RNAi in the Eusocial Wasp Polistes Figure 1. Colony cycle of an annual Polistes paper wasp, exemplified by P. metricus. Foundress Phase. The annual cycle begins (arrow) when a female emerges from quiescence with undeveloped ovaries. Feeding at flowers enables ovary development, which precedes nest founding. Oviposition occurs immediately upon initiation of each nest cell by the now-maternal foundress (crown icon) that performs all maternal behaviors in addition to oviposition, including foraging, feeding larvae, and nest construction. Worker Phase. The first female offspring to emerge in early summer are workers (hard hat icon) that forage, feed larvae, and construct the expanding nest. The foundress transitions into a true social queen that limits her activities to oviposition and feeding larvae using foods brought to the nest by workers. Reproductive Phase. Female offspring emerging in mid- summer are non-working future foundresses called gynes (tiara icon). Males, not shown, emerge synchronously with gynes. Decline Phase. In late summer the queen and workers die. Gynes and males depart the nest, which now is empty and unattended. Quiescence Phase. Gynes and males mate and feed at fall flowers. With the decline in flower availability and onset of cooler weather, males die, and gynes enter a torpor-like quiescence in sheltered concealment. Workers live only from their emergence until the end of the reproductive phase. A gyne from the reproductive phase of one year has the potential to become a foundress and then queen the following year, thus the tiara to crown transition can represent single individuals that pass from gyne to foundress to queen. Larval treatments and sampling reported in this experiment were conducted in the Reproductive Phase of the colony cycle. doi:10.1371/journal.pone.0026641.g001 Reticulitermes flavipes [22], and by dsRNA microinjection in adult honey bees [23,24], the red imported fire ant Solenopsis invicta Buren [25], and the termite Cryptotermes secundus [26]. The majority of these studies were initiated in a laboratory situation. To develop RNAi for use in natural settings could advance the research utility of RNAi for learning mechanisms that underlie important features of social insects’ behavioral ecology. P. metricus may provide an ideal opportunity for this initiative. ESTs for P. metricus are now available, and paper wasps’ ecology with easily observed nests has made them the subject of extensive natural history studies [8] and a smaller number of experimental studies in captive rearing (e.g. [27]), laboratory settings (e.g. [28]), and natural environments (e.g. [29,30]). Adults feed nectar directly to larvae [8], and this can be mimicked by feeding nectar-like liquids to larvae, providing a vehicle for dsRNA. Specific larvae can be tracked through time by means of ‘nest maps’, enabling individual marking of newly-emerged adults. These adults can then be tracked on their nests in field situations or collected for subsequent analyses. Here, we develop RNAi for P. metricus for experimental applications in natural environments. This approach enables testing of causal relationships between candidate genes (genotype) and their association with behavior and/or caste ecology (phenotype). We exemplify this potential by exploring a central developmental question with significance in behavioral ecology: the role of specific storage proteins in developmental bias toward workers and gynes (future queens of the next generation). Storage proteins are ecologically important gene products that play a role in diapause not only in Polistes [31] but in a broad range of insects [32,33]. Polistes metricus has two putative hexameric storage proteins, Hexamerin 1 and Hexamerin 2. Of these, Hexamerin 1 levels in late-larval and early-pupal stages provide a significant predictor of caste bias in adults, whereas Hexamerin 2 does not [31], leaving the roles(s) of the latter protein obscure. Fragments of putatively Hexamerin-encoding transcripts are available as ESTs, but it has been unclear how these relate to the two largely uncharacterized Polistes proteins. We report successful cloning of the Hexamerin 2 encoding gene of P. metricus from its available ESTs, and proteomic analyses of the P. metricus Hexamerin 1 and Hexamerin 2 proteins. The proteins were identified as separate products with largely diverging peptide sequence and different biochemical properties. Subsequently, PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e26641