Gut content analysis using molecular techniques can help elucidate predator-prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species-specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores' main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS50), which depended on the predator-prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5-fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator-prey interactions in tiny species such as mites, which include important agricultural pests and their predators.

Predatory mites of the Phytoseiidae family are considered one of the most important groups of natural enemies used in biological control. The behavioral patterns of arthropods can differ greatly daily and seasonally; however, there is a lack of literature related to Phytoseiidae diel and seasonal predation patterns. The predatory activity of three phytoseiid species (two Tetranychidae-specialists, Phytoseiulus persimilis and Neoseiulus californicus, and one omnivore, Euseius stipulatus) that occur naturally in Spanish citrus orchards was observed under laboratory conditions in winter and summer. The temperature and photoperiod of the climatic chamber where the mites were reared did not change during the experiment. Our study demonstrates that phytoseiids can exhibit diel and seasonal predatory patterns when feeding on Tetranychus urticae (Acari: Tetranychidae). Neoseiulus californicus was revealed to be a nocturnal predator in summer but diurnal in winter. In contrast, P. persimilis activity was maximal during the daytime, and E. stipulatus showed no clear daily predation patterns. The predatory patterns described in this study should be taken into account when designing laboratory studies and also in field samplings, especially when applying molecular techniques to unveil trophic relationships.

Acari harbor numerous minute species of agricultural economic importance, mainly Tetranychidae and Phytoseiidae. Great efforts have been established by means of recovering morphological, molecular, and phylogenetic traits for species identification. Traditional identification still relies on external diagnostic characters, which are limited and usually exhibit large phenotypic plasticity within the species, rendering them useless for species delimitation and identification. We decided to increase the number of sequences of the Acari mitochondrial COI (Cytochrome C oxidase I) marker and ITS nuclear ribosomal DNA region for species identification in Tetranychidae and Phytoseiidae. The molecular data allow us to establish species boundaries and phylogenetic relationships among several clades of Acari, mainly Tetranychidae and Phytoseiidae. Sequence comparisons between complete COI and the Acari mitochondrial COI, ITS1-5,8S-ITS2, and ITS2 among all Acari sequences have demonstrated that the selected regions, even small, gave enough informative positions for both species’ identification and phylogenetic studies. Analyses of both DNA regions have unveiled their use as species identification characters, with special emphasis on Acari mitochondrial COI for Tetranychidae and Phytoseiidae species in comparison with the Folmer fragment, which has been universally used as a barcode marker. We demonstrated that the Acari mitochondrial COI region is also a suitable marker to establish a barcode dataset for Acari identification. Our phylogenetic analyses are congruent with other recent works, showing that Acari is a monophyletic group, of which Astigmata, Ixodida, Mesostigmata, Oribatida, and Prostigmata are also monophyletic.