University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications: Department of Entomology Entomology, Department of 11-2013 DNA Barcoding Distinguishes Pest Species of the Black Fly Genus Cnephia (Diptera: Simuliidae) I. M. Confitti University of Toronto K. P. Pruess University of Nebraska-Lincoln A. Cywinska Ingenomics, Inc. T. O. Powers University of Nebraska-Lincoln D. C. Currie University of Toronto and Royal Ontario Museum, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/entomologyfacpub Part of the Entomology Commons Confitti, I. M.; Pruess, K. P.; Cywinska, A.; Powers, T. O.; and Currie, D. C., "DNA Barcoding Distinguishes Pest Species of the Black Fly Genus Cnephia (Diptera: Simuliidae)" (2013). Faculty Publications: Department of Entomology. 616. http://digitalcommons.unl.edu/entomologyfacpub/616 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. MOLECULAR BIOLOGY/GENOMICS DNA Barcoding Distinguishes Pest Species of the Black Fly Genus Cnephia (Diptera: Simuliidae) 1,2 3 4 5 1,2,6 I. M. CONFLITTI, K. P. PRUESS, A. CYWINSKA, T. O. POWERS, AND D. C. CURRIE J. Med. Entomol. 50(6): 1250Ð1260 (2013); DOI: http://dx.doi.org/10.1603/ME13063 ABSTRACT Accurate species identiÞcation is essential for cost-effective pest control strategies. We tested the utility of COI barcodes for identifying members of the black ßy genus Cnephia Enderlein (Diptera: Simuliidae). Our efforts focus on four Nearctic Cnephia speciesÑCnephia dacotensis (Dyar & Shannon), Cnephia eremities Shewell, Cnephia ornithophilia (Davies, Peterson & Wood), and Cnephia pecuarum (Riley)Ñthe latter two being current or potential targets of biological control programs. We also analyzed one Palearctic species, Cnephia pallipes (Fries). Although Cnephia adults can be identiÞed anatomically to species, control programs target the larval stage, which is difÞcult or impossible to distinguish morphologically. By using neighbor- joining, maximum parsimony, and Bayesian methods, we found that COI barcodes successfully identiÞed three Nearctic Cnephia species, but not C. pecuarum. The Palearctic C. pallipes was also successfully identiÞed. Despite nonmonophyly of C. pecuarum, we show that data from COI barcoding, in combination with geographical and ecological information, can be used to distin- guish all four Nearctic species. Finally, we discussed 1) possible reasons for paraphyly in C. pecuarum, 2) topological concordance to previously reported chromosomal dendrograms, and 3) evolution of diverse feeding strategies within the genus Cnephia. KEY WORDS biological control, COI gene, mitochondrial DNA, monophyly, paraphyly Species identiÞcation is important for pest control pect that hitherto intractable species or life-history programs that must consider both the costs and efÞ- stages can now be identiÞed (Hebert et al. 2003a,b). cacy of pest mitigation efforts. One group of organisms Preliminary research reveals that DNA barcoding that have been proven especially difÞcult to identify holds sufÞcient promise for black ßy identiÞcation to are black ßiesÑa worldwide family of nematocerous warrant further study (Day et al. 2010, Hunter et al. Diptera that is infamous for the bloodsucking habits of 2008, Ilmonen et al. 2009, Pramual et al. 2011, Rivera females and transmission of the causal agents of par- and Currie 2009). asitic diseases to birds and mammals. Members of the Cnephia Enderlein is a small genus of black ßies with family are structurally homogenous, making routine eight species distributed throughout the Holarctic Re- “hard parts” identiÞcation difÞcult or even impossible gion (Adler and Crosskey 2012). Four species occur in in many instances. Moreover, cytological studies re- the Nearctic Region, including the northern Holarctic veal that many nominal species consist of two or more species Cnephia eremities Shewell, and three endemic reproductively isolated (but structurally identical) species: Cnephia dacotensis (Dyar & Shannon), Cne- sibling species, or “cytospecies.” Cytological identiÞ- phia ornithophilia (Davies, Peterson & Wood), and cation is typically possible only through band-by-band Cnephia pecuarum (Riley). Although represented by analyses of the giant polytene chromosomes of larval few species, the North American Cnephia exhibit the black ßies, meaning that other life-history stages are full range of feeding habits of female black ßies. Two often impossible to identify. The emergence of DNA species are obligately autogenous (C. eremites and C. barcodingÑthe use of a short DNA sequence to iden- dacotensis), one is ornithophilic (C. ornithophilia), tify organisms to a particular speciesÑoffers the pros- and one is mammalophilic (C. pecuarum). C. ornitho- philia seeks blood from a wide variety of avian hosts 1 Department of Ecology and Evolutionary Biology, University of and is a vector of Leucocytozoon Ziemann protozoa to Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada. woodland birds (Fallis and Bennett 1961, 1962; Khan 2 Department of Natural History, Royal Ontario Museum, 100 and Fallis 1970). They pose a particular threat to the QueenÕs Park, Toronto, ON M5S 2C6, Canada. 3 Department of Entomology, University of Nebraska, 103 Ento- endangered AttwaterÕs Prairie-Chicken (Tympanu- mology Hall, Lincoln, NE 68583. chus cupido attwateri Bendire) through the bloodsuck- 4 Ingenomics Inc., 3164 Candela Dr., Mississauga, ON L5A 2T8, ing activity and possible transmission of Leucocytozoon Canada. (Adler et al. 2007). Control measures against C. orni- 5 Department of Plant Pathology, University of Nebraska, 406 Plant Science Hall, Lincoln, NE 68583. thophilia were suggested as a possibility, if warranted, 6 Corresponding author, e-mail: [email protected]. in streams and rivers in the vicinity of the Attwater 0022-2585/13/1250Ð1260$04.00/0 ᭧ 2013 Entomological Society of America November 2013 CONFLITTI ET AL.: DNA BARCODES DISTINGUISH Cnephia PEST SPECIES 1251 Prairie-Chicken National Wildlife Refuge in Colorado larval specimens of C. dacotensis and C. ornithophilia, Co., Texas (Adler et al. 2007). C. pecuarumÑthe which are morphologically identical, identiÞcations southern buffalo gnat of the Mississippi River Val- were based either on distributional data (i.e., speci- leyÑis among the most notorious bloodsucking pests mens were assigned to a species if it occurred deci- of humans and other mammals in North America sively within that speciesÕ range and was also clearly (Adler et al. 2004). Massive mortality of livestock was separated from the known ranges of all other species) attributed to this species during the American Civil or on cytologically screened specimens from the same War, when levees on the Mississippi River deterio- sites. For pupae, identiÞcations were based either by rated and river water ßowed onto the brushy alluvial using the same distributional criteria as described for zone, creating ideal breeding sites (Riley 1887). Mor- larvae, or on fully developed pupae (i.e., pharate tality was probably the result of acute toxemia and adults) whose genitalia could be examined. Vouchers anaphylactic shock caused by the injection of saliva for specimens are held in the entomological collec- during blood feeding (Adler and McCreadie 2002). tions of the University of Nebraska State Museum and Simuliotoxicosis on such a scale is now rare; however, Royal Ontario Museum. Extractions were performed occasional outbreaks of C. pecuarum make them the by using the GenElute Mammalian Genomic DNA target of control programs in localized areas through- Miniprep Kit (SigmaÐAldrich Co., St. Louis, MO) or out their range (Adler et al. 2004). the Gentra Puregene Kit (Qiagen, Germantown, Although Cnephia adults can be identiÞed anatom- MD), following manufacturerÕs instructions. Standard ically to species, the immature stages of most species DNA barcoding primers (LCO1490 and HCO2198, are difÞcult or impossible to distinguish morphologi- Folmer et al. 1994) were used to target a 658-bp region cally. This is an impediment to biological studies that of the mitochondrial COI gene. Each 25 ␮l polymerase require accurate identiÞcation of larvae and pupae; chain reaction (PCR) consisted of 0.92ϫ PCR buffer and it is especially problematical for control programs, pH 8.3 (10 mM Tris-HCl pH 8.3, 50 mM KCl, and 0.01% ␮ ␮ which focus mainly on larval control. The foremost NP-40), 2.6 mM MgCl2, 200 M of each dNTP, 0.3 M biological control agent for black ßiesÑthe bacterium of each primer, 1 U TaqDNA polymerase, 1Ð5 ␮l tem- Bacillus thuringiensis variety israelensis (Bti)Ñmust plate DNA, and the remaining volume of dH2O. PCR be applied to breeding sites in a timely and judicious cycling parameters were as follows: 1 min at 95ЊC; 35 fashion to increase efÞcacy and reduce costs (Molloy cycles of 1 min at 94ЊC, 1 min at 55ЊC, and 1.5 min at et al. 1981). As the two pest species (C. pecuarum and 72ЊC; and Þnally 7 min at 72ЊC. Samples were se- C. ornithophilia) are sympatric, it is impossible to quenced with the ABI Big Dye 3.1 Terminator Kit know which breeding sites to target unless 1) rela- (Applied Biosystems Inc., Carlsbad, CA). Sequencing tively mature larvae are available for cytotyping, or 2) primers were the same as those used