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PCR-based species identification of Agriotes larvae

  • K. Staudacher (a1), P. Pitterl (a1), L. Furlan (a2), P.C. Cate (a3) and M. Traugott (a1)...

Click beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.

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Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403410.
Antonini, G., Coletti, G., Serrani, L., Tronci, C., Cristofaro, M. & Smith, L. (2009) Using molecular genetics to identify immature specimens of weevil Ceratapion basicorne (Coleoptera: Apionidae). Biological Control 51, 152157.
Cate, P.C. (2007) Elateridae (Cebrioninae, Lissominae, Subprotelaterinae). pp. 94209 in Löbl, I. & Smetana, A. (Eds) Catalogue of Palaearctic Coleoptera, vol. 4. Stenstrup, Denmark, Apollo Books.
Ellis, J.S., Blackshaw, R., Parker, W., Hicks, H. & Knight, M.E. (2009) Genetic identification of morphologically cryptic agricultural pests. Agricultural and Forest Entomology 11, 115121.
Felsenstein, J. (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783791.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3, 294299.
Furlan, L. (1996) The biology of Agriotes ustulatus Schäller (Col., Elateridae). I. Adults and oviposition. Journal of Applied Entomology 120, 269274.
Furlan, L. (1998) The biology of Agriotes ustulatus Schäller (Col., Elateridae). II Larval development, pupation, whole cycle description and practical implications. Journal of Applied Entomology 122, 7178.
Furlan, L. (2004) The biology of Agriotes sordidus Illiger (Col., Elateridae). Journal of Applied Entomology 128, 696706.
Furlan, L. & Tóth, M. (2007) Occurence of click beetle pest spp. (Coleoptera, Elateridae) in Europe as detected by pheromone traps: survey results of 1998–2006. Integrated Control of Soil Insect Pests. IOBC/wprs Bulletin 30, 1925.
Greenstone, M.H., Rowley, D.L., Heimbach, U., Lundgren, J.G., Pfannenstiel, R.S. & Rehner, S.A. (2005) Barcoding generalist predators by polymerase chain reaction: carabids and spiders. Molecular Ecology 14, 32473266.
Hajibabaei, M., Singer, G.A.C., Hebert, P.D.N. & Hickey, D.A. (2007) DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends in Genetics 23, 167172.
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 9598.
Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B 207, 313321.
Hinomoto, N., Muraji, M., Noda, S., Shimizu, T. & Kawasaki, K. (2004) Identification of five Orius species in Japan by multiplex polymerase chain reaction. Biological Control 31, 276279.
Hosseini, R., Keller, M.A., Schmidt, O. & Framenau, V.W. (2007) Molecular identification of wolf spiders (Araneae: Lycosidae) by multiplex polymerase chain reaction. Biological Control 40, 128135.
Juen, A. & Traugott, M. (2005) Detecting predation and scavening by DNA gut-content analysis: a case study using a soil insect predator-prey system. Oecologia 142, 344352.
Kabanov, V.A. (1975) Über Vorkommen und Entwicklung von Agriotes lineatus (Coleoptera, Elateridae) im europäischen Teil der UDSSR. Pedobiologia 15, 98105.
King, R.A., Traugott, M., Read, D.S. & Symondson, W.O.C. (2008) Molecular analysis of predation: a review of best practice for DNA-based approaches. Molecular Ecology 17, 947963.
Klausnitzer, B. (1994) Familie Elateridae. pp. 118189 in Klausnitzer, B. (Ed.) Die Larven der Käfer Mitteleuropas. Band 2, Myxophaga/Polyphaga, Teil 1. Jena, Germany, Gustav Fischer Verlag.
Langenbuch, R. (1932) Beiträge zur Kenntnis der Biologie von Agriotes lineatus L. und Agriotes obscurus L. Zeitschrift für angewandte Entomologie 19, 278300.
Lindahl, T. (1991) Instability and decay of the primary structure of DNA. Nature 362, 709715.
Lindroth, E. & Clark, T.L. (2009) Phylogenetic analysis of an economically important species complex of wireworms (Coleoptera: Elateridae) in the Midwest. Journal of Economic Entomology, 102, 743749.
Lohse, G.A. (1979) Familie Elateridae. pp. 103186 in Freude, H., Harde, K.W. & Lohse, G.A. (Eds) Die Käfer Mitteleuropas. Band 6, Diversicornia (Lycidea-Byrrhidae). Krefeld, Germany, Goecke & Evers.
Macfadyen, S., Gibson, R., Raso, L., Sint, D., Traugott, M. & Memmott, J. (2009) Parasitoid control of aphids in organic and conventional farming systems. Agriculture, Ecosystems and Environment 133, 1418.
Miller, L.J., Allsopp, P.G., Graham, G.C. & Yeates, D.K. (1999) Identification of morphologically similar canegrubs (Coleoptera: Scarabaeidae: Melolonthini) using a molecular diagnostic technique. Australian Journal of Entomology 38, 189196.
Mitchell, A. (2008) DNA barcoding demystified. Australian Journal of Entomology 47, 169173.
Nei, M. & Kumar, S. (2000) Molecular Evolution and Phylogenetics. New York, USA, Oxford University Press.
Noel, S., Tessier, N., Angers, B., Wood, D.M. & Lapointe, F.-J. (2004) Molecular identification of two species of myiasis-causing Cuterebra by multiplex PCR and RFLP. Medical and Veterinary Entomology 18, 161166.
Parker, W.E. & Howard, J.J. (2001) The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the U.K. Agricultural and Forest Entomology 3, 8598.
Ratnasingham, S. & Hebert, P.D.N. (2007) BOLD: The Barcode of Life Data System ( Molecular Ecology Notes 7, 355364.
Roehrdanz, R., Olson, D., Fauske, G., Bourchier, R., Cortilet, A. & Sears, S. (2009) New DNA markers reveal presence of Aphthona species (Coleoptera: Chrysomelidae) believed to have failed to established after release into leafy spurge. Biological Control 49, 15.
Rugman-Jones, P.F., Morse, J.G. & Stouthamer, R. (2009a) Rapid molecular identification of armored scale insects (Hemiptera: Diaspididae) on Mexican ‘Hass’ Avocado. Journal of Economic Entomology 102, 19481953.
Rugman-Jones, P.F., Wharton, R., Van Noort, T. & Stouthamer, R. (2009b) Molecular differntiation of the Psyttalia concolor (Szépligeti) species complex (Hymenoptera: Braconidae) associated with olive fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), in Africa. Biological Control 49, 1726.
Saccaggi, D.L., Krüger, K. & Pietersen, G. (2008) A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae). Bulletin of Entomological Research 98, 2733.
Simon, Ch., Frati, F., Beckenbach, A., Crespi, B., Liu, H. & Flook, P. (1994) Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Entomological Society of America 87, 651701.
Subchev, M., Toshova, T., Furlan, L. & Tóth, M. (2005) Click beetles (Coleoptera, Elateridae) and their seasonal swarming as established by pheromone traps in different plant habitats in Bulgaria. 2. Maize. Acta Zoologica Bulgarica 57, 321332.
Sumer, F., Tuncbilek, A.S., Oztemiz, S., Pintureau, B., Rugman-Jones, P. & Stouthamer, R. (2009) A molecular key to the common species of Trichogramma of the Mediterranean region. BioControl 54, 617624.
Swofford, D.L. (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Ver. 4. Sunderland, MA, USA, Sinauer Associates.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 15961599.
Tóth, M. & Furlan, L. (2005) Pheromone composition of European click beetle pests (Coleoptera, Elateridae): common components - selective lures. Integrated Control of Soil Insect Pests. IOBC/wprs Bulletin 28, 133142.
Tóth, M., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Imrei, Z., Tolasch, T., Francke, W. & Jossi, W. (2003) Identification of pheromones and optimization of bait composition for click beetle pests (Coleoptera: Elateridae) in Central and Western Europe. Pest Management Science 59, 417425.
Tóth, M., Furlan, L., Xavier, A., Vuts, J., Toshova, T., Subchev, M., Szarukán, I. & Yatsynin, V. (2008) New sex attractant composition for the click beetle Agriotes proximus: Similarity to the pheromone of Agriotes lineatus. Journal of Chemical Ecology 34, 107111.
Traugott, M., Zangerl, P., Juen, A., Schallhart, N. & Pfiffner, L. (2006) Detecting key parasitoids of lepidopteran pests by multiplex PCR. Biological Control 39, 3946.
Traugott, M., Schallhart, N., Kaufmann, R. & Juen, A. (2008a) The feeding ecology of elaterid larvae in central European arable land: New perspectives based on naturally occuring stable isotopes. Soil Biology & Biochemistry 40, 342349.
Traugott, M., Bell, J.R., Broad, G.R., Powell, W., Van Veen, F.J.F., Vollhardt, I.M.G. & Symondson, W.O.C. (2008b) Endoparasitism in cereal aphids: molecular analysis of a whole parasitoid community. Molecular Ecology 17, 39283938.
Vernon, R.S. & Tóth, M. (2007) Evaluation of pheromones and a new trap for monitoring Agriotes lineatus and Agriotes obscurus in the Fraser valley of British Columbia. Journal of Chemical Ecology 33, 345351.
Vernon, R.S., La Gasa, E. & Philip, H. (2001) Geographic and temporal distribution of Agriotes obscurus and A. lineatus (Coleoptera: Elateridae) in British Columbia and Washington as determined by pheromone trap surveys. Journal of the Entomological Society of British Columbia 98, 257265.
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Bulletin of Entomological Research
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