Specific and sensitive primers for the detection of predated olive fruit flies, Bactrocera oleae (Diptera: Tephritidae)

Esther Lantero, Beatriz Matallanas, Maria Dolores Ochando, Susana Pascual, Carmen Callejas


Bactrocera oleae, the olive fruit fly, is a major pest of olive (Olea europaea L.) trees worldwide. Its presence can cause important losses, with consequences for the economies of countries that produce and export table olives and olive oil. Efforts to control olive fruit fly populations have, however, been insufficient. Now more than ever, environmentally friendly alternatives need to be considered in potential control programs. Generalist predators could provide a way of managing this pest naturally. However, the identification of candidate predator species is essential if such a management system is to be introduced. The present paper describes a set of species-specific primers for detecting the presence of B. oleae DNA in the gut of predatory arthropods. All primers were tested for checking cross-reactive amplification of other fruit fly DNA and evaluated in heterospecific mixes of nucleic acids. All were found to be very sensitive for B. oleae. Subsequent feeding trials were conducted using one of the most abundant species of ground dwelling carabids in olive groves in south-eastern Madrid, Spain. These trials allowed determining that 253F-334R and 334F-253R primer pairs had the highest detection efficiency with an ID50 of around 78 h. These primers therefore provide a very useful tool for screening the gut contents of potential predators of B. oleae, and can thus reveal candidate species for the pest's biological control


Olea europaea, predation; Carabidae; species-specific and sensitive primers; feeding-trials; cytochrome oxidase subunit I

Full Text:



Besnard G, Khadari B, Navascués M, Fernández-Mazuecos M, El Bakkali A, Arrigo N, Baali-Cherif D, Brunini-Bronzini de Caraffa V, Santoni S, Vargas P, Savolainen V, 2013. The complex history of the olive tree: from Late Quaternary diversification of Mediterranean lineages to primary domestication in the northern Levant. Proc R Soc B 280: 20122833. https://doi.org/10.1098/rspb.2012.2833

Boreau de Roincé C, Lavigne C, Richard JM, Franck P, Bouvier JC, Garcin A, Symondson WOC, 2012. Predation by generalist predators in the codling month versus a closely related emerging pest the oriental fruit moth: a molecular analysis. Agric Forest Entomol 14: 260-269. https://doi.org/10.1111/j.1461-9563.2011.00564.x

Brown PMJ, Ingels B, Wheatley A, Rhule EL, de Clercq P, van Leeuwen P, Thomas A, 2015. Intraguild predation by Harmonia axyridis (Coleoptera: Coccinellidae) on native insects in Europe: molecular detection from field samples. Entomol Sci 18: 130-133. https://doi.org/10.1111/ens.12092

Daane KM, Johnson MW, 2010. Olive fruit fly: Managing an ancient pest in modern times. Annu Rev Entomol 55: 151-169. https://doi.org/10.1146/annurev.ento.54.110807.090553

Dinis AM, Pereira JA, Pimenta MC, Oliveira J, Benhadi-Marín J, Santos SAP, 2015. Suppression of Bactrocera oleae (Diptera: Tephritidae) pupae by soil arthropods in the olive grove. J Appl Entomol 140: 677-687. https://doi.org/10.1111/jen.12291

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. Mol Mar Biol Biotechnol 3: 294-299.

Gagnon AE, Doyon SJ, Heimpel Ge, Brodeur J, 2011. Prey DNA detection success following digestion by intraguild predators: influence of prey and predator species. Mol Ecol Res 11: 1022-1032. https://doi.org/10.1111/j.1755-0998.2011.03047.x

Gkisakis V, Volakakis N, Kollaros D, Bàrberi P, Kabourakis EM, 2016. Soil arthropod community in the olive agroecosystem: Determined by environment and farming practices in different management systems and agroecological zones. Agric Ecosyst Environ 218: 178-189. https://doi.org/10.1016/j.agee.2015.11.026

Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleid. Acids Symposium. Series 41: 95-98.

Harper GL, King RA, Dodd CS, Harwood JD, Glen DM, Bruford MW, Symondson WOC, 2005. Rapid screening of invertebrate predators for multiple prey DNA targets. Mol Ecol 14: 819-827. https://doi.org/10.1111/j.1365-294X.2005.02442.x

Harwood JD, Desneux N, Yoo HJ, Rowley DI, Greenstone MH, Obrycki JJ, O'Neil RJ, 2007. Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach. Mol Ecol 16: 4390-4400. https://doi.org/10.1111/j.1365-294X.2007.03482.x

King RA, Read DS, Traugott M, WOC Symondson, 2008. Molecular analysis of predation: a review of best practice for DNA based approaches. Mol Ecol 17: 947-963. https://doi.org/10.1111/j.1365-294X.2007.03613.x

King RA, Moreno-Ripoll R, Agusti N, Shayler SP, Bell JR, Bohan DA, Symondson WOC, 2010. Multiplex reactions for the molecular detection of predation on pest and non pest invertebrates in agroecosystems. Mol Ecol Resour 11: 370-373. https://doi.org/10.1111/j.1755-0998.2010.02913.x

León JH, Fournier V, Hagler JR, Daane KM, 2006. Development of molecular diagnostic markers for sharpshooters Homalodisca coagulate and Homalodisca liturata for use in predator gut content examinations. Entomol Exp Appl 119: 109-119. https://doi.org/10.1111/j.1570-7458.2006.00399.x

MacDonald AJ, Young MJ, Lintermans M, Sarre SD, 2014. Primers for detection of Macquarie perch from environmental and trace DNA samples. Conserv Genet Resour 6: 551-553. https://doi.org/10.1007/s12686-014-0196-6

Monzó C, Sabater-Mu-óz B, Urbaneja A, Casta-era P, 2010. Tracking medfly predation by the wolf spider, Pardosa cribata Simon, in citrus orchards using PCR-based gut-content analysis. Bull Entomol Res 100: 145-152. https://doi.org/10.1017/S0007485309006920

Monzó C, Sabater-Mu-óz B, Urbaneja A, Casta-era P, 2011. The ground beetle Pseudophonus rufipes revealed as predator of Ceratitis capitata in citrus orchards. Biol Control 56: 17-21. https://doi.org/10.1016/j.biocontrol.2010.09.004

Moreno-Ripoll R, Gabarra R, Symondson WOC, King RA, Agustí N, 2012. Trophic relationships between predators, whiteflies and their parasitoids in tomato greenhouses: a molecular approach. Bull Entomol Res 102: 415-423. https://doi.org/10.1017/S0007485311000836

O'Rorke R, Lavery S, Jeffs A, 2012. PCR enrichment techniques to identify the diet of predators. Mol Ecol Res 12: 5-17. https://doi.org/10.1111/j.1755-0998.2011.03091.x

Pascual S, Cobos G, Series E, González-Nú-ez M, 2010. Effects of processed kaolin on pest and non- target arthropods in a Spanish olive grove. J Pest Sci 83: 121-133. https://doi.org/10.1007/s10340-009-0278-5

Pentisaari M, Salmela H, Mutanen, Roslin T, 2016. Molecular evolution of a widely adopted taxonomic marker (COI) across the animal tree of life. Sci Rep 6: art 35275.

Pereira-Castro I, Van Asch B, Trinidade Rei F, Texeira da Costa L, 2015. Bactrocera oleae (Diptera: Tephritidae) organophosphate resistance alleles in Iberia: Recent expansion and variable frequencies. Eur J Entomol 112: 20-26.

Ratnasingham S, Hebert PDN, 2007. BOLD: the barcode of life data system (www.barcodinglife.org). Mol Ecol Notes 7: 355-364. https://doi.org/10.1111/j.1471-8286.2007.01678.x

Rejili M, Fernandes T, Dinis AM, Pereira JA, Baptista P, Santos SAP, Lino-Neto T, 2016. A PCR based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods. Bull Entomol Res 106 (5): 695-699. https://doi.org/10.1017/S000748531600050X

Rosenheim JA, Limburg DD, Colfer RG, 1999. Impact of generalist predators on a biological control agent, Chrysoperla carnea: direct observations. Ecol Appl 9: 409-417. https://doi.org/10.1890/1051-0761(1999)009[0409:IOGPOA]2.0.CO;2

Sint D, Raso L, Kaufmann R, Traugott M, 2011. Optimizing methods for PCR-based analysis of predation. Mol Ecol 11: 759-801. https://doi.org/10.1111/j.1755-0998.2011.03018.x

Symondson WOC, 2002. Molecular identification of prey in predator diets. Mol Ecol 11: 627-641. https://doi.org/10.1046/j.1365-294X.2002.01471.x

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The Clustal X window interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876-4882. https://doi.org/10.1093/nar/25.24.4876

DOI: 10.5424/sjar/2017152-9920