Treatment of Rapamycin and Evaluation of an Autophagic Response in the Gut of Bactericera cockerelli (Sulč)

Abstract

Autophagy is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic content, abnormal protein aggregates, and excess of/or damaged organelles to promote cell survival. Autophagy is also a component of innate immunity in insects and is involved in the clearance of pathogens, including bacteria. The potato psyllid, Bactericera cockerelli, transmits the plant bacterial pathogen 'Candidatus Liberibacter solanacearum' (Lso) in the Americas and causes serious damage to solanaceous crops. Our previous studies showed that autophagy could be involved in the psyllid response to Lso and could affect pathogen acquisition. However, the tools to evaluate this response have not been validated in psyllids. To this end, the effect of rapamycin, a commonly used autophagy inducer, on potato psyllid survival and the expression of autophagy-related genes was evaluated. Further, the autophagic activity was assessed via microscopy and by measuring the autophagic flux. Artificial diet-feeding assays using rapamycin resulted in significant psyllid mortality, an increase in the autophagic flux, as well as an increase in the amount of autolysosomes. This study represents a stepping stone in determining the role of autophagy in psyllid immunity.

Keywords: autophagy; potato psyllid; programmed cell death; zebra chip; ‘Candidatus Liberibacter solanacearum’.

Figure 1

Analysis of potato psyllid mortality caused by feeding on rapamycin-containing diets. (A). Apparatus for the feeding assays. The liquid diet was placed between two Parafilm sheets that covered the chambers. (B). Mortality of potato psyllids following 24 h feeding on artificial diet without (control) or with 10 µM rapamycin. The reported p-value refers to the log-rank test. (C). Analysis of the daily mortality of psyllids. All values are represented as mean ± SD (* p < 0.05).

Figure 2

Relative expression of autophagy-related genes in midguts of potato psyllids after exposure to control and rapamycin-containing diets for 24 h. All values are represented as mean ± SD.

Figure 3

(A). Staining of lysosomes and autolysosomes (green) and nuclei (blue) in psyllid guts. More Lysotracker signals indicating increased lysosomal activity were observed in the midguts of psyllids fed on rapamycin-containing diets for 24 h compared to the control midguts. Left panel: control diets; right panel: rapamycin-containing diets. (B). Average intensity per 50 µm² of Lysotracker in control and rapamycin-treated midguts (n = 18 fields). Arrow indicates lysosomal signals. All values are represented as mean ± SD (**** p < 0.0001). Scale bar = 50 µm.

Figure 4

(A). Western blot analysis with anti-ATG8 antibody from protein extracts of 20 guts; bands corresponding to ATG8-I and ATG8-II were detected. (B). Ratio of ATG8-II/ATG8-I showing the conversion rate of ATG8-I to ATG8-II shown in A. ATG8-II represents lipidated ATG8-I. All values are represented as mean ± SD (* p < 0.05).