Longitudinal dynamics of intestinal bacteria in the life cycle and their effects on growth and development of potato tuber moth

Abstract

Potato tuber moth (PTM), Phthorimaea operculella (Lepidoptera: Gelechiidae), is an oligophagous pest that damages potatoes. Intestinal microorganisms play important roles in regulating the life activities of host insects. The gut of PTM is rich in microbials, but it is unclear that the dynamics of the structure and diversity of intestinal bacteria in the different development period of potato tuber moth. In this study, the dynamics of the intestinal bacterial community across the whole life cycle of PTM were evaluated using single molecule real-time sequencing. The intestinal microbiota of PTM is predominantly composed of Proteobacteria and Firmicutes, and it is different with the difference of development stages. Wolbachia endosymbionts were the dominant species of intestinal symbiotic bacteria in eggs and the first-instar larvae. Enterococcus mundtii was the dominant species of intestinal symbiotic bacteria in the second, third, and the fourth instar larvae, as well as in both male and female pupae. Moreover, the predominant species of intestinal symbiotic bacteria in female adults is Enterobacter ludwigii, while the dominant bacterial species is Serratia rubidaea in male adults. Principal component analysis and non-metric Multi-dimensional scaling analysis confirmed the differences in intestinal symbiotic bacteria structure at different developmental stages. In addition, after reintroducing the bacteria following antibiotic treatment, it was found that the antibiotics significantly inhibited the development of the potato tuber moth, whereas the gut bacteria appeared to facilitate its growth. The findings of this study will enhance our understanding of intestinal microorganisms on the development of their host insects across the life cycle. Moreover, it will establish a foundation for elucidating the physiological functions of key microorganisms in the intestinal tract of the potato tuber moth, while also offering new insights and strategy to the biological control of this pest.

Keywords: 16S rRNA sequencing; different life stages; gut microbiota; holometabolous development; potato tuber moth.

FIGURE 1

The diversity and comparison of the intestinal microbial community compositions in different PTM instars. (A) Rarefaction curve of bacterial diversity in different PTM instars. (B) Venn plots of common and unique bacterial species in the intestines of different PTM instars. (C) The diversity metrics ACE, Chao 1, Shannon, and the Simpson indexes of different PTM ages were estimated. Different lowercase letter marks above each group indicated that there were significant differences between different stages groups (one-way ANOVA, LSD post-hoc test, P < 0.05). Error bars represent ± SE of the mean. E, Egg; A, 1st instar; B, 2nd instar; C, 3rd instar; D, 4th instar; PF, Female pupae; PM, Male pupae; AF, Female adult; AM, Male adult.

FIGURE 2

The top 20 of the relative abundance of microbial composition at the level of PTM. Samples representing different developmental stages of the entire life cycle are listed, and different colors indicate annotations for different bacterial genera. E, Egg; A, 1st instar; B, 2nd instar; C, 3rd instar; D, 4th instar; PF, Female pupae; PM, Male pupae; AF, Female adult; AM, Male adult.

FIGURE 3

The dynamic changes of core flora in the life cycle of PTM at the species level. Columns represent the various developmental stages of the entire life cycle, and different colors represent the corresponding strains. E, Egg; A, 1st instar; B, 2nd instar; C, 3rd instar; D, 4th instar; PF, Female pupae; PM, Male pupae; AF, Female adult; AM, Male adult.

FIGURE 4

Gut microorganism dynamics across the life cycle of PTM. (A) Two-dimensional PCA of microbial communities using the Bray-Curtis distance calculation to measure different stage samples. Each point in the figure represents a sample, different colors represent different samples, the elliptical ring represents it as a 95% confidence ellipse. The abscissa represents the first principal component, and the percentage represents the contribution value of the first principal component to the sample difference. The ordinate represents the second principal component, and the percentage represents the contribution value of the second principal component to the sample difference. (B) NMDS based on the gut microbiota composition of PTM. Each point in the figure represents a sample, different colors represent different groups; the elliptical circle indicates that it is a 95% confidence ellipse, and the distance between points indicates the degree of difference. The where samples closer to each other on the coordinate plot exhibit higher similarity. (C) The heat map of the top 30 species in relative abundance at the species level. Each column represents the different developmental stages of the whole life cycle, and the vertical is the species classification group, which is clustered according to the similarity of the microbial abundance spectrum. The clustering tree on the left side is the species clustering tree, and the clustering tree above is the sample clustering tree, which reflects the similarity of community composition between samples. The value corresponding to the heat map is the Z-score standardization of the same species using the R scale function between different samples, and the color gradient from blue to red indicates the abundance from low to high between samples. E, Egg; A, 1st instar; B, 2nd instar; C, 3rd instar; D, 4th instar; PF, Female pupae; PM, Male pupae; AF, Female adult; AM, Male adult.

FIGURE 5

The interaction network among the top 30 bacterial species in the relative abundance of PTM. The node represents the group at the species level, the connection between each two nodes represents the correlation between the classification unit pairs, the thickness of the connection represents the strength of the correlation, and the size of the node represents the number of related objects. The purple solid line indicates positive correlation, and the gray dotted line indicates negative correlation.

FIGURE 6

KEGG function prediction of PTM intestinal bacteria. (A) The difference analysis diagram of KEGG metabolic pathways between eggs and 3rd instar larvae, 4th instar larvae and female adults at the second level was analyzed. Different colors in the diagram represent different groups. The left diagram shows the abundance ratio of different functions in two samples or two groups of samples, the middle is the difference ratio of functional abundance in the 95% confidence interval, and the rightmost value is p-value. (B) Heatmap of correlation between high-abundance bacterial species and metabolic pathways at different developmental stages of PTM (Spearman’s method, ***P < 0.001; **0.001 ≤ P < 0.01; *0.01 ≤ P < 0.05).

FIGURE 7

The influences of EM, SR, and EL on the growth and development of PTM. Different lowercase letter marks above each group indicated that there were significant differences between different stages groups (one-way ANOVA, LSD post-hoc test, P < 0.05). Error bars represent ± SE of the mean. (A) Mortality rate of PTM larvae inoculated with different bacterial strains. (B) Emergence rate of PTM adult inoculated with different bacterial strains. (C) Pupa weight of PTM inoculated with different bacterial strains. (D) Pupa duration of PTM inoculated with different bacterial strains. (E) Female adult duration of PTM inoculated with different bacterial strains. (F) Male adult duration of PTM inoculated with different bacterial strains.