From the bellies of babes: a larval-based approach to ant molecular gut content analysis

Abstract

We used high-throughput sequencing molecular gut content analysis (HTS-MGCA) to examine diets of the red imported fire ant (Solenopsis invicta; hereafter fire ant). Because adult fire ant workers rely on their larvae (brood) to digest prey and share it via trophallaxis with the rest of the colony, we conducted a study to determine if we could sequence the gut contents of final instar ant brood instead of adult workers to better capture colony diet. We sequenced all samples on the Illumina HiSeq 2500 using general primers to amplify and identify arthropod DNA within larval guts. We first verified our methods in a controlled-feeding experiment in the laboratory using crickets (Gryllodes sigillatus), beet armyworm (Spodoptera exigua), or a mixture of both species, and then we analyzed the diets of field-collected fire ants. The sequences from our controlled-feeding lab samples were very accurate, validating our analysis pipeline. We identified a total of 45 diet events from six field-collected fire ant samples. All fire ant field samples contained sequences from both detritus-based food webs (arthropods that consume decaying material) and plant-based food webs (arthropods that consume living plant material). HTS-MGCA of fire ant larvae was highly successful and provided novel insights into the community-level impacts of ant foraging. Our approach should be widely applicable to the study of resource use and trophic interactions in other eusocial insects.