The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation

Abstract

The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 μg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.

Fig 1. Relative proportion (%) of phylotypes isolated from the larval gut of insecticide-resistant lines of Spodoptera frugiperda afterr RFLP-PCR analysis and partial sequence (550 pb) of the 16S rDNA.

Putative identification obtained after heuristic search of 1350 bp of 16S rDNA against sequences available in the NCBi and EzTaxon-e databases.

Fig 2. Neighbor-joining phylogenetic tree of bacterial isolates obtained from the larval gut microbiota of insecticide-resistant lines of Spodoptera frugiperda.

Tree was built using 1350 bp sequences of the 16S rDNA. Support bootstrap values are shown in the branches. Scale bar indicates 0.02 substitutions per nucleotide position.

Fig 3. Growth of insecticide-degrading bacterial isolates obtained from the gut microbiota of strain of Spodoptera frugiperda resistant to the insecticides chlorpyrifos ethyl (A), lambda-cyhalothrin (B), deltamethrin (C), spinosad (D), and lufenuron (E) when cultured in minimum medium M9 added of 10 μg/mL of the insecticide the host insect strain was resistant to.

Fig 4. Growth of selected insecticide-degrading bacterial isolates obtained from the gut microbiota of insecticide-resistant lines of Spodoptera frugiperda at different insecticide concentrations (10, 20, 40, 80 or 160 μg/mL).

Isolates A) IIL-Cl29 (Leclercia adecarboxylata), B) IIL-Lc09 (Pseudomonas stutzeri), C) IIL-Dm05 (Arthrobacter nicotinovorans), D) IIL-Sp19 (Pseudomonas psychrotolerans), and E) IIL-Luf14 (Microbacterium arborescens) were isolated and cultivated on insecticide-based media containing, respectively, chlorpyrifos ethyl, lambda-cyhalothrin, deltamethrin, spinosad and lufenuron.