Germ‐line transformation and RNAi of the ladybird beetle, Harmonia axyridis

Abstract

To elucidate the molecular mechanisms underlying the tremendous diversity of insect wing colour patterns, it is imperative to identify and functionally characterize the genes involved in this developmental process. Here we report the first successful germ-line transformation using the transposable element vector piggyBac in the ladybird beetle Harmonia axyridis, which demonstrates typical genetic polymorphism in its wing colour patterns. The transformation efficiency by piggyBac was 3.7% per fertile G(0). We investigated the effectiveness of RNAi in Harmonia by injecting EGFP (enhanced green fluorescent protein) dsRNA into early transgenic EGFP-expressing embryos and observed substantial reduction of EGFP fluorescence in 87.2% of hatched larvae. Application of these new genetic tools to non-model insects such as Harmonia will facilitate the broad understanding of developmental mechanisms and evolutionary processes that are inaccessible using established model systems.

Tissue-specific gene silencing by RNA interference in the whitefly Bemisia tabaci (Gennadius)

“…This mechanism, active through a RNA interference mechanism (RNAi), was recently described in several model and non-model organisms that belong to different taxonomic groups. In insects, significant reduction in gene and protein expression was demonstrated in the fruitfly Drosophila melanogaster (Kennerdell and Carthew, 1998), the mosquito Anopheles gambiae (Paskewitz et al, 2006), the caterpillar Manduca sexta (Vermehren et al, 2001), the milkweed bug Oncopeltus fasciatus (Hughes and Kaufman, 2000), the German Cockroach Blatella germanica (Cruz et al, 2006), the triatomine bug Rhodnius prolixus (Araujo et al, 2006), the light apple brown moth Epiphyas postvittana (Turner et al, 2006), the ladybird beetle Harmonia axyridis (Kuwayama et al, 2006) and the pea aphid Acyrthosiphon pisum (Mutti et al, 2006). Gene silencing was achieved using germ-line transformation, injection of dsRNA, feeding with bacteria expressing dsRNA, or simply by dsRNA feeding.…”

Genetic transformation of the codling moth, Cydia pomonella L., with piggyBac EGFP

“…There have been a number of motivating reasons to genetically transform insects, including: (1) the need to develop a model for basic research into physiology, behavior, etc. (Robinson et al 2000); (2) the desire to create mutants in order to learn more about the genetics of the insect (Daborn et al 2007;Kuwayama et al 2006;Park et al 2002;Wilson et al 1989;Bellen et al 1989); (3) the wish to develop and/or improve genetic sexing strains (Marec et al 2005;Michel et al 2001;Handler et al 1998); (4) the ability to make a disease vector inhospitable to disease-causing organisms (Marrelli et al 2007); (5) the need to improve the viability/survival/adaptability of a beneficial arthropod (Presnail and Hoy 1992); and (6) the desire to create a conditionally sterile strain of a pest species as a means, through sterile insect release, of controlling pest populations (Fryxell and Miller 1995;Heinrich and Scott 2000;.…”

Establishment of Transgenic Lines for Jumpstarter Method Using a Composite Transposon Vector in the Ladybird Beetle, Harmonia axyridis

“…Transgenic ladybird beetles were generated by microinjection of 500 ng/µl composite vector without a helper plasmid into the posterior pole of embryos during the syncytium stage of development as described by [27]. The G 0 adults were crossed with non-injected wild type adults.…”

“…Inverse PCR for a mutator strain was performed as described by [27]. Genomic DNA were digested with HaeIII or MspI .…”