Hostname: page-component-76fb5796d-vfjqv Total loading time: 0 Render date: 2024-04-26T14:16:36.325Z Has data issue: false hasContentIssue false
  • English
  • Français

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Published online by Cambridge University Press:  13 December 2007

D.L. Saccaggi ,
K. Krüger  and
G. Pietersen
D.L. Saccaggi
Affiliation:
Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa
K. Krüger*
Affiliation:
Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa
G. Pietersen
Affiliation:
Citrus Research International, c/o Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
*
*Author for correspondence Fax: +27 12 362 5242 E-mail: kkruger@zoology.up.ac.za
Get access Rights & Permissions [Opens in a new window]

Abstract

Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.

Keywords

insectmealybugmultiplex PCRdiagnostic molecular identification
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 98 , Issue 1 , February 2008 , pp. 27 - 33
Copyright
Copyright © Cambridge University Press 2008

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Bermingham, N. & Luettich, K. (2003) Polymerase chain reaction and its applications. Current Diagnostic Pathology 9, 159–164.Google Scholar
Beuning, L.L., Murphy, P., Wu, E., Batchelor, T.A. & Morris, B.A.M. (1999) Molecular-based approach to the differentiation of mealybug (Hemiptera: Pseudococcidae) species. Journal of Economic Entomology 92, 463–472.Google Scholar
Charles, J.G., Froud, K.J. & Henderson, R.C. (2000) Morphological variation and mating compatibility within the mealybugs Pseudococcus calceolariae and P. similans (Hemiptera: Pseudococcidae), and a new synonymy. Systematic Entomology 25, 285–294.CrossRefGoogle Scholar
Clary, D.O. & Wolstenholme, D.R. (1983) Genes for cytochrome c oxidase subunit I, URF2, and three tRNAs in Drosophila mitochondrial DNA. Nucleic Acids Research 11, 6859–6872.Google Scholar
Clary, D.O. & Wolstenholme, D.R. (1985) The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. Journal of Molecular Evolution 22, 252–271.CrossRefGoogle ScholarPubMed
Daane, K.M., Bentley, W.J. & Weber, E.A. (2004) Vine mealybug: a formidable pest spreads throughout California vineyards. Practical Winery and Vineyard Magazine 3, 35–40.Google Scholar
de Borbón, C.M., Gracia, O. & Gómez Talquenca, G.S. (2004) Mealybugs and grapevine leafroll-associated virus 3 in vineyards of Mendoza, Argentina. American Journal of Enology and Viticulture 55, 283–285.CrossRefGoogle Scholar
Demontis, M.A., Ortu, S., Cocco, A., Lentini, A. & Migheli, Q. (2007) Diagnostic markers for Planococcus ficus (Signoret) and Planococcus citri (Risso) by random amplification of polymorphic DNA-polymerase chain reaction and species-specific mitochondrial DNA primers. Journal of Applied Entomology 131, 59–64.CrossRefGoogle Scholar
Downie, D.A. & Gullan, P.J. (2004) Phylogenetic analysis of mealybugs (Hemiptera: Coccoidea: Pseudococcidae) based on DNA sequences from three nuclear genes, and a review of the higher classification. Systematic Entomology 29, 238–259.CrossRefGoogle Scholar
Engelbrecht, D.J. & Kasdorf, G.G.F. (1990) Transmission of grapevine leafroll disease and associated closteroviruses by the vine mealybug, Planococcus ficus. Phytophylactica 22, 341–346.Google Scholar
Fukatsu, T. (1999) Acetone preservation: a practical technique for molecular analysis. Molecular Ecology 8, 1935–1945.Google Scholar
Gariepy, T.D., Kuhlmann, U., Haye, T., Gillott, C. & Erlandson, M. (2005) A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp. Biocontrol Science and Technology 15, 481–495.Google Scholar
Golino, D.A., Sim, S.T., Gill, R. & Rowhani, A. (2002) California mealybugs can spread grapevine leafroll disease. California Agriculture 56, 196–201.Google Scholar
Gullan, P.J. & Kosztarab, M. (1997) Adaptations in scale insects. Annual Review of Entomology 42, 23–50.Google Scholar
Harper, G.L., Sheppard, S.K., Harwood, J.D., Read, D.S., Glen, D.M., Bruford, M.W. & Symondson, W.O.C. (2006) Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators. Bulletin of Entomological Research 96, 295–304.Google Scholar
Hebert, P.D.N., Cywinska, A., Ball, S.L. & DeWaard, J.R. (2003a) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, series B: Biological Sciences 270, 313–321.Google Scholar
Hebert, P.D.N., Ratnasingham, S. & DeWaard, J.R. (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, series B (Suppl.): Biological Sciences 270, S96–S99.Google Scholar
Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. & Gibson, T.J. (1998) Multiple sequence alignment with Clustal X. Trends in Biochemical Sciences 23, 403–405.Google Scholar
Koekemoer, L.L., Kamau, L., Hunt, R.H. & Coetzee, M. (2002) A cocktail polymerase chain reaction assay to identify members of the Anopheles funestus (Diptera: Culicidae) group. The American Journal of Tropical Medicine and Hygiene 6, 804–811.Google Scholar
La Notte, P., Buzkan, N., Choueiri, E., Minafra, A. & Martelli, G.P. (1997) Acquisition and transmission of grapevine virus A by the mealybug Pseudococcus longispinus. Journal of Plant Pathology 78, 79–85.Google Scholar
Millar, I.M. (2002) Mealybug genera (Hemiptera: Pseudococcidae) of South Africa: identification and review. African Entomology 10, 185–233.Google Scholar
Post, R.J., Flook, P.K. & Millest, A.L. (1993) Methods for the preservation of insects for DNA studies. Biochemical Systematics and Ecology 21, 85–92.Google Scholar
Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. & Flook, P. (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a complication of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87, 651–701.CrossRefGoogle Scholar
Tayutivutikul, J., Pongprasert, W., Royce, L.A. & Ruangrit, K. (2003) Comparison of preservation techniques for silkworm (Bombyx mori L.) DNA based on polymerase chain reaction (PCR) products. CMU Journal 2, 107–114.Google Scholar
Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.Google Scholar
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.Google Scholar
Watson, G.W. & Kubiriba, J. (2005) Identification of mealybugs (Hemiptera: Pseudococcidae) on banana and plantain in Africa. African Entomology 13, 35–47.Google Scholar
Weeto, M.M., Koekemoer, L.L., Kamau, L., Hunt, R.H. & Coetzee, M. (2004) Evaluation of a species-specific PCR assay for the Anopheles funestus group from eleven African countries and Madagascar. Transactions of the Royal Society of Tropical Medicine and Hygiene 98, 142–147.CrossRefGoogle ScholarPubMed
Zehner, R., Amendt, J., Schütt, S., Sauer, J., Krettec, R. & Povolný, D. (2004) Genetic identification of forensically important flesh flies (Diptera: Sarcophagidae). International Journal of Legal Medicine 118, 245–247.CrossRefGoogle ScholarPubMed