2023 Volume 38 Issue 3 Article ID: ME22110
The sorghum plant bug, Stenotus rubrovittatus (order Heteroptera: family Miridae), is a notorious insect pest in Japan that causes pecky rice. In the present study, we sampled this insect pest in the northern part of Honshu Island in Japan and investigated its associated microbiota. The results obtained showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. The dominant Pantoea were phylogenetically close to rice pathogens. The present results suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious insect pest, but also as a potential vector of rice pathogenic Pantoea spp.
Insect-microbe symbiotic interactions are ubiquitous in nature (Buchner, 1965; Bourtzis and Miller, 2003; Douglas, 2015). Insect-associated microbes often affect the physiology and ecology of their hosts. In some cases, symbiotic microbes supply their host insects with essential nutrients for survival, such as essential amino acids and vitamins that cannot be obtained from the diet (Douglas, 2015), while in others, they change the host’s food availability and detoxify insecticides and phytotoxins (Hosokawa et al., 2007; Itoh et al., 2018; Sato et al., 2021).
Rice is a major and important crop in Japan and Asia. The sorghum plant bug Stenotus rubrovittatus (order Heteroptera: family Miridae) (Fig. 1A) is a notorious insect pest in Japan that feeds on rice grains and causes pecky rice (Yasunaga et al., 2001). This species has recently expanded its habitats from the southern to northern part of Japan, which includes famous regions for rice production, possibly due to global warming (Osawa et al., 2018). Therefore, this insect pest has been attracting increasing attention in Japan (Tabuchi et al., 2015).
Maximum likelihood phylogeny of Pantoea clones/isolates derived from Stenotus rubrovittatus based on the 16S rRNA gene. (A) S. rubrovittatus. (B) The maximum likelihood phylogeny of the five Pantoea OTUs associated with S. rubrovittatus, the seven Pantoea isolates from S. rubrovittatus, and related Pantoea species/clones. The multiple alignments of the 1,338 nucleotide sites were analyzed. Phylogenetic relationships based on the maximum likelihood method were reconstructed with RAxML v8.2.12 using the general time reversible model with gamma distribution (GTR+Γ) (Stamatakis, 2014). The bootstrap values of 1,000 replicates were calculated with a rapid bootstrapping algorithm (Stamatakis et al., 2008). A phylogenetic analysis based on the neighbor joining method was also performed with MEGAX (Stecher et al., 2020). Accession numbers in the DDBJ/EMBL/GenBank DNA database are shown in brackets. The Pantoea OTUs/isolates associated with S. rubrovittatus and Pantoea symbionts associated with other insects are shown in red and blue, respectively. The monophyletic clade containing the five Pantoea OTUs and seven Pantoea isolates is highlighted in yellow. Bootstrap support values higher than 70% are shown on the internal branches in the order of the maximum likelihood/neighbor joining method.
In close relatives of the sorghum plant bug, S. binotatus, “Rickettsia-like symbionts” were found in the nuclei and cytoplasm of some epithelial cells of the digestive tract using transmission electronic microscopy (Chang and Musgrave, 1970). Microbial community analyses were recently performed on some mirid bugs (Dally et al., 2020; Luo et al., 2021; Xue et al., 2021). Despite the increasing attention on this insect pest, the associated microbiota of the sorghum plant bug remains unknown. Therefore, we herein investigated the microbiota associated with the sorghum plant bug S. rubrovittatus to obtain a more detailed understanding of the physiology and ecology of this insect pest. Refer to the Supplementary Materials and Methods for details on the materials and methods performed in the present study. The primers used in this study are listed in Supplementary Table S1.
Insect sampling was performed at 24 locations over seven prefectures (29 samples) in the northern part of Honshu Island in Japan (Supplementary Table S2). Between 0.11 and 5.7 μg DNA (mean±SD, 2.2±0.15 μg) was extracted from the whole insect (Supplementary Table S2). A clone library analysis targeting a 1.5-kb fragment of the bacterial 16S rRNA gene was performed on the six samples collected in Akita city (three females and three males). Ninety-two clones were obtained and sequenced (15 or 16 clones from each sample) (Supplementary Table S2). These clones were classified into operational taxonomic units (OTUs) with vsearch 2.15.0 with a 99% identity threshold (Rognes et al., 2016). Eleven OTUs were generated and subsequent homology searches of the represented clone sequences revealed that 5 of the 11 OTUs were affiliated with the genus Pantoea (Supplementary Table S3). The genus Pantoea (Gammaproteobacteria: Enterobacteriaceae) consists of versatile and diverse species and includes plant pathogenic species, clinical and environmental isolates, and symbiotic species of various insects, particularly pentatomid stink bugs (Walterson and Stavrinides, 2015; Duron and Noël, 2016; Hosokawa et al., 2016, 2019; Itoh et al., 2017; Lv et al., 2022). Two of the five Pantoea OTUs were obtained from all six samples, independent of sex, and the five OTUs accounted for most clones (80 clones) (Supplementary Table S3).
We then attempted to isolate Pantoea bacteria from the digestive tract of the sorghum plant bug by plating the homogenate of each component of the digestive tract, namely, the midgut, first, second, and third sections, and hindgut (Supplementary Fig. S1). Emerging yellowish colonies, which is a characteristic of Pantoea spp. (Walterson and Stavrinides, 2015), were randomly selected and taxonomically identified by their 16S rRNA sequences. Seven Pantoea isolates were successfully cultured from all components of the digestive tract of the sorghum plant bug (Supplementary Table S4). The 16S rRNA sequences of the isolates suggested that these isolates were close relatives of the Pantoea OTUs detected in the clone library analysis (Supplementary Table S3 and S4).
To understand the phylogenetic position of the five Pantoea OTUs and the seven Pantoea isolates derived from the sorghum plant bug, representative clone sequences from these OTUs, sequences of the isolates, those of representative Pantoea type strains, and those of Pantoea symbionts derived from other insects were subjected to a phylogenetic analysis based on the maximum likelihood method (Fig. 1B). The five OTUs and seven isolates formed a monophyletic clade with several clones/isolates derived from other insects and several type strains, such as P. ananatis and P. agglomerans.
To examine the prevalence and relative abundance of Pantoea in wild populations of the sorghum plant bug, we performed amplicon sequencing targeting the V3–V4 region of the bacterial 16S rRNA gene with the next-generation sequencer for all 29 samples. Raw reads produced with Illumina Miseq (2×300 bp) were subjected to quality filtering, resulting in 16,501–63,999 (mean: 30,467) qualified and merged amplicon sequences per sample (Supplementary Table S2). These sequences were assigned to the genus level with RDP classifier 2.13 with an 80% confidence threshold (Wang et al., 2007). The sequences assigned to Chloroplast were removed from subsequent analyses. Consistent with the results of the clone library analysis, in the six samples collected in Akita city, Pantoea sequences accounted for more than 60% of bacterial sequences (Fig. 2 and Supplementary Table S2). In all 29 samples, Pantoea was the sole genus accounting for more than 1%; however, the relative abundance of Pantoea sequences for the 29 samples varied at 1.4–95.7% (mean±SD, 56.1±29.9%) (Fig. 2 and Supplementary Table S2). “Unclassified_Enterobacterales” was detected in most samples; in samples with a low abundance of Pantoea, e.g., OGT-F and NKH-F, genera, such as Lactococcus, Staphylococcus, and Corynebacterium, dominated (Fig. 2 and Supplementary Table S5). No sequences of Rickettsia, which had been indicated as symbionts in the mirid bug S. binotatus based on transmission electronic microscopic observations (Chang and Musgrave, 1970), were detected, except in one sample (Supplementary Table S5).
Relative abundance of the Stenotus rubrovittatus-associated microbiota at the genus level. The inset under the right shows a map of Japan, except for its small islands. Twenty-nine insect samples were collected at 24 locations across seven prefectures in the northern part of Honshu Island in Japan, which are indicated in gray in the map. The pie chart shows the relative abundance of the eight major taxa estimated by 16S rRNA amplicon sequencing. The sample name, number of sequences analyzed after removing Chloroplast sequences, and the percentage of Pantoea are shown in the center of the pie chart.
The size of the bacterial population associated with the sorghum plant bug was estimated by performing quantitative PCR targeting the bacterial 16S rRNA gene. The estimated size varied among samples from 2.9×103 to 8.0×106 (mean±SD, 8.3×105±1.6×106 gene copies insect–1) (Supplementary Table S2). Population sizes based on the number of bacterial 16S rRNA gene copies may have been overestimated because several bacteria had multiple copies in their genome, e.g., Pantoea spp. were previously reported to have six copies (Stoddard et al., 2015). A correlation was observed between the population size and relative abundance of Pantoea (Pearson’s correlation coefficient r=0.456).
The results of our microbial community analyses showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. Previous studies reported that some pentatomid stink bugs, e.g., the brown-winged green stink bug Plautia stali and the sloe bug Dolycoris baccarum, develop midgut crypts, the lumen of which is colonized by Pantoea symbionts to establish obligate symbiosis (Hosokawa et al., 2016; Itoh et al., 2017). Some of these Pantoea symbionts formed a monophyletic clade with the Pantoea clones and isolates derived from the sorghum plant bug (Fig. 1B). The dominance and high prevalence in microbial community analyses and the monophyletic relationship of the clones and isolates with Pantoea symbionts of the pentatomid stink bug suggest that the sorghum plant bug S. rubrovittatus also establishes a symbiotic interaction with Pantoea bacteria. However, the sorghum plant bug lacks midgut crypts, similar to many other mirid bugs (Supplementary Fig. S1) (Yanai and Iga, 1956; Chang and Musgrave, 1970). Although the successful culturing of Pantoea from other components of the digestive tract demonstrated their presence in the digestive tract, it is still possible that they were just transient via feeding. Their primary habitat may not be the digestive tract, but the body surface or salivary glands. In addition, mirid bugs are more likely to have a complex and unstable microbiota rather than established symbiotic interactions with a specific partner, such as the pentatomid stink bugs (present study; Dally et al., 2020; Luo et al., 2021; Xue et al., 2021). Therefore, since we cannot conclude that Pantoea is a stably colonized symbiont of the sorghum plant bug, further investigations are needed on this relationship. In future studies, we will examine the localization of Pantoea inside the host body using fluorescence in situ hybridization and perform rearing experiments on the host insect with and without Pantoea and other associated microbes to assess their benefit on the host’s fitness. The reasons and factors causing the large size dispersion of associated bacterial populations and the relative abundance of Pantoea also warrant further study. The large size dispersion may have been caused by the timing and contents of feeding (Luo et al., 2021). Differences in nutrition in the environments inhabited by insects may also affect population sizes and relative abundance. Further research efforts from the view of the associated microbiota are needed to obtain a more detailed understanding of the physiology and ecology of the sorghum plant bug and to achieve the control of this insect pest.
It is also important to note that P. agglomerans and P. ananatis, which formed the monophyletic clade with the OTUs and isolates derived from the sorghum plant bug, have been identified as rice pathogens (Cother et al., 2004; Lee et al., 2010). Although we need to carefully examine their pathogenicity, this finding indicates that these rice pathogens are brought into rice fields via the sorghum plant bug. The migration of plant pathogenic Pantoea spp. for other crops via various insects has been reported (Wielkopolan et al., 2021; Coolen et al., 2022). These findings suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious pest, but also as a potential vector of rice pathogenic Pantoea spp. In P. agglomerans, a lifestyle transition from a commensal to plant pathogenic style occurs via the acquisition of a plasmid-borne pathogenicity island (Barash and Manulis-Sasson, 2007). A transition between plant pathogenic and insect mutualistic lifestyles has also been reported in Burkholderia associated with Largia beetles (Flórez et al., 2017). Lifestyle transitions may happen in the future or even now. Therefore, we will continue to examine the relationship between the sorghum plant bug and its associated Pantoea spp.
The nucleotide sequences of the 16S rRNA genes identified in the present study have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accession numbers LC743641 to LC743732 and LC757513 to LC757519. The sequence reads of amplicon sequencing have been deposited in the DDBJ Sequence Read Archive under accession numbers DRR425141 to DRR425169 (also see Supplementary Table S2 and S4 for details).
Sato, Y., Akao, T., and Takeshita, K. (2023) High Prevalence of Pantoea spp. in Microbiota Associated with the Sorghum Plant Bug Stenotus rubrovittatus (Heteroptera: Miridae). Microbes Environ 38: ME22110.
https://doi.org/10.1264/jsme2.ME22110
The present study was supported by Nippon Life Insurance Foundation.
