GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant, Solenopsis invicta Walter S. Leal*, Yuko Ishida Honorary Maeda-Duffey Laboratory, Department of Entomology, University of California Davis, Davis, California, United States of America Abstract The red imported fire ant (RIFA), Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla. Citation: Leal WS, Ishida Y (2008) GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant, Solenopsis invicta. PLoS ONE 3(11): e3762. doi:10.1371/journal.pone.0003762 Editor: Hany A. El-Shemy, Cairo University, Egypt Received September 23, 2008; Accepted October 30, 2008; Published November 19, 2008 Copyright: ß 2008 Leal 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: This work was supported in part by the National Institutes of Health (1U01AI05826-01), a research agreement with Bedoukian Research Inc., the National Research Initiative of the U.S. Department of Agriculture (USDA-CSREES 2003-35302-13648) and National Science Foundation (0234769). 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: wsleal@ucdavis.edu Introduction The red imported fire ant (RIFA), Solenopsis invicta Buren (synonym of S. wagneri Santschi), is an invasive species native to South America, which was accidentally introduced in the United States through the port of Mobile, AL, sometime between 1933 and 1945 [1], probably even earlier (http://www.invasivespeciesinfo. gov/animals/rifa.shtml). RIFA is now widespread in the South- eastern United States and has been introduced into Southern California (http://www.aphis.usda.gov/plant_health/plant_pest_ info/fireants/downloads/fireant.pdf). This fire ant has two social forms. Monogyne colonies are those with a single functional queen, which may start by a single or few queens but only one survives the battle after the first workers appear. Polygyne colonies, on the other hand, have at least two functional queens and sometimes as many as twenty thousand. While polygyne societies are known to accept foreign queens of the monogyne- and polygyne-type, monogyne colonies may accept polygyne queens, but kill encroaching monogyne-type queens [2]. This colony social organization has been associated with a gene (Gp-9), which has two electrophoret- ically detectable alleles in introduced populations in the USA, with the B allele being the only allele found in the monogyne form, and the b allele occurring together with the B allele in the polygyne form [3]. Krieger and Ross [4] isolated GP-9s from the thoraces of monogyne and polygyne queens, cloned the genes encoding GP-9B and GP-9b, and based on modest amino acid sequence assigned them as putative pheromone-binding proteins (PBPs), which they suggested regulate social behavior in this fire ant. While polygyne expresses both GP-9B and GP-9b, monogyne queens expressed only GP-9B [4]. On the other hand, Fletcher and Blum [5] and Vander Meer and Alonso [6] elegantly demonstrated that worker aggression is mediated by a queen-produced recognition primer pheromone. While introduction of newly-mated queen intruders into queenright monogyne colony residents resulted in immediate investigation and eventual alarm and attack, queenless workers often aggregated around and walked over intruders [6]. The apparent conundrum of monogyne workers devoid of GP-9b perceiving a queen-produced recognition primer pheromone (with the implication that polygyne workers expressing GP-9B and GP-9b are anosmic to this pheromone) prompted us to revisit the putative role of GP-9s. Here, we report (1) that polygyne workers express one or two forms of GP-9s, which are demonstrated to be general hemolymph proteins unlikely involved in olfaction, and (2) on the identification of a chemosensory protein specifically expressed in olfactory tissues PLoS ONE | www.plosone.org 1 November 2008 | Volume 3 | Issue 11 | e3762