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. 2025 Jul 17;34(6):960–969. doi: 10.1111/imb.70001

Establishment of transgenic Drosophila suzukii lines that express phiC31 integrase and carry the sepia gene as a marker for transformation

Kalindu Ramyasoma Hewawasam 1, Akihiko Yamamoto 1, Maxwell J Scott 1,✉
PMCID: PMC12604457  PMID: 40673377

Abstract

Many eye colour mutants have been identified in Drosophila melanogaster. Mutations in the sepia gene result in brown eyes due to a lack of PDA synthase, which is essential for production of the red drosopterin eye pigment. We previously used CRISPR/Cas9 to target the PDA synthase gene to establish sepia mutant strains for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an invasive global pest of soft skinned fruits. The fecundity and fertility of some of the sepia mutant strains were similar to wild‐type. The goal of this study was to determine if the sepia gene could be used as a marker to identify transgenic D. suzukii. By using the sepia gene as a marker, we successfully developed lines expressing Streptomyces phage phiC31 integrase in the germline. For most of these lines, hemizygotes exhibited complete rescue of the sepia eye colour and relatively high levels of phiC31 RNA in ovaries. In contrast, lines with partial rescue showed low levels of sepia RNA in heads and phiC31 RNA in ovaries. These findings suggest that the sepia gene is an effective marker for D. suzukii transgenesis and its relatively small size (1.8 kb) makes it advantageous when assembling large gene constructs. The phiC31 integrase lines established in this study should serve as a valuable resource for future genetic research in D. suzukii, including the further development of strains for genetic biocontrol.

Keywords: Drosophila suzukii, eye colour, phiC31 integrase, piggyBac, sepia

  • Germline transformation of the Drosophila suzukii se 1 strain with the wild‐type sepia gene (~1.8 kb) using a piggyBac vector.

  • Most lines show full rescue of sepia mutant eye colour.

  • The lines express phage phiC31 integrase in the germline using a Dsnanos promoter. The lines with partial rescue of the eye colour have low phiC31 RNA levels in ovaries.

graphic file with name IMB-34-960-g004.jpg

INTRODUCTION

The application of transgenic techniques has greatly facilitated studies on gene function, insect physiology and development of genetic‐based pest management strategies (Benedict & Scott, 2022; Handler & James, 2000). Central to transgenic approaches is the use of selectable marker genes, which enable researchers to identify successfully modified organisms. Germline transformation in Drosophila melanogaster (Diptera: Drosophilidae) Meigen 1830 has utilised mutant rescue selection to identify transgenic flies, including the first report of successful germline transformation that used the rosy eye colour gene (Rubin & Spradling, 1982). Similarly, the first report of successful transformation of a non‐drosophilid insect used the white eye colour gene to identify transgenic Ceratitis capitata (Diptera: Tephritidae) Wiedemann, 1824 (Loukeris et al., 1995). The recipient eye colour mutant lines are typically easy to rear in the laboratory, comparable to wild‐type. Further, the relatively small size of some of the eye colour genes (<3 kb) and ease of distinguishing transformants from mutant have made eye colour genes highly suitable selectable markers for insect transgenesis (Horn et al., 2002). Another advantage of using an eye colour gene as a selectable marker is that fluorescent protein genes can then be used for other purposes such as the green fluorescent protein (GFP)‐based GCaMP proteins for detecting neuronal activity in live insects (Bykhovskaia, 2022; Coutinho‐Abreu & Akbari, 2023). The D. melanogaster white (Pirrotta, 1988), rosy (Rubin & Spradling, 1982) and vermilion (Fridell & Searles, 1991) eye colour genes are widely used to identify transgenic adults. The sepia gene in D. melanogaster is essential for the biosynthesis of the red drosopteridin pigment and consequently sepia mutant flies have a distinctive brown eye colour (Gramates et al., 2022; Kim et al., 2006). Although the sepia gene, which encodes PDA (6‐acetyl‐2‐amino‐3,7,8,9‐tetrahydro‐4H‐pyrimido[4,5‐b]‐[1,4]diazepin‐4‐one or pyrimidodiazepine) synthetase (Kim et al., 2006), is small (transcribed region is less than 1000 bp), to our knowledge it has not been used as a marker for transformation.

Drosophila suzukii (Diptera: Drosophilidae) Matsumura, 1931, commonly known as the spotted‐wing drosophila, has gained attention as a significant agricultural pest (Tait et al., 2021). Unlike other Drosophila species that feed on decaying fruit, D. suzukii can infest fresh fruit, causing extensive damage to crops such as cherries, strawberries and raspberries (Walsh et al., 2011). Since its introduction to North America and Europe, this species has posed a severe threat to fruit industries, necessitating innovative pest control solutions (Asplen et al., 2015). We previously reported on using CRISPR/Cas9 gene editing to obtain mutant strains of D. suzukii homozygous for loss‐of‐function mutations in the white, cinnabar, and sepia eye colour genes (Yadav et al., 2024). The fecundity and fertility of some of the sepia mutant strains were similar to wild‐type and thus would be highly suitable to use as parental strains for germline transformation. In contrast, white mutant strains are unsuitable as most white eye males are sterile (Yadav et al., 2024; Yan et al., 2020). Compared with cinnabar mutants, sepia mutants are slightly easier to distinguish from wild‐type.

Current methods for managing D. suzukii populations rely heavily on chemical pesticides, which pose risks to both the environment and non‐target species (Tait et al., 2021). Genetic approaches, such as conditional female‐lethal and gene drive systems, offer promising alternatives for sustainable pest control (Buchman et al., 2018; F. Li et al., 2021; Schetelig et al., 2021; Yadav et al., 2023; Yan et al., 2025). However, the success of such strategies depends on efficient genetic systems for generating and tracking genetically modified individuals.

Transgenic D. suzukii strains have been made using the piggyBac transposase (Buchman et al., 2018; Schetelig & Handler, 2013) but the efficiency of transformation is strain‐dependent (Ahmed et al., 2020). Further, there is no control over where the transgene integrates in the genome. Site‐specific recombination systems provide precise control over transgene location in the genome. The Streptomyces phage phiC31 integrase mediates recombination between the bacterial attachment site (attB) and the phage attachment site (attP) (Bischof & Basler, 2008; Venken & Bellen, 2012). It has the advantage that it only mediates integration and has been used for very large gene constructs (>100 kb). The integrase can also be used for recombination‐mediated cassette exchange (RMCE), where the gene constructs are flanked by inverted attP or attB sites (Bateman et al., 2006).

The main aim of this study was to evaluate the use of the sepia gene as a selectable marker for identifying transgenic D. suzukii. A secondary aim was to generate transgenic lines that express phiC31 integrase in the germline for targeted transgenesis and RMCE. This work has implications for the development of novel gene‐editing tools in pest control and broader applications in Drosophila genetic research.

RESULTS

Rescue of the sepia phenotype

Two gene constructs were made based on the piggyBac transformation vector pBAC2 (Concha et al., 2011), with both constructs employing the sepia gene as a selectable marker (Figure 1). For the current assembly (Paris et al., 2020), the sepia gene (LOC108013534) consists of three exons and two introns (67 and 57 bp) spanning 949 bp (Figure 1). The current annotation shows a 5′UTR of 36 bp in the 64 bp first exon and a 3′UTR of 57 bp in the 192 bp final exon. The sepia gene fragment used in this study includes 801 bp upstream of the translation start codon and 210 bp downstream of the stop codon, resulting in a total fragment length of 1867 bp. The first construct, designated SE+, includes the phiC31 integrase coding sequence driven by the D. suzukii nanos (Dsnos) promoter and followed by the Dsnos 3′UTR/polyadenylation terminator (Figure 1). The same Dsnos promoter/terminator combination was previously used for the expression of Cas9 in the germline (Yadav et al., 2023). The second construct, designated SE + attP, is identical to SE+ with the addition of inverted attP sites flanking the Dsnos‐phiC31 and sepia genes for future RMCE experiments (Figure 1).

FIGURE 1.

FIGURE 1

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Schematic maps of the constructs used in germline transformation experiments. Both constructs carry the sepia gene and the phiC31 coding sequence driven by the Drosophila suzukii nanos promoter. For the sepia gene, exons are shown as boxes and introns as lines. SE+ DNA construct, without inverted attP sites. SE + attP DNA construct, having the inverted attP sites to serve cassette exchange.

For germline transformation, the sepia mutant line se 1 (Yadav et al., 2024) was crossed with the H7 piggyBac jumpstarter line (Chu et al., 2018) and bred to homozygosity for the piggyBac transposase and sepia eye colour mutant. Purified SE+ and SE + attP plasmid DNAs were injected into embryos from the H7; se 1 line along with a hyperactive piggyBac helper plasmid as an additional source of transposase and a plasmid (PUbDsRed, Figure 1) for constitutive expression of DsRed. The G0 first instar that showed transient expression of DsRed was picked and transferred to diet. The adult G0 that developed from the selected larvae all showed the sepia mutant eye colour. Thus, transient expression of the sepia gene from plasmid DNA injected at the posterior end is not sufficient to rescue the sepia eye colour. The G0 were crossed to se 1 and transgenic G1 adult offspring identified by partial or full rescue of the sepia eye phenotype (Figure 2 and Figure S1). A total of four independent injections of the SE+ construct led to the identification of 12 independent NPS (NPS is for nos‐phiC31 sepia) lines, yielding a transformation efficiency that ranged from 3% to 17% (Table 1). Eight transgenic INPS (INPS is for inverted attP nos‐phiC31 sepia) lines were obtained from three independent injections of the SE + attP construct, corresponding to a transformation efficiency of 6%–13% (Table 2). The G1 were crossed with se 1 and offspring that lacked the H7 piggyBac transgene identified through loss of the 3XP3‐DsRed fluorescent protein marker gene. After establishing the lines, transgenic males were crossed with se 1 females and transgenic offspring collected and photographed (Figure 2). All flies should carry only one copy of the sepia transgene. For comparison, wild‐type was also crossed with se 1 to obtain progeny with one wild‐type copy of sepia. The NPS4A48, INPS2 and INPS3 lines showed a partial rescue of the sepia eye colour (Figure 2). NPS4A48 and INPS2 eyes showed small patches of darker pigment (Figure 2). The INPS3 eye colour was darker than wild‐type but brighter than se 1 homozygotes. For the hemizygotes that showed a partial rescue of eye colour, it was possible to breed to homozygosity through selection of flies with a brighter red eye. However, this was not so straightforward for the other lines where hemizygotes showed a wild‐type eye colour, and consequently the lines were maintained as a mix of hemi‐ and homozygotes.

FIGURE 2.

FIGURE 2

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The sepia gene provides rescue of the sepia mutant phenotype. (a) Male and female flies having one copy of the SE+ DNA construct. (b) Male and female flies having one copy of the SE + attP DNA construct. NPS4A48, INPS2 and INPS3 show partial rescue of the sepia eye colour. Dark patches in the eyes are indicated by arrows. The se 1 fly is homozygous for a null mutation in the sepia gene. Wild‐type is heterozygous for se 1 and thus carries one copy of the wild‐type sepia gene. INPS is for inverted attP nos‐phiC31 sepia; NPS is for nos‐phiC31 sepia.

TABLE 1.

Germline transformation of Drosophila suzukii with the SE+ gene construct.

Experiment number #Embryos Hatching rate L1 larvae Transient expression of DsRed #G0 adults #G0 fertile #G0 with positive G1 Transformation rate
♂ ♀ Total ♂ ♀ Total
1 125 32.00% 40 20 9 20 29 9 20 29 5 17%
2 201 63.68% 128 61 26 27 53 20 20 40 4 10%
3 203 59.11% 120 63 15 15 30 12 14 26 2 8%
4 268 41.79% 112 49 25 26 51 19 21 40 1 3%
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TABLE 2.

Germline transformation of Drosophila suzukii with the SE + attP gene construct.

Experiment number #Embryos Hatching rate L1 larvae Transient expression of DsRed #G0 adults #G0 fertile #G0 with positive G1 Transformation rate
♂ ♀ Total ♂ ♀ Total
1 202 38.61% 78 40 15 21 36 13 18 31 4 13%
2 92 34.78% 32 19 9 11 20 9 10 19 2 11%
3 148 43.92% 65 23 16 16 32 15 16 31 2 6%
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Chromosomal location and transgene expression

Third chromosome linkage of the transgenes was determined by crosses to a line that had a constitutively expressed DsRed fluorescent protein gene at the location of the previously characterised FL19 line (F. Li et al., 2021). Insertions occurring on the X and Y chromosomes were identified by crossing transgenic males with se 1 females (Table 3). Transgenes not on the X, Y or third chromosomes were inferred to likely be on the second chromosome as the fourth is much smaller. Splinkerette PCR is an adaptor‐ligation‐based method for determining the precise genomic location of piggyBac transgenes in the genome (Potter & Luo, 2010). The splinkerette adaptor is unphosphorylated and contains a stable hairpin loop, which helps reduce non‐specific products following two rounds of nested PCR (Devon et al., 1995; Potter & Luo, 2010). We used this method to determine the exact location of the transgenes within the genome (Figure 3 and Figure S2) which were consistent with the linkage analyses. For three of the X‐linked lines, NPS2A46, INPS2 and INPS3, the transgene mapped to an unassigned contig in the reference assembly (Paris et al., 2020).

TABLE 3.

Insertion site analysis of transgenic lines.

Construct Name of the line G0 sex Eye colour Linkage Genome location Gene disrupted?
SE+ NPS1A4 ♀ WT 3rd chr3L_NW_023496812v1:7,604,588 First intron of DsUDPGP3
NPS1A13 ♂ WT 2 or 4 chr2R_NW_023496808v1:5,113,434 No
NPS2A46 ♂ WT X chrUn_NW_023497026v1:45,352 No
NPS3A23 ♀ WT 2 or 4 chr2R_NW_023496807v1:12,288,240 No
NPS4A48 ♀ Partial rescue X chrX_NW_023496847v1:194,181 No
SE + attP INPS2 ♂ Partial rescue X chrUn_NW_023496985v1:42,415 No
INPS3 ♀ Partial rescue X chrUn_NW_023496987v1:42,004 No
INPS4 ♂ WT Y Unknown Unknown
INPS6 ♂ WT 2 or 4 chr2L_NW_023496800v1:10,608,399 No
INPS8 ♂ WT 3rd chr3R_NW_023496835v1:6,485,895 DsKeap1 upstream promoter region
INPS10 ♂ WT 2 or 4 chr2L_NW_023496800v1:18,034,569 DsCG4415 upstream promoter region
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Abbreviations: INPS is for inverted attP nos‐phiC31 sepia; NPS is for nos‐phiC31 sepia; WT, wild‐type.

FIGURE 3.

FIGURE 3

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Genome locations of the transgenes in the NPS (SE+) and INPS (SE + attP) lines (a) Overview of transgene locations on the major chromosomes. (b) Locations of the SE+ DNA constructs in the NPS lines relative to annotated genes. (c) Locations of the SE + attP DNA constructs in the INPS lines relative to annotated genes. INPS4 is located on the Y chromosome. INPS is for inverted attP nos‐phiC31 sepia; NPS is for nos‐phiC31 sepia.

To compare the expression of the sepia and phiC31 transgenes between lines, transgenic flies were backcrossed to the se 1 parental strain, and offspring selected that had inherited the sepia transgene. Thus, all flies assayed had one copy of the sepia and phiC31 genes. RNA was extracted from heads and ovaries and analysed by qPCR. The sepia primer pairs were designed so that RNA from the se 1 allele, which carries a 6 bp deletion, would not be detected (Figure S4). The relative expression levels of phiC31 RNA in ovaries and sepia RNA in heads are shown in Figure 4 and Table S1. The control wild‐type for sepia was heterozygous for the se 1 allele and thus also contained one copy of the wild‐type sepia gene. The INPS3 line, which exhibits a partial rescue of the sepia phenotype (Figure 2), exhibited significantly lower levels of phiC31 and sepia RNA expression compared with their fully rescued counterparts (Student's t‐test, p < 0.05). The INPS10 line showed the highest levels of phiC31 and sepia among the SE + attP transgenic lines, though sepia expression was significantly different from the wild‐type flies. The NPS4A48 transgenic line exhibited the lowest phiC31 and sepia expression among the SE+ flies (Figure 4). Hemizygous NPS4A48 flies initially exhibited partial rescue of the sepia eye phenotype (Figure 2), which facilitated breeding to homozygosity. In contrast, the NPS1A13 and NPS3A23 lines showed significantly higher expression of phiC31 and sepia compared with the other SE+ transgenic lines.

FIGURE 4.

FIGURE 4

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Relative expression of phiC31 in ovaries and sepia in heads. All lines were crossed to se 1 and se + offspring with a single copy of the transgene selected for RNA extractions. (a, b) Relative phiC31 RNA levels in NPS (a) and INPS (b) lines. An Actin gene was used as a reference for normalising RNA levels. NPS lines were relative to NPS1A4 and INPS lines were relative to INPS2, which were set at one. (c, d) Relative sepia RNA levels in NPS (c) and INPS (d) lines. All relative to se 1 heterozygotes that carry one copy of the wild‐type gene. Results are presented as means ± standard error mean (SEM) of three biological replicates, and each biological replicate represents ovaries or heads from five flies. Data points followed by the same lowercase letter do not differ significantly from each other by Student t‐test (p ≤ 0.05). INPS is for inverted attP nos‐phiC31 sepia; NPS is for nos‐phiC31 sepia.

DISCUSSION

The present study demonstrates the successful rescue of the sepia eye phenotype in transgenic D. suzukii that carried a 1867 bp size fragment containing the wild‐type sepia gene. The observed transformation efficiencies of 3%–17% for the SE+ construct and 6%–13% for the SE + attP construct align with previous results using piggyBac vectors with fluorescent protein marker genes (Ahmed et al., 2020; F. Li et al., 2021; Schetelig & Handler, 2013). Transformation efficiencies in Drosophila have been reported to vary widely due to factors such as injection technique and genetic background (Handler & James, 2000). An advantage of piggyBac vectors is the high carrying capacity (>100 kb) (R. Li et al., 2013). CRISPR/Cas9 could potentially have been used for targeted transgenesis of the nos‐phiC31 constructs developed in this study. However, the transformation efficiency may have been lower than obtained with piggyBac given the sizes of the constructs (up to 6.6 kb) (He et al., 2016). These results not only confirm the efficacy of the piggyBac transposon system in generating stable transgenic lines but also highlight its utility in functional studies of gene rescue.

The eye colour mutant se 1 used in this study is likely a null allele as it contains a 6 bp deletion that removes the translation start codon for PDA synthase (Yadav et al., 2024), which catalyses the conversion of 6‐PTP (2‐amino‐4‐oxo‐6‐pyruvoyl‐5,6,7,8‐tetrahydropteridine; also known as 6‐pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis (Kim et al., 2006). Here, we were able to rescue the phenotype using the wild‐type sepia gene by transposition to sites on the 2nd, 3rd and X chromosomes. We did not obtain any insertion on the 4th chromosome, but we obtained one Y‐linked line (INPS4) with the SE + attP construct. Y‐linkage was determined as only the sons of transgenic males crossed with wild‐type females inherited the sepia transgene (Table 2 and Figure S2). As the reference genome was assembled using only female DNA (Paris et al., 2020), we were unable to use it to determine the genome location of the Y‐linked transgene (Figure S3). The sepia gene could be a suitable target for identifying successful knockins. Wild‐type embryos would be injected with Cas9/se gRNA and plasmid DNA containing the gene of interest flanked by homology arms from the sepia gene. Potential knockins could be identified by crossing G0 adults with se 1 flies and selecting for G1 with a sepia eye colour. Molecular methods would be used to distinguish successful knockins from loss‐of‐function mutations due to NHEJ repair. An advantage of this approach is that there would be no need for a selectable marker gene such as GFP, which would reduce the size of the knockin construct.

A noteworthy finding of our study was the presence of partial rescue phenotypes in hemizygotes for three of the lines, INPS2, INPS3 and NPS4A48. These individuals, characterised by a mosaic pattern of sepia eye coloration, exhibited the lowest levels of sepia RNA in heads and phiC31 RNA in ovaries. This suggests that, while the insertion of the transgene can restore some function, it may not always achieve the full expression levels necessary for complete phenotypic recovery. Other lines, such as INPS8, also had relatively low sepia gene expression in heads compared with wild‐type but nevertheless fully rescued the sepia mutant eye colour (Figure 2). This suggests that relatively low levels of sepia gene activity are sufficient for full rescue, which could explain why we were able to identify one Y‐linked line. The heterochromatic Drosophila Y chromosome is rich in repetitive sequences with few protein coding genes (Marsano et al., 2019). Interestingly, the INPS2 and INPS3 transgenes map to the same contig only 411 bp apart; yet, phiC31 and sepia RNA levels in INPS3 and eye colour (Figure 2) are significantly decreased compared with INPS2 (Student t‐test, p < 0.05). INPS2 adults show a brighter eye colour than INPS3 but with some dark spots within the eye (Figure 2). The INPS2, INPS3 and NPS4A48 lines are X‐linked and map to gene poor, unassigned contigs in the assembly. Together with the observed low transgene expression levels, this suggests that the transgenes may have inserted into the proximal heterochromatin of the X chromosome (Hoskins et al., 2002). In the sheep blowfly Lucilia cuprina (Diptera: Calliphoridae) Wiedemann 1830, we have also noted low expression for transgenes located in the mostly heterochromatic X chromosome (Williamson et al., 2021).

In the previous study on the identification of the D. melanogaster sepia gene, confirmation was obtained by using the GAL4/UAS system to express the coding region in the eye (Kim et al., 2006). Although the strong GMR‐Gal4 driver was used to activate expression in the eye, only partial rescue of the sepia phenotype was observed. The eyes showed patches of brown pigment. Here we used the wild‐type sepia gene including exons and introns and obtained full rescue of the sepia phenotype in most lines. This suggests that the correct spatial and temporal expression was achieved by using the native promoter and perhaps the inclusion of introns. However, the expression level of the sepia transgene in transgenic flies was lower than in wild‐type. This suggests that the fragment we used to rescue the sepia phenotype may be missing an element required for full expression. One disadvantage of using the sepia gene as a transformation marker was that it was difficult to distinguish hemizygotes from homozygotes for most lines since a single copy of the sepia gene was sufficient to rescue the sepia phenotype at most genome locations. If the location of the gene construct in the genome is known, molecular methods can be used to identify homozygotes to establish a stable line.

In general, the lines that showed the highest levels of sepia RNA in the head had higher levels of phiC31 RNA in ovaries (Figure 4, Table S2). Thus, full rescue of sepia eye colour can provide an initial screen for lines that will likely not show low expression of the linked transgene. For phiC31 integrase‐mediated site‐specific recombination, the NPS lines would need to be combined with additional transgenic lines that carry an attP or attB site (Bischof & Basler, 2008; Venken & Bellen, 2012). With phiC31 transgenes located on each of the major chromosomes (Figure 3), establishing combined stocks should be relatively straightforward. The NPS lines could also be used to mediate integration of gene constructs into existing lines that carry an attP or attB site. For example, the FL19 male‐only strain has an attP site adjacent to one of the piggyBac ends (F. Li et al., 2021). This could facilitate the assembly of more complex genetic systems at genome locations that provide the desired level of gene expression. The INPS lines with inverted attP sites could be used for RMCE. Embryos from INPS lines would be injected with plasmid DNA carrying inverted attB sites flanking a marker gene and a gene of interest. RMCE would cause loss of the sepia and phiC31 genes and insertion of the marker and gene of interest. Consequently, without the sepia marker gene, adults would show a sepia eye colour. INPS lines 6, 8 and 10 had the highest level of phiC31 RNA in the ovaries and would enable targeting to either of the major autosomes. The Y‐linked line could be particularly advantageous for developing Y‐linked editor strains for suppression of D. suzukii populations (Burt & Deredec, 2018). The NPS and INPS lines developed in this study add to the lines developed by others for site‐specific recombination in D. suzukii (Ahmed et al., 2020; Schetelig et al., 2019).

In conclusion, our results underscore the effectiveness of the sepia gene as an excellent natural marker for Drosophila transgenesis. The sepia gene is only expressed in the adult head and has no function other than pigment synthesis in the eye (Kim et al., 2006). Drosophila melanogaster sepia mutant strains appear to maintain normal physiological functions without detrimental effects on overall fitness (Gramates et al., 2022; Kim et al., 2006). These characteristics make sepia advantageous compared with other eye colour genes that are expressed in multiple tissues or have a fitness cost such as white (Yadav et al., 2024; Yan et al., 2020). The identification of the sepia gene (Kim et al., 2006) more than 20 years after the white (Levis et al., 1982) and rosy (Rubin & Spradling, 1982) genes were identified may be why the sepia has not previously been used as a marker for Drosophila transgenesis. The strains developed in this study should facilitate future genetic studies in D. suzukii.

EXPERIMENTAL PROCEDURES

Design and synthesis of DNA constructs

PCR reactions were performed using Q5 Hot Start High Fidelity 2× Master Mix (NEB, M0494S) with the following conditions: initial denaturation for 5 min at 98°C, followed by 35 cycles consisting of denaturation for 10 s at 98°C, annealing for 20 s at the annealing temperature calculated using the NEB Tm calculator (https://tmcalculator.neb.com), and extension at 72°C, with the extension time adjusted based on the size of the amplified fragment. Primer sequences are given in Table S3. The NosPhiC31 gene was amplified from the plasmid Dsnos‐phiC31‐Dshsp83‐mTagBFP2‐pBAC2 (gift of Anand Patil) using primers SePro‐NosUTRR and pBAC_Nos_fwd, and the piggyBac arms with the vector backbone plasmid were amplified from the same plasmid using primers SeUTR‐pBacF and Nos_pBac_rev. The sepia gene was amplified from genomic DNA isolated from a NC strain of D. suzukii using primers NosUTR‐SeProF and pBac‐SeUTRR. These fragments were ligated using NEBuilder HiFi DNA Assembly (cat# E2621S, New England Biolabs, MA, USA) to synthesise the SE+ DNA construct (GenBank# PV232306). To construct SE + attP (GenBank#PV232307), an intermediate plasmid was generated by amplifying the backbone plasmid from Dsnos‐phiC31‐Dshsp83‐mTagBFP2‐pBAC2 using primers SeUTR‐pBacF and Nos_pBac_rev. The PUbDsRed gene with inverted attP site ends was amplified from the plasmid pUC57‐iattP‐FL19HA‐PUbDsRed (gift of Amarish Yadav) using primers iattp‐PUbDsRed_fwd and iattp‐PUbDsRed_rev, and these fragments were ligated to create the intermediate plasmid pBAC‐PUbDsRed‐iattP. The intermediate plasmid was then digested with ApaI to remove the PUbDsRed gene and excise the piggyBac elements containing inverted attP sites. The NosPhiC31 gene, along with the sepia gene DNA fragment, was isolated from the SE+ plasmid using primers PBRight‐NosPhic31Sepia_rev and PBRight‐NosPhic31Sepia_fwd, and the isolated fragments were ligated using NEBuilder HiFi DNA Assembly to complete the final SE + attP plasmid (Figure 1). A hyperactive piggyBac transposase helper plasmid was assembled by isolating the Dshsp83 promoter and 3′UTR fragments using the primer sets hsp83_HiFifwd and hyphspKozak_rev, and hyphsp83UTR_fwd and hsp83 3′UTR_HiFirev from the pBAC2[Dshsp83‐Kozak‐NLS‐ZsGreen‐3′Dshsp83] plasmid (Yadav et al., 2023). The hyperactive piggyBac protein coding sequence was amplified from the plasmid pS6[Dmhsp70‐iPB7] (Eckermann et al., 2018) using primers hspHypPBac_fwd and hspHypPBac_rev. The isolated fragments were then ligated using NEBuilder HiFi DNA Assembly to generate the final Dshsp83‐HybPBAChelper plasmid (GenBank#PV232308). The complete construct was then cloned into the pUC19 vector. Additionally, a transient expression marker plasmid was created by amplifying the PUbDsRed marker gene using pUC19‐Pub_fwd/pUC19SV40_rev primers and cloned to BamHI digested pUC19 vector by NEbuilder(R) Hifi DNA assembly (GenBank#PV232309). Whole plasmid sequencing was performed to confirm the plasmid sequences, and sequences were uploaded to NCBI.

piggyBac‐ based germline transformation

To obtain a line that expressed piggyBac transposase and had a sepia eye colour, the H7 piggyBac jumpstarter line, which carries a Dmhsp70‐piggyBac transgene (Chu et al., 2018), was crossed with se 1 and offspring that showed red fluorescence in the ocelli selected. Further crosses established a line homozygous for the 3XP3‐DsRed marker (and piggyBac transgene) and se 1. Embryos from the H7; se 1 line were microinjected at the posterior end with purified plasmid DNA extracted from Escherichia coli cultures with the Zymo Midi Kit (Cat#D4201) and subsequently ethanol‐precipitated to achieve a final concentration of 1.25 μg/μL. For piggyBac transformation, the final injection solution comprised donor and helper plasmid concentrations of 0.5 and 0.3 μg/μL, respectively, along with a transient expression marker plasmid at 0.1 μg/μL. Prior to microinjection, the plasmid DNA was filtered through a 0.45‐μm filter (MilliporeSigma, Cat#UFC30HV25). Post‐injection, the G0 adult flies were individually crossed with se 1, and the offspring were screened for rescue of sepia eye colour using a light microscope. The piggyBac transposase gene was removed by selecting flies without red fluorescence in the ocelli to prevent further repositioning of the piggyBac construct. Independent lines were created from individual G1 flies.

Identification of transgene insertion sites

The location of each transgene in the genome was determined using splinkerette PCR, as outlined in Potter and Luo (Potter & Luo, 2010). Genomic DNA was digested with BstY1 and then ligated to the double stranded splinkerette oligonucleotide, formed by annealing SPLNK‐GATC‐TOP and SPLNK‐BOT (Table S3). This was followed by two rounds of nested PCR as described by Potter and Luo (Potter & Luo, 2010). In the first round, SPINK#1 was used with 5′SPLNK‐PB#1 (5′ PBAC) or 3′SPLNK‐PB#1 (3′ PBAC). In the second round, SPINK#2 was used with 5′SPLNK‐PB#2 (5′ PBAC) or 3′SPLNK‐PB#2 (3′ PBAC). The DNA sequences of the PCR fragments obtained were analysed using the piggyBac terminal repeat sequences to map the insertion site within the D. suzukii female genome (UCSC Genome Browser on LBDM_Dsuz_2.1.pri Jun. 2020 fly D. suzukii [GCF_013340165.1]). The mapping and identification of the insertion site were conducted using the Blat tool (UCSC Genome Browser) to identify the locations in the genome.

phiC31 and sepia gene expression analysis

RNA was extracted from the ovaries or heads of 5–6‐day‐old female flies, each carrying a single copy of the transgene (wild‐type flies also carried a single copy of the sepia gene), using the Quick‐RNA Miniprep Plus Kit (Cat# R1054, Zymo Research). cDNA synthesis was performed using the qScript® UltraFlex Kit (Cat# 95215–025, Quantabio, Beverly, USA). To measure the expression levels of phiC31 and sepia, primer pairs PhiC31qPCR1F/PhiC31qPCR1R, Seg2RTF/Seg2RTR, and ActinRTF3/ActinRTR3 were designed, as specified in Table S4. The sepia primers were designed to amplify and detect the active sepia mRNA by using a forward primer that ends in a sequence deleted in se 1 and by taking advantage of the lack of 3′ to 5′ exonuclease activity in Taq polymerase (Figure S4). Quantitative PCR was conducted using the Luna® Universal qPCR Master Mix (Cat# M3003L, New England Biolabs) and cDNA synthesised in a CFX384 Touch™ real‐time PCR detection system (Bio‐Rad, Hercules, CA, USA). All reactions were performed in triplicate for both biological and technical replicates. The 2−ΔΔCt method (Livak & Schmittgen, 2001) was used for data analysis, with actin used as a reference gene (Zhai et al., 2014) to normalise sepia and phiC31 RNA levels. For the relative expression levels shown in Figure 4, normalised sepia expression was relative to se 1/+ heterozygote and phiC31 expression was relative to the normalised RNA level found in the INPS2 line. Statistical differences between samples were evaluated using Student's t‐test. All statistical analyses were performed using GraphPad Prism (version 10.4.1).

AUTHOR CONTRIBUTIONS

Kalindu Ramyasoma Hewawasam: Writing – original draft; writing – review and editing; conceptualization; methodology; investigation; data curation; formal analysis; visualization. Akihiko Yamamoto: Writing – review and editing; methodology; investigation. Maxwell J. Scott: Writing – original draft; writing – review and editing; conceptualization; methodology; funding acquisition; supervision; project administration.

FUNDING INFORMATION

This work was supported by the National Institute of Food and Agriculture's (NIFA) Biotechnology Risk Assessment Research (BRAG) program project award 2020‐33522‐32317 and by a Research Capacity Fund (HATCH) project award no. 7008054 from NIFA. This research was also supported by an interdisciplinary seed grant from N.C. State University's Genetic and Genomics Academy.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Figure S1. Transgenic flies showing full and partial rescue of sepia eye colour phenotype. Male (A) and females (B) from the NPS lines that have the SE+ DNA construct. Male (C) and females (D) from the INPS lines that have the SE + attP DNA construct. NPS4A48, INPS2 and INPS3 shows a partial eye colour rescue phenotype compared with the other fly lines. The mutant flies with the sepia eye colour are from the se 1 line and WT is wild‐type.

IMB-34-960-s002.tif (1.2MB, tif)

Figure S2. Sanger sequencing results of DNA fragments obtained by splinkerette PCR. SE+ lines (A) SE + attP lines (B). 5′ PBAC or 3′ PBAC sequences were identified in the sequences and are indicated.

IMB-34-960-s007.pdf (506.3KB, pdf)

Figure S3. The INPS4 Y‐linked line shows partial rescue of the sepia eye colour in males. (A) Eyes of the INPS4 line males and females. (B) Sanger sequencing results of DNA fragments obtained by splinkerette PCR. 5′ PBAC sequence was identified in the sequences and is indicated.

IMB-34-960-s004.tif (1.3MB, tif)

Figure S4. Seg2RTF specific primer designed to amplify the wild‐type sepia gene and not the se 1 mutant. Alignment of wild‐type (WT) and se 1 mutant DNA sequences with location of the Seg2RTF primer shown.

IMB-34-960-s003.pdf (110.1KB, pdf)

Table S1. Real‐time PCR Ct values for sepia and actin and relative expression calculations.

IMB-34-960-s001.xlsx (16.9KB, xlsx)

Table S2. Real‐time PCR Ct values for phiC31 and actin and relative expression calculations.

IMB-34-960-s005.xlsx (15.7KB, xlsx)

Table S3. Oligonucleotide primers were used in this study.

IMB-34-960-s008.docx (17.8KB, docx)

Table S4. Oligonucleotide primers used for qPCR.

IMB-34-960-s006.docx (15.9KB, docx)

ACKNOWLEDGEMENTS

We thank Amarish Yadav and Anand Patil for plasmids DNAs, Esther Belikoff for biosafety training and our colleagues in the Scott lab for helpful discussions.

Hewawasam, K.R. , Yamamoto, A. & Scott, M.J. (2025) Establishment of transgenic Drosophila suzukii lines that express phiC31 integrase and carry the sepia gene as a marker for transformation. Insect Molecular Biology, 34(6), 960–969. Available from: 10.1111/imb.70001

Associate Editor: Gareth Lycett

DATA AVAILABILITY STATEMENT

The datasets generated for this study are available in Dryad at https://doi.org/10.5061/dryad.b2rbnzst9 (Sequences for the four plasmids made in this study have been deposited at NCBI (https://www.ncbi.nlm.nih.gov/), accession numbers PV232306, PV232307, PV232308, PV232309. [Correction added on 13 September 2025, after first online publication: The Dryad data link has been included].

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Transgenic flies showing full and partial rescue of sepia eye colour phenotype. Male (A) and females (B) from the NPS lines that have the SE+ DNA construct. Male (C) and females (D) from the INPS lines that have the SE + attP DNA construct. NPS4A48, INPS2 and INPS3 shows a partial eye colour rescue phenotype compared with the other fly lines. The mutant flies with the sepia eye colour are from the se 1 line and WT is wild‐type.

IMB-34-960-s002.tif (1.2MB, tif)

Figure S2. Sanger sequencing results of DNA fragments obtained by splinkerette PCR. SE+ lines (A) SE + attP lines (B). 5′ PBAC or 3′ PBAC sequences were identified in the sequences and are indicated.

IMB-34-960-s007.pdf (506.3KB, pdf)

Figure S3. The INPS4 Y‐linked line shows partial rescue of the sepia eye colour in males. (A) Eyes of the INPS4 line males and females. (B) Sanger sequencing results of DNA fragments obtained by splinkerette PCR. 5′ PBAC sequence was identified in the sequences and is indicated.

IMB-34-960-s004.tif (1.3MB, tif)

Figure S4. Seg2RTF specific primer designed to amplify the wild‐type sepia gene and not the se 1 mutant. Alignment of wild‐type (WT) and se 1 mutant DNA sequences with location of the Seg2RTF primer shown.

IMB-34-960-s003.pdf (110.1KB, pdf)

Table S1. Real‐time PCR Ct values for sepia and actin and relative expression calculations.

IMB-34-960-s001.xlsx (16.9KB, xlsx)

Table S2. Real‐time PCR Ct values for phiC31 and actin and relative expression calculations.

IMB-34-960-s005.xlsx (15.7KB, xlsx)

Table S3. Oligonucleotide primers were used in this study.

IMB-34-960-s008.docx (17.8KB, docx)

Table S4. Oligonucleotide primers used for qPCR.

IMB-34-960-s006.docx (15.9KB, docx)

Data Availability Statement

The datasets generated for this study are available in Dryad at https://doi.org/10.5061/dryad.b2rbnzst9 (Sequences for the four plasmids made in this study have been deposited at NCBI (https://www.ncbi.nlm.nih.gov/), accession numbers PV232306, PV232307, PV232308, PV232309. [Correction added on 13 September 2025, after first online publication: The Dryad data link has been included].

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