Gene Editing of the ABC Transporter/White Locus Using Crispr/Cas9-Mediated Mutagenesis in the Indian Meal Moth

Shirk, Bryce D.; Shirk, Paul D.; Furlong, Richard B.; Scully, Erin D.; Wu, Ke; Siegfried, Blair D.

doi:10.2139/ssrn.4192442

Cited by 4 publications

(6 citation statements)

References 48 publications

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Methodsmentioning

confidence: 99%

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Efficienthyperactive piggyBactransgenesis inPlodiapantry moths

Heryanto

Mazo‐Vargas

Martin

2022

Preprint

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WhilepiggyBactransposon-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-optimizedhyperactive piggyBactransposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry mothPlodia interpunctella. Co-injection ofhyPBasemRNA 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 the3xP3promoter. 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 G0founders transmitted single-insertion copies of3xP3::EGFPand3xP3::EYFPand generated stable isogenic lines. Random in-crossing of a small cohort of G0founders expressing3xP3::DsRedyielded a stable transgenic line segregating for more than one transgene insertion site. We discuss howhyPBasecan be used to generate stable transgenic resources inPlodiaand other moths.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Efficienthyperactive piggyBactransgenesis inPlodiapantry moths

Heryanto

Mazo‐Vargas

Martin

2022

Preprint

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“…All experiments in this paper were performed using a lab population of P. interpunctella that was started at the USDA-ARS in Gainesville, FL, USA (see Shirk et al, 2022 for further information on the history of this colony). In 2018, several individuals were transferred to the Florida Museum of Natural History, where a new colony was started for this project.…”

Section: Methodsmentioning

confidence: 99%

Effect of behavioral conditions on silk characteristics in the Indian meal moth (Plodia interpunctella)

Barve

Godfrey

Storer

et al. 2022

Preprint

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Lepidopteran silks are produced during the larval stage and are used for mobility and protection from predators, parasitoids, and pathogens. Our knowledge of silk structure and production in Lepidoptera is based largely on the biology of the domestic silk moth (Bombyx mori), but recent comparative evidence suggests that silk production and structure vary widely across moth taxa. Some species like the Indian meal moth (Plodia interpunctella) are becoming important biological models to study silk for its potential application to materials science and medicine, but many aspects of silk production in this species remain unknown. Here we characterize the silk of P. interpunctella by measuring the width of wandering and pupal silk strands and find that pupal silk is significantly thicker than the latter. We then report individual variation in pupal silk production in our lab-reared colony with a very small number of individuals forgoing pupal silk (< 4%) and find that overcrowding had no effect on this, whereas exposure to elevated temperatures reduced rates of pupal silk production.

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“…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 eyescreening 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).…”

Section: Other Practical Considerations For Transgenesis In Lepidopteramentioning

confidence: 99%

“…The Pi_wFog strain (Heryanto et al, 2022) consists of an introgression of the recessive w-mutation (Shirk, 2021) from the Pi wstrain (origin: USA, kind gift of Paul Shirk), into the genetic background of the "Dundee" strain (origin: United Kingdom, kind gift of Mike Boots). Genome assemblies of both Pi wand Pi_ Dundee parental strains are available (Roberts et al, 2020;Kawahara et al, 2022).…”

Section: Plodia Strains and Rearingmentioning

confidence: 99%

Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

2022

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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 G0 injected generation was detectable from 72-h 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 G0 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.

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Gene Editing of the ABC Transporter/White Locus Using Crispr/Cas9-Mediated Mutagenesis in the Indian Meal Moth

Cited by 4 publications

References 48 publications

Efficienthyperactive piggyBactransgenesis inPlodiapantry moths

Efficienthyperactive piggyBactransgenesis inPlodiapantry moths

Effect of behavioral conditions on silk characteristics in the Indian meal moth (Plodia interpunctella)

Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

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