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

Abstract

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 speciesspecific 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 78h. 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.

PCR Species-Specific Primers for Molecular Gut Content Analysis to Determine the Contribution of Generalist Predators to the Biological Control of the Vector of Xylella fastidiosa

“…Both primer pairs were very sensitive given that P. spumarius DNA was amplified in all the heterospecific DNA mixes, even when potential predator DNA was in great excess (Ratio 1:4000) (Figure 4). Outcomes returned a detection limit of 2.5 pg/µL of P. spumarius DNA, surpassing that obtained for the same gene on sharpshooters (6 pg/µL [34]), equaling the one obtained for Haplodiplosis marginata [35] and slightly higher than that for the olive pest, Bactrocera oleae (1 pg/µL [26]). phylogeny and DNA barcoding of P. spumarius and its related species is found in the literature [14].…”

“…The amplicon size is crucial for postmortem gut content analyses to be successful. Given the preys' DNA degradation during the digestive processes, amplifying small fragments from multicopy genes improves the detection rates of these minute quantities of preys' DNA in the predators' gut [19,20,26]. The sensitivity assays were carried out in PCR reactions performed as in the specificity assay.…”

“…Sixty-three spiders were collected by hand as potential predators of P. spumarius in an olive-growing area in the southeast of Madrid, located near the locality (Villarejo de Salvanés, Spain) of Xylellas' outbreak in Madrid [6] at the beginning of May, expecting high levels of vectors' infestation. The DNA from the spiders' gut was isolated and amplified as described in [26]. PCR reactions were conducted at least twice using the two primer pairs selected in the previous tests.…”

“…Altea Calabuig (Elytra Agroscience Services). We collected the remaining specimens from a previous study [26].…”

“…The quality of all DNA extracted was examined on a first set of PCR reactions using the universal COI primers LCO1490 and HCO2198 [27] under the conditions reported in Lantero et al [26]. Amplified products with these primes from Aphrophoridae nymphs were used as templates for sequencing reactions.…”