A visible dominant marker for insect transgenesis

Abstract

Transgenesis of most insects currently relies on fluorescence markers. Here we establish a transformation marker system causing phenotypes visible to the naked eye due to changes in the color of melanin pigments, which are widespread in animals. Ubiquitous overexpression of arylalkylamine-N-acetyl transferase in the silkworm, Bombyx mori, changes the color of newly hatched first-instar larvae from black to a distinctive light brown color, and can be used as a molecular marker by directly connecting to baculovirus immediate early 1 gene promoter. Suppression of black pigmentation by Bm-arylalkylamine-N-acetyl transferase can be observed throughout the larval stages and in adult animals. Alternatively, overexpression in another gene, B. mori β-alanyl-dopamine synthetase (Bm-ebony), changes the larval body color of older instars, although first-instar larvae had normal dark coloration. We further show that ectopic Bm-arylalkylamine-N-acetyl transferase expression lightens coloration in ladybird beetle Harmonia axyridis and fruit fly Drosophila melanogaster, highlighting the potential usefulness of this marker for transgenesis in diverse insect taxa.

Figure 1. Bm-aaNAT overexpression alters cuticle coloration in neonatal silkworm larvae.

(a) Structures of UAS-NAT/3xP3EGFP, UAS-ebony/3xP3EGFP and actin A3-GAL4/3xP3DsRed2 (not to scale). 3xP3, eye-specific promoter; ITR, inverted terminal repeat of the piggyBac transposon. Effect of ectopic expression of Bm-aaNAT (b) and Bm-ebony (c) using the GAL4/UAS binary system (w1-pnd background). Left, bright field; middle, GFP; and right, red fluorescent protein (RFP). White arrowheads indicate fluorescence in the ommatidia.

Figure 2. Bm-aaNAT overexpression lightens pigmentation in the black striped silkworm strain.

The fifth-instar larvae shown here have the dominant striped (p^S) genetic background. (a) Dorsal view of the larvae. The positions of the markings magnified in b are indicated by the arrowheads. (b) Magnified images of the larval markings.

Figure 3. Bm-ebony overexpression alters silkworm larval pigmentation in older instars.

The black striped silkworm (p^S) strain, which the effect on melanin pigmentation is easy to observe is used. The dorsal view of third-instar larvae is shown. Ubiquitous overexpression of Bm-ebony changed most areas of black pigmentation.

Figure 4. Ectopic expression of Bm-aaNAT and Bm-ebony lightens adult antenna coloration.

Moths carrying only actin A3-GAL4/3xP3DsRed2 were used as controls.

Figure 5. Development of a dominant marker IE1-NAT that alters neonatal larval coloration.

(a) Structure of the IE1-NAT/3xP3EGFP transgene. Bm-aaNAT expression was under the direct control of the constitutive baculovirus IE1 promoter. (b) Neonate larvae carrying IE1-NAT transgene can be easily distinguished by its light coloration (white arrowheads), whether w1-pnd (kynurenine 3-mono oxygenase (KMO)-deficient strain, left panels) or commercial strain C146 (with intact KMO gene, right panels) is used as the host strain for transgenesis. Top panels, bright field; bottom panels, GFP filter.

Figure 6. Bm-aaNAT lightens coloration in other insect orders.

(a,b) Effect of ectopic Bm-aaNAT expression in D. melanogaster. The flies are at least 4 days (100 h) old. Scale bar, 1 mm (a), 0.5 mm (b). (a) Shows the effect of Bm-aaNAT driven by hsp-GAL4. The fly shows overall lighter pigmentation, and as shown in the bottom panels (the magnified view of the square in the top panel) bristle coloration are also suppressed by Bm-aaNAT overexpression. (b) Shows the effect of Bm-aaNAT driven by pannier-GAL4, which drives expression in a broad stripe along the dorsal midline (brackets). Abdomen of adult flies is shown. (c) Effect of hsp-NAT transgene on H. axyridis fourth-instar larvae. Left, insect with no transgene; middle, insect with hsp-NAT transgene; right, insect with hsp-NAT transgene with heat shock treatment. Scale bar, 1 mm.

Figure 7. Effects of overexpression of Bm-aaNAT and Bm-ebony on silkworm pigmentation.

The striped strain p^S is used in the model for larval coloration. The melanin synthesis pathway is adapted from Futahashi et al. The melanic pigmentation of silkworm larvae and adult antennae is affected by Bm-aaNAT and Bm-ebony, which use dopamine as substrate and produce transparent and yellowish cuticle, respectively. Notably, Bm-aaNAT effect on melanic pigmentation can be easily detected at the first instar, whereas Bm-ebony does not have significant effect in this stage. TH, tyrosine hydoxylase; DDC, dopa decarboxylase.