PSEUDACTEON DECAPITATING FLIES:POTENTIAL VECTORS OF A FIRE ANT VIRUS - PDF document

90(1)March 2007PSEUDACTEON DECAPITATING FLIES:POTENTIAL VECTORS OF A FIRE ANT VIRUS?Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS1600 SW 23rd Drive, Gainesville, Florida 32608, USAvirus (SINV-1) is a positive-stages of the red imported Þre ant, (Valles et al. 2004; Valles & Strong 2005).SINV-1 and a second genotype have been tenta-(Mayo2002). Infected individuals or colonies did not ex-hibit any immediate, discernible symptoms in theÞeld. However, under stress from introductioninto the laboratory, brood death was often ob-served among infected colonies, ultimately lead-ing to the death of the entire colony (Valles et al.2004). These characteristics are consistent withruses. They often persist as inapparent, asymp-tomatic infections that, under certain conditions,induce replication within the host, resulting in& Scotti 1998; Fernandez et al. 2002). The SINVinfection rate among colonies was reported to bearound 25% in Gainesville, Florida (Valles et al.2004; Valles & Strong 2005). SINV vertical andRT-PCR detection of virus genome in eggs andditions (Valles et al. 2004). However, the exactmechanisms by which the virus is spread fromAmerica (Porter 1998) and these ßies have beenreleased as a biological control in the U.S. Be-cause egg laying by female ßies is an intrusiveant host while consuming internal tissues, weparasitoids that oviposited or developed in SINV-nies. Laboratory and Þeld experiments were con-tectable levels of SINV.whether SINV was detectable in or oviposited in SINV-infected containing SINV-infected Þre ants were identiÞedfrom around Gainesville, Florida, by plunging 20collect a sample of workers. Total RNA was ex-tracted from 30 to 40 worker ants by the TRIZOLmethod (Invitrogen, Carlsbad, CA). cDNA wasOne-Step RT-PCR kit (Invitrogen) with oligonu-leotide primers p114 and p116 (SINV-1-speciÞc)and p117 and p118 (SINV-1A-speciÞc) (Valles &Strong 2005). Samples were positive for each vi-(646 nt for SINV-1 and 153 nt for SINV-1A) wasstained with ethidium bromide. Every RT-PCRreaction included a positive and negative control.or simplicity, positive infection by either virusgenotype was labeled as SINV-infected. RT-PCRas conducted in a PTC 100 thermal cycler (MJResearch, Waltham, MA) under the following op-timized temperature regime: 1 cycle at 45¡C for30 min, 1 cycle at 94¡C for 2 min, 35 cycles of 94¡Cfor 15 s, 54¡C for 15 s, 68¡C for 30 s, followed by aThree SINV-positive nests were excavatedthe soil by the ßoating method (Jouvenaz et al.1977). Ants from a SINV-infected nest were(Porter 1998). Samples ofevaluated for the presence of SINV-1 and SINV-1A. RNA was extracted from pooled groups of 3= 14 groups) workers and RT-PCRas conducted as described on each of theseSINV infection. After a suitable nest of ants wasidentiÞed and sieved, the appropriate sizedSINV-infected workers were presented to newly-eclosed into a ßy attack box (Vogt et al. 2003) containingnewly-eclosed ßies, respectively. Flies were allowed to ovipositin the ants for 1 d. A sample of ßies from eachspecies was collected about 2 h after being ex-ants. Parasitized worker ants were removedfrom the attack boxes and maintained as de-scribed by Vogt et al. (2003). Approximately 2weeks after oviposition, parasitized ants (exposure; RT-PCR. The remainder exhibited symptomsconsistent with parasitization, including twitch- ing/uncoordination, morbidity, and decapitation.to contain a parasite. Fly pupae were observeddaily for the next month. All ßies emerging fromSINV-1 or SINV-1A by RT-PCR.ßy populationsdetermine whether the virus was present inphorid ßies in their natural setting. mounds (10 nests at each site) were opened witha shovel in 3 areas around Gainesville, Florida,SINV infection in these areas was determined byRT-PCR within 2 weeks of sampling for ßies.ration while they attacked worker ants. The ßiesassayed for the presence of SINV. Flies werefor sex. RT-PCR was conducted on all of the collec-tions for the presence of SINV-1 and SINV-1A. ßfeeding upon SINV-infected ants do not acquirethe virus. SINV was not detectable by RT-PCR intheir larval development in SINV-infected Þre antworkers (Table 1). SINV infection of host ants didplete their development (Table 2). The rate ofranged from 71 to 93%. SINV was not detected inhigh a incidence of SINV infection. The lack of vi-play a broad host range.decapitating ßies, horizontal transmis-sion of SINV could occur mechanically from con-tamination of the ovipositor. This type of viruscoviruses in Lepidoptera (Stasiak et al. 2000).However, SINV infection of be limited to cells of the digestive tract, especiallythe midgut (Valles unpublished data). Further,we have not detected SINV in the hemolymph ofinfected Þre ants. Thus, it is unlikely that oviposi-the ovipositor. Indeed, ßies allowed to oviposit inSINV-infected Þre ants for several hours were all= 77; Table 1). However, me-hanical transmission still could have occurred,but was not detectable by the RT-PCR methodemployed; the limit of detection of SINV by RT-(Valles unpublished data).normally from SINV-infected workers.Mechanical transmission of SINV to uninfectedants by oviposition appears unlikely.1.EFly speciesDays after ovipositionDevelopment (groups)*SINV (+/-)Ovipositing 102Ñ34-46Emerging689ÑOvipositing674Ñ26-32Emerging708Ñ 2.RAnt workers parasitized byAnt conditionBefore oviposition217100arasitized391493 ±27Before oviposition21771 ±49arasitized17771 ±49 90(1)March 2007, P. D. 1998. Picornalike vi-ruses of insects, pp. 301-336 The Insect Viruses,Plenum Publishing Corporation, New York., J., I. Y2002. Regulation of internal ri- Biol. Chem. 277: 19198-19205., D. P., G. E. A1977. A survey for pathogens of Þre ants,spp., in the southeastern United States.Florida. Entomol. 60: 275-279., M. A. 2002. A summary of taxonomic changes re-cently approved by ICTV. 2002. Arch. Virol. 147:, S. D. 1998. Biology and behavior of decapitating ßies (Diptera: Phoridae) that par-ormicidae). Florida Entomol. 81: 292-309., K., M. V. D2000. Phylogenetic position of the mus pulchellusviruses with large double-stranded DNA genomes. J.Gen. Virol. 81: 3059-3072., S. M., C. A. S2004. A picorna-like virus fromthe red imported Þre ant, discovery, genome sequence, and characterization.irology 328: 151-157., S. M. 2005. virus-1A (SINV-1A): Distinct species or geno-type of SINV-1? J. Invertebr. Pathol. 88: 232-237., J. T., S. D. P2003. A modiÞed rearing system for pro-(Diptera: Phoridae),a parasitoid of imported Þre ants. Biol. Cont. 28: 346-