
==== Front
Microb Ecol
Microb Ecol
Microbial Ecology
0095-3628
1432-184X
Springer US New York

39249553
2426
10.1007/s00248-024-02426-8
Research
Geographical, Seasonal, and Growth-Related Dynamics of Gut Microbiota in a Grapevine Pest, Apolygus spinolae (Heteroptera: Miridae)
http://orcid.org/0009-0003-7823-080X
Morimura Hiroyuki morimura.hiroyuki@aist.go.jp

1
http://orcid.org/0000-0002-8670-5858
Ishigami Kota 12
http://orcid.org/0009-0008-2427-5144
Sato Tomoyuki 34
Sone Teruo 3
http://orcid.org/0000-0002-1651-2532
Kikuchi Yoshitomo y-kikuchi@aist.go.jp

15
1 https://ror.org/01703db54 grid.208504.b 0000 0001 2230 7538 Bioproduction Research Institute, Hokkaido Center, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, 062-8517 Japan
2 https://ror.org/02z1n9q24 grid.267625.2 0000 0001 0685 5104 Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa 903-0213 Japan
3 https://ror.org/02e16g702 grid.39158.36 0000 0001 2173 7691 Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
4 Corporate Planning Department, Hokkaido Wine Co., Ltd. 1-130 Asarigawa Onsen, Otaru, 047-8677 Japan
5 https://ror.org/02e16g702 grid.39158.36 0000 0001 2173 7691 Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
9 9 2024
9 9 2024
2024
87 1 1129 4 2024
10 8 2024
© The Author(s) 2024
2024
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A number of insects are associated with gut symbiotic microorganisms, wherein symbiotic partners play pivotal metabolic roles for each other such as nutrient supplementation, diet degradation, and pesticide detoxification. Despite the ecological and evolutionary importance of gut microbial communities in insects, their diversity and dynamics remain unclear in many species. The green plant bug Apolygus spinolae, a notorious grapevine pest in Japan, damages grape shoots and severely reduces grape berry yield and quality. The plant bug possesses a simple tubular gut housing ~ 104 bacteria. Here, we investigated geographic, seasonal, and growth-related dynamics of gut microbiota by high-throughput sequencing in 82 individuals (11 nymphs and 71 adults) from five locations in Hokkaido, Japan. In plant bugs, gut microbiota changed dynamically depending on region, season, and developmental stage. Among the gut bacteria, Serratia was consistently and abundantly detected and was significantly affected by seasonal changes. In addition, Caballeronia, known as a specific symbiont in some stinkbug species, was abundantly detected, especially in insects collected in late summer despite A. spinolae complete lack of midgut crypts known as symbiotic organ harboring Caballeronia in other stinkbug species. Considering their prevalence among host bug populations, it is possible these gut microorganisms play a pivotal role in the adaptation of the green plant bug to grapevine fields, although further confirmation through rearing experiments is needed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00248-024-02426-8.

Keywords

Plant bug
Gut symbiosis
Caballeronia
Serratia
Grapevine
http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 22KJ0057 21K20579 Ishigami Kota Sato Tomoyuki http://dx.doi.org/10.13039/501100007697 Northern Advancement Center for Science and Technology S-3-7 Kikuchi Yoshitomo issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

Many insects harbor diverse microorganisms inside their body which play essential metabolic roles for the host [1, 2]. In particular, recent studies have revealed that gut microbiota provide various benefits to the host insect in immune system activation, provision of essential nutrients, digestion of indigestible foods, and/or degradation of plant toxins and insecticides [2–4]. Despite the growing body of knowledge, the gut microbial community of several insect species remains unclear, and their biological functions remain under investigation.

Studies of gut microbiota have been performed on a wide range of insect species, especially investigating the gut microbes involved in agriculture and hygiene pest insects [2, 5, 6]. In several cases, gut microbiota changes dynamically depending on season, location, and growth stages, relating to host adaption to heterogenic environments [6–8]. These studies provide novel knowledge about how insects take advantage from gut symbiosis. Additionally, a detailed investigation of agricultural pest microbiota dynamics could lead to the development of eco-friendly pest management [7] and carry a potential for industrial application [5, 8].

The family Miridae (order Heteroptera: infraorder Cimicomomorpha), or plant bugs, include more than 11,000 species worldwide [9] which cause serious damage to agricultural crops [10]. While specific gut symbionts have been reported in members of the infraorder Pentatomomorpha, based on their anatomical features, members of the Miridae are not thought to be associated with any specific gut symbiont. Recent studies, however, revealed that plant bugs possess diverse microorganisms in their gut [11–15], some of which are commonly found in or even specific to host plant bug species [14]. Considering the diverse functions of gut bacteria in insects, elucidating the diversity and biological functions of gut microbiota in plant bugs is pivotal for controlling these pest insects.

The green pale plant bug Apolygus spinolae, distributed in a wide range of temperate zones in Eurasia, is a notorious pest of various crops including sunflowers, asparagus, tea and apple trees, and grapevines [16, 17]. In addition to these crops, reports of feeding damage to wine grapes by A. spinolae have increased in recent years and are becoming problematic, especially in northern Japan (personal communication). In Europe, the occurrence of the green pale plant bug in vineyards has also been reported in Slovenia [18]. While adults suck and damage grapevine leaves, nymphs cause more severe damage because they feed on shoots during their budding period, resulting in ragged leaves with numerous holes (Fig. 1A and B), and eventually leading to the loss of grape quality and quantity. Although A. spinolae has been recognized as an important agricultural pest of grapevines, their ecology, development, and gut morphology, as well as their symbiotic association with microbes, are still unclear.Fig. 1 Apolygus spinolae and its midgut. A Healthy leaves of wine grapevine. B Damaged leaves of wine grapevine. C Each developmental stage of A. spinolae. D Midgut structure of adult A. spinolae. M1, midgut first section; M2, midgut second section; M3, midgut third section; H, hindgut

In this study, we investigated geographical, seasonal, and growth-related dynamics of gut microbiota in A. spinolae populations of vineyards in Hokkaido, northern Japan, by high-throughput amplicon sequencing of bacterial 16S rRNA variable gene fragment V4. This study reveals dynamic community changes in A. spinolae gut microbiota, suggesting the environment affects the gut microbiota of the pest plant bug species. The findings of this study potentially contribute to controlling this serious pest of wine grapes via modulation of gut symbiosis.

Materials and Methods

Insects

A. spinolae were collected at five vineyards in Hokkaido, Japan: Tsurunuma, Furano, Obihiro, Niseko, and Hakodate (Table S1). Nymphs (3rd, 4th, and 5th instar) and adult insects (both male and female) were collected from the fields, transported alive to the laboratory, and stored in acetone at room temperature for DNA extraction at a later date. At Tsurunuma, the plant bugs were caught from June to September 2022, and the bugs collected at the other sites were sampled in September 2022. Insects were captured by sweeping more than 20 times between six hedges of grapevines at the collection sites. To clarify basic ecological and anatomical information about the plant bug, some adults and hatchlings were reared in our laboratory on sunflower seeds, buckwheat, wheat seeds, and distilled water containing 10% honey at 25 °C under long-day condition (light:dark = 18 h:6 h). To investigate gut morphology, adults were dissected in phosphate-buffered saline (PBS: 0.14 M NaCl, 0.0027 M KCl, 0.01 M PO43−, pH 7.4) using forceps under a stereo microscope (Leica S8AP0 stereo microscope; Leica, Germany).

DNA Extraction and PCR Amplification

After volatilizing the acetone, the whole bodies of adult stinkbugs were homogenized in sterile water before performing DNA extraction. Overall, four to 15 insects from each vineyard were used in this study. In addition to the adult insects collected at the above sites, DNA was also extracted from three samples of 3rd, two samples of 4th, and six samples of 5th instar nymphs. DNA extraction was performed using Qiagen DNA extraction kits (Qiagen, Netherlands) following company protocols. For checking extracted DNA quality, Nanodrop (ThermoFisher MA, USA) was used and PCR amplification of the stinkbug’s mitochondrial COI gene. The COI gene was amplified using Amplitaq Gold polymerase (Applied Biosystems CA, USA) with primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [19] under the following condition: initial denaturation at 95 °C for 10 min, followed by 30 cycles of 95 °C for 30 s, 48 °C for 60 s, and 72 °C for 90 s, and a final extension at 72 °C for 10 min. Amplification of the COI gene was observed in all individuals subjected to the DNA extraction.

The variable region V4 of bacterial 16S rRNA gene was amplified using Amplitaq Gold polymerase (Applied Biosystems CA, USA) with the degenerate PCR primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [20] under the following PCR condition: initial denaturation at 95 °C for 10 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 60 s, and 72 °C for 90 s, and a final extension at 72 °C for 120 s. The PCR products were purified with Wizard SV Gel and PCR Clean-Up system (Promega, WI, USA). PCR of the V4 region was performed using the elution buffer instead of the samples, but no amplification was observed.

Quantitative PCR

In a preliminary investigation, A. spinolae samples were dissected in PBS to separate the gut from other body parts, before subjecting them separately to qPCR to measure their bacterial content. The preliminary results showed the bacterial content outside the gut was negligible (Table S2); therefore, we decided to use the whole body of plant bugs to estimate their gut bacterial content. To indirectly estimate the number of bacterial cells, the copy number of 16S rRNA variable region V4 was measured by quantitative PCR (qPCR), which was performed using a reaction master mix including the following: primers 515F and 806R and the KAPA SYBR FAST qPCR Master Mix Kit (KAPA Biosystems, MA, USA). The Roche LightCycler 96 System (Roche, Switzerland) was used to perform qPCR, with a temperature profile as follows: 45 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. The gene copy number was calculated based on the standard curve for the 16S V4 region gene containing 10, 102, 103, 104, 105, and 106 copies of the PCR product per reaction.

Amplicon Sequencing

Prepared DNA was subjected to PCR amplification of 16S rRNA V4 region for deep sequencing. The reaction master mix contained 50 µM of each dNTP, 0.4 µM 515F with Illumina P5 sequences, 0.4 µM 806R with Illumina P7 sequences (Illumina, CA, USA) [19], Q5 High-Fidelity DNA polymerase (New England Biolabs, MA, USA), and the extracted DNA template. The PCR cycling was as follows: initial denaturation at 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s, and a final extension at 72 °C for 2 min. The DNA library containing tagged amplicons and the internal control PhiX were used for sequencing on an Illumina iSeq100 sequencer (Illumina) using iSeq100 Reagent kit V2 (Illumina). The library generated 2 × 151 bp paired-end reads. All sequence data obtained in this study were deposited in the DDBJ/GenBank/EBI, under accession number PRJDB17462.

The paired-end reads were uploaded to EzBioCloud 16S-based MTP (ChunLab, Inc., Seoul, South Korea) [21] to check data quality. The EzBioCloud software application filtered out low-quality sequences with averaged Q values of less than 30. Moreover, low-quality amplicons were removed from filtered total amplicons, and reads with low validity containing non-target amplicons and chimeras were filtered out by using EzBioCloud 16S chimera-free database applied with the UCHIME algorithm [22]. EzBioCloud 16S database used with the VSEARCH program [23] performed the taxonomic assignment to detect and calculate sequence similarities of the query paired-ends reads. EzBioCloud sequencing reads were clustered into operational taxonomic units (OTUs) at 97% sequence similarity using UPARSE [24].

Data Analysis

Microbial richness was measured by ACE and Chao1 using OTUs generated in this microbiome taxonomic profile. Shannon and Simpson α-diversity indices were applied to estimate the evenness for each group. Statistical analysis for ACE, Chao1, Shannon, and Simpson was performed using the Kruskal–Wallis test, one-way analysis of variance (ANOVA), and Mann–Whitney U test conducted by GraphPad Prism v. 9. 5. 1 (Graph Pad Software, MA, US). Beta diversity was assessed using the Bray–Curtis distance between groups and their ordination visualized by principal coordinate analysis (PCoA) using the vegdist function of vegan package in R [25]. Statistical difference in community structure between groups was tested with permutational multivariate analysis of variance (PERMANOVA) generated using the prcomp function of ape package in R [25]. Generalized liner model (GLM) and correlation analysis were also conducted in R [25]. Climatic factors analyzed by GLM were obtained from the Automated Meteorological Data Acquisition System (AMeDAS) administrated by the Japan Meteorological Agency (https://www.jma.go.jp).

Phylogenetic Analysis

More than 2% of bacterial OTUs were used to construct a phylogenetic tree of the insect-associated bacteria using the MAFFT program [26] which aligned the V4 regions (250 bp) of the OTUs acquired in this study. In addition, bacterial 16S rRNA sequences of related genera were obtained from the NCBI nucleotide (nr) database and subsequently aligned with the OTUs using CLUSTAL W [27]. The phylogenetic analysis was conducted using the maximum likelihood method (1000 bootstraps) through the MEGA X software [28]. Kimura’s two-parameter model, evaluated with the best-fit method, was applied for the calculation [29].

Results

Basic Information About A. spinolae and Its Gut

The field collection and laboratory rearing confirmed that A. spinolae has five nymphal stages before reaching adulthood (Fig. 1C). The midgut of A. spinolae was anatomically divided into three sections, M1–M3. The boundaries of each section are unclear, and M4, the crypt-bearing gut symbiotic organ in other stinkbugs, is completely absent (Fig. 1D).

Regional Diversity of Gut Bacterial Community

In the late summer season (September), A. spinolae were collected from five geographically separated vineyards (Table S1), and their gut bacterial communities were investigated by high-throughput sequencing. The size of the bacterial population in A. spinolae was estimated by qPCR targeting bacterial 16S rRNA gene. The gene copy sizes of the gut and remaining body parts excluding the gut, indicated here as mean ± SD, were of 1.6 × 104 ± 1.7 × 104 gene copies insect−1 and 1.1 × 101 ± 1.5 × 101 gene copies insect−1 for both groups, respectively. To analyze the gut microbiota, we decided to investigate the whole body of A. spinolae due to the small amount of 16S rRNA gene copies in body parts other than the gut. The size of the bacterial population from whole homogenized adult bodies varied among samples with means of 2.4 × 104 ± 2.5 × 104 gene copies per insect (Table S2).

We obtained raw 16S rRNA V4 region reads from adults (mean, 13,273 ± 8031 reads) using Illumina iSeq100 for 71 adult insects (28 males and 43 females from the five Hokkaido regions) (Table S2). Good’s coverage of all clean reads was above 99%, which indicates that the sequencing effort was enough to capture total diversity (Table S2). At the phylum level, the beta diversity analysis showed no significant differences among vineyard samples (Fig. S1). Microbiota of all vineyard samples included the genera Serratia, Lactococcus, and Caballeronia in different proportions (Fig. 2A), and the genus level diversities among the vineyards showed significant differences by PERMANOVA using a PCoA plot based on Bray-Cutis distance (Fig. 2B). Among the five populations, microbiota of plant bugs collected in Tsurunuma, Niseko, and Hakodate were remarkably similar to each other (Fig. 2B), wherein all three microbiota included a high percentage of Caballeronia (Fig. 2). In Furano and Obihiro vineyards, Serratia showed the highest proportional abundance in adult insect guts (Fig. 2). Turning to the Hakodate site, the A. spinolae microbiota had a unique composition in which Spiroplasma was most abundant (Fig. 2A). The phylogenic analysis suggested that the OTUs generated from Hakodate samples were relatively close to the insect-associated Spiroplasma platyhelix group and the plant pathogen S. citri group (Fig. S2), although the phylogenetic analysis was based on only the 16S rRNA V4 region. Alpha-diversity analysis indicated significant differences in the richness and evenness scores (Fig. S3), while the diversity analysis between females and males showed no significant difference (Fig. S4).Fig. 2 Diversity and specificity of Apolygus spinolae–associated microbiota in adults collected from five vineyards in September. A Relative abundance of the A. spinolae–associated microbiota at the genus level. The insect samples were collected at five locations. Black dots indicate geographical locations for sampling in this study. Bacterial genera of profound relevance to insects are marked as follows: Lactococcus, plus sign ( +); Serratia, asterisk (*); Acinetobacter, black diamond (◆); Enterobacterales, black circle (●); Caballeronia, black star (✭). B Principal coordinate analysis (PCoA) plot generated by Bray–Curtis dissimilarity. The confidence ellipsoids of different sites provided by lines indicates 95% confidence intervals

Growth-Related Diversity of Gut Bacterial Community

The growth-related diversity of the microbial community was investigated in A. spinolae nymphs collected in the Tsurunuma vineyard from August to September. For the homogenized nymph’s body, gene copy numbers were calculated as 6.7 × 103 ± 4.3 × 103 gene copies insect−1 (Table S2). For 11 nymphs, we obtained raw reads of nymph stages (mean, 18,473 ± 8594 reads) from deep sequencing (Table S2). At the phylum level, the beta diversity analysis did not show significant differences among each stage (Fig. S5). Among the detected bacterial genera, Hymenobacter, Methylobacterium, and Pseudomonas were consistently present from the 3rd to 5th instar, while in adults, these bacterial communities were negligible (Fig. 3A and Fig. S6). In 3rd instar nymphs, Caballeronia was not detected. Still, Caballeronia increased gradually from 4th instar to adult (Fig. 3A). Focusing on the adult stage, a change in the bacterial proportion of the microbiota was observed, in which Caballeronia, Acinetobacter, Cutibacterium, Flavobacterium, and Aquabacterium increased relatively. Serratia was consistently detected in high proportion through the developmental stages. To compare the bacterial community among different stages, a PCoA plot was generated and analyzed by PERMANOVA; results indicated that bacterial community structure differed significantly among the growth stages (3rd instar nymph vs 5th instar nymph: F = 3.01, r2 = 0.333, P = 0.036; 3rd instar nymph vs adult: F = 4.013, r2 = 0.223, P = 0.018; 4th instar nymph vs 5th instar nymph: F = 4.049, r2 = 0.405, P = 0.072; 4th instar nymph vs adult: F = 3.12, r2 = 0.194, P = 0.048; 5th instar nymph vs adult: F = 7.52, r2 = 0.307, P < 0.0001) (Fig. 3B). Alpha-diversity analysis showed that the richness was not significantly different, but evenness showed a significant difference among the samples by month (Fig. S7).Fig. 3 Bacterial diversity at each stage, 3rd instar (n = 3), 4th instar (n = 2), 5th instar (n = 6), and adult (n = 13). A Relative abundance of dominant OTUs with more than 2.0% proportion. B Beta diversity comparisons at the genus level between the different growth stages. Principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity was tested using PERMANOVA with 999 permutations. The confidence ellipsoids of different stages provided by lines indicates 95% confidence intervals

Seasonal Diversity of Gut Bacterial Community

To reveal the seasonal population dynamics of A. spinolae and associated gut bacteria, we monitored the seasonal occurrence of adult A. spinolae from May to September in the Tsurunuma vineyard and investigated their gut microbiota (Fig. 4). The vineyard has hedge-style viticulture and is located on a mountain with rich vegetation. In May, A. spinolae adults were not detected, while over 100 adults were collected in June before decreasing in number in the midsummer, but drastically increasing with more than 300 individuals detected in September (Fig. 4A). Although we collected only a small number of nymphs and adults of A. spinolae inside vineyards in the midsummer season, our preliminary investigation suggests this plant bug proliferates outside vineyards and migrates into vineyards in late summer (data not shown).Fig. 4 Monthly change in bacterial diversity in each A. spinolae adult, June (n = 15), July (n = 11), and Sep (n = 13). A Number of adult A. spinolae collected at a vineyard located in Tsurunuma. B Relative abundance of dominant OTUs with more than 2.0% proportion. C Beta diversity comparisons at the genus level between adult samples from July, June, and September. Principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity tested using PERMANOVA with 999 permutations. The confidence ellipsoids of different months provided by lines indicates 95% confidence intervals

To reveal the seasonal diversity of microbiota in adult A. spinolae, we examined and compared community structure for adults collected in June, July, and September (not enough adults were collected in August for analysis). PCoA plots based on Bray–Curtis showed that microbiota at the phylum level of monthly samples was significantly different between seasons (June vs July: F = 19.918, r2 = 0.454, P < 0.0001; July vs September: F = 3.599, r2 = 0.141, P = 0.0049; June vs September: F = 4.018, r2 = 0.134, P = 0.0002) (Fig. S8). In the bacterial genera, the beta diversity analysis result indicated that the microbiota changed over the months (Fig. 3B and Fig. S9). In June, Methylobacterium, Pseudomonas, Sphingomonas, Enterobacterales, Acinetobacter, and Serratia appeared in many of the insect samples. However, the proportion of Enterobacterales and Acinetobacter decreased in July. Instead of Enterobacterales and Acinetobacter, the proportions of Caballeronia and Flavobacterium increased in the samples. Finally, the microbiota of insects collected in September included a large proportion of the genera Caballeronia, Aquabacterium, Acinetobacter, Cutibacterium, Flavobacterium, and Serratia. However, the proportion of Methylobacterium, Pseudomonas, Sphingomonas, and Enterobacterales decreased gradually from June to September. Otherwise, the genera Caballeronia, Aquabacterium, Acinetobacter, and Cutibacterium increased over the months. Beta-analysis by PCoA plot generated from Bray–Curtis dissimilarity indicated that monthly samples were significantly differed by PERMANOVA (June vs July: F = 5.3452, r2 = 0.185, P < 0.0001; July vs September: F = 7.427, r2 = 0.252, P < 0.0001; June vs September: F = 16.124, r2 = 0.383, P < 0.0001) (Fig. 3C). Alpha-diversity analysis based on generated OTUs showed that the richness and evenness were not significantly different among the samples by month (Fig. S10).

Environmental Factors Affect Caballeronia and Serratia Proportions in A. spinolae

Caballeronia and Serratia were consistently highly detected in investigated individuals (Figs. 2, 3 and 4), but most bacterial groups seem negatively correlated with Serratia following GLM analysis (Table S3). To investigate important factors influencing Serratia proportions, ANOVA was performed and indicated that the proportion of Caballeronia and Serratia from June to September differed significantly (Caballeronia: F = 20.120, r2 = 0.528, P < 0.001; Serratia: F = 3.339, r2 = 0.182, P = 0.047) (Fig. S9 and Table S4). Focusing on the environmental factor, GLM indicated that temperature, precipitation, humidity, and day length had a significant impact on the OTUs numbers of Serratia (Table S5). On the other hand, the climatic conditions did not influence Caballeronia (Table S5).

Phylogenetic Diversity of Caballeronia and Serratia Associated with A. spinolae

To analyze the phylogenetic diversity, more than 2% of bacterial OTUs were selected. Notably, not all but many A. spinolae individuals collected in vineyards consistently possess a certain proportion of Caballeronia (Figs. 2, 3 and 4). The genus Caballeronia is well characterized as a highly-specialized gut symbiotic bacteria of the bean bug Riptortus pedestris and allied stinkbug species [30]. Caballeronia includes over 60 described species and is currently divided into four clades based on phylogenetic analyses: SBE-α, SBE-β, SBE-γ, and Coreoidea-clade [30]. Phylogenetic analysis of Caballeronia OTUs detected from the Tsurunuma population suggested that they belong to the SBE-β clade of Caballeronia (Fig. S11). Additionally, OTUs detected from A. spinolae collected at other vineyards were placed into the clades SBE-α and SBE-β (Fig. S11).

With respect to Serratia, the genus is classified into omnivorous insect-associated group and insect-symbiont group [31–33]. Since the omnivorous insect-associated group includes entomopathogens and phytopathogens, it is often known as an important bacterial group in agriculture [34]. The phylogenetic analysis suggested that the OTUs detected in A. spinolae belong to the omnivorous insect-associated group Serratia (Fig. S12).

Discussion

To clarify community dynamics of associated microbiota in a grapevine pest Apolygus spinolae, we performed high-throughput sequencing of bacterial 16S rRNA gene, revealing that the microbiota was dynamically changed depending on geographic region, season, and insect developmental stage (Figs. 2, 3 and 4). Considering that A. spinolae lacks a gut symbiotic organ (Fig. 1D) and the remarkable diversity of their microbiota, it is plausible that the plant bug has no specific symbionts and acquires its microbiota from the environment every generation, although the insect looks likely to have an affinity for a certain bacterial group such as Caballeronia and Serratia. Even though the whole body of insects was homogenized and subjected to high-throughput sequencing in this study, most of the detected bacteria are thought to be gut bacteria because almost no PCR amplification was detected in the insect tissues after removing the gut. However, some bacterial species such as Spiroplasma, detected only in Hakodate samples (Fig. 2 and Fig. S2), are probably associated with body tissues other than the luminal region of the gut, as reported previously in other insects [35]. There are few studies investigating the geography, season, and insect developmental stages that affect the gut bacterial composition of insect pests, and thus, this report provides an example of how these factors impact insect gut microbiota. However, there was variation in the number of individuals at each collection site, especially for early-stage nymphs which were difficult to collect in the fields due to their small body size. For a general conclusion, further surveys as well as rearing experiments are still needed.

In recent years, the gut microbiota has been actively investigated in some members of the heteropteran family Miridae, including the sorghum plant bug Stenotus rubrovittatus, the small green mirid Nesidiocoris tenuis, the tropical plant bug Monalonion velezangeli, Adelphocoris suturalis, and Apolygus lucorum [12–15, 36, 37]. These studies have led to the finding that gut microbiota composition varies with climate, habitat, diet, and developmental stage, as well as results suggesting that some plant bugs may be vectors of phytopathogen and endophytes [13–15, 36, 38]. As shown here in A. spinolae, Proteobacteria consistently dominate the gut bacterial community in Miridae species, although the composition and dominant bacterial genus are remarkably diverse. While the feeding habits of A. spinolae are not thoroughly investigated, feeding habits of the Miridae are generally diverse from phytophagous to carnivorous through omnivorous, and change throughout their life cycle [10], which may be a factor causing their diverse gut community. Indeed, rearing experiments in Adelphocoris suturalis confirmed the diet-dependent effects on gut microbiota composition in this plant bug species [36].

In the rearing experiments on Adelphocoris suturalis, the proportion of Serratia increased when the insect was reared under carnivorous or omnivorous conditions [36], suggesting a metabolic function of this bacterial group when plant bugs feed on other insects. In addition, Serratia species are commonly detected in the gut of carnivorous and omnivorous insects, including mosquitoes (Aedes aegypti), fruit fly (Drosophila), and ladybugs (Harmonia axyridis) [39–41]. Serratia species are known to have urea metabolism and secrete highly active proteases and chitinases in insect bodies, suggesting that Serratia support host digestion [42]. If so, it may be notable that Serratia shows seasonal changes in A. spinolae (Fig. 4, Table S4, and Table S5). Although speculative, the dynamics of Serratia suggest the seasonal changes in the feeding habits of A. spinolae in grapevine fields. On the other hand, considering the Serratia OTUs detected in this study were closely related to S. marcescens, a well-known entomopathogenic bacterium [43, 44] (Fig. S12), this bacterial group is possibly a seasonal gut pathogen of A. spinolae. It should be noted that, in A. suturatis, dietary change from herbivorous to omnivorous/carnivorous enhanced S. marcescens in the gut, which increased mortality [36]. Although the biological functions of Serratia in A. spinolae are still unclear at this stage, rearing experiments of the insect inoculated with S. marcescens would clarify how the bacterial species influences the grapevine pest bug.

In addition to Serratia, members of Caballeronia were also consistently detected in A. spinolae. The genus Caballeronia is a well-known gut symbiont in several stinkbug species of the infraorder Pentatomomorpha [8, 30, 45], wherein the symbionts are acquired from soil every generation and associated with the crypt-bearing M4 midgut region [45]. Although A. spinolae has no crypts in its gut, the consistent and abundant detection of Caballeronia probably indicates their biological functions in the plant bug. For instance, in pentatomomorphan stinkbugs, Caballeronia symbionts play important roles in recycling metabolic wastes and in insecticide detoxification [46–48], and this bacterial group may play similar biological functions in the plant bug host. The evolutionary process of the specific stinkbug-Caballeronia gut symbiosis is totally unknown because insects that are prevalently associated with Caballeronia are scarcely reported, other than pentatomomorphan stinkbugs, and their lack of an intermediate state. In this context, the consistent and frequent association of Caballeronia in A. spinolae may provide a clue as to how the stinkbug-Caballeronia gut symbiosis evolved from a preliminary gut association to the highly-evolved crypt-associating symbiosis. Not only to manage the pest plant bug but also to reveal the evolutionary process of gut symbiosis in heteropteran insects, it would be of great interest to clarify microbial dynamics and biological functions of Caballeronia in A. spinolae in the future.

Among the other bacteria detected in A. spinolae, it is notable that plant bugs collected in Hakodate had a high percentage of Spiroplasma (Fig. 2). S. citri, causing citrus stubborn disease (CSD), and S. kunkelii, causing corn stunt in maize, are known as typical phytopathogenic bacteria in the Spiroplasma family [49, 50]. Some of the Spiroplasma infecting insects cause the well-known male killer phenomenon in Drosophila [35]. Among the Spiroplasma species detected in this study, not only were insect parasites detected, but also OTUs classified in the same group as phytopathogens, S. citri and S. kunkelii (Fig. S4). This preliminary result based on phylogenetic analysis of 16S rRNA V4 region, suggests that, although speculative, A. spinolae may be a vector of phytopathogenic Spiroplasma. However, further field survey and experimental investigation should be conducted to confirm this possibility.

In this study, we comprehensively analyzed the gut microbiota of vineyard populations of A. spinolae. To develop an integrated pest management (IPM) strategy, it is necessary to elucidate this pest’s detailed developmental status and life cycle in vineyards. The clarification of the relationship and function between insects and gut symbiotic bacteria from this study not only leads to elucidating the basic ecology of the plant bug but also has great potential to be developed into agricultural technology.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (PPTX 9983 KB)

Supplementary file2 (XLSX 11 KB)

Supplementary file3 (XLSX 12 KB)

Supplementary file4 (XLSX 24 KB)

Supplementary file5 (XLSX 14 KB)

Supplementary file6 (XLSX 12 KB)

Supplementary file7 (XLSX 13 KB)

Acknowledgements

We thank the researchers at the National Institute of Advanced Industrial Science and Technology (AIST) and local vineyard farmers for their help with collecting A. spinolae samples. Hideomi Ito and Kazumori Mise gave valuable advice about bioinformatic analysis. Madoka Miyazaki contributed insect survey and PCR assay. Antoine-Olivier Lirette verified the use of English in this paper.

Author Contribution

HM, T. Sato, T. Sone, and YK designed and developed the study. HM, KI, T. Sato, T. Sone, and YK collected insect samples. HM and KI performed 16S rRNA amplicon sequencing and data analysis. HM and KI performed statistical analysis. HM and YK wrote the manuscript with input from all authors.

Funding

This study was supported by NOASTEC (S-3–7) to YK, JSPS KAKENHI to T. Sato (21K20579), and JSPS research fellowship for Young Scientists to KI (22KJ0057).

Data Availability

All sequence data obtained in this study were deposited in the DDBJ/GenBank/EBI, under accession number PRJDB17462.

Declarations

Competing Interests

The authors declare no competing interests.
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