A methodology to generate aposymbiotic and gnotobiotic Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Abstract

As aposymbiotic and gnotobiotic hosts are important for the study of the relationship between gut bacteria and their hosts, the feasibility of flour disc to deliver antibiotics and bacterial isolates to the gut to generate aposymbiotic and gnotobiotic Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) was evaluated in this study. To minimize the negative effects of an antibiotic on beetle development when applied at a high dosage, two broad-spectrum antibiotics, ciprofloxacin and tetracycline, with the complementary antimicrobial spectrum was combined to eliminate whole gut bacteria of T. castaneum. The results showed that wheat flour disc was a feasible material for gastrointestinal delivery of antibiotics and bacterial isolates to the gut of adult T. castaneum. Furthermore, compared with the treatment of a higher antibiotic dosage for a shorter duration, the treatment of a lower antibiotic dosage for a longer duration was more effective to deplete gut bacteria. Compared with the whole gut bacteria, a single bacterial strain was easily removed by moderate antibiotic treatment. The treatment with 0.03 mg/g of the mixture of ciprofloxacin and tetracycline (1: 1, w: w) for 72 h was effective to eliminate most gut bacteria of adult T. castaneum, while had no significant negative effects on beetle development. However, the efficacy of antibiotic treatment was time-dependent, and the relative abundance of gut bacteria recovered to the normal level since the 17th day after the antibiotic treatment. Reinoculation of beetles with a gut bacterial isolate for 3 d was enough to generate aposymbiotic beetles. These results establish a protocol to generate aposymbiotic and gnotobiotic adult T. castaneum. This protocol is worth verifying in other symbiotic systems with powder-feeding habits. The success of this protocol is essential to reveal the function of symbiotic bacteria on their hosts, which will offer valuable insights for development of new pest control strategies.