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

Abstract

Abstract:The European Union (EU) is facing the recent arrival of the bacterium Xylella fastidiosa. Its fast spread has caused great alarm because of the economic impact it implies for the agroeconomy of European countries. Among its insect vectors, Philaenus spumarius has been demonstrated to transmit the bacterium from infected to uninfected trees in the EU, where different measures have been established to control it. One of the proposals to manage this vector is the augmentation of natural enemy populations. 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 designed to detect the presence of P. spumarius DNA in soil arthropod fauna generalist predators' gut which can reveal candidate species for the pest's biological control. Such primers have been proven to be a useful and reliable taxonomic tool for P. spumarius identification at any life stage, i.e., nymphs. This rapid and accurate identification is essential for control strategies designed to avoid the spread of the pest and consequently the considerable economic losses it causes in crops.

A novel molecular diagnostic method for the gut content analysis of Philaenus DNA

“…the standard barcode for invertebrates, cytochrome c oxidase subunit I (COI_Ph71F/COI_Ph941R), and the cytochrome b (cytB_Ph85F/cytB_Ph635R) genes. Currently, the COI gene is one of the most used markers for PCR-based gut-content analysis in arthropods 17 , 18 , 23 , 30 – 32 , including in Philaenus detection 24 . On the contrary, the cyt B has rarely been used for species identification of invertebrates but is widely used within vertebrates 16 .…”

“…naturally occurring in its ecosystem. Lantero et al 24 have previously designed specific primers for P. spumarius based on the COI sequence region. In their work, no feeding assay was developed to establish the prey detectability over time, and the DNA was extract from the gut.…”

“…Also, COI primers designed in our study were developed to amplify a larger fragment than those from Lantero et al . 24 , since an unequivocal identification of Philaenus at the species level may not be feasible by using small COI fragments (Seabra et al 44 ; see also Supplementary Fig. S2 ).…”

“…Indeed, as in Lantero et al . 24 we did not evaluate the primers on other Philaenus species. So, we cannot assure that the primers from this study are species-specific for P. spumarius , although the Primer-BLAST results indicated a good likelihood for it.…”

“…Another methodological aspect to be considered on PCR-based analysis of predation includes the type of predator tissue to be used to extract DNA 19 . The predator's digestive tract is the preferred source to extract DNA from ingested preys 18 , 22 – 24 . However, gut dissection in spiders is not possible.…”