sign in sign up
<<
Home , Hippoboscidae, Lutzomyia, Lutzomyia shannoni

Journal of the American Mosquito Control Association, 24(3):444–447, 2008 Copyright E 2008 by The American Mosquito Control Association, Inc.

PATHOGEN SCREENING AND BIONOMICS OF APACHE (DIPTERA: ) IN WYOMING, USA

WILL K. REEVES,1 CECILIA Y. KATO1 AND TRAVIS GILCHRIEST2

ABSTRACT. Phlebotomine sand are vectors of bacteria, parasites, and . Lutzomyia apache, a North American sand , was incriminated as a of vesicular stomatitis viruses (VSV) due to overlapping ranges of the sand fly and recent outbreaks of VSV. We report on the discovery of 2 populations of L. apache in Wyoming from Albany and Fremont counties. We attempted to isolate VSV and phleboviruses from sand flies from Albany County and screened select flies by polymerase chain reaction for Bartonella and blood meals. We did not isolate viruses or detect DNA from hosts or Bartonella. Flies were also tested for pathogens and other microbes. We detectedaRickettsia sp. in all flies that were examined and a parasitic protozoon, Ascogregarina sp., from the midgut of a . Eustigmaeus lirella, a stigmaeid mite, parasitized 2 field-caught females.

KEY WORDS Lutzomyia, Wyoming, vesicular stomatitis , Rickettsia, Stigmaeidae, Ascogregarina

INTRODUCTION 1984, Alsuhaibani 1990, Schmidtmann et al. 2002), but geographic information system–based Phlebotomine sand flies are vectors of bacteria, predictive models extended its range to include parasites, and viruses. In the , portions of California, Idaho, Nevada, Texas, Lutzomyia spp. were incriminated as the vectors Utah, and Wyoming (Herrero et al. 2004). These and reservoirs of vesicular stomatitis viruses models were not evaluated by field collections. (VSV) (Comer et al. 1990). The goal of our study was to determine if the Dyar was a vector of VSV on Ossabaw Island, population of L. apache predicted by Herrero et Georgia (Comer et al. 1990). In the laboratory, L. al. (2004) existed in Wyoming. We also wanted to shannoni was shown to transmit VSV by both bite determine if L. apache was naturally infected with and by transovarial (Comer et al. viruses or pathogens that might be used in 1990). In the western USA, a sand fly, Lutzomyia biological control. (Helcocyrtomyia) apache Young and Perkins, was initially associated with VSV when sand flies were trapped at sites of a VSV outbreak (Schmidtmann MATERIALS AND METHODS et al. 2002). Later work indicated that the range Herrero et al. (2004) predicted a population of of L. apache overlapped with epizootics of VSV L. apache near Sybille Canyon, WY. Updraft (Herrero et al. 2004). In addition to VSV, light traps (Clarke Mosquito Control, Roselle, Lutzomyia spp. are the primary vectors of 26 of IL) were set out in Sybille Canyon, Albany the known 38 serotypes of phleboviruses, some of County, WY, for sand flies. Individual traps were which cause fevers in humans (Tesh placed on the ground near and lagomorph 1989). Rio Grande virus is the only known burrows or in areas that had rodent or lago- phlebovirus in the USA, and serve as morph activity approximately 1 h before sunset the vertebrate of this virus (McLean et al. and retrieved the following morning. 1982). Neotropical of Lutzomyia transmit caught in the traps were anesthetized with carbon abacterium, Strong, dioxide and sorted from the traps on a chill table. Tyzzer, and Sellards, which causes Sand flies were sorted into 3 groups: gravid in humans (Caceres 1993). Lutzomyia apache is in females, females with blood meals, and all other the subgenus Helcocyrtomyia, which contains flies. During sorting, all adult flies were examined many reptile feeders. Some species of Helcocyrto- for ectoparasites. In addition to material from myia are anthropophilic and 3 of the species in Sybille Canyon, we examined material from the Neotropics were incriminated as vectors of Centers for Disease Control and Prevention (Young and Duncan 1994). (CDC) suction traps for additional Wyoming Lutzomyia apache was collected in Arizona, records. Representative adult sand flies were Colorado, and New Mexico (Young and Perkins cleared in 80% lactic acid and slide mounted in Euperal or Hoyer’s media and identified with the keys by Young and Perkins (1984). 1 Agricultural Research Service, -Borne Animal Diseases Research Laboratory, College of Gravid females of L. apache were held over- Agriculture, Department 3354, 1000 E University night in sealed tubes with moist paper towels for Avenue, Laramie, WY 82071. oviposition. Eggs on the paper towels were placed 2 University of Wyoming, Department of Renewable in rearing chambers similar to those described by Resources, Laramie, WY 82071. Modi and Rowton (1999). Larvae were reared

444 SEPTEMBER 2008 LUTZOMYIA APACHE IN WYOMING 445 following the protocols described by Modi and passed onto fresh Vero cells without SAM. Cells Rowton (1999), with modifications to use aged were passed 3 times and examined daily for CPE. goat dung instead of rabbit dung. Larvae did not Three 2nd instars were carefully dissected in feed on aged rabbit dung in our laboratory but distilled water on a microscope slide to remove ate aged goat dung. their midguts and hindguts. The peritrophic We extracted DNA from 38 engorged females matrix was gently teased from the midgut with with a DNeasy Blood & Tissue Kit (Qiagen, a pin. The guts and peritrophic matrix were Valencia, CA) and resuspended it in nuclease-free examined by light microscopy at 4003 for water. The DNA extracts were screened for DNA parasites such as gregarines or trichomycetes. from vertebrate hosts, Rickettsiaceae, and Bar- Voucher specimens of male and female flies tonella by polymerase chain reaction (PCR). We were deposited in the University of Wyoming screened DNA from the vertebrate blood meals, Insect Museum. Both the 1,281-bp and 329-bp using the primers described by Malmqvist et al. sequences from the 16S rRNA and gltA genes (2004) for detecting blood meals in black flies. We from a Rickettsia sp. detected in L. apache were screened for DNA from Rickettsiaceae, using the deposited in GenBank (Accession # EU223247, EC12A and HE3 primers (Reeves et al. 2006) and EU368001). for Bartonella with the QHEV1 and QHEV4 primers (Houpikian and Raoult 2001). Rickettsi- RESULTS aceae were further characterized by amplifying ,1,300 base pairs (bp) of the 16S rRNA and Both male and female L. apache were trapped ,400 bp of the citrate synthase (gltA) gene using (4–50/night) in Sybille Canyon, Albany County, primers described by Reeves (2005) and Norman WY, from August 7–31, 2007. Flies were most et al. (1995). All PCR products were separated by active on nights without precipitation and with electrophoresis on 2% agarose gels and visualized temperatures above freezing. No flies were under ultraviolet light with GelStar (Cambrex trapped in September. In addition to the flies in BioScience Rockland, Inc., Rockland, ME). Sybille Canyon, we trapped a female and 2 male Products were purified with a QIAquick PCR L. apache in a CDC light trap used for mosquito Purification Kit (Qiagen). Sequencing reactions collections. The trap from July 20, 2007, was set were performed with a BigDye Terminator v3.1 approximately 5 km north of Lander on the Cycle Sequencing Kit (Applied Biosystems, Fos- Wind River Reservation side of the Popo Agie ter City, CA) using PCR primers, and excess dye River, Fremont County, WY. It was baited with was removed by ethanol precipitation. Sequences dry ice but operated without a light. were determined using an ABI 3700 capillary We did not isolate VSV from any sand flies, but sequencer (Applied Biosystems), aligned and local transmission in Wyoming had not been assembled with Chromas Lite 2.01 (Technely- reported in over a year. A 1,281-bp sequence of sium, Tewantin, Australia) and ClustalW (Kyoto the 16S rRNA gene from a novel Rickettsia sp. was University Bioinformatics Center, Kyoto, Japan), amplified by PCR from 38/38 (100%) sand flies. and compared to sequences in GenBank using the Ectoparasitic mites were discovered on 2 BLAST 2.0 program (National Center for Bio- female flies from August 13 and 22, 2007. These technology Information, Bethesda, MD). mites were slide mounted and identified as Virus isolation in Vero cell cultures is a Eustigmaeus lirella (Summers and Price) (Acari: commonly used assay for phleboviruses (Tesh Stigmaeidae). Trophozoites of an aseptate greg- 1989) and can be used to isolate VSV from arine were dissected from the midgut of 1 of 3 Lutzomyia (Corn et al. 1990). We attempted to larvae. isolate viruses from adult L. apache. A total of 151 individual flies (90 males, 61 females) were DISCUSSION individually homogenized in 600 ml of sand fly antibiotic solution (SAM). The SAM used The sand flies from Fremont County were consists of 400 U/ml penicillin, 400 mg/ml strep- morphologically similar to L. apache from Sybille tomycin, 200 mg/ml gentamicin, 25 mg/ml Cipro- Canyon and reference specimens from Colorado. floxacin (Serologicals, Inc., Clarkston, GA), and However, in these 3 specimens the ascoids on the 5 mg/ml Fungizone (Bristol-Myers, Squibb, New second antennal flagellomere were shorter than York, NY) that were prepared in Medium 199 on other L. apache. The ascoids reached the end (Sigma-Aldrich, St. Louis, MO) with Earle’s salts of the flagellomere and the genitalia matched in 10% fetal bovine serum. An inoculum of those of L. apache as described by Young and 100 ml of sand fly homogenate and SAM were Perkins (1984). More study is needed to deter- added to 100 ml of Vero cells (2.5 3 105 cells/ml). mine whether this variation is within the normal This was done in triplicate. These cells were range for the species, or whether this might be a incubated at 37uC with 5–6% CO2 for 14 days. new species. Cells were checked for cytopathic effects (CPE) Our collections extend the range of L. apache daily. After 14 days, the cells were disrupted and into Wyoming. Herrero et al. (2004) predicted 446 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL.24,NO.3 that a small population L. apache might exist near or by consuming a dead fly. The larvae from the Sybille Canyon, WY. However, they did not rearing container with Ascogregarina died on predict that the range of L. apache could extend October 6–10, 2007. They appeared to have a to Fremont County, WY. The validation of the bacterial or fungal infection, but the role of the predicted population of L. apache in Sybille Ascogregarina in weakening the larvae is un- Canyon and range extension of L. apache into known. Fremont County, WY, is significant, because of We did not amplify DNA from Bartonella, the correlation between L. apache and VSV Wolbachia, other microbes, or from the blood (Herrero et al. 2004). There was an outbreak of meals. DNA in the blood meals might have VSV in central Wyoming in 2005 and 2006 and degraded or have been digested before we trapped the overwintering mechanism for this outbreak is the flies. Female flies had dark red material in unknown. Some Lutzomyia spp. can vertically their midgut. The dark red material in the transmit VSV (Comer et al. 1990) and diapausing abdomens of female flies could have been a plant larval sand flies might provide a mechanism for product or honeydew. In addition to the lack of the virus to survive the winter. detectable blood meals, we did not isolate viruses The 1,281-bp sequence of the 16S rRNA gene on Vero cells. from this Rickettsia was 99% similar to a Rickettsia sp. discovered in a crane fly, Limonia chorea (Meigen) (GenBank Accession # AF322443). ACKNOWLEDGMENTS It was also 99% similar to a Rickettsia sp. from leeches (GenBank Accession # AB066352) de- We thank Andrew Fabian, Linda McHolland, scribed by Kikuchi et al. (2002). Campbell et al. and William Yarnell for their valuable assistance (2004) reported a Rickettsia from Culicoides with field and laboratory research. This research sonorensis Jones and Wirth that was 99% similar could not have been conducted without the to those from the same leeches. Campbell et al. assistance of Terry Kreeger who allowed access (2004) did not submit their gene sequences in to his facility. We thank Ron Ochoa and Eddie GenBank or deposit voucher specimens, so it is Ueckermann for identifying the parasitic mites. impossible to determine if the Rickettsia reported The use of trade names in this document does not from C. sonorensis was identical to the microbe constitute an official endorsement or approval of identified in L. apache. The 329-bp sequence of the the use of such commercial hardware or software. gltA gene was 92% similar to the Rickettsia Do not cite this document for advertisement. endosymbiont of a spider, Pityohyphantes phry- gianus (Koch) (GenBank Accession # DQ231491), REFERENCES CITED reported by Goodacre et al. (2006). When this gene sequence was translated into amino acids, it was Alsuhaibani SM. 1990. Field and laboratory studies of sand flies in Larimer County, Colorado [Ph.D. thesis]. 100 identical to that of the Rickettsia of P. % Colorado State University, Fort Collins, CO. phrygianus. The Rickettsia of P. phrygianus and Caceres AG. 1993. Distribucion geografica de Lutzo- most other spiders are the sister taxa to the myia verrucarum (Townsend, 1913) (Diptera, Psy- spotted fever and typhus group Rickettsia (Good- chodidae, ) vector de la bartonellosis acre et al. 2006). One hypothesis of the origin of humana en el . Rev Inst Med Trop Sa˜o Paulo these endosymbiotic Rickettsia could be that 35:485–490. spiders acquired the Rickettsia from fly hosts. Campbell CL, Mummey DL, Schmidtmann ET, Wilson Unfortunately, the gltA gene of the endosymbi- WC. 2004. Culture-independent analysis of midgut onts of leeches or Culicoides has not been microbiota in the vector Culicoides sonor- sequenced and could not be compared. ensis (Diptera: Ceratopogonidae). J Med Entomol 41:340–348. We collected stigmatid mites from female sand Comer JA, Tesh RB, Modi GB, Corn JL, Nettles VF. flies. Stigmatid mites have been reported as 1990. Vesicular stomatitis virus, New Jersey serotype: ectoparasites of phlebotomine sand flies (Zhang replication in and transmission by Lutzomyia shan- andGerson1995).Eustigmaeus lirella was noni (Diptera: Psychodidae). Am J Trop Med Hyg previously reported from wood rat nests (Sum- 42:483–490. mers and Price 1961). In addition to the mites on Corn JL, Comer JA, Erison GE, Nettles VF. 1990. adult flies we dissected gregarine trophozoites Isolation of vesicular stomatitis virus New Jersey from larvae. The trophozoites were morpholog- serotype from phlebotomine sand flies in Georgia. ically similar to Ascogregarina chagasi (Adler and Am J Trop Med Hyg 42:476–482. Mayrink), an endoparasite of Neotropical Lutzo- Goodacre SL, Martin OY, Thomas CFG, Hewitt GM. myia spp. (Wu and Tesh 1989). Larvae of 2006. Wolbachia and other endosymbiont infections in spiders. Mol Ecol 15:517–527. Lutzomyia spp. are infected by Ascogregarina Herrero MV, Yarnell WE, Schmidtmann ET. 2004. spp. when they consume infective oocysts released Landscape associations of the sand fly, Lutzomyia by infected female flies (Wu and Tesh 1989). Our (Heleocyrtomyia) apache (Diptera: Psychodidae), in larvae must have acquired the gregarines by the southwestern United States: a geographic infor- feeding on oocysts released by infected adult flies mation system analysis. JVectorEcol29:205–211. SEPTEMBER 2008 LUTZOMYIA APACHE IN WYOMING 447

Houpikian P, Raoult D. 2001. 16S/23S rRNA intergenic reptile breeding facility in the USA. J Med Entomol spacer regions for phylogenetic analysis, identifica- 43:1099–1101. tion, and subtyping of Bartonella species. J Clin Schmidtmann ET, Craig ME, English LM, Herrero Microbiol 39:2768–2778. MV. 2002. Sampling for sand flies (Diptera: Psy- Kikuchi Y, Sameshima S, Kitade O, Kojima J, Fukatsu chodidae) among prairie colonies on ranches T. 2002. Novel clade of Rickettsia spp. from leeches. with histories of vesicular stomatitis in New Mexico Appl Environ Microbiol 68:999–1004. and Colorado. J Med Entomol 39:680–684. Malmqvist B, Strasevicius D, Hellberg O, Adler PH, Summers FM, Price DW. 1961. New and redescribed Bensch S. 2004. Vertebrate host specificity of wild- species of Ledermuelleria from North America caught blackflies revealed by mitochondrial DNA in (Acarina: Stigmaeidae). Hilgardia 31:369–387. blood. Proc R Soc Lond B (Suppl) Biol Let Tesh RB. 1989. Phlebotomus fevers. In: Monath TP, ed. 271:S152–S155. The : epidemiology and ecology. Volume 4. McLean RG, Szymd DM, Calisher CH. 1982. Exper- Boca Raton, FL: CRC Press. p 15–27. imental studies of Rio Grande virus in rodent hosts. Young DG, Duncan MA. 1994. Guide to the identifi- Am J Trop Med Hyg 31:569–573. cation and geographic distribution of Lutzomyia sand Modi GB, Rowton ED. 1999. Laboratory maintenance flies in Mexico, the West Indies, Central and South of phlebotomine sand flies. In: Maramorosch K, America (Diptera: Psychodidae). Mem Am Entomol Mahmood F, eds. Maintenance of human, animal, and Inst 54:1–881. plant pathogen vectors. Enfield, NH: Scientific Pub- Young DG, Perkins PV. 1984. Phlebotomine sand flies lishers. p 109–121. of North America (Diptera: Psychodidae). Mosq Norman AF, Regnery R, Jameson P, Greene C, Krause DC. 1995. Differentiation of Bartonella-like isolates News 44:263–304. at the species level by PCR-restriction fragment Wu WK, Tesh RB. 1989. Experimental infection of length polymorphism in the citrate synthase gene. Old and New World phlebotomine sand flies J Clin Microbiol 33:1797–1803. (Diptera: Psychodidae) with Ascogregarina chagasi Reeves WK. 2005. Molecular genetic evidence for a (Eugregarinorida: Lecudinidae). J Med Entomol novel bacterial endosymbiont of americana 26:237–242. (Diptera: Hippoboscidae). Entomol News 116:263–265. Zhang ZQ, Gerson U. 1995. Eustigmaeus johnstoni, new Reeves WK, Durden LA, Dasch GA. 2006. A spotted species (Acari: Stigmaeidae), parasitic on phleboto- fever group Rickettsia from an exotic tick species, mine (Diptera: Psychodidae). Tijdschr En- Amblyomma exornatum Koch (Acari: Ixodidae), in a tomol 13:297–301.