Effects of a protease inhibitor protein on Buchnera aphidicola and gene expression in pea aphids (Acyrthosiphon pisum)

Aphids are serious insect pests for agricultural and horticultural crops and may cause major economic losses. Insecticides used to control aphids have caused environmental pollution and insecticide residues in agricultural products. A new protease inhibitor gene from bacterium Xenorhabdus bovienii (Xbpi-1) and the protease inhibitor protein expressed by the gene against the pea aphid (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) have been reported, however, effects of the PIP on the symbiotic bacteria, Buchnera aphidicola, and gene expression in pea aphids are unknown. By assessing the quantity of B. aphidicola, the primary symbiotic bacterium, in the aphid fed on an artificial diet containing 100 and 500 μg/ml Xbpi-1, we observed a substantial reduction in its population by 27% and 46%, respectively, as analyzed by real-time quantitative reverse transcription PCR (qRT-PCR). Furthermore, the growth of other aphid-associated bacteria was also significantly inhibited by Xbpi-1. To elucidate the mechanisms at the gene level, we conducted transcriptome analysis and identified differentially expressed genes (DEGs). Subsequent Gene Ontology (GO) analysis of the 213 DEGs shed light on the impact of Xbpi-1 on aphid metabolism processes and gene expression. Notably, the results highlighted several aphid nutrient metabolism pathways affected by Xbpi-1, which are relevant to vector-borne diseases. These pathways encompass crucial factors such as heat shock proteins, cuticle proteins, and proteases. The results from this study revealed that the PIP had a novel mechanism against pea aphids by having adverse effects on the primary symbiotic bacteria in pea aphids and affecting aphid gene expression, showing that the PIP may be a promise bioinsecticide for aphid control in the future.

Methods
1. Previous studies reported that B. aphidicola was related to aphid nutrition and important for aphid growth and survival (Muller et al., 2001; Nishikor et al., 2009). Therefore, the effects of the PIP on the quantity of B. aphidicola in pea aphids were further investigated by qRT-PCR analysis. The expression level of the GROEL gene was chosen to reflect the number of B. aphidicola as that of the previous report (Sakurai et al., 2005). Relative numbers of B. aphidicola in aphids fed on the artificial diet containing different concentrations of the PIP were calculated. The GROEL gene from B. aphidicola was used for this quantitative analysis. The primer sequences were as follows: GROEL-F (5'-CGTTTCAGATCCATTGGATTCA-3') and GROEL-R (5'-AGCTCAAATGGTAAAAGAAGTTGCA-3').

2. Three-day-old aphids were placed in a glass tube with both ends closed by sealing film. Artificial feed was added into the sealing film at one end, and then the glass tube was placed in a light incubator for feeding. The aphids were fed on artificial feed for 3 days, and the mortality rate of aphids was calculated. There are two treatment groups in the experiment, which were artificial feed containing 100 µg/ml and 500 µg/ml Xbpi-1 respectively. The control group used artificial feed with the same volume of PBS solution instead.