Gene silencing of arginine kinase in tissues of the squash bug, Anasa tristis (De Geer) (Heteroptera: Coreidae)

The Squash bug, Anasa tristis (De Geer), is a major pest of squash, pumpkin, and other cucurbits throughout North America.  A. tristis is a piercing/sucking feeder which causes extensive foliar wilting, fruit scarring, and in addition transmits plant pathogens.  Current biological control agents available for control of A. tristis infestations are extremely limited in efficacy, thus newer control methods must be developed.  Gene silencing by application of double stranded RNA to insects, or RNA interference (RNAi) is a recently introduced technology with the potential to reach pest insects via either the food plant (in planta RNAi expression) or by field applications of encapsulated RNA.  In order to determine whether A. tristis is a suitable candidate for control via RNAi we undertook an examination of insect-specific gene silencing in this pest by injection of dsRNAi against arginine kinase (ATP: L-arginine phosphotransferase, EC 2.7.3.3) which catalyzes the reversible phosphorylation of Arg by ATP yielding phosphoarginine.  A. tristis arginine kinase (AtAK) transcript was identified within the recently completed survey of immune-stimulated A. tristis transcripts (Genbank Accession NCBI#JQ266746.1).  Expression of AtAK is significantly upregulated in A. tristis adults and nymphs following bacterial elicitation with a septic puncture.  As a control for activation of the immune response upregulated expression of the antibacterial peptide anasin (KF578378.1) was concurrently measured.  An AtAK dsRNAi-producing amplimer was constructed for generation of dsRNAi by in vitro transcription.  Injection of up to 1 ug AtAK RNAi into A. tristis adults and nymphs resulted in significant reduction in AtAK transcripts compared to water injected control insects.