Journal of INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 96 (2007) 18–27 www.elsevier.com/locate/yjipa Phenology, distribution, and host specificity of Solenopsis invicta virus-1 Steven M. Valles a,*, Charles A. Strong a, David H. Oi a, Sanford D. Porter a, Roberto M. Pereira a, Robert K. Vander Meer a, Yoshifumi Hashimoto a, Linda M. Hooper-Bu`i b, Hussein Sa´nchez-Arroyo c, Tim Davis d, Vedham Karpakakunjaram e, Karen M. Vail f, L.C. ‘‘Fudd’’ Graham g, Juan A. Briano h, Luis A. Calcaterra h, Lawrence E. Gilbert i, Rufina Ward j Kenneth Ward j, Jason B. Oliver k, Glenn Taniguchi l, David C. Thompson m a Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608, USA b Department of Entomology, 404 Life Science Building, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA c Colegio de Postgraduados, Instituto de Fitosanidad, km 36.5 Carr. Los Reyes-Texcoco, Montecillo, Texcoco, Edo. de Mexico 56230, Mexico d Sandhills Research and Education Center, 900 Clemson Road, Columbia, SC 29224-3205, USA e Division of Agricultural Sciences and Natural Resources, Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA f Entomology and Plant Pathology, 2431 Joe Johnson Drive, 205 Plant Science Building, University of Tennessee, Knoxville, TN 37996, USA g Alabama Fire Ant Management Program/Pesticide Safety Education Program, 301 Funchess Hall, Auburn University, AL 36849, USA h USDA-ARS South American Biological Control Laboratory, Bolivar 1559, Hurlingham, Buenos Aires Province, Argentina i School of Biological Sciences, Section of Integrative Biology, University of Texas, Austin, TX 78712, USA j Department of Plant and Soil Science, Alabama A&M University, Normal, AL35762, USA k Tennessee State University, Institute for Agricultural and Environmental Research, TSU Otis L. Floyd Nursery Research Center, 472 Cadillac Lane, McMinnville, TN 37110, USA l Department of Plant and Environmental Protection Sciences, University of Hawaii, 3190 Maile Way Honolulu, HI 96822, USA m New Mexico State University, MSC 3BE, Box 30003, Skeen Hall #N220, Las Cruces, NM 88003, USA Received 20 October 2006; accepted 5 February 2007 Available online 25 February 2007 Abstract Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p < 0.0005, r = 0.82) and SINV-1A (p < 0.0001, r = 0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV- 1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Flor- ida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV- 1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S. Published by Elsevier Inc. * Corresponding author. Fax: +1 352 374 5818. E-mail address: [email protected]fl.edu (S.M. Valles). 0022-2011/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.jip.2007.02.006 S.M. Valles et al. / Journal of Invertebrate Pathology 96 (2007) 18–27 19 Keywords: Solenopsis invicta; Dicistroviridae; RNA virus; Host specificity 1. Introduction 2. Materials and methods Escape from natural enemies during the founding bot- 2.1. Phenology tleneck is considered the most likely reason why the red imported fire ant, Solenopsis invicta Buren, has become a During initial experiments and genome sequencing, we major pest in the United States (Porter et al., 1997). Com- noticed that the prevalence of the SINV-1 infection among parative ecological studies among populations in native S. invicta collected from Florida seemed to change during (Argentina) and introduced (United States) regions have the course of the year. Because of these observations and revealed that red imported fire ant populations are greater previous reports of seasonal variation of related single- in number, have higher mound densities, possess larger stranded, RNA viruses in other Hymenopteran insects mound volumes and comprise a larger fraction of the ant (Bailey, 1967; Berenyi et al., 2006), we conducted a study community in the U.S. (Porter et al., 1992, 1997). There- to formally monitor the SINV-1 infection rate in S. invicta fore, it is not surprising that discovery, characterization, during the course of a year in Gainesville, Florida. Two and utilization of fire ant pathogens and parasites are roadside sites were chosen. The first site was located on among the goals of U.S. fire ant researchers to achieve State Route 441, north of Paynes Prairie Nature Preserve self-sustaining control of this aggressive, territorial ant (N29° 35.2420; W82° 20.3300) and the second site at the pest. crossroads of State Route 26 and East University Avenue Several years ago, the Center for Medical, Agricul- (N29° 39.1080; W82° 15.6500). From December 2004 to tural and Veterinary Entomology, USDA-ARS, began December 2005, 10 fire ant colonies per site were sampled to search for viruses infecting S. invicta. In pursuit of monthly and evaluated by reverse transcriptase-polymerase this goal, this laboratory created an expressed sequence chain reaction (RT-PCR) for the presence of SINV-1. Ant tag (EST) library from S. invicta. Bioinformatics analyses collection was accomplished by plunging a scintillation vial of approximately 2000 ESTs revealed several sequences (20 ml) into a nest for several minutes allowing fire ants to exhibiting strong homology to RNA viruses. Further fall into the vial. Ant samples were taken to the laboratory investigation resulted in the discovery, characterization and evaluated for SINV-1 infection. Total RNA was and complete genome sequence of a virus that has been extracted from 20 to 30 worker ants by the Trizol method assigned to the Dicistroviridae (Mayo, 2002) and named according to the manufacturer’s instructions (Invitrogen, Solenopsis invicta virus-1 [SINV-1] (Valles et al., 2004; CA). Valles and Strong, 2005). SINV-1 is a positive-strand Two genotypes have been reported to infect S. invicta, RNA virus found in all stages of S. invicta. Its RNA SINV-1 (Valles et al., 2004) and SINV-1A (Valles and genome is single-stranded, monopartite and comprised Strong, 2005). Therefore, cDNA was synthesized and sub- of 8026 nucleotides encoding two, non-overlapping open sequently amplified using the One-Step RT-PCR kit (Invit- reading frames (ORFs). Genomic comparisons with other rogen) with genotype specific oligonucleotide primers (p117 known dicistroviruses revealed that SINV-1 encodes 6 and p118, SINV-1A-specific and p114 and p116, SINV-1- copies of the 50, covalently-bound, genome-linked pro- specific) (Valles and Strong, 2005). Samples were consid- tein, Vpg (Nakashima and Shibuya, 2006), and a type ered positive for each virus when a visible amplicon of II internal ribosomal entry site (Jan, 2006). These charac- anticipated size (646 nt for SINV-1 and 153 nt for SINV- teristics are hypothesized to facilitate efficient SINV-1 1A) was present after separation on a 1.2% agarose gel multiplication in host cells. Although SINV-1-infected stained with ethidium bromide. RT-PCR was conducted fire ants or colonies did not exhibit any immediate, dis- in a thermal cycler (PTC 100, MJ Research, Waltham, cernible symptoms in the field, brood die-off was often MA) under the following optimized temperature regime: observed when infected colonies were reared under lab- 1 cycle at 45 °C for 30 min, 1 cycle at 94 °C for 2 min, 35 oratory conditions (Valles et al., 2004). These charac- cycles of 94 °C for 15 s, 54 °C for 15 s, 68 °C for 30 s, fol- teristics are consistent with other insect-infecting lowed by a final elongation step of 68 °C for 5 min. Two positive-strand RNA viruses; they frequently persist as controls were included in all assays, one with RNA from unapparent, asymptomatic infections that, under certain infected ants (positive control) and one devoid of RNA conditions, induce replication within the host resulting (non-template or negative control). in observable symptoms and often death (Christian The percentage of colonies infected with SINV-1 (geno- and Scotti, 1998). In an effort to learn more about types-1 and -1A) for each month was correlated with the the biology of SINV-1, we conducted experiments average monthly temperature and rainfall using Pearson’s to examine its phenology, distribution and host correlation analysis (SAS Correlation Procedure [Cary, specificity. NC]). Meteorological data were obtained for the Gaines- 20 S.M. Valles et al. / Journal of Invertebrate Pathology 96 (2007) 18–27 ville, FL, airport at the National Weather Service Forecast Gainesville and Williston, FL, 15 pitfall traps were buried Office website: www.srh.noaa.gov/jax/climate.shtml.