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.

A Pathway Analysis of Melanin Patterning in a Hemimetabolous Insect

“…This is in contrast with previous studies in Drosophila, Tribolium, and Bombyx, in which depletion of ebony, black, tan, aaNAT, or yellow resulted in alteration of melanin patches throughout the body (Wright 1987;Walter et al 1996;Wittkopp et al 2002b;True et al 2005;Gibert et al 2007;Jeong et al 2008;Arakane et al 2009;Tomoyasu et al 2009;Zhan et al 2010;Osanai-Futahashi et al 2012). Hence, the insight from Oncopeltus is that the entire melanin pathway can be split into different sections that are used in different body regions.…”

“…More specifically, the phenotypes observed in the abdomen and hindwings of RNAi adults were different from those in the head, thorax, and forewings. These observations are unexpected because RNAi depletion of ebony, black, or aaNAT in holometabolous species (Tribolium and Bombyx) caused alterations in color patterns over the entire body (Tomoyasu et al 2009;Arakane et al 2010;Zhan et al 2010;Osanai-Futahashi et al 2012). Results presented here from Oncopeltus suggest that different melanin genes are used to form black patterns in distinct body regions.…”

“…Here we observed that the role of aaNAT is restricted to the anal lobe region of the hindwing ( Figure 3D2), which correlates with its expression profile ( Figure 4B1). This observation is in contrast to the depletion of aaNAT in the silkworm Bombyx, which causes a global darkening of the adult body (Zhan et al 2010;Osanai-Futahashi et al 2012). Thus, Oncopeltus appears to preferentially activate aaNAT expression in wings to create the colorless tissue in the anal lobe region.…”

“…Assays in Drosophila have provided functional evidence associating ebony, yellow, and tan with body and wing pigmentation (Walter et al 1996;Wittkopp et al 2002b;Gompel et al 2005;True et al 2005;Jeong et al 2008). In addition, functional studies in Tribolium have also reported the essential involvement of ebony, black, and yellow Tomoyasu et al 2009;Arakane et al 2010), whereas the pigmented phenotype of aaNAT was reported only in Bombyx (Zhan et al 2010;Osanai-Futahashi et al 2012). A systematic functional profile of these melanin genes is lacking in any of the more basal hemimetabolous insect lineages.…”

“…In addition, functional studies in Tribolium have also reported the essential involvement of ebony, black, and yellow Tomoyasu et al 2009;Arakane et al 2010), whereas the pigmented phenotype of aaNAT was reported only in Bombyx (Zhan et al 2010;Osanai-Futahashi et al 2012). A systematic functional profile of these melanin genes is lacking in any of the more basal hemimetabolous insect lineages.…”