Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Abstract

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of obligate endosymbiotic bacteria possibly involved in nutrient distributions. I attempted to first identify what bacteria species are in the guts of larvae followed by antibiotic and microscopy work to further study their roles in the ants. The composition of the bacterial community in fire ant larvae was described with culture-independent methods utilizing 16S rDNA sequencing. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. The colony from Baton Rouge had 34 identified species, 25 identified species from the Rosepine colony, and nine from the Bogalusa colony. None of the identified bacteria were closely related to known coadapted endosymbionts from other insect species. Bacterial inventories from each location provided little evidence of common bacteria among them. Even though Klebsiella pneumoniae appeared in all three colonies, its biology suggested that it came from the environment. Obligate symbiotic bacteria, if present should be present in all larvae regardless of physical location. Antibiotic treatments indicated that ants were not affected by clearing the guts of microorganisms. Clearing of obligate symbiotic bacteria should have detrimental effects on ant mortality. Since no significant change was found, obligate symbiosis is not likely to be present between fire ants and their gut bacteria. I was unable to detect bacteriocytes in the guts of larvae using light and electron microscopy, providing further data against an obligate symbiotic relationship. In this study I did not detect the presence of common symbiotic bacteria in the guts of RIFA larvae among the colonies. Bacteria communities appeared to be unique to each colony and were determined by the food and environment.