Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

Abstract

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase ( hyPBase ) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella . Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G 0 injected generation was detectable from 72-hr embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G 0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G 0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

Full text

Efficient hyperactive piggyBac transgenesis in Plodia
pantry moths
Christa Heryanto1, Anyi Mazo-Vargas1, Arnaud Martin*,1
1Department of Biological Sciences, The George Washington University, Science & Engineering Hall, Suite 6000,
800 22nd St. NW, Washington, DC 20052, USA
*Author for correspondence: arnaud@gwu.edu
ORCID
CH: 0000-0002-9917-5710
AMV: 0000-0001-9644-2871
AM: 0000-0002-5980-2249
While piggyBac transposon-based transgenesis is widely used in various emerging model organisms,
its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic
transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive
piggyBac transposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the
genome of the pantry moth Plodia interpunctella. Co-injection of hyPBase mRNA with donor plasmids
successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed,
or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene
in the G0injected generation was detectable from 72-hr embryos and onward in larvae, pupae and
adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into
transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0
founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable
isogenic lines. Random in-crossing of a small cohort of G0founders expressing 3xP3::DsRed yielded a
stable transgenic line segregating for more than one transgene insertion site. We discuss how
hyPBase can be used to generate stable transgenic resources in Plodia and other moths.
Keywords: Plodia, Lepidoptera, transgenesis, piggyBac, microinjection, germline transformation, transposon
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INTRODUCTION
Lepidoptera is a large insect order that comprises 160,000 species (Kristensen et al., 2007; Roskov et al., 2013),
including a wide range of agricultural pests and ecosystem service providers, as well as important model systems for
research in conservation biology, ecology, and evolutionary biology. In order to foster the potential of lepidopteran
insects for functional genetics beyond the silkworm flagship system, for which transgenic resources already exist, we
are developing the pantry moth Plodia interpunctella (hereafter Plodia; abbr. Pi), or Indianmeal moth, as an
alternative laboratory organism amenable to routine genome editing and transgenesis. Plodia is a worldwide pest of
stored food products, and exhibits convenient laboratory features that make it a promising system for the long-term
maintenance of isogenic lines. In addition to its relatively short life cycle (25 days at 28°C) and ease of culture on a
low-cost diet (Silhacek and Miller, 1972), Plodia cultures are resilient to inbreeding (Bartlett et al., 2018). Mass
egg-laying can be stimulated by exposing their highly fecund females (Mbata, 1985) to CO2gas, a property that
allows the collection of synchronized embryos within the time frame of the first cell divisions, thus facilitating
genetic transformation by microinjection (Dyby and Silhacek, 1997; Bossin et al., 2007). Finally, several genome
assemblies and several transcriptomic resources have been published in this species (Harrison et al., 2012; Tang et
al., 2017; Roberts et al., 2020; Heryanto et al., 2022; Kawahara et al., 2022).
Transgenesis techniques based on the piggyBac transposase (PBase) have been successfully implemented in a wide
variety of insect model organisms and beyond (Handler, 2002; Gregory et al., 2016; Laptev et al., 2017). Butterflies
and moths were shown to have transposition rates an order of magnitude lower than in beetles, mosquitoes and flies
(Gregory et al., 2016), making routine transgenesis more challenging in the Lepidoptera order. A modified version of
the transposase dubbed hyperactive piggyBac (hyPBase) was isolated from a mutant screen in 2011 (Yusa et al.,
2011). hyPBase was later shown to dramatically increase transformation rates in flies and honeybees compared to its
native version (Eckermann et al., 2018; Otte et al., 2018), and was also shown to provide practical transformation
rates in Spodoptera noctuid moths (Chen and Palli, 2021).
Previously, delivery of the original PBase as a helper plasmid into Plodia syncytial embryos resulted in somatic
transformation of fluorescent markers, but its efficiency for germline transformation was not reported (Bossin et al.,
2007). Here, we extend the assessment of hyPBase transgenesis in Lepidoptera with a focus on the pyralid moth P.
interpunctella, a pest of stored foods that is amenable to genome editing and genetic transformation (Bossin et al.,
2007; Heryanto et al., 2022). In the current study, we injected an insect codon-optimized hyPBase as a mRNA (Otte
et al., 2018) and monitored both the somatic and germline transformation rates of fluorescent markers driven by the
3xP3 promoter, a canonical promoter with strong activity in the ocular and glial tissues in Lepidoptera and other
insects (Berghammer et al., 1999; Horn et al., 2002; Thomas et al., 2002). This approach robustly generated
transgenic lines carrying various fluorescent protein markers, illustrating the suitability of hyPBase for routine genetic
transformation in Plodia pantry moths. We discuss future strategies for establishing transgenic lines in emerging
laboratory systems for lepidopteran functional genomics.
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FIGURE 1 | Microinjection procedure and transgenic constructs for the testing of hyperactive piggyBac transformation in
Plodia.(A) Microinjection of P. interpunctella syncytial embryos. Gravid females oviposit en masse after CO2narcosis, and
eggs are collected and oriented on a parafilm strip in a tissue culture dish. A wet brush is used to position eggs, with water
contact helping firm adhesion to the parafilm (I). Microinjection is performed on the side opposite to the micropyle (II).
Peripheral droplets of water are used to periodically flush the injection capillary of yolk. Eggs are sealed with glue following
injection (III). (B) Expression cassettes of donor plasmids carrying 3xP3 eye and glia fluorescent markers. IR = piggyBac
internal repeats (L, left ; R, right). (C) Transposon-mediated random integration following the injection of donor plasmid
and hyPBase mRNA. (D) Somatic transformation efficiency (%) is equivalent to the number of potential G0founders
obtained out of 100 injected eggs. Germline transformation efficiency (%) factors proportion of transgenic G1broods
obtained from G0outcrosses. Ninj = number of injected eggs. Made with Biorender.
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RESULTS
We tested the suitability of hyPBase for transgenesis, using three donor plasmids that drive the expression of the
fluorescent markers EGFP, DsRed, and EYFP. For each experiment, we report the levels of somatic transformation observed
in the G0injected generation, as well as our observations carrying the transgenes into further G1-3 generations.
hyPBase delivery of a 4.4 kb insert expressing 3xP3::EGFP
A practical transgenesis method must allow the delivery of relatively large cargos of several kilobases. To test the
efficiency of hyPBase, we generated a piggyBac donor plasmid with a 4.4 kb insert with both a transgene and a
transgenesis marker (Fig. 1B). The cassette consisted of the mScarlet red fluorescent protein flanked by 3’ and 5’UTR
regions of the nanos-O gene Plodia homolog, a germline determinant selected on its apparent specificity to gonadic
tissues (Nakao and Takasu, 2019; Xu et al., 2022). As a transgenesis marker, we used a 3xP3::EGFP marker that
labeled ocular tissues during previous somatic piggyBac transformation attempts in Plodia (Bossin et al., 2007).
First, we injected this plasmid without hyPBase mRNA to control for episomal expression of the 3xP3::EGFP driver.
These injections showed strong EGFP expression in large internal cells 48 h post-injection, suggesting episomal
expression from the embryo vitellophages (Fig 2A). However, this signal was lost in 72-h old embryos, which only
showed background levels of fluorescence or external autofluorescence artifacts at injection sites (Fig 2B). Thus,
episomal expression of injected plasmids dissipates by 72 h of embryonic development and should not interfere with
the screening of successful integration events at this stage and onwards.
We then co-injected the donor plasmid pBac[3xP3::EGFP; nosO::mScarlet] with a hyPBase mRNA and
monitored somatic transformation efficiencies throughout the G0generation. In order to facilitate the screening of
fluorescence, all experiments were performed in the Pi_wFog white-eyed strain that is devoid of screening pigments
in eye tissues and also shows increased larval translucency (Heryanto et al., 2022).Transformed embryos and first
instar hatchlings showed ocellar and glial EGFP fluorescence (Fig. 2C-D), with 23.7% of injected eggs showing EGFP
in 72-h embryos (Table 1). Injections produced viable larvae with persistent ocellar fluorescence, as well as eye
fluorescence in pupae and adults (Fig. 2E-F). Over several replicated experiments, we found that 16% of injected
eggs resulted in pupae, of which 18.6% were EGFP+. Taking into account occasional pupal failure observed in normal
rearing conditions, we determined that 2.5-3% of injected eggs become viable and fertile G0somatic transformants.
Next, we tested germline transmission by back-crossing G0EGFP+individuals to uninjected stock (Table 2).
Out of 6 fertile pairs, 50% yielded EGFP+G1progeny, suggesting a practical level of germline mobilization among G0
founders. This result is mitigated by the fact that only 6 out a total of 16 single-pair matings (37.5%) generated in our
conditions. This establishes a germline efficiency rate of 0.94% (Fig. 1D, GTE = 6/16 x 2.5% G0founders), meaning
that for 1000 G0embryos injected, 9.4 embryos will survive as fertile founders passing the transgene to the G1
generation.
As we wanted to assess whether hyPBase would allow the rapid isolation of single-insertion lines, we needed
to test if transgenes were integrated into multiple copies per G0gamete, or if they could cause sterility. EGFP+G1
individuals (N= 3) were back-crossed (Table 2) and produced a mean of 61 EGFP+adults out of 124 emerged G2per
cross (49.3%), showing no statistical difference from an expected 50% ratio of a single insertion event (0.06<
2<0.46; df =1; 0.10< p< 0.80). Likewise, a total of five subsequent in-crosses (G2EGFP+x G2EGFP+) each resulted in
positive offspring ratios close to the expected 75% (0.06< 2<0.44; df =1; 0.507< p< 0.80). Of note, the
Plodia-nosO::mScarlet transgene failed to drive red fluorescent signals detectable by epifluorescent and confocal
microscopy in dissected ovaries, and we will explore the activity of alternative germline-driving promoters in the
future (Nakao and Takasu, 2019; Xu et al., 2022). Overall, these data demonstrate that hyPBase provides practical
transformation rates for a relatively large cargo insert, with at least 2.5% of injected zygotes yielding potential
founders ready for isogenic line establishment after only one or two generations of backcrossing.
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FIGURE 2 | Phenotype of transgenic Plodia expressing EGFP in eyes and putative glia. (A) Control injections of
pBac[3xP3::EGFP] show variable levels of green autofluorescence (af), most markedly at the injection wound site (ws).
Episomal expression of EGFP in vitellophages (vp) is intense 24 h post-injection, reduced to background level after 48 h.
(B) Donor pBac[3xP3::EGFP] + hyPBase mRNA injections resulted in 3xP3::EGFP expression, emerging as nervous system
markings around 72 h post-injection (arrowheads). 23.7% of injected G0eggs (262/1104) showed a similar fluorescence
during screening. (C) 3xP3::EGFP expression in a first instar larva, in ganglia of the Central Nervous System (consistent
with an expected glial reporter activity of 3xP3), and in ocellar stemmata (circled, magnified in C’). (D) G0mosaics of
3xP3::EGFP expression in pupal eyes. An EGFP-negative pupa is shown on the left for reference. (E) 3xP3::EGFP expression
in a G1Plodia adult with non-mosaic expression of EGFP in the eye (bottom right). EGFP is also visible in the brightfield
(bottom right), with a green tint of the compound eyes in the Pi_wFog recessive white-eyed strain. Scale bars: A-C’ = 200
µm; D-E = 500 µm.
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TABLE 1 | G0phenotypes of Plodia injected with pBac donor plasmids and hyPBase transposase mRNA.
Plasmid
Trial
Embryos
Larvae
Pupae
Adults
Injected
F+
F-
Total
F+
F-
Total
F+
F+
pBac[3xP3::EGFP;
nosO::mScarlet]
1
275
112
163
-
-
-
67
12
7
2
433
92
335
72
37
35
45
16
16
3
396
58
338
60
18
42
38
5
5
Total
1104
262
836
-
-
-
150
33
28
pBac[3xP3::DsRed]
1
381
55
326
87
15
72
25
11
11
2
479
101
378
121
42
79
39
19
19
Total
860
156
704
208
57
151
64
30
30
pBac[3XP3::EYFP; attP]
1
384
-
-
39
-
-
26
13
9
2
413
88
325
74
32
42
37
5
5
Total
797
-
-
113
-
-
63
18
14
F+: number of individuals with fluorescent signal
F-: number of individuals with no fluorescent signal
- : missing data.
TABLE 2 | Subsequent crossing of transgenic Plodia G0founders.
G1experiments
No. of G0crosses
+ strategy
No. of fertile G1broods
Crossing
success rate
with F+
no F+
pBac[3xP3::EGFP; nosO::mScarlet]
16
BC to wFog
3
3
37.5% (6/16)
pBac[3xP3::DsRed]
18
BC to wFog
0
2
11.1% (2/18)
1-4
G0 in-cross
1
NA
container of 5 G0s (F+)
pBac[3XP3::EYFP; attP]
14
BC to wFog
1
6
50% (7/14)
G2experiments
No. of G1fertile
crosses + strategy
Total no. of G2progeny
Expected ratio if G1
heterozygous at
single-insert
F+pupae
F-pupae
pBac[3xP3::EGFP; nosO::mScarlet]
3
BC to wFog
184
189
yes (1:1)
pBac[3xP3::DsRed]
3
BC to wFog
167
92
no
pBac[3XP3::EYFP; attP]
2
BC to wFog
101
87
yes (1:1)
G1 in-cross
55
23
yes (3:1)
G3experiments
No. of G2fertile
crosses + strategy
Total no. of G3progeny
Expected ratio if G2
heterozygous at
single-insert
F+pupae
F-pupae
pBac[3xP3::EGFP; nosO::mScarlet]
5
G2 in-cross
102
34
yes (3:1)
pBac[3xP3::DsRed]
mixed
G2 in-cross
189
58
yes (3:1)
pBac[3XP3::EYFP; attP]
mixed
G2 in-cross
189
51
yes (3:1)
F+: number of individuals with fluorescent signal
F-: number of individuals with no fluorescent signal
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Evaluation of a 3xP3::DsRed donor vector
FIGURE 3 | Somatic and germline transgenesis of 3xP3:DsRed.(A) Two control eggs (top and bottom rows) injected with
only pBac[3xP3::DsRed] show background autofluorescence levels in the DsRed channel (af, magenta) at 72 h
post-injection, including residual signal in vitellophages (vp). Wound site (ws) autofluorescence is limited to the EGFP
channel (af, green). (B-C) hyPBase mRNA and pBac[3xP3::DsRed] result in glial expression of 3xP3::DsRed (magenta) in
injected embryos. Ocellar expression was not observed in these experiments at the G0phase. (D) G0pupae showing
various fluorescent signals in the abdomen(DsRedbody), a phenomenon not observed with other constructs. Expression in
the head (DsRedeye) was occasionally seen at the G0phase. (E) G1transgenic embryo with non-mosaic expression of
3xP3::DsRed.(E’) G1pupae showing weak eye fluorescent signals. These signals did not expand to the entire eye as the
pupae developed, suggesting possible epigenetic effects. (F) G2Plodia transgenic pupae obtained from G1outcrosses
resulted in pupae with bright 3xP3 fluorescence patterns that expanded throughout development. Variable intensity may
be due to transgene copy number variation in this line. Scale bars: A-C, E = 200 µm; D, E’, F = 500 µm.
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To expand the toolkit of transgenesis markers, we sought to test the activity of a pBac[3xP3::DsRed] donor vector for
the screening of red eye fluorescence. We used the pHD-DsRed plasmid available through Addgene (Gratz et al.,
2014, 2015), which carries a 1,146 bp 3xP3::DsRed-SV40 cassette tightly flanked by piggyBac internal repeats.
Control injections without hyPBase mRNA revealed weak episomal expression in vitellophages and red background
fluorescence (Fig. 3A). Injection sites, which show nonspecific autofluorescence under EGFP filter sets, do not
fluoresce in the red channel. hyPBase-mediated insertion of pBac[3xP3::DsRed] resulted in glial signals in 14.4% of
injected 72-h AEL (after egg-laying) embryos , but intriguingly, no signal in the head region. Likewise, larval
transformants showed sporadic signals in abdominal regions, seemingly nervous ganglia, but these patterns were
always mosaic (Fig. 3C). About 25/30 G0DsRed+pupae (83%) exhibited DsRed expression in the body (Fig. 3D, G0
DsRedbody). DsRed fluorescence in the head region was observed in only 5 G0pupae (Fig. 3D, G0DsRedeye), but its
expression failed to reproduce the 3xP3::EGFP signal pattern in ocelli and eye tissues (Fig. 2D).
The presence of DsRed in abdominal regions suggested successful integration of the donor plasmid
including in tissues close to the germline. To evaluate the germline transmission in G0DsRedeye individuals, we
backcrossed DsRedbody individuals to the uninjected stock. Only 2 out of 14 G0DsRedeye backcrossed pairs gave G1
progeny (Table 2), and none inherited any DsRed fluorescence expression. In contrast, we recovered eggs from 5 G0
DsRedbody individuals that were incrossed liberally in a container, and showed full embryonic 3xP3::DsRed signals (Fig
3E). This salvaged stock resulted in 6 G1pupae with DsRed expression in the eyes (Fig. 3E’) out of 52 isolated G1
pupae (11.5%), with no body phenotype observed. These six G1DsRed+Plodia were then individually crossed with
Pi_wFog, 3 of which generated 83%, 70%, and 67% G2DsRed+progeny (Fig. 3F). As these ratios deviate from the 1:1
ratio expected in these crosses, we conclude that more than one insert occured in the parental G0founder germline.
The 3xP3 activity in this DsRed donor plasmid showed inconsistent results not seen with the EGFP donor,
including absence of activity in G0eye tissues, unusual abdominal fluorescent patches in G0pupae, and reduced
activity in G1eyes. Intriguingly, full 3xP3::DsRed activity was recovered in G2pupae, suggesting possible epigenetic -
effects of transient nature in earlier generations. This unusual behavior may be due to minor differences in the
cassette proximal promoter (Fig. S1), to the compact design of this cassette (Fig. 1B), or to other sequence features
making the insert prone to abnormal expression.
Generation of 3xP3::EYFP transgenic lines carrying an attP docking site
We co-injected the pBac[3XP3::EYFP; attP] plasmid (Stern et al., 2017) with hyPBase mRNA into Pi_wFog. This donor
includes an attP docking site (Fig. 1B), a feature that may facilitate genetic engineering using site-specific
recombination, if successfully integrated into the Plodia genome. Control injections show little background
autofluorescence and vitellophage signals under the EYFP filter set (Fig. 4A). Transgenic G0embryos and larvae
showed strong somatic 3xP3 activity consistent with ocular and glial expression (Fig. 4B-C), with expected mosaic
variations such as unilateral expression in one side of ocellus glia and ocelli-only expression. We recovered 14 pupae
with mosaic G0EYFP expression (Fig. 2D) from a total of 63 surviving pupae, out of 797 embryos injected over two
trials (Table 1).
To estimate the efficiency of germline integration from these mosaic founders, we individually backcrossed
the 14 EYFP+G0adults to single Pi_wFog individuals (Fig. 4E, Table 2). Seven pairs gave progeny, among which only 1
cross generated progeny with 22 G1EYFP+pupal phenotypes out of 37 total isolated pupae, a ratio statistically close
to the 50% proportion expected from a germline tissue heterozygous for a single insertion in the G0founder
(2=1.32; df =1, p=0.25). To test if positive G1individuals were heterozygous carriers for a single insertion, we
simultaneously backcrossed 12 G1EYFP+to Pi_wFog and in-crossed 5 pairs of G1EYFP+. Three of these crosses
resulted in G2EYFP+progenies, with 59% and 48% positive ratios matching the 50% expected from backcrossing
(0.18< 2< 3.17; df =1; 0.07< p< 0.67), and a 71% positive ratio matching the expected 75% in the in-cross (2= 0.84,
df =1 p= 0.36). In summary, injection of pBac[3XP3::EYFP; attP] had a somatic transformation efficiency of 1.8%.
The high level of mosaicism in G0resulted in only 1 out of 14 successful backcrosses, resulting in a germline
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transformation efficiency of 0.13% (Fig. 1D, GTE = 7.1.% x 1.8% G0founders), but this event was successfully carried
into a stable transgenic line.
FIGURE 4 | Somatic and germline transgenesis of 3xP3::EYFP in Plodia.(A) Weak background autofluorescence in the
EYFP observation channel following control injection of the donor plasmid only. (B) Somatic activity of 3xP3::EYFP
transgenes at 72 h and 120 h post injection in the late egg stage (C) Mosaic G03xP3::EYFP expression in a first instar
larva, marking glia and ocellar stemmata (arrowheads). (D) Mosaic G03xP3::EYFP expression in pupal eyes. (E) 3xP3::EYFP
expression in G1Plodia pupae. (E) Ventral (left) and lateral views (right) of 3xP3::EYFP expression in G2Plodia adults. Scale
bars: A-C = 200 µm; D-F = 500 µm.
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DISCUSSION
Transformation efficiency rates of hyPBase in Plodia
In this study, we carried out somatic and stable germline transformation in Plodia interpunctella using the
hyperactive piggyBac transposase (Yusa et al., 2011), and achieved high rates of somatic transformation with 3
independent piggyBac donor plasmids. We injected the transposase as a mRNA and used hyPBapis,a version of
hyPBase codon-optimized for honeybees (Otte et al., 2018). Because Apis and Plodia both have an average GC
content of around 35% (Jørgensen et al., 2007; Kawahara et al., 2022), we can reasonably expect compatibility in
their codon usage biases.
Our study is the second to use an hyPBase mRNA as a transposase for transgenesis in a lepidopteran insect
(Chen and Palli, 2021). Plodia injections generated 15-40% of G0somatic transformants when observed in 72-h
embryos (mean of 22%), suggesting highly efficient integration.
Across different trials, a mean of 2.5% of injected eggs expressed the transgene marker as adults,
representing 30% of surviving adults. However, somatic fluorescence in the injected generation does not guarantee
that the transgene has transposed into the germline, or that transgenic gametes are fertile. To assess transgene
inheritability into the G1generation, we backcrossed G0fluorescent founders to non-transgenic individuals. We
obtained 3 independent G1lines expressing 3xP3::EGFP out of 6 fertile G0crosses, and 1 line expressing 3xP3::EYFP
out of 7 fertile G0crosses. Founders expressing 3xP3::DsRed showed unusual patterns of G0mosaicism, possibly due
to epigenetic regulatory effects (see Results section), and failed to propagate the transgene when mated in single
outcrossing pairs (N=14), but we recovered a stable insertion from G1eggs that had been laid in a container where 5
G0founders had been left to mate randomly, meaning that 1 out of 19 G0transmitted 3xP3::DsRed.
In summary, our hyPBase mRNA-based injections in Plodia resulted in germline transformation efficiency
rates of 0.18% (DsRed), 0.25% (EYFP), and 0.94% (EGFP). For comparison, Plutella transgenic experiments using
PBase have efficiency rates of 0.43-0.65% (Gregory et al., 2016). Our Plodia injection protocol has a median pharate
survival of 9%, much lower than the published Plutella adult survival rate of 27.8% (Gregory et al., 2016). Indeed, our
injection methods favor speed and quantity over precision, using relatively wide-open needle bores that avoid
clogging during injections, as well as a rapid but aggressive glue-based egg sealing procedure (Heryanto et al.,
2022). Only 10-25% of eggs injected with piggyBac reagents hatched across trials in our conditions — as opposed to
21-60% in a previous Plodia microinjection report conducted by another group (Bossin et al., 2007) — but this is
balanced by the fact that a single experimenter can inject about 400 pre-blastoderm embryos in a 2 h session with
our procedure. Overall, the germline efficiency rates reported here mean that one fertile G0founder was obtained for
every 106 (EGFP), 555 (DsRed), and 777 (EYFP) injected embryos, making a 2-4 h injection effort (400-800 eggs)
reasonably well suited for initiating each transgenic line attempt. Ultimately, practicality boils down to a trade-off
between the number of injected embryos and their survival, and our data suggest that the high efficiency of hyPBase
(Yusa et al., 2011; Eckermann et al., 2018) can make transgenesis feasible if one of these two factors is not optimal.
Other practical considerations for transgenesis in Lepidoptera
Mendelian segregation patterns observed at the G2generations indicate that all 4 out of 5 stable lines
originated as single-insertion events, with G0founders likely carrying a single copy (Table 2). This feature can be
used by experimenters to use various crossing strategies in the future, but we must highlight that single-mating
strategies and crossing conditions resulted in few successful pairings in our initial attempts (e.g. 11-50% of G0
crosses, Table 2). This artificially lowered germline transmission rates, likely due to founders failing to mate in small
containers in suboptimal condition. As we gained experience with Plodia husbandry during these experiments, we
increased mating success rates to 66-78% in subsequent generations (see Methods for the optimized procedure).
Furthermore we recommend to mix one transgene carrier with 2-3 wild-type unmated adults of the opposite sex
instead of one, as this maximizes the likelihood of successful mating in this system (Brower, 1975; Huang and
Subramanyam, 2003).
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Each of the three constructs we tested provided complementary information. The EGFP construct was the
largest and delivered the highest germline transformation rate. Of note, the compact DsRed construct resulted in
unusual G0fluorescent patterns. While circumstantial, these observations bode well for using large inserts, and we
caution that the pBac[3xP3::DsRed] (pHD-DsRed, Addgene #64703) has a more compact minimal promoter that
might also explain its weaker expression (Fig. S1). The candidate germline driver of mScarlet, consisting of the
proximal promoter and 3’UTR of nanos-O (Nakao and Takasu, 2019; Xu et al., 2019) cloned from the Plodia genome,
failed to drive detectable fluorescence in ovarian tissues. We will investigate alternative germline promoters in the
future (Xu et al., 2022), for instance by testing the PhiC31 site-specific integrase at the attP docking site from our
new EYFP transgenic line (Yonemura et al., 2013; Haghighat-Khah et al., 2015; Stern et al., 2017; Stern, 2022). Both
EGFP and EYFP showed robust and strong 3xP3-driven expression at all generations, without noticeable decrease
over time in adult eyes (Das Gupta et al., 2015), with EYFP benefiting from lower autofluorescence effects than EGFP
at various stages. We strategically used a white mutant strain deficient for eye-screening pigment, as routinely done
in other insects to facilitate the screening of 3xP3-driven fluorescence (Stern et al., 2017; Klingler and Bucher, 2022),
and this mutation also increases the translucency of Plodia larvae (Shirk, 2021; Heryanto et al., 2022). Of note, white
mutations can be recessive-lethal in some lepidopteran species (Khan et al., 2017). Until alternative way to generate
depigmented eyes are found, this may limit the usefulness of 3xP3 drivers, especially in species where eggs and
larvae are opaque and where screening becomes limited to narrow developmental windows (Das Gupta et al., 2015;
Özsu et al., 2017). In such species, we suggest that stronger, more ubiquitous promoters of viral origin such as
Op-ie2 and Hr5-ie1 may be more practical for transgenic screening (Martins et al., 2012; Xu et al., 2019, 2022).
MATERIALS AND METHODS
Plodia strains and rearing
The Pi_wFog strain (Heryanto et al., 2022) consists of an introgression of the recessive w- mutation (Shirk, 2021)
from the Pi w- strain (origin: USA, kind gift of Paul Shirk), into the genetic background of the “Dundee” strain (origin :
UK, kind gift of Mike Boots). Genome assemblies of both Pi w- and Pi_Dundee parental strains are available (Roberts
et al., 2020; Kawahara et al., 2022). The resulting hybrid Pi_wFog strain has been maintained in inbred state for 3
years and used throughout this study. All rearing used previously published methods (Heryanto et al., 2022), using
special containers and a wheat bran-sucrose-glycerol diet (Silhacek and Miller, 1972). A rearing temperature of 28°C
resulted in a generation time of 28 d.
Plasmid constructs
The pBac[3xP3::EGFP; Tc'hsp5'-Gal4Delta-3'UTR] (Addgene plasmid # 86449) was used as a donor plasmid with
Piggybac insertion repeats and the 3xP3::EGFP reporter (Schinko et al., 2010). To generate pBac[3xP3::EGFP;
nosO_prom::mScarlet-nosO_3’UTR], an mScarlet cassette preceded by 2 kb of 5’UTR sequence immediately
upstream of the Plodia nanos-O start codon, was synthesized in the pUC-GW-Amp backbone by Genewiz (South
Plainfield, NJ) and sub-cloned into the FseI and AscI restriction sites of pBac[3xP3::EGFP;
Tc'hsp5'-Gal4Delta-3'UTR]. The pBac[3xP3::DsRed] (pHD-DsRed) and pBac[3XP3::EYFP; attP] plasmids were
obtained from Addgene (#64703, and #86860) and used without modification (Gratz et al., 2014, 2015; Stern et al.,
2017). All the 3xP3-driven fluorophore genes included an SV40 termination sequence.
Transposase mRNA and injection mixes
The pGEM-T_hyPBapis plasmid encodes a hyPBase that was codon-optimized for honeybees (Otte et al., 2018). The
source plasmid was purified using the QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD), linearized with
NcoI-HF (NEB, Ipswich, MA) and concentrated using acetate/ethanol precipitation. Around 500 ng of linearized
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template were transcribed using the Invitrogen™ mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Invitrogen,
Carlsbad, CA) and purified using the MEGAclear™ Transcription Clean-Up Kit (Invitrogen, Carlsbad, CA). After
quantification with Nanodrop (Thermofisher, Waltham, MA), the solution was divided into 1050 ng/µL one-time use
aliquots and stored at -80°C.
Microinjections
Injection mixes consisted of 400 ng/µl hyPBase mRNA, 200 ng/µl donor plasmid, and 0.05% cell-culture grade
Phenol Red (Sigma-Aldrich, Burlington, MA). Donor plasmids without hyPBase mRNA were injected in separate
experiments as controls. Microinjection procedures (Figure 1A) followed a previously described procedure (Heryanto
et al., 2022), with all embryo injections performed within 40 min after egg laying (AEL). Injected embryos were
counted and kept in a rearing container with a small damp Kimwipe at 28°C. For the first 72 h, the container vent was
covered with tape in order to maintain humidity saturation, a parameter that prevents egg desiccation. After 72 h, the
vent was opened and the Kimwipe removed, and about five flakes of Plodia food added next to the eggs, in order to
keep the emerging larvae within the injection dish. Mean emergence time of the Pi_wFog strain is 83 h AEL at 28°C
for uninjected eggs, and is delayed by injection stress to 100-115 h AEL. Because of this variability, we report times
of observation after injection in hours rather than in relative percentages.
Fluorescent microscopy
Larvae and adult Plodia were anesthetized in tissue culture dishes positioned over a cold metal block during
microscopy observation. All pictures were taken under the Olympus SZX16 stereomicroscope equipped with a
Lumencor SOLA Light Engine SM 5-LCR-VA lightsource or standard stereomicroscope brightfield lamp, and with a
trinocular tube connected to an Olympus DP73 digital color camera. Separation of fluorescent channels was
performed using Chroma Technology filter sets ET-EGFP 470/40x 510/20m, ET-EYFP 500/20x 535/30m , and
AT-TRICT-REDSHFT 540/25x, 620/60m.
Survival and G0somatic transformation rates
Embryonic survival rates (“egg hatching” rates) were determined by the ratio of hatched eggs at 120 h AEL over the
number of injected eggs (Ninj). Empty egg shells were counted for this purpose instead of first-instar hatchlings,
which are difficult to count accurately in the presence of food. Pharate survival rates were determined by the ratio of
pupae obtained from a given injection experiment, divided by Ninj, and thus accounts for mortality occurring at
embryonic and larval stages. Pupal mortality was negligible, making pharate survival rates a reasonable proxy for
overall adult survival, and is more convenient to couple to fluorescent screening than in mobile adults. G0
transformation rates were independently measured in embryos and in pupae. For embryos, eggs with bright, internal
fluorescent signals consistent with an ocellar or glial expression were counted as positive (fluorescent, F+ in Table 1)
around 72 h AEL, and non-fluorescent eggs were counted as negative (F-). To isolate individual pupae, cardboard
strips that are preferentially used as pupation sites (“hotels”) were added into containers containing fifth instar
larvae, allowing a convenient isolation of individual Plodia pupae. Pupae were then extirpated from these lodges and
aligned on double-sided tape for fluorescence screening. Pupae with any glial or eye signal were counted as positive,
while others were counted as negative. G0somatic transformation efficiency rate was determined as the number of
healthy adult individuals emerged from fluorescent pupae, and normalized by Ninj.
Controlled crosses for germline transmission
Germline transformation efficiency rates factored the somatic transformation efficiency rate by the proportion of
attempted G0backcrosses yielding transgenic offspring. G0transgenic adults or late pupae exhibiting positive
fluorescent signals (G0F+) were crossed to a single unmated Pi_wFog adult of the opposite sex, by mixing in a 1.25
oz Plastic Souffle Cup (Solo) containing ~ 0.2 g of diet and ~ 1 cm2of paper towel. Pi_wFog outcrossing mates were
replaced if found dead before any visible egg laying. These cups were monitored for up to 2 weeks for any larval
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emergence, after which they were transferred to a vented rearing container with a bed of Plodia food (modified
LocknLock containers described in Heryanto et al., 2022; 177 mL and 350 mL formats). At the wandering L5 stage,
cardboard "hotels" were added into the containers for pupal isolation. G1pupae with positive fluorescent signal were
counted and backcrossed to an unmated Pi_wFog with the same procedure stated above. The resulting G2pupae
were in-crossed as sib-matings and maintained as isogenic stock in the G3generations and henceforth.
DATA AVAILABILITY STATEMENT
The pBac[3xP3::EGFP; nosO_prom::mScarlet-nosO_3’UTR] plasmid generated in this study is available upon request.
AUTHOR CONTRIBUTIONS
CH and AM designed the study and wrote the manuscript. AMV and AM advised on the methodology. CH performed
the experiments and analyzed the data.
FUNDING
This work was supported by National Science Foundation Grant under NSF/IOS grant IOS-1923147.
CONFLICT OF INTEREST
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or materials discussed in the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://____
FIGURE S1 | Alignment of the region spanning, from left to right, the 3’ end of the 3xP3 promoter and Kozak sequence
preceding the fluorophore genes in 3 pBac donor plasmids.
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