PBAN gene architecture and expression in the fire ant, Solenopsis invicta

Abstract

The PBAN/pyrokinin peptides are a major neuropeptide family characterized by a common FXPRLamide at the C-termini. These peptides are distributed ubiquitously in the Insecta and are involved in many essential endocrine functions, e.g. pheromone production. We report the gene architecture of the fire ant Solenopsis invicta PBAN (Soi-PBAN) gene, including the exon and intron boundaries. Furthermore, we quantified expression of the Soi-PBAN mRNA in the head, thorax and abdomen of the fire ant. The Soi-PBAN gene is comprised three exons and two introns, all composed of 13,358 nucleotides, which is 2–4 times larger than lepidopteran PBAN genes. The overall pattern of the PBAN immunoreactive neuron number and localization was similar for female and male alates, inseminated female delates, workers and queens. The Soi-PBAN mRNA expression level was highest in the head, followed by the thorax, and abdomen of adult ants. Expression in the abdominal tissues was expected to be similar to the head, or at least higher than thorax because strong PBAN immunoreactive neurons were detected previously in brain–subesophageal and abdominal ganglia. This result suggests that another FXPRL gene could be dominant in the abdomen rather than Soi-PBAN gene.

Introduction

Insect neurohormones function as intercellular communicators that regulate a variety of physiological and behavioral events during development and reproduction. One major class of neuropeptide hormones is the pheromone biosynthesis activating neuropeptide (PBAN)/pyrokinin peptide family, which is defined by a common FXPRLamide or similar amino acid fragment at the C-terminal end. The PBAN/pyrokinin family of peptides is produced in the central nervous system (CNS) of insects, with homologous peptides and receptors in vertebrates. These peptides have been conserved throughout evolution, because they are expected to be found in all insects (reviewed by Rafaeli, 2009), in Crustacea (Torfs et al., 2001), as well as in Caenorhabditis elegans (Lindemans et al., 2009). PBAN has been reported to regulate pheromone biosynthesis in a variety of moths. Pyrokinins are peptides that activate visceral muscle contraction of mid- and/or hind-guts in a variety of insects. In addition, the family of peptides containing the FXPRL sequence in the C-terminus have other physiological roles in insects, including egg and pupal diapause, cuticle melanization, and puparium formation (reviewed by Rafaeli, 2009).

Two decades ago, the first PBAN molecule identified was a 33-amino acid peptide from Helicoverpa zea, Hez-PBAN (Helicoverpa zea PBAN) (Raina et al., 1989). Subsequently PBAN amino acid sequences were determined through direct isolation and purification of peptides, or DNA cloning methods (Choi et al., 2010, Rafaeli, 2009). The results yielded additional PBAN encoding genes from 18 lepidopteran moths and several non-lepidopteran insects (summarized by Choi et al., 2010). In addition to PBAN, a diapause hormone and three additional FXPR/KL neuropeptides (NPs: α, β, γ) were deduced from the same gene and are well conserved in moths. The neurohormonal action of PBAN for pheromone biosynthesis in lepidopteran moths has been well studied (Rafaeli, 2009, Rafaeli and Jurenka, 2003), but the physiological functions of PBAN peptides in the other species are unknown. PBAN is synthesized in the subesophageal ganglion (SG) and is released into the hemolymph via the corpora cardiaca, a neurohemal organ, in moth species.

The red imported fire ant, S. invicta, is among world's 100 worst invasive alien species (Lowe et al., 2000). The affected economic sectors are broad ranging and include households, electric service and communications, agriculture, schools and recreation areas (Lard et al., 2006). Workers (sterile females) may number 250,000 in mature colonies, female alate sexuals (winged female potential queens) and male alate sexuals (winged males) represent the reproductive offspring of the colony queen(s). The fire ant is probably the most studied invasive ant species in the world and a great deal is known about the pheromone systems used to reduce reproductive competition, recruit resources, and maintain colony social structure and territoriality (Vander Meer and Alonso, 1998, Vander Meer and Alonso, 2002, Vargo, 1998). In spite of decades of study on fire ant pheromones, virtually nothing is known about how pheromone production and release are regulated, nor whether protein hormones, especially neuropeptides, are involved in key physiological and endocrinal processes during development. Recently, we demonstrated the presence of PBAN/pyrokinin immunoreactive neurons in the central nervous system of the fire ant, S. invicta, and identified and characterized PBAN and three additional F/PXPRL peptides from the Soi-PBAN cDNA (Choi et al., 2009, Choi and Vander Meer, 2009). In the present study we have mapped the Soi-PBAN gene structure and demonstrated similar PBAN immunoreactive neuron patterns from the different sexual forms, including female and male alates, workers and queens. We determined the tissue expression level of the Soi-PBAN mRNA using RT- and Q-PCRs from the head, thorax, and abdomen. We also discovered the PBAN gene structure and immunoreactivity in the different sexual forms, and discuss the possibility of different genes encoding PBAN/pyrokinin family like peptides in the central nervous system of the fire ant.