 38,607,090,382 95504 949 40.7 Short read WGS NovaSeq 6000, S4 55,815,810 16,856,374,620 17.8 Hi-C NovaSeq 6000, S4 261,079,890 78,323,967,000 82.5 Anastrepha ludens Long read WGS PacBio, Sequel 2,354,154 29,476,521,382 28586 949 31.1 Short read WGS HiSeq 4000 62713989 18486278987 19.5 Hi-C NovaSeq 6000, S4 172,962,558 51,888,767,400 54.7 Bactrocera dorsalis Long read WGS PrometION R9 4,135,925 74,158,935,467 27457 949 78.1 Short read WGS HiSeq 4000 49,171,609 14,511,740,112 15.3 Hi-C NovaSeq 6000, S4 288,365,720 86,509,716,000 91.2 Bactrocera zonata Long read WGS PrometION R9 2,172,892 38,457,368,805 137042 949 40.5 Short read WGS MGI DNB-G400 110,367,330 33,110,199,000 34.9 Hi-C MGI DNB-G400 154,157,696 46,247,308,800 48.7 Zeugodacus cucurbitae Long read WGS PrometION R9 21,084,151 87,690,269,635 28588 949 92.4 Short read WGS NovaSeq 6000, S4 60,261,930 18,199,102,860 19.2 Hi-C NovaSeq 6000, S4 154,157,696 46,247,308,800 48.7 Supplementary Table S2. 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