Minos-mediated transgenesis in the pantry moth Plodia interpunctella

Plasmids

The pMi[3xP3::EGFP-SV40] donor plasmid and the Minos transposase transcription template plasmid pBlueSKMimRNA (Pavlopoulos et al., 2004) were sourced from Addgene (RRID:Addgene_102540; RRID:Addgene_102535). The pMi[3xp3::mCherry-SV40, Plodia-FibL::mBaojin-P10] plasmid was produced using Gibson Assembly, by replacing the EGFP-SV40 section of pMi[3xP3::EGFP-SV40] with a synthetic (mCherry-SV40, FibL::mBaoJin-P10) gene fragment synthesized by Twist Bioscience, and is available from Addgene (RRID:Addgene_240410). The FibL promoter sequence corresponds to 1 kb of sequence upstream of the P. interpunctella FibL gene (https://www.ncbi.nlm.nih.gov/gene/?term=LOC128678647).

Preparation of Minos transgenesis reagents

Plasmids were amplified in 50 mL liquid cultures of Luria-Bertani broth with 100 μg/mL Ampicillin, and plasmid DNA was purified with the ZymoPURE II Plasmid Midiprep Kit (Cat# D4200). Plasmids were eluted in 120 μL of elution buffer, yielding concentrations >4,800 ng/μL of plasmid DNA.

To produce Minos transposase mRNA, the pBlueSKMimRNA plasmid was linearized using the NotI-HF restriction enzyme (New England Biolabs, Ipswich, MA, USA), purified by gel extraction using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA), and further purified using the Zymo DNA Clean & Concentrator-25 kit with an elution volume of 13 μL of DNA elution buffer. Subsequently, the HiScribe T7 ARCA mRNA with tailing kit (New England Biolabs, Ipswich, MA, USA) was used for transcription and capping of Minos transposase mRNA, followed by purification using the MEGAClear Transcription Clean-Up Kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified mRNA was eluted in 50 μL of the provided elution buffer, which was pre-heated to 65 °C and incubated on the filter column for 10 min in a 65 °C oven. After quantification with Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), the transposase mRNA was divided into >520 ng/μL one-time use aliquots and stored at −70 °C.

Injection mixes were freshly prepared before injection and consisted of 400 ng/μL Minos transposase mRNA, 200 ng/μL donor plasmid, and 0.05% Phenol Red (1:10 dilution of a 0.5% cell-culture grade Phenol Red solution; Sigma-Aldrich, St. Louis, MO, USA), brought to a 5 μL final volume with 1x Bombyx injection buffer (pH = 7.2, 0.5 mM NaH₂PO₄, 0.5 mM Na₂HPO₄, 5 mM KCl).

Plodia strain

The white-eyed wFog laboratory strain of Plodia interpunctella was used to facilitate the screening of 3xP3-driven fluorescence in pupal and adult eyes (Heryanto et al., 2022; Heryanto, Mazo-Vargas & Martin, 2022). This strain consists in the genome background of the Dundee strain (NCBI genome accession GCA_001368715.1), into which a white gene c.737delC mutation originating from the Piw3 strain (NCBI genome accession: GCA_022985095.1) was introgressed during two generations of backcrossing and several rounds of sib-sib inbreeding (Heryanto et al., 2022).

Plodia husbandry

The wFog strain and the transgenic broods were reared in the laboratory from egg to adulthood in an incubator set at 28 °C with 40–70% relative humidity and a 14:10 h light:dark cycle, following methods made available via the Open Science Framework repository (Heryanto et al., 2025). Briefly, egg laying was induced by CO₂ narcosis of adult stocks in an oviposition jar, and a weight boat containing 10–13 mg eggs was placed in a rearing container containing 45–50 g of wheat bran diet with 30% glycerol (Table S1). This life cycle spans 29 days from fertilization to a reproductively mature adult stock at 28 °C.

Microinjection of Plodia syncytial embryos

The general procedure for microinjection as well as a list of suggested equipment is available online (Heryanto, Mazo-Vargas & Martin, 2022) and summarized below. Minos capped mRNA and the donor plasmid were mixed into a 5 μL aliquot (400:200 ng/μL mRNA:DNA) and kept on ice shortly before the procedure. Eggs were collected in an oviposition jar following CO₂ narcosis of a stock of wFog strain adults, aligned on parafilm, and microinjected between 20–50 min after egg laying (AEL) using borosilicate pulled glass needles and an air pressure microinjector. Eggs were then sealed with cyanoacrylate glue and left to develop at 25 °C or 28 °C in a closed tupperware with a wet paper towel.

Fluorescent screening equipment

All life stages were screened under an Olympus SZX16 stereomicroscope equipped with a Lumencor SOLA Light Engine SM 5-LCR-VA lightsource, mounted to a trinocular tube connected to an Olympus DP73 digital color camera. Fluorophore expression was observed using Chroma Technology filter sets ET-EGFP 470/40× 510/20m green fluorescent protein (GFP) and AT-TRITC-REDSHFT 540/25X, 620/60m red fluorescent protein (RFP). Autofluorescence patterns are shown for uninjected individuals, using the GFP and RFP filter sets and exposure times consistent with the other images in this study (Fig. S1). It is worth adding that a yellow fluorescent protein filter set (e.g., Chroma ET-EYFP 500/20× 535/30m) reduces cuticle autofluorescence and can be advantageous to use in addition to a GFP filter set for screening EGFP and mBaoJin expression. For small mosaic clones, or weak fluorescent signals, the YFP filter on its own may lead to false negatives.

Screening of embryos and first instars larvae

To obtain synchronized embryos, mated adults are anesthetized by CO₂ narcosis and transferred to a glass jar with a mesh lid insert (Heryanto et al., 2025). CO₂ exposure triggers egg release in Plodia, and embryos are collected after 2–4 h, transferred to a glass petri dish, and incubation at 28 °C and saturating humidity (e.g., in a sealed tupperware with a wet paper towel), until time of screening. The screening of embryos is most convenient when using crosses performed in relatively large pools, for example with more than 10 mated females. Screening of transgenic embryos is optimal at 68–74 h AEL if incubated at 28 °C, a time window where 3xP3-driven fluorescence in presumptive glia and ocelli is discernible in the larval head and body. Earlier times of screening show weaker fluorescence and confounding signals, likely due to the episomal expression of residual plasmids in large vitellophages (Heryanto, Mazo-Vargas & Martin, 2022). Hatching occurs at 78–82 h AEL in uninjected embryos and can be delayed by up to 12 h in G₀ embryos, due to injection stress.

Screening of pupae and adults

The screening of pupae is ideal to verify the transgenic status of imagos in preparation of a new generation, and can also be the method of choice when screening offspring from small crosses (less than 10 females), where isolating synchronized embryos by CO₂ narcosis is not optimal. As pupae can be difficult to isolate from the larval diet, it is helpful to add strips of cardboard to larval containers that contain fifth instar larvae, as the corrugations within this material are the preferred pupation site that facilitates the collection of pupae (Shim & Lee, 2015). Pupae are then isolated from these lodges, as well as from those located within the diet, transferred to tissue culture dishes for screening and sex sorting, and subsequently combined according to the cross design in fresh containers to allow adult emergence and mating prior to initiating the next generation. Fluorescent and brightfield images of pupae were acquired using the same fluorescent microscopy set-up using the Olympus DP74 camera, and overlaid using the Screen blending mode in Adobe Illustrator. Brightfield images of transgenic G₂ adult eyes (Fig. 1F) were taken using a Keyence VHX-5000 digital microscope. Pupal stages (%, Figs. 1C, 2G) were estimated based on eye, wing, tarsal, and cuticle pigmentation (Zimowska et al., 1991). Adult moths can be screened in a petri dish held on ice to limit moth activity.

Characterization of silk gland fluorescence

The FibL::mBaojin transgene drives strong silk gland fluorescence visible directly through the cuticle of all larval instars. The silk glands of a G₂ FibL::mBaojin transgenic fifth instar larva were dissected, fixed, and mounted following a previously described procedure (Alqassar & Martin, 2025), and imaged on an Olympus SZX16 fluorescence stereomicroscope, as well as on an Olympus FV1200 confocal microscope mounted with a 20x objective.

Somatic and germline transformation of a test Minos donor plasmid

To test for the efficacy of Minos-mediated transgenesis in Plodia embryos, we initially microinjected 1,317 eggs between 20–50 mins AEL, with the pMi[3xP3::EGFP-SV40] test donor plasmid (Pavlopoulos et al., 2004). This injection time window, as well as the microinjection technique and rearing conditions were comparable to our previous experiments using the Hyperactive piggyBac transposase (HyPBase) in Plodia (Heryanto, Mazo-Vargas & Martin, 2022). The injection mix consisted of capped poly-A tailed Minos mRNA, mixed with a donor plasmid for the integration of the 3xP3::EGFP transgenesis marker cassette.

We conducted three injection trials using the pMi[3xP3::EGFP-SV40] plasmid. All three attempts resulted in detectable activity in a portion of G₀ embryos and pupae. Embryonic activity of 3xP3::EGFP, observed as of bright fluorescent puncta in the presumptive glia and larval ocelli (Thomas et al., 2002; Bossin et al., 2007; Heryanto, Mazo-Vargas & Martin, 2022; Pearce et al., 2024), was respectively detected in 10.3%, 7.8% and 4.3% of G₀ embryos in Experiments #1, #2 and #3 (Fig. 1A, Table 1). Pupal activity of 3xP3::EGFP, consisting of bright fluorescent stripes of expression in the pupal eyes of the wFog white-eyed strain used here (Heryanto, Mazo-Vargas & Martin, 2022), was observed in 7.1%, 15.7%, and 16.7% of pupae in Experiments #1, #2 and #3 (Fig. 1B, Table 1). Overall, these data indicate that Minos transposase can drive transgene insertion into the genome of somatic cells.

Next, we assessed whether Minos could efficiently transpose the test insert into the germline, and enable its transmission over subsequent generations. Following Experiment #1, we in-crossed all the 14 G₀ adults by pooling them for random mating and rearing in a single container. We recovered 250 G₁ pupae, 23.6% of which showed transgene activity (Fig. 1C, Table 2). Following Experiment #2, we similarly in-crossed 41 G₀ adults, but then directly screened embryos instead of pupae. This in-cross generated 470 G₁ embryos, of which 11.5% were transgenic (Table 2). It is worth noting that in these two crosses, 13 out of 14 of the G₀ founders from Experiment #1, and all of the G₀ founders from Experiment #2, lacked a detectable somatic activity of 3xP3::EGFP at the pupal stage. For comparison, we out-crossed 5 EGFP⁺ G₀ males from Experiment #2 to 10 females from the uninjected wFog strain, and obtained 10.6% transgenic embryos out of 113 G₁ eggs. In summary, both G₀ negative and positive individuals can be used as founders for germline transmission of Minos transgenes, as somatic transformants do not necessarily reflect whether germline transformation has occurred.

Stable transgene transmission over several generations

Further examination of the germline-integrate 3xP3::EGFP transgene activity revealed fluorescence patterns identical to the ones previously reported with piggyBac-mediated integration (Heryanto, Mazo-Vargas & Martin, 2022), consistent with glial expression in embryos, and ocelli expression in larvae (Figs. 1D, 1E, Video S1). In the depigmented eyes from the wFog strain, the retinal expression of EGFP was strong enough to reveal a green tint of the eyes, visible without fluorescent microscopy equipment (Fig. 1F).

In addition, out of the 59 G₁ pupae with eye fluorescence generated from Experiment #1, 10 showed an additional, unexpected fluorescence in pupal oenocytes (Stendell, 1912; Shirk & Zimowska, 1997; Makki, Cinnamon & Gould, 2014), visible as ventral metameric belts in the second to seventh abdominal segments (Fig. 1C’, Fig. S2). This activity is likely due to an identical insertion shared between these 11 siblings. Below we refer to this randomly captured oenocyte activity as coming from a single EGFP^+/oeno allele, and dub the transgene alleles that lack it EGFP⁺. To further test the transmissibility of these G₁ insertion alleles, we generated four crosses each consisting of a single EGFP⁺ or EGFP^+/oeno G₁ founder mixed with four non-transgenic individuals from the opposite sex. These crosses generated 60.2% transgenic G₂ offspring individuals on average (Table 2), including transmission of the EGFP^+/oeno allele in the two corresponding crosses.

We then continued these experiments over the G₃ and G₄ generations to further assess transgene stability (Table 2). To obtain G₃ offspring, we performed two backcrosses each consisting of one positive G₂ female mixed with four non-transgenic white-eyed males, yielding 51.3% [EGFP⁺] G₃ pupae in the first cross, and 46.0% [EGFP^+/oeno] pupae in the second cross, each consistent with their donor parent carrying a single copy of a EGFP⁺ or EGFP^+/oeno insertion allele (Chi-square test p > 0.7; no significant deviation from 50% transmission). Pooled in-crossing of positive G₃ individuals yielded 100% G₄ positive progeny in both cases, with [EGFP⁺] or [EGFP^+/oeno] fluorescent expression patterns consistent with their G₂ donor grand-parent. The persistence of ectopic oenocyte activity across four generations suggests that the location of the expression cassette was stable and had not been subjected to re-transposition.

The FibL promoter enables fluorescent marking of the silk glands

Next, to provide an alternative marker to eye/glia in future experiments, we designed a new donor plasmid aimed at labeling the silk glands. In Bombyx, the promoter of the Fibroin-L silk gene (FibL) drives fluorescent protein expression in the posterior region of the silk glands, enabling detection through the cuticle. This promoter has notably been used as a transgenesis marker in experiments where the labeling of neural tissues is unwanted (Imamura et al., 2003; Fujiwara et al., 2014). To implement a similar strategy, we cloned a 1 kb fragment of the Plodia FibL proximal promoter to drive the expression of mBaoJin—a recently developed monomeric fluorophore with remarkable brightness and photostability (Zhang et al., 2024) followed by a P10 3′UTR sequence (Xu et al., 2022). This cassette was cloned upstream of a 3xP3::mCherry red fluorescence eye marker in order to monitor successful integrations.

Injections of the resulting Minos donor plasmids yielded positive hatchling larvae and pupae expressing distinguishable mosaic patches of 3xP3::mCherry expression in the eye/glia (Figs. 2A, 2B). We conducted two independent injections trials using this plasmid, Experiments #4 and #5. Experiments #4 generated only one somatic transformant out of 77 pupae (Table 1), and random in-crossing of this large pool of founders successfully generated transgenic 22 transgenic G₁ larvae, out of more than a hundred screened larvae (we failed to record an exact number of screened larvae, and can not provide a germline transmission rate in this experiment). These G₁ individuals successfully transmitted transgenes into G₂ backcrosses in a pattern consistent with the segregation of single-copies (Table 3). In Experiment #5, five out of 32 G₀ pupae showed mosaic 3xP3::mCherry expression (Table 1), but these somatic transformants failed to pass a transgene into a G₁ generation, confirming that somatic mosaicism in the pupal eyes is a poor indicator of germline transformation. In contrast, a pooled in-cross of the 17 remaining negative G₀ individuals yielded a high-rate of germline transmission, with 51 out 518 embryos showing strong 3xP3 activity (Fig. 2C, Table 3).

In G₁ larvae, we detected FibL::mBaojin activity in sections of two inner tubes on either side of the gut, spanning the A3-A6 segments, directly screenable through the cuticle (Figs. 2C, 2D, Videos S2). Dissected glands show that FibL::mBaoJin fluorescence is confined to the Posterior Silk Gland (PSG, Figs. 2F, 2F’). This compartment of the silk gland is specialized in the expression of the fibroin genes FibL and FibH (Figs. 2F, 2F’), and is thus consistent with the FibL promoter recapitulating its endogenous expression in Plodia (Alqassar et al., 2025). Notably, in G₁ larvae, FibL expression in the PSG was also visible when screening with an RFP filter (Video S3), suggesting that the FibL promoter may be interacting with mCherry in addition to mBaojin, which could be due to the absence of insulator sequences between the two expression cassettes. G₁ donor successfully established G₂ lines in controlled crosses, including in four backcrosses where segregation ratios indicate that a single-insertion allele is present. Even in these G₂ individuals carrying a single-copy of the transgene, fluorescence of both the mCherry and FibL markers provided extremely bright signals that enable fast screening (Videos S4, S5).

Overall, these data further confirm the efficiency of Minos transgenesis for a second donor plasmid, and the FibL::mBaoJin cassette provides a marker that is easily screenable in Plodia throughout the entire larval stage (Fig. 3A). The FibL promoter could be used in the future to drive the expression of other fluorescent markers or ectopic silk factors in the Plodia Posterior Silk Gland.

Recommendations for successful transgenesis in Plodia and beyond

The feasibility of transgenesis experiments depends not only on the germline integration rate of the transposase system, but also on other logistic criteria such as the injection, rearing and crossing efforts. Below, we elaborate on these aspects and provide experimental considerations with the overarching goal to facilitate implementation in Plodia and other lepidopteran organisms.

We detected lower rates of G₀ mosaic fluorescence with Minos compared to previous HyPBase experiments, with 9.0% of the G₀ injected eggs and 9.6% of surviving pupae showing a 3xP3-driven signal, compared to 22% of injected eggs and 32% of surviving pupae with HyPBase (Heryanto, Mazo-Vargas & Martin, 2022). However, here we established that G₀ injectees devoid of visible somatic activity are effective germline carriers of the transgene. As a consequence, we propose that the random in-crossing of these negative G₀ injectees, performed in pools of adults, offers a practical route to the generation of transgenic lines. For example, in Experiment #1, 341 embryos were microinjected (an effort of only 3 h for two experimenters). In order to establish a G₁ line, all 14 surviving adults from the injected batch were transferred to a larval diet container and allowed to randomly mate. This approach generated a large proportion of G₁ individuals with 3xP3-driven fluorescence, with 23.6% of 250 screened pupae showing EGFP expression in their eyes, which were then used to establish two transgenic lines (EGFP⁺ and EGFP^+/oeno). In other words, we recommend in-crossing all G₀ individuals to ensure germline transmission and the generation of G₁ transformants. Indeed, crossing of small numbers of G₀ individuals can be time-consuming or yield unsuccessful matings, and selecting G₀ somatic transformants as founders entails the risk that the transgene has not been integrated into the germline. This approach is supported by our results from in-crossing all phenotypically G₀ negative individuals across several experiments, which all resulted in large G₁ cohorts with >10% transgenic individuals to select from to establish stable lines (Tables 2, 3). In contrast, backcrosses of positive G₀ individuals was successful in Experiment #2, but failed to produce any G₁ transformants in Experiment #5.

The pooled G₀ crossing scheme presents some caveats. First, not being able to trace how many individuals reproduced, we could not accurately measure germline integration rates as previously done in other insects, including our report of successful transgenesis in Plodia using the Hyperactive piggyBac transposase (Gregory et al., 2016; Heryanto, Mazo-Vargas & Martin, 2022). With the current data, it is unclear which of the Minos or Hyperactive piggyBac transposases drives the highest rates of germline transformation. Second, pooling G₀ siblings risks creating G₁ individuals inheriting several insertion sites. While having several insertions segregating in a line is not always an issue, e.g., if a transgene is designed to fluorescently label structures for qualitative imaging, or for testing the ability of select promoters to drive tissue specific expression (e.g., the FibL promoter), this could be a concern for quantitative or expression-reporter assays that may be influenced by positional effects. In such cases, we recommend sorting G₀ pupae by sex (Heryanto et al., 2025), and mix a pool of G₀ males to an equivalent number of uninjected females, and vice-versa (Fig. 3B). Generating a synchronized stock can facilitate this, simply by leaving a portion of the collected embryos uninjected at the beginning of the experiment.

Alternatively, if G₁ broods originate from an in-cross of pooled G₀ siblings, mixing single G₁ transgenic individuals with non-transgenic individuals should generate a majority of G₂ lines where a single transgene insert is segregating. It is worth noting that while we observed 50% segregation ratios consistent with single-inserts among our G₂-to-G₄ generations (Tables 2 and 3), we have not determined whether these lines include transcriptionally silent transgene copies, which might exist if Minos-mediated insertions can occur in heterochromatin regions. Inverse/splinkerette polymerase chain reaction (PCR) or sequencing techniques could be used in the future to map the position of inserts in the genome (Potter & Luo, 2010; Pavlopoulos, 2011; Stern, 2016).

Flexible marker choice using 3xP3 and FibL drivers and various fluorophores

The 3xP3:EGFP and 3xP3:mCherry markers provide a reliable labeling on larval ocelli, glial tissue, and eyes across insects, including the hemimetabolous crickets and firebrats (Gonzalez-Sqalli, Caron & Loppin, 2024; Inada, Ohde & Daimon, 2025). This signal was strong enough to be screened throughout the cuticle throughout the entire larval stage of the Plodia wFog strain, in which a white gene null mutation not only results in white eyes but also in increase translucency of the larval cuticle (Heryanto et al., 2022). In species where opaque cuticle may block fluorescence, or where a white-eyed mutant line is not available, it is still be possible to screen for 3xP3 activity in the larval lateral ocelli (Thomas et al., 2002), or in early pupal eyes before melanization occurs (Özsu et al., 2017).

This said, these features make the 3xP3 constraining to use, and we expect FibL-driven fluorescence to provide an interesting alternative in some species, particularly those with a reasonably translucent or clear cuticle in the first larval instars. And not only FibL may be more convenient to screen, it may also be preferred for experimental reasons, particularly in neurogenetics studies where tagging neural tissues may be undesirable (Fujiwara et al., 2014).

Last, we tested mBaoJin for the first time in a lepidopteran insect, as a monomeric version of the StayGold fluorophores, which show improved photostability and brightness over existing green fluorescent proteins (Zhang et al., 2024). Its successful tagging of the silk gland makes it a promising tool as a fusion-protein or expression reporter for Lepidoptera in-vivo studies. A plasmid carrying Minos repeats, 3xP3:mCherry, and the Plodia-derived FibL:mBaoJin marker is available on the Addgene repository for academic use (Addgene #240410).