Plant defenses interact with insect enteric bacteria by initiating a leaky gut syndrome

Abstract

Plants produce suites of defenses that can collectively deter and reduce herbivory. Many defenses target the insect digestive system, with some altering the protective peritrophic matrix (PM) and causing increased permeability. The PM is responsible for multiple digestive functions, including reducing infections from potential pathogenic microbes. In our study, we developed axenic and gnotobiotic methods for fall armyworm (Spodoptera frugiperda) and tested how particular members present in the gut community influence interactions with plant defenses that can alter PM permeability. We observed interactions between gut bacteria with plant resistance. Axenic insects grew more but displayed lower immune-based responses compared with those possessing Enterococcus, Klebsiella, and Enterobacter isolates from field-collected larvae. While gut bacteria reduced performance of larvae fed on plants, none of the isolates produced mortality when injected directly into the hemocoel. Our results strongly suggest that plant physical and chemical defenses not only act directly upon the insect, but also have some interplay with the herbivore's microbiome. Combined direct and indirect, microbe-mediated assaults by maize defenses on the fall armyworm on the insect digestive and immune system reduced growth and elevated mortality in these insects. These results imply that plant-insect interactions should be considered in the context of potential mediation by the insect gut microbiome.

Keywords: Lepidoptera; chitinase; maize; microbiome; trichome.

Fig. 1.

Impacts of plant genotype and bacterial inoculation on the performance of fall armyworm. Axenically reared insects were inoculated with 1 of 3 bacterial isolates on artificial diet and transferred to gamma-irradiated maize leaves of susceptible (Tx601) or resistant (Mp708) genotypes. (A) Growth of the fall armyworm was negatively impacted by plant genotype (F_1,380 = 385; P < 0.001), bacteria (F_3,380 = 20.5; P < 0.001), and their interaction 7(F_3,380 = 3.99; P = 0.008). Different letters and capitalization represent statistically significant differences between bacterial treatments in resistant maize. (B) Mortality of fall armyworm was increased with specific bacterial inoculations only on resistant maize (Z = 2.91; P = 0.016). Lines connecting symbols in A and B are present for visual clarity between bacterial treatments. (C) SEM imaging of the PM of fall armyworm feeding on resistant maize demonstrates perforations of the PM, and the opportunity for bacteria to access the body cavity. Combining the 3 isolates resulted in similar growth responses (SI Appendix, Fig. S2). Full ANCOVA results are reported in SI Appendix, Table S3.

Fig. 2.

Impacts of maize genotypes (S, susceptible; R, resistant) and Enterobacter oral inoculation on fall armyworm baseline phenoloxidase activity (A), activatable (pro) phenoloxidase activity (B). There was a significant effect of plant genotype (F_1,39 = 8.0, P = 0.007) and bacterial colonization (F_1,39 = 39.3, P < 0.001) on baseline PO activity. Likewise, there were genotype (F_1,40 = 5.3, P = 0.026) and bacteria (F_1,40 = 33.2, P < 0.001) effects on activatable PO. (C) Total hemocytes were not influenced by plant genotype (F_1,40 = 0.0002, P = 0.989), but were by the presence of bacteria (F_1,40 = 30.5, P < 0.001). Colony forming units (CFU·μL⁻¹) in the hemolymph (D) were higher in insects feeding on resistant maize compared with susceptible maize (P = 0.009). Different letters and asterisks represent statistically significant differences among treatments (P < 0.05). Enterobacter inoculation of larvae fed on artificial diet also elevated the level of PO activity in hemolymph, but not to the extent that was induced by plants (SI Appendix, Table S6). Full ANOVA tables are reported in SI Appendix, Table S5.

Fig. 3.

Interactions between chitinases, plant physical defenses, and gut bacteria on fall armyworm growth. (A) Recombinant chitinases (F_1,35 = 55.1, P < 0.001) and their interaction with plant genotype (F_1,35 = 43.5, P < 0.001) interacted with the different plant genotypes to influence fall armyworm growth. Susceptible plants with more abundant large trichomes (S6) had greater impacts on the growth of fall armyworm than those with smaller trichomes (F_1,35 = 169, P < 0.001), particularly when there was topical application of chitinases. (B) Accompanying this reduced growth, plant chitinases resulted in significantly higher numbers of bacteria in the hemolymph of the insects compared with other treatments. (C) The presence of Enterobacter increased the negative effects of plant defenses on larval growth. Using the genotype with long trichomes (Tx601), we observed an effect of chitinase (F_1,35 = 27.5, P < 0.001), the presence of bacteria (F_1,35 = 29.2, P < 0.001), and their interaction (F_1,35 = 4.42, P = 0.043). Different letters above the bars represent statistically significant differences among treatments (P < 0.05). Full ANCOVA results are reported in SI Appendix, Tables S8 and S9.