Development of an RNA Interference Tool, Characterization of Its Target, and an Ecological Test of Caste Differentiation in the Eusocial Wasp Polistes James H. Hunt1*., Navdeep S. Mutti2., Heli Havukainen3,4, Michael T. Henshaw5, Gro V. Amdam2,3 1 Departments of Biology and Entomology and W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America, 2 School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America, 3 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway, 4 Department of Biomedicine, University of Bergen, Bergen, Norway, 5 Department of Biology, Grand Valley State University, Allendale, Michigan, United States of America Abstract Recent advancements in genomics provide new tools for evolutionary ecological research. The paper wasp genus Polistes is a model for social insect evolution and behavioral ecology. We developed RNA interference (RNAi)-mediated gene silencing to explore proposed connections between expression of hexameric storage proteins and worker vs. gyne (potential future foundress) castes in naturally-founded colonies of P. metricus. We extended four fragments of putative hexamerin-encoding P. metricus transcripts acquired from a previous study and fully sequenced a gene that encodes Hexamerin 2, one of two proposed hexameric storage proteins of P. metricus. MALDI-TOF/TOF, LC-MSMS, deglycosylation, and detection of phosphorylation assays showed that the two putative hexamerins diverge in peptide sequence and biochemistry. We targeted the hexamerin 2 gene in 5th (last)-instar larvae by feeding RNAi-inducing double-stranded hexamerin 2 RNA directly to larvae in naturally-founded colonies in the field. Larval development and adult traits were not significantly altered in hexamerin 2 knockdowns, but there were suggestive trends toward increased developmental time and less developed ovaries, which are gyne characteristics. By demonstrating how data acquisition from 454/Roche pyrosequencing can be combined with biochemical and proteomics assays and how RNAi can be deployed successfully in field experiments on Polistes, our results pave the way for functional genomic research that can contribute significantly to learning the interactions of environment, development, and the roles they play in paper wasp evolution and behavioral ecology. Citation: Hunt JH, Mutti NS, Havukainen H, Henshaw MT, Amdam GV (2011) Development of an RNA Interference Tool, Characterization of Its Target, and an Ecological Test of Caste Differentiation in the Eusocial Wasp Polistes. PLoS ONE 6(11): e26641. doi:10.1371/journal.pone.0026641 Editor: Peter K. Dearden, University of Otago, New Zealand Received June 3, 2011; Accepted September 30, 2011; Published November 1, 2011 Copyright: 2011 Hunt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research at NCSU was supported by NSF grant #0819807 to JHH and C.M. Grozinger. PROBE received support from the National Program for Research in Functional Genomics, which is funded by the Research Council of Norway (RCN). GVA was supported by RCN 180504, 185306, and 191699 and the PEW Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jim_hunt@ncsu.edu . These authors contributed equally to this work. Introduction Experimental studies conducted in natural environments are important for understanding ecological patterns and processes [1]. Experimental studies conducted on laboratory model organisms are important for understanding the selective basis of evolution [2]. Polistes paper wasps offer an opportunity to pursue a third realm of experimental studies now in its infancy – the implementation of genomics-based methods to experimentally study evolutionary mechanisms in natural environments. The paper wasp genus Polistes has long been an important taxon for field studies of its ecology [3] and behavior [4,5,6]. It is an especially important taxon for understanding the evolution of insect sociality [7,8] and as a model system in behavioral ecology [9,10]. The paper wasp Polistes metricus Say, distributed in the Midwestern and Southeastern U. S., has for several decades been the specific focus of diverse studies on aspects of Polistes biology. Early work includes physiology [11], inclusive fitness [12], behavior [13], and sex ratio [14]. To extend and expand these and other research areas at both the species and genus levels, P. metricus was the first Polistes species subjected to analysis of its expressed sequence tags (ESTs – its expressed mRNA) [15], which then were annotated by reference to the genome of a related species, the honey bee Apis mellifera [16]. Using this EST resource, Toth et al. [15] documented differences in gene expression among the four categories of adult females that occur in an annual colony cycle of P. metricus (Fig. 1). Similarly, Hunt et al. [17] used the EST resource to document differences in gene expression between larvae destined to become workers and larvae destined to become gynes (pre-queens). These studies were ‘natural experiments’ that were based on collection of specimens from natural environments at particular points in the colony cycle, but they were not manipulative experiments designed to test causal connections. The manipulative experimental approach of RNA interference (RNAi)-mediated gene targeting occurs post-transcriptionally and involves specific degradation of target mRNA. RNAi is triggered by introduction of double stranded RNA (dsRNA) and can be a near-universal method of gene silencing [18,19]. Among social insects, RNAi has been reported for dsRNA feeding in larval honey bees A. mellifera [20,21] and nymphs of the termite PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26641