Diet Analysis of Wrinkle-Lipped Free-Tailed Bat (Chaerephon plicatus Buchannan, 1800) Using Direct-PCR DGGE Technique / Kantima Thongjued

Abstract

Globally insectivorous bats have been reported as a biological pest control agent. Chaerephon plicatus may play an important role for rice pest suppression. Diet analysis is used to reveal this ecosystem service. However, fecal examination using microscopic method have never provided reliable species prey list due to the possibility of thorough mastication for some insects. In this study, first, we developed and validated a direct PCR protocol for fast and effective universal insect species identification. Second, we tested applicability of the well-optimized protocol in various sample types regularly encountered in ecological studies. Third, we employed direct PCR protocol together with Denaturing Gradient Gel Electrophoresis (DGGE) (called direct PCR-DGGE technique) to identify insect preys in bat guano samples, and fourth, the ecosystem service of C. plicatus in regulating insect pest and also its foraging behavior in the surrounding agricultural landscapes was assessed. The developed direct PCR protocol that incorporates a 2-min sample preparation in PBS-buffer step achieved 100% success rates for amplification in six insect orders: Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea, and Orthoptera. High and moderate success rates were obtained for five other groups: Lepidoptera (97.3%), Coleoptera (93.8%), Diptera (90.5%), Hemiptera (81.8%), and Hymenoptera (75.0%). High-quality sequencing data were obtained from these amplifiable products, allowing confidence in species identification. The method was sensitive down to 1⁄4 of a 1-mm2 fragment of leg or body and its success rates with oven-dried, ethanol-preserved, food, bat guano, and museum specimens were 100%, 98.6%, 90.0%, 86.3%, and 30.0%, respectively. Two hundreds and seven of 240 bat guano pellets collected monthly from bat caves surrounded by rice fields were successfully amplified and provided 325 bands on DGGE gel. Sequencing confirmed that these bands comprised 42 identified OTU of insects and could be assigned to 7 orders, 25 families, 24 genera, and 26 species. The results showed that C. plicatus diet was shaped by agricultural landscape, and also relied on availability of insect preys in their foraging range. Potential rice pest species, e.g. brown planthoppers (Nilaparvata lugens), and medical important insects, e.g. mosquitoes (Culex sp.) were consumed by C. plicatus, indicating its function as pest suppressing agent. This is the first time direct PCR-DGGE has been successfully used to analyze bat diet from guano samples. Diet of the bat was revealed genetically down to species level resulting in a more complete picture of ecosystem service, which allows further understanding of predator-prey interaction. These findings also provide basic data which could further benefit conservation and sustainable management of bat caves adjacent to the farmland to protect their habitat and prevent population decline, which may help to improve productivity, profitability of the agriculture industry, and consequently promote human well-being.