RNA interference against gut osmoregulatory genes in phloem-feeding insects Vered Tzin a,1 , Xiaowei Yang b,1 , Xiangfeng Jing b , Kai Zhang b , Georg Jander a , Angela E. Douglas b,c,⇑ a Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, USA b Department of Entomology, Cornell University, Ithaca, NY 14853, USA c Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA article info Article history: Received 18 April 2015 Received in revised form 23 May 2015 Accepted 9 June 2015 Available online 10 June 2015 Keywords: Aquaporin Bactericera cockerelli Myzus persicae RNAi Sucrase Sugar transporter abstract In planta RNAi (i.e. plants engineered to synthesize active RNAi molecules) has great potential as a strat- egy to control insect crop pests. This study investigated the impact of RNAi against osmoregulatory genes expressed in the gut of two phloem-feeding species, the green peach aphid Myzus persicae and the potato/tomato psyllid Bactericera cockerelli. The target genes comprising candidate gut sucrase, aquaporin and sugar transporter genes were identified by mining insect genomic and transcriptomic datasets for genes orthologous to empirically-tested osmoregulatory genes of the pea aphid Acyrthosiphon pisum. Insects feeding on plants with RNAi against the target genes exhibited elevated hemolymph osmotic pressure (a predicted effect of perturbed osmotic function) and some reduction in performance, espe- cially offspring production in M. persicae and mortality in B. cockerelli, associated with up to 50% reduc- tion in mean expression of the target genes. The effects were particularly pronounced for insects treated with RNAi against multiple osmoregulatory genes, i.e. combinatorial RNAi, suggesting that the partial silencing of multiple genes with related roles can yield greater functional impairment than RNAi against a single gene. These results demonstrate the potential of RNAi against osmoregulatory genes, but further advances to improve the efficacy of RNAi in phloem-feeding insects are required to achieve effective pest control. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Insects that feed on plant phloem sap are generally deleterious to their plant hosts by both direct damage (including chlorosis, necrosis, wilting, stunting and gall formation) and transmission of plant viruses and phytoplasmas (Dolling, 1991; Jones, 2014; Sugio and Hogenhout, 2012). All phloem-feeding insects are mem- bers of the order Hemiptera (Douglas, 2003), and many, including various species of aphids, whiteflies, psyllids, scale insects, leafhoppers, planthoppers and heteropteran bugs, are important crop pests (Edwards and Gatehouse, 2007). The increasing inci- dence of resistance to chemical insecticides (Bass et al., 2014; Whalon et al., 2010) is creating a strong demand for novel strate- gies to control these insect pests. This demand is compounded by the limited success of traditional plant breeding programs to generate durably resistant cultivars (Ragsdale et al., 2011; Alvarez et al., 2006; Merrill et al, 2014) and the dearth of naturally-occurring Bt toxins that are active against hemipterans, although recombinant Bts with greater toxicity are being devel- oped (Chougule et al., 2013; Porcar et al., 2009). In planta RNAi (i.e. plants engineered to synthesize active RNAi molecules) has great potential for the control of agricultural insect pests, including phloem-feeding hemipterans (Burand and Hunter, 2013; Scott et al., 2013; Xue et al., 2012). As first demonstrated for chewing insect pests, the western corn rootworm Diabrotica vir- gifera virgifera (Baum et al., 2007) and the cotton bollworm Helicoverpa zea (Mao et al., 2007), insects can suffer high mortality when feeding on plants producing RNAi constructs targeting essen- tial insect genes. Statistically significant effects of in planta RNAi on gene expression of hemipteran target genes have been demon- strated, for example for the potato/tomato psyllid Bactericera cock- erelli (Wuriyanghan and Falk, 2013), the planthopper Nilaparvata lugens (Zha et al., 2011), the aphid Myzus persicae (Elzinga et al., 2014; Pitino et al., 2011) and the whitefly Bemisia tabaci (Thakur et al., 2014). These studies have variously used virus-induced gene silencing (VIGS), transient Agrobacterium-mediated transformation http://dx.doi.org/10.1016/j.jinsphys.2015.06.006 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. E-mail addresses: vt223@cornell.edu (V. Tzin), xy322@cornell.edu (X. Yang), xj43@cornell.edu (X. Jing), kz49@cornell.edu (K. Zhang), gj32@cornell.edu (G. Jander), aes326@cornell.edu (A.E. Douglas). 1 These authors contributed equally to this work. Journal of Insect Physiology 79 (2015) 105–112 Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys