Citrus Huanglongbing (HLB) is the most destructive disease in citrus production worldwide. The Asiatic citrus psyllid (Diaphorina citri Kuwayama) is currently the only insect vector identified to transmit the citrus HLB pathogen and is one of the most significant pests in citrus production. Although D. citri is being controlled chemically, alternative biorational strategies should be developed. Predators play an important role in the biological control of agricultural pests, and arthropod predators have been identified as an important factor in pest population growth control. Traditionally, approaches to measure insect predation include direct observation, predator enhancement or exclusion, and direct or biochemical estimation of gut contents. However, predators and prey are often small, mobile or live under dense vegetation. Thus, arthropod predation is generally difficult to observe and estimate directly. These evaluations require a heavy workload in complicated ecological environments, which can influence their accuracy. How to evaluate predation accurately is one of the problems yet to be solved in ecology.
The cytochrome oxidase subunit I gene (COⅠ) is known as a conservative protein-coding gene that has been widely used in systematic studies of invertebrates. To understand the predation of key predators on D. citri in citrus orchards, a DNA marker, COⅠ of D. citri, was developed in this study. Specific primers can be used in polymerase chain reactions to amplify prey DNA from the gut content of generalist predators with high specificity and sensitivity. A prerequisite for applying this approach to field studies is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from the predator species or from the wide range of potential alternative prey found under natural conditions. Our study was not confined to a species-specific molecular systematics study, but used the population of pests and their enemies in their citrus orchard environment. The sequence-characterized primers were designed according to the COⅠ gene sequence of D. citri. The specificity test results performed with the primers showed that the primers were specific and efficient to detect the species-specific DNA of D. citri. The positive detection of D. citri DNA in the predators collected in field samples showed that Propylea japonica, Oxyopes sertatus and Chrysopa formosa had a significant predation on D. citri; the detection rates of these predators were 58.06%, 57.89%, and 48.00%, respectively. The time that residual prey DNA can be detected by molecular analysis of a predator's gut-content is very important, and it decides the practical feasibility of this DNA analysis technique. The lab digestion analysis of P. japonica adults on D. citri indicated that detection decreased with the extension of digestion time. The half-lives of DNA detectability for a single D. citri 4^th instar nymph and adult consumed by a P. japonica adult were 4.93 h and 12.98 h, respectively. DNA marker technology has the advantages of a simple protocol and a quick inspection over the previously described detection procedures for predators of D. citri. It provides an important theoretical basis to investigate the spectrum of predatory natural enemies and the sustainable control of D. citri.