Journal of Insect Physiology 58 (2012) 1159–1165 Contents lists available at SciVerse ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea ⇑ ⇑ Man-Yeon Choi a, , Robert K. Vander Meer a, , Monique Coy b, Michael E. Scharf b,c a U. S. Department of Agriculture, Agricultural Research Service, Center of Medical, Agricultural and Veterinary Entomology (CMAVE), 1600 SW, 23rd Drive, Gainesville, FL 32608, USA b Department of Entomology and Nematology, University of Florida, Gainesville, FL 32608, USA c Department of Entomology, Purdue University, West Lafayette, IN 47907, USA article info abstract Article history: Insect neuropeptide hormones represent more than 90% of all insect hormones. The PBAN/pyrokinin fam- Received 30 January 2012 ily is a major group of insect neuropeptides, and they are expected to be found from all insect groups. Received in revised form 1 June 2012 These species-specific neuropeptides have been shown to have a variety of functions from embryo to Accepted 5 June 2012 adult. PBAN is well understood in moth species relative to sex pheromone biosynthesis, but other poten- Available online 13 June 2012 tial functions are yet to be determined. Recently, we focused on defining the PBAN gene and peptides in fire ants in preparation for an investigation of their function(s). RNA interference (RNAi) technology is a Keywords: convenient tool to investigate unknown physiological functions in insects, and it is now an emerging PBAN method for development of novel biologically-based control agents as alternatives to insecticides. This Neuropeptide RNAi could be a paradigm shift that will avoid many problems associated with conventional chemical insecti- dsRNA cides. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target from two Fire ant insect groups; a social insect, the fire ant (Solenopsis invicta) and a non-social insect, the corn earworm Hymenoptera (Helicoverpa zea). Both insects are economically important pests. We report negative impacts after PBAN Corn earworm dsRNA treatment to suppress PBAN gene transcription during developmental and adult stages of both Lepidoptera species, e.g. increased adult and larval mortality, delayed pupal development and decreased sex phero- Pest control mone production in the moth. This is an important first step in determining the multiple functions of the PBAN gene in these two insects. This work illustrates the variety of phenotypic effects observed after RNAi silencing of the PBAN gene and suggests the possibility of novel biologically-based insect pest con- trol methods. Published by Elsevier Ltd. 1. Introduction moths (Raina et al., 1989); (2) induction of melanization in moth larvae (Matsumoto et al., 1990; Raina et al., 2003); (3) induction Insect neuropeptides are the largest group of insect hormones. of embryonic diapause in Bombyx mori (Suwan et al., 1994; Uehara A variety of peptide families have been identified from insects et al., 2011); (4) stimulation of visceral muscle contraction in cock- (Gäde, 1997). One of these families is the Pheromone Biosynthesis roaches (Predel and Nachman, 2001); (5) acceleration of puparium Activating Neuropeptide (PBAN)/pyrokinin family defined by a formation in the flesh fly (Zdarek et al., 1997); and (6) termination conserved C-terminal pentapeptide (e.g: FXPRLamide) that is the of development of pupal diapause in heliothine moths (Xu and active core fragment for peptide function (Kuniyoshi et al., 1992; Denlinger, 2003). Raina and Kempe, 1992). During the past two decades, PBAN has In moth species, PBAN is synthesized in the subesophageal gan- been one of the most intensively studied insect neuropeptide, be- glion (SG), released into the hemolymph, and acts on sex phero- cause it regulates sex pheromone biosynthesis in many lepidopter- mone production in the pheromone gland. PBAN plus four or an moths. PBAN was first identified from Helicoverpa zea adults three additional peptides are encoded by the PBAN/pyrokinin gene. (Raina et al., 1989), and now, the PBAN/pyrokinin peptide family These additional peptides also share a common C-terminal has been identified from many insects and other arthropods. The FXPRLamide motif and have been named diapause hormone studies have shown a broad range of physiological functions, for (DH), neuropeptide (NP)–a,–b, and –c homologues (Choi et al., example: (1) stimulation of sex pheromone biosynthesis in female 2011). PBAN mRNA was expressed at similar levels in both female and male adult moths, although it is unclear if male moths produce pheromones or if these peptides elicit other functions (Bober and ⇑ Corresponding authors. Rafaeli, 2010; Choi et al., 1998; Jing et al., 2007; Lee and Boo, E-mail addresses: [email protected] (M.-Y. Choi), bob.vandermeer@ars. 2005; Wei et al., 2008, 2004). The physiological mechanism of usda.gov (R.K. Vander Meer). 0022-1910/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jinsphys.2012.06.005 1160 M.-Y. Choi et al. / Journal of Insect Physiology 58 (2012) 1159–1165 PBAN control over pheromone production is well understood for pupae or virgin adults were used for dsRNA injection or sex pher- sex pheromone biosynthesis in a number of lepidopteran moths omone analysis. (Rafaeli, 2009; Rafaeli and Jurenka, 2003). However, thus far no other insect group has been shown to regulate pheromone biosyn- 2.3. Cloning of Soi-PBAN gene and construction of dsRNA thesis using PBAN, although PBAN/pyrokinin peptides have been found in all insect groups thus far investigated. We recently iden- Br-SGs were dissected from S. invicta female alates to isolate tified and characterized the PBAN mRNA encoding four FXPRLa- mRNA using the MicroFast mRNA purification kit (Invitrogen, mide peptides in the fire ant, Solenopsis invicta and sibling Carlsbad, CA, USA), and used to synthesize cDNA with the GeneR- invasive ants (Choi et al., 2011, 2009; Choi and Vander Meer, 2009). acer cDNA synthesis kit (Invitrogen). The detailed cloning proce- There are 17 land invertebrates listed among the world’s 100 dure was described previously (Choi and Vander Meer, 2009). worst invasive alien species (Lowe et al., 2000). Ants represent The S. invicta PBAN (Soi-PBAN) dsRNA was synthesized from the 28% of these invertebrates, including fire ants. In the United States, full length Soi-PBAN cDNA (531-bp) using specific 50-T7-appended imported fire ants infest more than 130 million hectares in 13 PCR primers (50-TAATACGACTCACTATAGGGACCGTCGACAACCGAC southern tier states and Puerto Rico and are continuing their TTAC-30 and 50-TAATACGACTCACTATAGGGGACTCTCAAGAGGTGG spread (USDA-APHIS, 2009). The fire ant is estimated to be respon- TGGC-30) to amplify a 496-bp Soi-PBAN DNA fragment. This frag- sible for over $6 billion annually in damage repair, medical care, ment served as the template for dsRNA synthesis using the MEGA- and control costs. The affected economic sectors are broad-ranging script RNA kit (Ambion, Austin, TX, USA). Green fluorescence and include households, agriculture, and electric and communica- protein (GFP) dsRNA served as the negative dsRNA control and tions (Lard et al., 2006). Current control methods rely on chemical was either purchased (Ambion) or was synthesized from a 546- insecticides in the form of toxic baits or drenches. There is a need bp GFP DNA template amplified by 50-T7-appended primers, 50-TA for novel biologically-based control alternatives that are more spe- ATACGACTCACTATAGGGACGTAAACGGCCACAAGTTC-30 and 50-TAA cies-specific and have less impact on the environment. TACGA CTCACTATAGGGTGCTCAGGTAGTGGTTGTCG-30, using the Helicoverpa spp. are major insect pests of many economically same kit as above. important crops including corn, cotton, soybean, green and hot peppers, tomatoes, and potatoes throughout the world. The corn 2.4. Cloning of the Hez-PBAN gene and construction of dsRNA earworm, H. zea, is distributed in the New World, and is a well known moth species for the study of basic physiology and chemical Br-SGs of 1–3- day old H. zea female moths were dissected to ecology. Currently, control methods for Helicoverpa spp. rely on isolate mRNA for synthesis of cDNA using Invitrogen MicroFast chemical insecticides and/or Bt transgenic plants, but control is mRNA purification kits and GeneRacer cDNA synthesis kits to clone increasingly more difficult due to insecticide resistance and non- the full length H. zea PBAN (Hez-PBAN) cDNA. The primer set: target toxicity. sense primer (50-AAGATGTTCAATCAAACTCAGTTG-30) and anti- RNA interference (RNAi) technology is an emerging method in sense primer (50-AAATTATGTTGATCGCGTTTTGTTTGT-30), was de- insect pest management because it has high potential to provide signed from the sequence deposited in GenBank (Accession num- novel insect control methods for crop and other insect pests (Aso- ber: U08109). PCR was performed with the following kan, 2008; Huvenne and Smagghe, 2010). Although there are many temperature program: 33 cycles at 95 °C for 30 s, 52 °C for 30 s, technical and target gene selection challenges, RNAi for insect con- and 72 °C for 1 min. The PCR product was gel purified and cloned trol represents a potentially new direction for insect pest manage- using TOPO TA cloning kit (Invitrogen) and sequenced. The ment (Scharf et al., 2008). obtained full-length sequence information was aligned and The multifunctional roles already shown for PBAN/pyrokinin sequences compared with our partial sequence. To construct peptides translated by the PBAN mRNA provides an interesting Hez-PBAN dsRNA, a PCR primer set was designed, 50-T7-appended: RNAi target for both basic function studies and to assess potential 50-TAATACGACTCACTATAGGGGTGTTTGCATTGTGTACCGC-30, and for RNAi as a novel control method. We selected the PBAN gene for 50-TAATACGACTCACTATAGGGTATAGGAAGGGGTTGATGGC-30,to RNAi from two insect groups, the fire ant, S. invicta (social insect) amplify a 508-bp DNA fragment, which served as the template and the corn earworm, H.