Bacterial Community Structure in the Asian Rice Gall Midge Reveals a Varied Microbiome Rich in Proteobacteria

Abstract

The Asian rice gall midge (ARGM) has emerged as a model gall forming pest of rice. The ARGM infestation of rice results in failure of panicle formation and economic loss. Understanding the molecular basis of ARGM-rice interactions is very crucial in order to control this devastating pest of rice. The current investigation was devised to identify bacterial communities present in the ARGM and in addition the bacterial diversity in the maggots during their interaction with susceptible or resistant rice varieties. Sequencing of 16S rRNA bacterial gene (V3-V4 region) revealed differences in the microflora of the ARGM maggots feeding on susceptible or resistant rice hosts. Results revealed that Wolbachia was the predominant bacterium in pupae and adults while Pseudomonas was predominant in maggots. Further, we observed that members of proteobacteria were predominant across all the samples. There was high species diversity in maggots isolated from susceptible rice and a high representation of unclassified bacteria in maggots isolated from resistant rice. This is the first study that reports variation of microbiome of the ARGM, based on host phenotype from which it was isolated, and results suggest that these variations could have an important role in host's susceptibility.

Figure 1

(a) PCR amplification of hypervariable region (V3-V4) of the Asian rice gall midge biotype 1 (GMB1). 1, 1 kb DNA ladder, 2, GMB1M (adult male), 3, GMB1F (adult female), 4, GMB1P (pupa), 5, GMB1LS (maggots from susceptible host) and 6, GMB1LR (maggots from resistant host). (b) Comparative rarefaction curve analyses for observed microbial OTUs. The rarefaction curves of microbial community in GMB1 samples; GMB1F (red), GMB1LS (dark yellow), GMB1LR (blue), GMB1M (green) and GMB1P (violet). (Multiple exposure gels are presented in Supplementary Figure 1).

Figure 2

Heat map showing relative distribution and abundance of the OTUs among the GMB1 samples at phylum level.

Figure 3

Assessment of microbial diversity in the GMB1 sample using Principal Co-ordinate Analysis (PCoA) (a) unweighted 3-D visualization and (b) parallel visualization of UniFrac plots to reveal quantitative assessment of microbial diversity between the GMB1 samples. (c) Principal Co-ordinate Analysis (PCoA)-weighted 3-D visualization and (d) parallel visualization of UniFrac plots to reveal qualitative assessment of microbial diversity between the GMB1 samples.

Figure 4

Relative abundance of different bacterial communities among all the GMB1 samples at the level of (a) phyla and (b) genus. (c) Percentage of abundant and rare species among the different GMB1 samples.

Figure 5

Distribution of abundant and rare bacterial population (shared and specific) in the GMB1 samples. (a) Venn diagram and (b) bar chart showing distribution of abundant species (shared and specific), (c), Venn diagram and (d) bar chart showing distribution of rare species (shared and specific) among the GMB1 samples.

Figure 6

(a) Semi quantitative PCR and (b) image analyses of the agarose gel in ‘a’, for quantifying abundance of Pseudomonas and Wolbachia in different GMB1 samples. Actin gene served as the internal control.