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Effects of Host Plant and Insect Generation on Shaping of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis

Effects of Host Plant and Insect Generation on Shaping of the Gut Microbiota in the Rice... fmicb-13-824224 April 5, 2022 Time: 19:11 # 1 ORIGINAL RESEARCH published: 11 April 2022 doi: 10.3389/fmicb.2022.824224 Effects of Host Plant and Insect Generation on Shaping of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis 1 1,2 1 2 1 Yajun Yang , Xiaogai Liu , Hongxing Xu , Yinghong Liu * and Zhongxian Lu * State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, College of Plant Protection, Southwest University, Chongqing, China Gut microbes in insects may play an important role in the digestion, immunity and protection, detoxification of toxins, development, and reproduction. The rice leaffolder Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Crambidae) is a notorious insect pest that can damage rice, maize, and other gramineous plants. To determine the effects of host plants and generations on the gut microbiota of C. medinalis, we deciphered the bacterial configuration of this insect pest fed rice or maize for three generations Edited by: by Illumina MiSeq technology. A total of 16 bacterial phyla, 34 classes, 50 orders, 101 F. L. Consoli, University of São Paulo, Brazil families, 158 genera, and 44 species were identified in C. medinalis fed rice or maize for Reviewed by: three generations. Host plants, insect generation, and their interaction did not influence Abrar Muhammad, the alpha diversity indices of the gut microbiota of C. medinalis. The dominant bacterial Zhejiang University, China Bruna Laís Merlin, taxa were Proteobacteria and Firmicutes at the phylum level and Enterococcus and University of São Paulo, Brazil unclassified Enterobacteriaceae at the genus level. A number of twenty genera coexisted *Correspondence: in the guts of C. medinalis fed rice or maize for three generations, and their relative Yinghong Liu abundances occupied more than 90% of the gut microbiota of C. medinalis. A number [email protected] Zhongxian Lu of two genera were stably found in the gut of rice-feeding C. medinalis but unstably [email protected] found in the gut microbiota of maize-feeding C. medinalis, and seven genera were stably found in the gut of maize-feeding C. medinalis but unstably found in the gut of rice- Specialty section: This article was submitted to feeding C. medinalis. In addition, many kinds of microbes were found in some but not all Systems Microbiology, samples of the gut of C. medinalis fed on a particular host plant. PerMANOVA indicated a section of the journal Frontiers in Microbiology that the gut bacteria of C. medinalis could be significantly affected by the host plant and Received: 29 November 2021 host plant  generation. We identified 47 taxa as the biomarkers for the gut microbiota Accepted: 09 March 2022 of C. medinalis fed different host plants by LEfSe. Functional prediction suggested that Published: 11 April 2022 the most dominant role of the gut microbiota in C. medinalis is metabolism, followed Citation: Yang Y, Liu X, Xu H, Liu Y and by environmental information processing, cellular processes, and genetic information Lu Z (2022) Effects of Host Plant processing. Our findings will enrich the understanding of gut bacteria in C. medinalis and Insect Generation on Shaping and reveal the differences in gut microbiota in C. medinalis fed on different host plants of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis. for three generations. Front. Microbiol. 13:824224. doi: 10.3389/fmicb.2022.824224 Keywords: rice leaffolder, gut bacteria, host plant, Lepidoptera, rice, maize Frontiers in Microbiology | www.frontiersin.org 1 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 2 Yang et al. Gut Microbiota in C. medinalis colonization (Hammer et al., 2014). Nevertheless, microbiota INTRODUCTION are abundant and diverse in many species of Lepidoptera. Insects harbor numerous microorganisms in the gut (Douglas, Proteobacteria and Firmicutes were found to be dominant in 2015). Gut microorganisms in insects have been shown to the gut of the diamondback moth Plutella xylostella (L.) based contribute to digestion (Anand et al., 2010; Jing et al., 2020), on the high-throughput DNA sequencing data (Xia et al., detoxification (Ceja-Navarro et al., 2015; Beran and Gershenzon, 2013). Enterococcus and Lactococcus were dominant bacteria in 2016; Blanton and Peterson, 2020), development (Wang et al., a field population of Helicoverpa armigera Hubner, followed 2018b; Qiao et al., 2019; Pyszko et al., 2020), physiology (Engel by Flavobacterium, Acinetobacter, and Stenotrophomonas (Xiang and Moran, 2013; Xu et al., 2019; Liberti and Engel, 2020), et al., 2006). The composition of microbes in the insect gut pathogen resistance (Dillon and Dillon, 2004; Voirol et al., could be affected by many factors. The environmental habitat, 2018; Moore and Aparicio, 2022), immune response (Engel diet, developmental stage, and phylogeny of the host could and Moran, 2013; Li et al., 2020; Li et al., 2021), and the determine the bacterial diversity in the insect gut (Yun et al., production of essential vitamins and amino acids (Hansen and 2014). In the larvae of Spodoptera littoralis (Boisduval), bacterial Moran, 2014; Jang and Kikuchi, 2020; Jing et al., 2020). For communities were shown to be instar-specific (Chen et al., 2016). instance, some microorganisms with metabolic characteristics In addition, host plants were observed to have a considerable could promote insect adaptation to host plants (Voirol et al., effect on the composition of gut bacteria in Henosepilachna 2018). The gut microbiota was found to function in the protection vigintioctopunctata (F.) (Lü et al., 2019). of a European Bombus species against the intestinal pathogen The rice leaffolder Cnaphalocrocis medinalis (Guenée) Crithidia bombi (Koch and Schmid-Hempel, 2011). Another (Lepidoptera: Crambidae) is an important insect pest in Asia example in Helicoverpa zea (Boddie), Enterbacter ludwigii, a that can damage rice (Oryza sativa L.), maize, and other gut-associated bacterium, could indirectly trigger the defense gramineous plants (Barrion et al., 1991; Cheng, 1996; Yang et al., of tomato (Solanum lycopersicum L.) and maize (Zea mays L.) 2015). The heavy occurrence of this insect could cause serious (Wang et al., 2017, 2018a). Chung et al. (2013) documented economic loss to rice production (Yang et al., 2015). In 2015, that the Colorado potato beetle Leptinotarsa decemlineata C. medinalis damaged rice plants with an area of 15.5 million ha (Say) suppressed the defenses of tomatoes by exploiting orally and caused yield losses of 0.47 million tons in China (Yang et al., secreted bacteria. The gut microbiota of the pine weevil 2015; Lu, 2017). Based on the traditional isolation and culture (Hylobius abietis) degrades conifer diterpenes and increases methods, 25 species of 15 phyla of gut microbiota were obtained insect fitness (Berasategui et al., 2017). Gut microbes may from C. medinalis larvae (Yang, 2012). By comparison, a large facilitate insect herbivory to chemically defend plants (Hammer number of gut microbiota were obtained from C. medinalis and Bowers, 2015). Gut symbionts could enhance insecticide larvae through Illumina MiSeq technology (Liu et al., 2016). resistance in a significant pest, the oriental fruit fly Bactrocera Yang et al. (2020a) analyzed the gut microbiota composition dorsalis (Hendel) (Cheng et al., 2017). Insect symbionts could of C. medinalis across the developmental stages. Information influence insect–plant interactions at different levels through on the host-associated changes in gut bacteria will facilitate direct interactions and also through indirect plant-mediated the overall understanding of insect ecology and promote the interactions (Frago et al., 2012). Given the importance of the development of novel methods for pest management. This study associated microorganisms to host fitness and feeding ecology, illustrates the composition and diversity of the gut microbiota an effort to manipulate these partnerships and render insect pests in C. medinalis feeding on rice or maize for three generations by more vulnerable to broad-scale measures of population control Illumina MiSeq technology. The findings in this study will enrich by targeting the bacterial symbionts was one of the important the understanding of the gut microbiota in C. medinalis and applications in gut symbiont-driven pest control (Berasategui provide novel insight into the relationship between C. medinalis et al., 2016). The functions of gut microbes could provide a and its host plants. novel concept for the application of bacteria in pest control through the restraint of the insect immune response and the MATERIALS AND METHODS induction of plant defense (Kyritsis et al., 2017) and promote the understanding of gut symbiont-driven pest control (Frago et al., 2012; Berasategui et al., 2016). Insect Rearing and Sampling Lepidoptera is one of the largest insect orders and has Adults of C. medinalis were collected from paddy fields in approximately 160,000 described species (Mitter et al., 2017). Hangzhou, Zhejiang Province, East China and then cultured with Some of them can damage agricultural crops and cause large 10% honey solution in the laboratory under controlled conditions economic losses (Wagner, 2013). However, the evidence of the of 26  1 C temperature, 70  10% relative humidity, and a fundamental function of bacteria in lepidopteran biology is photoperiod of 16:8 (L:D) h. The neonates of the population were scarce. Furthermore, a recent study from Hammer et al. (2017) divided into two groups. One was reared using rice plants, and the reported that caterpillars lack a resident microbiome in the gut other was reared using maize plants. Every group was reared for compared with other insect orders. The authors of this study three generations. Rice and maize were planted in pots (one plant argued that caterpillars with rough environments may prevent per pot) in the greenhouse. The leaves of plants were collected and bacterial colonization. Lepidopteran reshaping the body structure rinsed with sterile ddH O and then air-dried before feeding them during metamorphosis also enhances the difficulty of bacterial to C. medinalis, and sufficient leaves were provided for the insects. Frontiers in Microbiology | www.frontiersin.org 2 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 3 Yang et al. Gut Microbiota in C. medinalis Guts of C. medinalis were dissected from the fifth instar larvae distances was applied among all the bacterial groups. Non-metric of both groups from every generation. A total of fifteen guts multidimensional scaling (NMDS) plots were constructed using were pooled into a biological sample, and three replicates were Bray–Curtis. Analysis of similarity (ANOSIM) was used to test prepared for each treatment. Before dissection, the whole larva the difference in the composition of microbiota among different was rinsed with sterile ddH O, disinfected with ethanol (75%) for group samples. Permutational multivariate analysis of variance 90 s, and rinsed again with sterile ddH O. Following dissection, (PerMANOVA) was generated using 999 permutations, and the the guts were collected into a 1.5-ml sterile tube and stored individual repeats were included in the model as a random effect. at –80 C until use. PCoA, NMDS, ANOSIM, and PerMANOVA were analyzed and graphed using R software. Linear discriminant analysis (LDA) was used to screen the biomarkers for significant differences DNA Extraction and PCR Amplification between different groups with LDA scores greater than two. The dissected guts were homogenized by shaking in a sterile A cladogram was drawn to show the distribution of these tube containing sterile glass beads (0.5 mm diameter) and 0.5 ml biomarkers at different taxonomic levels by Galaxy (accessed of PBS buffer (pH 7.5) for 15 min using a vortex. Total DNAs on 1 January 2022). Microbiota functions were predicted by were extracted from samples using the E.Z.N.A. bacteria DNA annotating pathways of OTUs against the Ref99NR database extract kit (OMEGA, United States) according to the instructions. using R software with the Tax4Fun2 package. The primers 515F 5’-GTGCCAGCMGCCGCGG-3’ and 907R 5’-CCGTCAATTCMTTTRAGTTT-3’ were used to amplify the V4-V5 regions of the bacterial 16S ribosomal RNA gene through PCR (95 C for 2 min, followed by 25 cycles at 95 C for 30 s, RESULTS 55 C for 30 s, and 72 C for 30 s and a final extension at 72 C for 5 min). Amplicons were generated in a 20 ml reaction system Reads Analyzed and Taxa Generated containing 4 ml of 5  FastPfu Buffer, 2 ml of 2.5 mM dNTPs, 0.8 We sequenced the gut microbes of C. medinalis fed on different ml of each primer (5 mm), 0.4 ml of FastPfu Polymerase, and 10 ng host plants for three generations and obtained 1,473,836 trimmed of template DNA. Blank DNA as a negative control was extracted, paired reads in total (Supplementary Table 1). Blank DNA and and products generated from no-template PCR were sequenced no-template PCR sequencing were used for decontamination, to assess what sequences are contaminants. and sequences of cyanobacteria or chloroplasts were found to be contaminants. After decontamination, 446 OTUs were obtained. Illumina MiSeq Sequencing The OTU numbers of C. medinalis from different samples varied Amplicons were extracted and purified using the AxyPrep from 49 to 194 (Table 1). The Ace index varied from 62.93 DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, to 252.14, the Chao1 index varied from 57.27 to 256.25, the United States) according to the manufacturer’s instructions and Shannon index varied from 0.47 to 1.36, and the Simpson index TM quantified using QuantiFluor -ST (Promega, United States). varied from 0.46 to 0.87 (Table 1). ANOVA indicated that alpha Then, they were pooled in equimolar amounts and paired-end diversity indices were not significantly affected by the host plant, sequenced (2  250) on an Illumina MiSeq platform according to generation, or their interaction (Supplementary Table 2). A total the standard protocols. of 16 bacterial phyla, 34 classes, 50 orders, 101 families, 158 genera, and 44 species were identified in C. medinalis fed rice or Bioinformatic and Statistical Analyses maize for three generations (Table 2). Raw FASTQ files were demultiplexed and quality-filtered using QIIME (version 1.17). According to the similarity of the Gut Microbiota of Cnaphalocrocis sequences, effective sequences were classified into multiple medinalis Fed Rice for Three operational taxonomic units (OTUs) at a similarity level of Generations 97% using UPARSE (version 7.1), and chimeric sequences were At the phylum level, Firmicutes, Proteobacteria, Actinobacteria, identified and removed using UCHIME. All the sequences were Bacteroidetes, and unclassified Bacteria were found in the gut annotated and blasted against the Silva (SSU115)16S rRNA microbiota of C. medinalis fed on rice plants through all database using a confidence threshold of 70% for each 16S rRNA samples of three generations. Among them, Firmicutes was the gene sequence analyzed by RDP Classifier. absolute dominant phylum with the highest relative abundance Alpha diversity was estimated through five indices: OTU in rice-feeding C. medinalis for three generations (70.62– number, ACE, Chao1, Shannon, and Simpson’s index. The 87.53%) (Figure 1A). The relative abundance of Proteobacteria alpha diversity and relative abundance data were analyzed using was 10.51–26.88%, followed by Actinobacteria (1.00–4.66%), one-way analysis of variance (ANOVA) with SPSS 26.0 (IBM Bacteroidetes (0.41–0.43%), and unclassified Bacteria (0.02– SPSS Statistics), and multiple comparisons were analyzed using 0.14%) (Figure 1A). At the family level, 18 families were found in Tukey’s test. Venn diagrams and stack bars were graphed by the gut microbiota of C. medinalis fed rice through all samples of R software. Principal coordinate analysis (PCoA) based on the three generations. Enterococcaceae and Enterobacteriaceae were matrices of pairwise weighted UniFrac distances and Bray–Curtis the two major families in the rice-feeding C. medinalis for three http://drive5.com/uparse/ 2 3 http://rdp.cme.msu.edu/ http://huttenhower.sph.harvard.edu/galaxy/ Frontiers in Microbiology | www.frontiersin.org 3 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 4 Yang et al. Gut Microbiota in C. medinalis generations, with relative abundance ranges of 70.55–87.27% and respectively. Enterococcus and unclassified Enterobacteriaceae are 9.12–24.75%, respectively (Figure 1B). The relative abundance of the majority. In addition to the microbes found in all samples Anaplasmataceae in the gut of the third generation of C. medinalis of rice-feeding C. medinalis for three generations, many kinds of fed rice was higher than that of the second generation of microbes were found in some but not all samples of rice-feeding C. medinalis, and the relative abundance of Nocardiaceae in C. medinalis gut. the gut of the first generation of C. medinalis fed rice was Gut Microbiota of Cnaphalocrocis higher than that of the other two generations of C. medinalis (Figure 1B). At the genus level, 21 genera were found in the medinalis Fed Maize for Three gut microbiota of C. medinalis fed rice through all samples of Generations three generations. Enterococcus, unclassified Enterobacteriaceae, Similar to rice-feeding C. medinalis, the same six phyla were Pectobacterium, Corynebacterium, Leucobacter, and Anaplasma found in the gut microbiota of maize-feeding C. medinalis occupied the top 10 in the gut microbiota of C. medinalis fed through all samples of three generations. The phylum with rice for three generations (Supplementary Table 3). Common the highest relative abundance was Firmicutes (68.49–80.25%), genera found in all three generations occupied 93.95, 98.51, followed by Proteobacteria (16.58–27.65%), Actinobacteria (1.62– and 97.78% of the first generation to the third generation, 2.01%), Bacteroidetes (0.41–0.83%), and unclassified Bacteria (0.004–0.29%) (Figure 2A). At the family level, 20 families found gut microbiota of C. medinalis fed maize through all samples TABLE 1 | Alpha diversity indices of gut bacterial communities in rice- or of three generations. Enterococcaceae and Enterobacteriaceae maize-feeding Cnaphalocrocis medinalis for three generations. were also the dominant families, with relative abundance ranges of 67.88–80.23% and 14.69–24.42%, respectively (Figure 2B). Sample Alpha diversity indices The relative abundance of Comamonadaceae in the gut of OTU number ACE Chao1 Shannon Simpson the third generation of C. medinalis fed maize was higher than that of the first generation of C. medinalis, the relative R1-1 201 252.14 256.25 1.33 0.60 abundance of Micrococcaceae in the second generation of R1-2 59 85.09 75.15 0.88 0.60 C. medinalis was higher than that of the first generation of R1-3 49 62.93 57.27 0.58 0.77 C. medinalis, and the relative abundance of Rhodocyclaceae in R2-1 162 210.16 225.14 0.52 0.84 the gut of the second generation of C. medinalis was higher R2-2 157 194.09 200 0.47 0.87 than that of the third generation of C. medinalis (Supplementary R2-3 183 221.45 228.12 0.90 0.62 Table 3). At the genus level, 26 genera were found in the gut R3-1 85 107.55 110.07 0.95 0.59 microbiota of C. medinalis fed maize through all samples of R3-2 61 117.04 119 0.94 0.56 three generations. Enterococcus, unclassified Enterobacteriaceae, R3-3 145 178.45 184.26 1.36 0.46 Corynebacterium, unclassified Comamonadaceae, Leucobacter, M1-1 194 242.48 231.66 1.20 0.52 Microbacterium, Anaplasma, and Sphingobacterium occupied the M1-2 69 95.25 81.67 0.81 0.65 top 10 in the gut microbiota of C. medinalis fed maize for three M1-3 135 184.21 197.67 0.52 0.85 generations (Supplementary Table 3). Common genera found in M2-1 171 207.09 212.25 1.17 0.52 all three generations occupied 97.75, 96.34, and 99.29% of the first M2-2 165 189 180.53 1.26 0.46 generation to the third generation, respectively. Enterococcus and M2-3 159 194.74 222.07 1.15 0.58 unclassified Enterobacteriaceae are the majority. In addition to M3-1 96 120.32 113.25 0.95 0.52 the microbes found in all samples of maize-feeding C. medinalis M3-2 110 144.6 153.5 0.94 0.58 for three generations, many kinds of microbes were found in M3-3 98 108.44 111.91 0.81 0.59 some but not all samples of maize-feeding C. medinalis gut. R1–R3: the first to third generation of C. medinalis fed on rice; M1–M3: the first to third generation of C. medinalis fed on maize. Influence of Host Plant and Insect Generation on the Gut Bacterial TABLE 2 | Number of identified gut bacterial taxonomic categories in rice- and Communities of Cnaphalocrocis maize-feeding Cnaphalocrocis medinalis for three generations. medinalis Treatments Phylum Class Order Family Genus Species Comparing the gut microbiota between C. medinalis fed R1 12 28 39 69 99 30 rice and maize, five phyla and 16 families were found in R2 13 30 42 86 118 36 all samples of the three generations. At the genus level, 19 R3 11 23 33 68 93 30 genera were found in the gut microbiota of C. medinalis fed M1 13 29 40 74 106 32 on rice or maize plants for three generations (Supplementary M2 12 26 42 85 115 37 Table 3). The relative abundance of these genera occupied M3 5 9 21 51 71 31 more than 90% of the gut microbiota of C. medinalis fed rice or Total 16 34 50 101 158 44 maize plants, and the two major genera were Enterococcus and a unclassified Enterobacteriaceae (Figure 3). Seven genera, Bacillus, R1–R3, the first to third generation of C. medinalis fed rice; M1–M3, the first to third generation of C. medinalis fed maize. Empedobacter, Flavobacterium, Rhizobium, Rhodococcus, Frontiers in Microbiology | www.frontiersin.org 4 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 5 Yang et al. Gut Microbiota in C. medinalis FIGURE 1 | Relative abundance of the gut microbiota from rice-feeding C. medinalis for three generations at the phylum (A) and family (B) levels. R1–R3: the first to third-generation of C. medinalis fed on rice. FIGURE 2 | Relative abundance of the gut microbiota from maize-feeding C. medinalis for three generations at the phylum (A) and family (B) levels. M1–M3: the first to third generation of C. medinalis fed on maize. Frontiers in Microbiology | www.frontiersin.org 5 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 6 Yang et al. Gut Microbiota in C. medinalis 77.73 and 71.25% of the total OTUs of the first generation of C. medinalis fed rice or maize, were shared by C. medinalis fed rice or maize (Figure 6A). The second generation of C. medinalis fed on rice or maize shared 243 OTUs, which accounted for 85.56 and 86.17% of the total OTUs of the second generation of C. medinalis fed on rice or maize, respectively (Figure 6B). The third generation of C. medinalis fed on rice or maize shared 82 OTUs, which accounted for 40.39 and 64.57% of the total OTUs of the third generation of C. medinalis fed on rice or maize, respectively (Figure 6C). To find the biomarkers with significant differences between different groups, LDA effect size (LEfSe) was used to screen out different taxa at various levels (kingdom, phylum, class, order, family, genus, and species) between different groups based on a standard LDA value greater than two (Figure 7). Meanwhile, the cladogram from phylum to genus was graphed to fully understand the distribution of these different taxa at various taxonomic levels (Figure 8). In the third generation of C. medinalis fed maize (M3), the gut microbiota had the most of the different taxa (LDA > 2). There were 60 taxa FIGURE 3 | Relative abundance of the genera in the gut microbiotas found in mainly belonging to Firmicutes, Bacteroidota, Acidobacteriota, all samples of C. medinalis. R1–R3: the first to third generation of C. medinalis Proteobacteria, Actinobacteria, and Ignavibacteriae. A total of fed rice; M1–M3: the first to third generation of C. medinalis fed maize. six taxa belonging to Actinobacteria were in the gut microbiota of the first generation of C. medinalis fed rice (R1). A total of two taxa belonging to Proteobacteria and one taxon belonging Sphingobacterium, and unclassified Beutenbergiaceae, were stably to Bacteroidetes were in the gut microbiota of the second found in all samples of maize-feeding C. medinalis for three generation of C. medinalis fed rice (R2). A total of two taxa generations, whereas Tsukamurella and Ochrobactrum were belonging to Proteobacteria were in the gut microbiota of the stably found in all samples of rice-feeding C. medinalis for three third generation of C. medinalis fed rice (R3). A total of five generations (Supplementary Table 4). taxa belonging to Proteobacteria were in the gut microbiota of Principal coordinate analysis based on the Bray–Curtis the first generation of C. medinalis fed maize (M1). A total distance and weighted UniFrac distance was used to compare of three taxa belonging to Proteobacteria, five taxa exclusive to the community similarities between samples. The PCoA scatter Ignavibacteriae, five taxa belonging to Firmicutes, and four taxa plot showed that the abscissa and ordinate represent the two belonging to Actinobacteria were in the gut microbiota of the characteristic values that contribute to the largest differences second generation of C. medinalis fed maize (M2). A total of between the samples, and their influence degrees were 74.09 fourteen taxa belonging to Proteobacteria, seven taxa belonging and 14.73% based on weighted UniFrac distance (Figure 4A) to Actinobacteria, and six taxa belonging to Bacteroidetes were and 65.61 and 18.16% based on the Bray–Curtis (Figure 4B), in the gut microbiota of the third generation of C. medinalis respectively. PerMANOVA showed that there were significant fed on maize (M3). LEfSe was also used to find the biomarkers differences in the gut microbiota of rice- and maize-feeding with significant differences between samples fed different host C. medinalis (Table 3; PerMANOVA: R = 0.35, p = 0.001). Host plants (Supplementary Figure 1). A total of forty-seven taxa were plant  generation significantly affected the gut microbiota of identified as the biomarkers in the gut microbiota of C. medinalis C. medinalis (Table 3; PerMANOVA: R = 0.28, p = 0.004). No fed on different host plants (Supplementary Figure 2). A total significant differences were observed between the samples from of six taxa belonging to Actinobacteria and one taxon belonging different generations of C. medinalis (Table 3; PerMANOVA: to Proteobacteria were in the gut microbiota of C. medinalis fed R = 0.02, p = 0.751). rice. Nineteen taxa belonging to Proteobacteria, 10 taxa belonging Non-metric multidimensional scaling analysis revealed to Bacteroidetes, and 11 taxa belonging to Actinobacteria were in significant differences between the gut microbiota of rice- the gut microbiota of C. medinalis fed maize. and maize-feeding C. medinalis (Figure 5). ANOSIM showed that there were significant differences in the gut microbiota of Functional Prediction of the Gut rice- and maize-feeding C. medinalis (R = 0.5538, p = 0.001) (Supplementary Table 5). There were no significant differences Microbitota of Cnaphalocrocis medinalis in the gut microbiota of rice- and maize-feeding C. medinalis in To better understand the important role of the gut microbiota the same generations (Supplementary Table 5). of C. mednialis, we used R software with Tax4Fun2 to predict Venn diagrams showed overlapping OTUs of C. medinalis fed the function in samples based on 16S rDNA sequencing data on rice or maize from the first generation to the third generation and compared them with the Ref99NR database (Figure 9). (Figure 6). The results indicated that 171 OTUs, which comprised The results showed that the most functional prediction Frontiers in Microbiology | www.frontiersin.org 6 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 7 Yang et al. Gut Microbiota in C. medinalis FIGURE 4 | PCoA bacterial communities of C. medinalis fed on different host plants over generations based on weighted UniFrac (A) and Bray–Curtis (B) distances. M1–M3: the first to third generation of C. medinalis fed on maize; R1–R3: the first to third generation of C. medinalis fed on rice. categories were related to metabolism (70.12–71.18%) followed DISCUSSION by environmental information processing (16.17–17.08%), cellular processes (5.39–5.94%), and genetic information This study profiled the gut bacterial community in C. medinalis processing (3.87–4.06%). In the metabolism category, global fed on different host plants for three generations. Considering and overview maps had the highest abundance (34.18– the limited gut bacterial information isolated and cultured by 34.73%) followed by carbohydrate metabolism (14.93–15.75%), traditional methods (Yang, 2012), we obtained the bacterial amino acid metabolism (5.08–5.45%), energy metabolism information of C. medinalis by MiSeq sequencing. Recently, we (3.00–3.10%), metabolism of cofactors and vitamins (2.16– reported that the composition of the gut bacterial community 2.40%), nucleotide metabolism (2.18–2.26%), lipid metabolism changes across the life cycle of C. medinalis, and the phyla (2.02–2.17%), xenobiotics biodegradation and metabolism Proteobacteria and Firmicutes were the dominant bacterial (1.34–1.55%), biosynthesis of other secondary metabolites (1.31– taxa (Yang et al., 2020a). In the guts of both C. medinalis 1.35%), metabolism of other amino acids (1.12–1.28%), glycan fed rice and maize, the phyla Proteobacteria and Firmicutes biosynthesis and metabolism (1.08–1.14%), and metabolism of were also the dominant bacterial taxa. In this study, host terpenoids and polyketides (0.74–0.85%). In the environmental plants, generation, and their interaction did not significantly information processing category, membrane transport had affect the alpha diversity indices of the gut microbiota in the highest abundance (12.05–12.87%) followed by signal C. medinalis. Ace and choa1 values indicated that community transduction (4.08–4.26%). In the cellular processes category, richness did not differ among the different groups. Shannon the cellular community had the highest abundance (3.85–4.21%) and Simpson values indicated that community diversity did followed by cell motility (0.88–1.06%), cell growth and death not differ among the different groups. The experimental results (0.49–0.52%), and transport and catabolism (0.14–0.15%). In the provide a more comprehensive understanding of the relationship genetic information processing category, replication and repair between C. medinalis and its microbiota. Our results revealed had the highest abundance (1.55–1.64%), followed by translation the influence of host plants and insect generation on the gut (1.45–1.53%) and folding, sorting, and degradation (0.76–0.79%). bacterial community in C. medinalis and provide a foundation for investigating gut microbe C. medinalis–host plant interactions. Diet is one of the important factors for insect development (Karley et al., 2002; Qubaiová et al., 2021), and it also plays an important role in shaping insect phenotypes and gut microbial TABLE 3 | PERMANOVA of the bacterial communities of C. medinalis fed rice or maize for three generations. communities (Colman et al., 2012; Xu et al., 2019; Luo et al., 2021; Mason et al., 2021). Host diet could influence the diversity, Source df SS MS Pseudo-F R p-value structure, or composition of the gut in many insects (Strano Host plant 1.17 1.5924 1.5924 8.66 0.35 0.001 et al., 2018; Lü et al., 2019; Leite-Mondin et al., 2021; Yuan Generation 1.17 0.1086 0.1086 0.39 0.02 0.751 et al., 2021). Leite-Mondin et al. (2021) discovered that the gut Host plant  Generation 1.17 1.2699 1.2699 6.22 0.28 0.004 microbiota composition of Trichoplusia ni (Hubner) altered by diet may influence its polyphagous behavior. An imbalanced PERMANOVA was generated using 999 permutations, and the individual repeat was included in the model as a random effect. diet-altered variation in gut microbiota is detrimental to mirid Frontiers in Microbiology | www.frontiersin.org 7 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 8 Yang et al. Gut Microbiota in C. medinalis FIGURE 5 | NMDS analysis of the bacterial communities of C. medinalis fed maize and rice for three generations. NMDS plots were constructed using Bray–Curtis with the main groups of host plants (A) and generations (B). Stress values (stress = 0.079) indicate a good fit in two dimensions. M: samples from C. medinalis fed maize; R: samples from C. medinalis fed rice. FIGURE 6 | Venn diagram of bacterial community OTUs of C. medinalis fed different host plants for three generations. M1–M3: the first to third generation of C. medinalis fed on maize; R1–R3: the first to third generation of C. medinalis fed on rice. (A) M1 vs. R1; (B) M2 vs. R2; (C) M3 vs. R3. bugs, Adelphocoris suturalis Jakovlev (Luo et al., 2021). In fed rice or maize, whereas their relative abundances occupied this study, at the family and genus levels, the composition of more than 90% of the gut microbiota of C. medinalis fed rice the gut microbiota of C. medinalis differed between the host or maize. In addition, we found that two genera (Tsukamurella plants. Among the genera found in the gut of C. medinalis fed and Ochrobactrum) were stable in the gut of rice-feeding different host plants, only 21 genera were found in all samples C. medinalis, but unstable in the gut microbiota of maize- of three generations of rice-feeding C. medinalis, and only 26 feeding C. medinalis, and seven genera (Bacillus, Empedobacter, genera were found in all samples of three generations of maize- Flavobacterium, Rhizobium, Rhodococcus, Sphingobacterium, and feeding C. medinalis. These results indicated that most kinds unclassified Beutenbergiaceae) were stable in the gut of maize- of microbes are not stably colonized in the gut of C. medinalis feeding C. medinalis, but unstable in the gut of rice-feeding fed a particular host plant. Hammer et al. (2017) reported C. medinalis. For example, some genera that were stable in the that caterpillars lack a resident gut microbiome. Jones et al. gut of maize-feeding C. medinalis were found in some but not (2019) found high variability in gut bacterial composition and all samples of rice-feeding C. medinalis. The gut bacteria that abundance between the individuals of the same insect species were stable in the gut of C. medinalis for three generations may even fed on the same food source. The reports from other have an important role in shaping the microbiota community lepidopteran species showed that gut microbial assemblages in C. medinalis. Through LEfSe, 47 taxa were found to be the differed between individuals (Priya et al., 2012; Staudacher et al., biomarkers for the gut microbiota of C. medinalis fed different 2016). In this study, only 19 genera coexisted in C. medinalis host plants. Stable host-related bacteria may function to help Frontiers in Microbiology | www.frontiersin.org 8 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 9 Yang et al. Gut Microbiota in C. medinalis FIGURE 7 | Bacterial taxa with LDA scores greater than two in the gut microbiota of C. medinalis fed different host plants for three generations. Frontiers in Microbiology | www.frontiersin.org 9 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 10 Yang et al. Gut Microbiota in C. medinalis FIGURE 8 | Cladogram of bacterial biomarkers, from the phylum (innermost ring) to genus (outermost ring) level, with an LDA score > 2. Differential bacterial taxa are marked by lowercase letters. Each small circle at different taxonomic levels represents a taxon at that level, and the diameter of the circle is proportional to the relative abundance. The coloring principle is to color the species with no significant difference as yellow and the other different species as the group with the highest abundance of the species. Different colors represent different groups, and nodes with different colors represent the communities that play an important role in the group represented by the color. C. medinalis to adapt to host plants. In addition to diet, there major morphological changes with dietary transformation, could are many factors that influence the gut microbiota in insects. also have a strong impact on the gut microbiota composition Life stage and environment could shape the insect gut microbial (Voirol et al., 2018). However, certain taxa can persist throughout community combined with diets as drivers (Colman et al., 2012). all the stages of the insect (Hammer et al., 2014; Yang Host plant and population sources could drive the diversity of the et al., 2020a). In insects, the gut microbiota can promote gut microbial community in two polyphagous insects (Jones et al., homeostasis (Buchon et al., 2013), and core microbes in the gut 2019). Different host genotypes and microbial sources could microbiota may reach homeostasis by interacting with the factors influence the gut bacterial communities in lepidopterans (Mason in the environment. Gut microbes coexisting in all samples of et al., 2021). In this study, host plant  insect generation may rice- and maize-feeding C. medinalis may compose the core be a factor influencing the gut microbiota in C. medinalis. In the microbes in C. medinalis. colonization of gut microbes, the interaction of the host plant and The gut microbiota could play a crucial role in the generation may play an important role. A recent study indicated whole life of insects. The lepidopteran gut microbiota could that diet is not the primary driver of gut bacterial community function in digestion and nutrient acquisition, protection against structure in wood- and litter-feeding cockroaches (Lampert entomopathogens, and counteraction to anti-herbivore plant et al., 2019). The phyllosphere microbiome in host plants defenses (Voirol et al., 2018). Jing et al. (2020) found that contributes more than leaf phytochemicals to the variation in the most dominant role of gut bacteria is essential nutrient the gut microbiome structure in Agrilus planipennis (Mogouong provisioning, followed by digestion and detoxification. In this et al., 2021). In lepidopterans, metamorphosis, which entails study, functional prediction indicated that the most dominant Frontiers in Microbiology | www.frontiersin.org 10 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 11 Yang et al. Gut Microbiota in C. medinalis FIGURE 9 | Comparison of predicted GO functions of the gut bacteria of C. medinalis fed different host plants for three generations. Frontiers in Microbiology | www.frontiersin.org 11 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 12 Yang et al. Gut Microbiota in C. medinalis role of the gut microbiota in C. medinalis is metabolism, followed both rice- and maize-feeding C. medinalis for three generations by environmental information processing, cellular processes, may play an important role in the development of insects, and genetic information processing. Distinct antimicrobials while stably colonized bacteria in C. medinalis fed a particular could alter gut microbial communities as a result of different plant may function in host adaptation. The most dominant mortalities of P. xylostella (Lin et al., 2015). The gut microbiota role of the gut microbiota in C. medinalis is metabolism, involved in P. xylostella susceptibility to Bt Cry1Ac protoxin followed by environmental information processing, cellular is associated with the host immune response (Li et al., 2021). processes, and genetic information processing. Furthermore, In the guts of both C. medinalis fed rice and maize, the further experiments should be performed to reveal the function Proteobacteria and Firmicutes phyla were the dominant bacterial of these microbes, which may promote the identification of new taxa. Proteobacteria and Firmicutes have also been reported as targets for the management of C. medinalis. Our results provide a dominant taxa in many insects’ gut microbiota, especially in theoretical basis for the study of gut microbes in C. medinalis. Lepidoptera (Chen et al., 2020; Liu et al., 2020). They may function in carbohydrate metabolism, amino acid metabolism, DATA AVAILABILITY STATEMENT and membrane transport pathways of the host (Liu et al., 2020; Wang et al., 2020a; Chen et al., 2021). In particular, The datasets presented in this study can be found in online stably colonized gut bacteria may be crucial for insects to repositories. The names of the repository/repositories and adapt to host plants (Yang et al., 2020b). Global and overview accession number(s) can be found below: NCBI—PRJNA785679, maps, carbohydrate metabolism, membrane transport, amino SRR17106748–SRR17106753 acid metabolism, signal transduction, and cellular community were the top six pathways in the functions of the gut microbiota in C. medinalis. Enterococcus is an important flora that exists in AUTHOR CONTRIBUTIONS both rice- and maize-feeding C. medinalis for three generations, followed by the unclassified Enterobacteriaceae, Pectobacterium, YY, YL, and ZL contributed to conceptualization of the study. YY and Corynebacterium. Enterococcus has also been reported to be and ZL contributed to funding acquisition. XL investigated the stably maintained in many insects, and it can protect insects study. HX contributed to methodology. YL and ZL contributed against pathogens, fix toxic molecules from plants, increase host to supervision. YY and XL visualized the study. YY wrote the fitness, and tolerate toxic diets (Shao et al., 2011; Johnston original draft and contributed to writing, reviewing, and editing and Rolff, 2015; Vilanova et al., 2016; Shao et al., 2017). the manuscript. All authors have read and agreed to the published Enterobacteriaceae is one of the important dominant taxa in version of the manuscript. the gut microbiota of many insects (Wang et al., 2014; Yun et al., 2018; Raza et al., 2020). Enterobacteriaceae are involved in insect metabolism (Pers and Hansen, 2021; Zhou et al., 2021), FUNDING insect resistance or susceptibility to parasites, and pathogens This research was funded by the Zhejiang Provincial and insecticides (Oliver et al., 2003; Álvarez-Lagazzi et al., 2021; Polenogova et al., 2021) and play an important role in the host Natural Science Foundation of China (grant no. LY20C140004), the earmarked fund for the China Agriculture adaptability and reproduction of insects (Shi et al., 2012; Wang et al., 2020b). Pectobacterium, a clade of Enterobacteriaceae, is Research System (grant no. CARS-01-39), and the State Key Laboratory for Managing Biotic and Chemical Treats to known as a function of nitrogen fixation (Behar et al., 2005, 2008). In addition to fixing nitrogen, the gut microbiota may the Quality and Safety of Agro-products (grant nos. 2010DS700124-ZZ2007 and 2010DS700124-KF1908). help recycle nitrogenous waste products into usable compounds, such as uric acid and ammonia (Behar et al., 2005, 2008). Corynebacterium-related bacteria grow on a variety of sugars, ACKNOWLEDGMENTS organic acids, and alcohols as the single or combined carbon and energy sources as a workhorse for the large-scale production of We are thankful to Josie Lynn Catindig for her generous help with amino acids (Eikmanns and Blombach, 2014). The detailed actual manuscript editing. functions of these microbes in the gut of C. medinalis need to be proven and verified in further investigations. SUPPLEMENTARY MATERIAL CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. In conclusion, our results indicated that the alpha diversity 2022.824224/full#supplementary-material indices of gut microbes in C. medinalis could not be affected by the host plant, generation, or host plant  generation. Supplementary Figure 1 | Bacterial taxa with linear discriminant analysis (LDA) score >2 in the gut microbiota of C. medinalis fed on different host plants. PerMANOVA indicated that the gut bacteria of C. medinalis could be significantly affected by the host plant and host Supplementary Figure 2 | Cladogram of bacterial biomarkers, from the phylum plant  generation. Coexisting bacteria that were found in (innermost ring) to genus (outermost ring) level, with an LDA score >2. Differential Frontiers in Microbiology | www.frontiersin.org 12 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 13 Yang et al. Gut Microbiota in C. medinalis bacterial taxa are marked by lowercase letters. Each small circle at different Supplementary Table 2 | ANOVA of Alpha diversity indices of gut bacterial taxonomic levels represents a taxon at that level, and the diameter of the circle is communities in Cnaphalocrocis medinalis fed rice or maize for three generations. proportional to the relative abundance. The coloring principle is to color the Supplementary Table 3 | Relative abundance (%) of genus in gut microbiota of species with no significant difference as yellow and the other different species as rice- or maize-feeding Cnaphalocrocis medinalis for three generations. the group with the highest abundance of the species. Different colors represent different groups, and nodes with different colors represent the communities that Supplementary Table 4 | List of genera of the gut microbiota only stable in all play an important role in the group represented by the color. samples of rice- or maize-feeding Cnaphalocrocis medinalis. Supplementary Table 1 | Sequencing statistics of gut microbiota from rice- or Supplementary Table 5 | Analysis of similarity (ANOSIM) between gut microbial maize-feeding Cnaphalocrocis medinalis for three generations. communities from within sample groups of Cnaphalocrocis medinalis. Chung, S. H., Rosa, C., Scully, E. D., Peiffer, M., Tooker, J. F., Hoover, K., et al. REFERENCES (2013). Herbivore exploits orally secreted bacteria to suppress plant defenses. Álvarez-Lagazzi, A. P., Cabrera, N., Francis, F., and Ramírez, C. C. (2021). Proc. Natl. Acad. Sci. 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Progresses in management technology of rice leaffolders in China. J. Plant Prot. Publisher’s Note: All claims expressed in this article are solely those of the authors 42, 691–701. and do not necessarily represent those of their affiliated organizations, or those of Yuan, X. Q., Zhang, X., Liu, X. Y., Dong, Y. L., Yan, Z. Z., Lv, D. B., et al. (2021). the publisher, the editors and the reviewers. Any product that may be evaluated in Comparison of gut bacterial communities of Grapholita molesta (Lepidoptera: this article, or claim that may be made by its manufacturer, is not guaranteed or Tortricidae) reared on different host plants. Int. J. Mol. Sci. 22:6843. doi: 10. endorsed by the publisher. 3390/ijms22136843 Yun, J. H., Jung, M. J., Kim, P. S., and Bae, J. W. (2018). Social status shapes Copyright © 2022 Yang, Liu, Xu, Liu and Lu. This is an open-access article distributed the bacterial and fungal gut communities of the honey bee. Sci. Rep. 8:2019. under the terms of the Creative Commons Attribution License (CC BY). The use, doi: 10.1038/s41598- 018- 19860- 7 distribution or reproduction in other forums is permitted, provided the original Yun, J. H., Roh, S. W., Whon, T. W., Jung, M. J., Kim, M. S., Park, author(s) and the copyright owner(s) are credited and that the original publication D. S., et al. (2014). Insect gut bacterial diversity determined by in this journal is cited, in accordance with accepted academic practice. No use, environmental habitat, diet, developmental stage, and phylogeny of distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 15 April 2022 | Volume 13 | Article 824224 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Unpaywall

Effects of Host Plant and Insect Generation on Shaping of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis

Yang, Yajun; Liu, Xiaogai; Xu, Hongxing; Liu, Yinghong; Lu, Zhongxian
Frontiers in Microbiology – Apr 11, 2022
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fmicb-13-824224 April 5, 2022 Time: 19:11 # 1 ORIGINAL RESEARCH published: 11 April 2022 doi: 10.3389/fmicb.2022.824224 Effects of Host Plant and Insect Generation on Shaping of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis 1 1,2 1 2 1 Yajun Yang , Xiaogai Liu , Hongxing Xu , Yinghong Liu * and Zhongxian Lu * State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, College of Plant Protection, Southwest University, Chongqing, China Gut microbes in insects may play an important role in the digestion, immunity and protection, detoxification of toxins, development, and reproduction. The rice leaffolder Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Crambidae) is a notorious insect pest that can damage rice, maize, and other gramineous plants. To determine the effects of host plants and generations on the gut microbiota of C. medinalis, we deciphered the bacterial configuration of this insect pest fed rice or maize for three generations Edited by: by Illumina MiSeq technology. A total of 16 bacterial phyla, 34 classes, 50 orders, 101 F. L. Consoli, University of São Paulo, Brazil families, 158 genera, and 44 species were identified in C. medinalis fed rice or maize for Reviewed by: three generations. Host plants, insect generation, and their interaction did not influence Abrar Muhammad, the alpha diversity indices of the gut microbiota of C. medinalis. The dominant bacterial Zhejiang University, China Bruna Laís Merlin, taxa were Proteobacteria and Firmicutes at the phylum level and Enterococcus and University of São Paulo, Brazil unclassified Enterobacteriaceae at the genus level. A number of twenty genera coexisted *Correspondence: in the guts of C. medinalis fed rice or maize for three generations, and their relative Yinghong Liu abundances occupied more than 90% of the gut microbiota of C. medinalis. A number [email protected] Zhongxian Lu of two genera were stably found in the gut of rice-feeding C. medinalis but unstably [email protected] found in the gut microbiota of maize-feeding C. medinalis, and seven genera were stably found in the gut of maize-feeding C. medinalis but unstably found in the gut of rice- Specialty section: This article was submitted to feeding C. medinalis. In addition, many kinds of microbes were found in some but not all Systems Microbiology, samples of the gut of C. medinalis fed on a particular host plant. PerMANOVA indicated a section of the journal Frontiers in Microbiology that the gut bacteria of C. medinalis could be significantly affected by the host plant and Received: 29 November 2021 host plant  generation. We identified 47 taxa as the biomarkers for the gut microbiota Accepted: 09 March 2022 of C. medinalis fed different host plants by LEfSe. Functional prediction suggested that Published: 11 April 2022 the most dominant role of the gut microbiota in C. medinalis is metabolism, followed Citation: Yang Y, Liu X, Xu H, Liu Y and by environmental information processing, cellular processes, and genetic information Lu Z (2022) Effects of Host Plant processing. Our findings will enrich the understanding of gut bacteria in C. medinalis and Insect Generation on Shaping and reveal the differences in gut microbiota in C. medinalis fed on different host plants of the Gut Microbiota in the Rice Leaffolder, Cnaphalocrocis medinalis. for three generations. Front. Microbiol. 13:824224. doi: 10.3389/fmicb.2022.824224 Keywords: rice leaffolder, gut bacteria, host plant, Lepidoptera, rice, maize Frontiers in Microbiology | www.frontiersin.org 1 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 2 Yang et al. Gut Microbiota in C. medinalis colonization (Hammer et al., 2014). Nevertheless, microbiota INTRODUCTION are abundant and diverse in many species of Lepidoptera. Insects harbor numerous microorganisms in the gut (Douglas, Proteobacteria and Firmicutes were found to be dominant in 2015). Gut microorganisms in insects have been shown to the gut of the diamondback moth Plutella xylostella (L.) based contribute to digestion (Anand et al., 2010; Jing et al., 2020), on the high-throughput DNA sequencing data (Xia et al., detoxification (Ceja-Navarro et al., 2015; Beran and Gershenzon, 2013). Enterococcus and Lactococcus were dominant bacteria in 2016; Blanton and Peterson, 2020), development (Wang et al., a field population of Helicoverpa armigera Hubner, followed 2018b; Qiao et al., 2019; Pyszko et al., 2020), physiology (Engel by Flavobacterium, Acinetobacter, and Stenotrophomonas (Xiang and Moran, 2013; Xu et al., 2019; Liberti and Engel, 2020), et al., 2006). The composition of microbes in the insect gut pathogen resistance (Dillon and Dillon, 2004; Voirol et al., could be affected by many factors. The environmental habitat, 2018; Moore and Aparicio, 2022), immune response (Engel diet, developmental stage, and phylogeny of the host could and Moran, 2013; Li et al., 2020; Li et al., 2021), and the determine the bacterial diversity in the insect gut (Yun et al., production of essential vitamins and amino acids (Hansen and 2014). In the larvae of Spodoptera littoralis (Boisduval), bacterial Moran, 2014; Jang and Kikuchi, 2020; Jing et al., 2020). For communities were shown to be instar-specific (Chen et al., 2016). instance, some microorganisms with metabolic characteristics In addition, host plants were observed to have a considerable could promote insect adaptation to host plants (Voirol et al., effect on the composition of gut bacteria in Henosepilachna 2018). The gut microbiota was found to function in the protection vigintioctopunctata (F.) (Lü et al., 2019). of a European Bombus species against the intestinal pathogen The rice leaffolder Cnaphalocrocis medinalis (Guenée) Crithidia bombi (Koch and Schmid-Hempel, 2011). Another (Lepidoptera: Crambidae) is an important insect pest in Asia example in Helicoverpa zea (Boddie), Enterbacter ludwigii, a that can damage rice (Oryza sativa L.), maize, and other gut-associated bacterium, could indirectly trigger the defense gramineous plants (Barrion et al., 1991; Cheng, 1996; Yang et al., of tomato (Solanum lycopersicum L.) and maize (Zea mays L.) 2015). The heavy occurrence of this insect could cause serious (Wang et al., 2017, 2018a). Chung et al. (2013) documented economic loss to rice production (Yang et al., 2015). In 2015, that the Colorado potato beetle Leptinotarsa decemlineata C. medinalis damaged rice plants with an area of 15.5 million ha (Say) suppressed the defenses of tomatoes by exploiting orally and caused yield losses of 0.47 million tons in China (Yang et al., secreted bacteria. The gut microbiota of the pine weevil 2015; Lu, 2017). Based on the traditional isolation and culture (Hylobius abietis) degrades conifer diterpenes and increases methods, 25 species of 15 phyla of gut microbiota were obtained insect fitness (Berasategui et al., 2017). Gut microbes may from C. medinalis larvae (Yang, 2012). By comparison, a large facilitate insect herbivory to chemically defend plants (Hammer number of gut microbiota were obtained from C. medinalis and Bowers, 2015). Gut symbionts could enhance insecticide larvae through Illumina MiSeq technology (Liu et al., 2016). resistance in a significant pest, the oriental fruit fly Bactrocera Yang et al. (2020a) analyzed the gut microbiota composition dorsalis (Hendel) (Cheng et al., 2017). Insect symbionts could of C. medinalis across the developmental stages. Information influence insect–plant interactions at different levels through on the host-associated changes in gut bacteria will facilitate direct interactions and also through indirect plant-mediated the overall understanding of insect ecology and promote the interactions (Frago et al., 2012). Given the importance of the development of novel methods for pest management. This study associated microorganisms to host fitness and feeding ecology, illustrates the composition and diversity of the gut microbiota an effort to manipulate these partnerships and render insect pests in C. medinalis feeding on rice or maize for three generations by more vulnerable to broad-scale measures of population control Illumina MiSeq technology. The findings in this study will enrich by targeting the bacterial symbionts was one of the important the understanding of the gut microbiota in C. medinalis and applications in gut symbiont-driven pest control (Berasategui provide novel insight into the relationship between C. medinalis et al., 2016). The functions of gut microbes could provide a and its host plants. novel concept for the application of bacteria in pest control through the restraint of the insect immune response and the MATERIALS AND METHODS induction of plant defense (Kyritsis et al., 2017) and promote the understanding of gut symbiont-driven pest control (Frago et al., 2012; Berasategui et al., 2016). Insect Rearing and Sampling Lepidoptera is one of the largest insect orders and has Adults of C. medinalis were collected from paddy fields in approximately 160,000 described species (Mitter et al., 2017). Hangzhou, Zhejiang Province, East China and then cultured with Some of them can damage agricultural crops and cause large 10% honey solution in the laboratory under controlled conditions economic losses (Wagner, 2013). However, the evidence of the of 26  1 C temperature, 70  10% relative humidity, and a fundamental function of bacteria in lepidopteran biology is photoperiod of 16:8 (L:D) h. The neonates of the population were scarce. Furthermore, a recent study from Hammer et al. (2017) divided into two groups. One was reared using rice plants, and the reported that caterpillars lack a resident microbiome in the gut other was reared using maize plants. Every group was reared for compared with other insect orders. The authors of this study three generations. Rice and maize were planted in pots (one plant argued that caterpillars with rough environments may prevent per pot) in the greenhouse. The leaves of plants were collected and bacterial colonization. Lepidopteran reshaping the body structure rinsed with sterile ddH O and then air-dried before feeding them during metamorphosis also enhances the difficulty of bacterial to C. medinalis, and sufficient leaves were provided for the insects. Frontiers in Microbiology | www.frontiersin.org 2 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 3 Yang et al. Gut Microbiota in C. medinalis Guts of C. medinalis were dissected from the fifth instar larvae distances was applied among all the bacterial groups. Non-metric of both groups from every generation. A total of fifteen guts multidimensional scaling (NMDS) plots were constructed using were pooled into a biological sample, and three replicates were Bray–Curtis. Analysis of similarity (ANOSIM) was used to test prepared for each treatment. Before dissection, the whole larva the difference in the composition of microbiota among different was rinsed with sterile ddH O, disinfected with ethanol (75%) for group samples. Permutational multivariate analysis of variance 90 s, and rinsed again with sterile ddH O. Following dissection, (PerMANOVA) was generated using 999 permutations, and the the guts were collected into a 1.5-ml sterile tube and stored individual repeats were included in the model as a random effect. at –80 C until use. PCoA, NMDS, ANOSIM, and PerMANOVA were analyzed and graphed using R software. Linear discriminant analysis (LDA) was used to screen the biomarkers for significant differences DNA Extraction and PCR Amplification between different groups with LDA scores greater than two. The dissected guts were homogenized by shaking in a sterile A cladogram was drawn to show the distribution of these tube containing sterile glass beads (0.5 mm diameter) and 0.5 ml biomarkers at different taxonomic levels by Galaxy (accessed of PBS buffer (pH 7.5) for 15 min using a vortex. Total DNAs on 1 January 2022). Microbiota functions were predicted by were extracted from samples using the E.Z.N.A. bacteria DNA annotating pathways of OTUs against the Ref99NR database extract kit (OMEGA, United States) according to the instructions. using R software with the Tax4Fun2 package. The primers 515F 5’-GTGCCAGCMGCCGCGG-3’ and 907R 5’-CCGTCAATTCMTTTRAGTTT-3’ were used to amplify the V4-V5 regions of the bacterial 16S ribosomal RNA gene through PCR (95 C for 2 min, followed by 25 cycles at 95 C for 30 s, RESULTS 55 C for 30 s, and 72 C for 30 s and a final extension at 72 C for 5 min). Amplicons were generated in a 20 ml reaction system Reads Analyzed and Taxa Generated containing 4 ml of 5  FastPfu Buffer, 2 ml of 2.5 mM dNTPs, 0.8 We sequenced the gut microbes of C. medinalis fed on different ml of each primer (5 mm), 0.4 ml of FastPfu Polymerase, and 10 ng host plants for three generations and obtained 1,473,836 trimmed of template DNA. Blank DNA as a negative control was extracted, paired reads in total (Supplementary Table 1). Blank DNA and and products generated from no-template PCR were sequenced no-template PCR sequencing were used for decontamination, to assess what sequences are contaminants. and sequences of cyanobacteria or chloroplasts were found to be contaminants. After decontamination, 446 OTUs were obtained. Illumina MiSeq Sequencing The OTU numbers of C. medinalis from different samples varied Amplicons were extracted and purified using the AxyPrep from 49 to 194 (Table 1). The Ace index varied from 62.93 DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, to 252.14, the Chao1 index varied from 57.27 to 256.25, the United States) according to the manufacturer’s instructions and Shannon index varied from 0.47 to 1.36, and the Simpson index TM quantified using QuantiFluor -ST (Promega, United States). varied from 0.46 to 0.87 (Table 1). ANOVA indicated that alpha Then, they were pooled in equimolar amounts and paired-end diversity indices were not significantly affected by the host plant, sequenced (2  250) on an Illumina MiSeq platform according to generation, or their interaction (Supplementary Table 2). A total the standard protocols. of 16 bacterial phyla, 34 classes, 50 orders, 101 families, 158 genera, and 44 species were identified in C. medinalis fed rice or Bioinformatic and Statistical Analyses maize for three generations (Table 2). Raw FASTQ files were demultiplexed and quality-filtered using QIIME (version 1.17). According to the similarity of the Gut Microbiota of Cnaphalocrocis sequences, effective sequences were classified into multiple medinalis Fed Rice for Three operational taxonomic units (OTUs) at a similarity level of Generations 97% using UPARSE (version 7.1), and chimeric sequences were At the phylum level, Firmicutes, Proteobacteria, Actinobacteria, identified and removed using UCHIME. All the sequences were Bacteroidetes, and unclassified Bacteria were found in the gut annotated and blasted against the Silva (SSU115)16S rRNA microbiota of C. medinalis fed on rice plants through all database using a confidence threshold of 70% for each 16S rRNA samples of three generations. Among them, Firmicutes was the gene sequence analyzed by RDP Classifier. absolute dominant phylum with the highest relative abundance Alpha diversity was estimated through five indices: OTU in rice-feeding C. medinalis for three generations (70.62– number, ACE, Chao1, Shannon, and Simpson’s index. The 87.53%) (Figure 1A). The relative abundance of Proteobacteria alpha diversity and relative abundance data were analyzed using was 10.51–26.88%, followed by Actinobacteria (1.00–4.66%), one-way analysis of variance (ANOVA) with SPSS 26.0 (IBM Bacteroidetes (0.41–0.43%), and unclassified Bacteria (0.02– SPSS Statistics), and multiple comparisons were analyzed using 0.14%) (Figure 1A). At the family level, 18 families were found in Tukey’s test. Venn diagrams and stack bars were graphed by the gut microbiota of C. medinalis fed rice through all samples of R software. Principal coordinate analysis (PCoA) based on the three generations. Enterococcaceae and Enterobacteriaceae were matrices of pairwise weighted UniFrac distances and Bray–Curtis the two major families in the rice-feeding C. medinalis for three http://drive5.com/uparse/ 2 3 http://rdp.cme.msu.edu/ http://huttenhower.sph.harvard.edu/galaxy/ Frontiers in Microbiology | www.frontiersin.org 3 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 4 Yang et al. Gut Microbiota in C. medinalis generations, with relative abundance ranges of 70.55–87.27% and respectively. Enterococcus and unclassified Enterobacteriaceae are 9.12–24.75%, respectively (Figure 1B). The relative abundance of the majority. In addition to the microbes found in all samples Anaplasmataceae in the gut of the third generation of C. medinalis of rice-feeding C. medinalis for three generations, many kinds of fed rice was higher than that of the second generation of microbes were found in some but not all samples of rice-feeding C. medinalis, and the relative abundance of Nocardiaceae in C. medinalis gut. the gut of the first generation of C. medinalis fed rice was Gut Microbiota of Cnaphalocrocis higher than that of the other two generations of C. medinalis (Figure 1B). At the genus level, 21 genera were found in the medinalis Fed Maize for Three gut microbiota of C. medinalis fed rice through all samples of Generations three generations. Enterococcus, unclassified Enterobacteriaceae, Similar to rice-feeding C. medinalis, the same six phyla were Pectobacterium, Corynebacterium, Leucobacter, and Anaplasma found in the gut microbiota of maize-feeding C. medinalis occupied the top 10 in the gut microbiota of C. medinalis fed through all samples of three generations. The phylum with rice for three generations (Supplementary Table 3). Common the highest relative abundance was Firmicutes (68.49–80.25%), genera found in all three generations occupied 93.95, 98.51, followed by Proteobacteria (16.58–27.65%), Actinobacteria (1.62– and 97.78% of the first generation to the third generation, 2.01%), Bacteroidetes (0.41–0.83%), and unclassified Bacteria (0.004–0.29%) (Figure 2A). At the family level, 20 families found gut microbiota of C. medinalis fed maize through all samples TABLE 1 | Alpha diversity indices of gut bacterial communities in rice- or of three generations. Enterococcaceae and Enterobacteriaceae maize-feeding Cnaphalocrocis medinalis for three generations. were also the dominant families, with relative abundance ranges of 67.88–80.23% and 14.69–24.42%, respectively (Figure 2B). Sample Alpha diversity indices The relative abundance of Comamonadaceae in the gut of OTU number ACE Chao1 Shannon Simpson the third generation of C. medinalis fed maize was higher than that of the first generation of C. medinalis, the relative R1-1 201 252.14 256.25 1.33 0.60 abundance of Micrococcaceae in the second generation of R1-2 59 85.09 75.15 0.88 0.60 C. medinalis was higher than that of the first generation of R1-3 49 62.93 57.27 0.58 0.77 C. medinalis, and the relative abundance of Rhodocyclaceae in R2-1 162 210.16 225.14 0.52 0.84 the gut of the second generation of C. medinalis was higher R2-2 157 194.09 200 0.47 0.87 than that of the third generation of C. medinalis (Supplementary R2-3 183 221.45 228.12 0.90 0.62 Table 3). At the genus level, 26 genera were found in the gut R3-1 85 107.55 110.07 0.95 0.59 microbiota of C. medinalis fed maize through all samples of R3-2 61 117.04 119 0.94 0.56 three generations. Enterococcus, unclassified Enterobacteriaceae, R3-3 145 178.45 184.26 1.36 0.46 Corynebacterium, unclassified Comamonadaceae, Leucobacter, M1-1 194 242.48 231.66 1.20 0.52 Microbacterium, Anaplasma, and Sphingobacterium occupied the M1-2 69 95.25 81.67 0.81 0.65 top 10 in the gut microbiota of C. medinalis fed maize for three M1-3 135 184.21 197.67 0.52 0.85 generations (Supplementary Table 3). Common genera found in M2-1 171 207.09 212.25 1.17 0.52 all three generations occupied 97.75, 96.34, and 99.29% of the first M2-2 165 189 180.53 1.26 0.46 generation to the third generation, respectively. Enterococcus and M2-3 159 194.74 222.07 1.15 0.58 unclassified Enterobacteriaceae are the majority. In addition to M3-1 96 120.32 113.25 0.95 0.52 the microbes found in all samples of maize-feeding C. medinalis M3-2 110 144.6 153.5 0.94 0.58 for three generations, many kinds of microbes were found in M3-3 98 108.44 111.91 0.81 0.59 some but not all samples of maize-feeding C. medinalis gut. R1–R3: the first to third generation of C. medinalis fed on rice; M1–M3: the first to third generation of C. medinalis fed on maize. Influence of Host Plant and Insect Generation on the Gut Bacterial TABLE 2 | Number of identified gut bacterial taxonomic categories in rice- and Communities of Cnaphalocrocis maize-feeding Cnaphalocrocis medinalis for three generations. medinalis Treatments Phylum Class Order Family Genus Species Comparing the gut microbiota between C. medinalis fed R1 12 28 39 69 99 30 rice and maize, five phyla and 16 families were found in R2 13 30 42 86 118 36 all samples of the three generations. At the genus level, 19 R3 11 23 33 68 93 30 genera were found in the gut microbiota of C. medinalis fed M1 13 29 40 74 106 32 on rice or maize plants for three generations (Supplementary M2 12 26 42 85 115 37 Table 3). The relative abundance of these genera occupied M3 5 9 21 51 71 31 more than 90% of the gut microbiota of C. medinalis fed rice or Total 16 34 50 101 158 44 maize plants, and the two major genera were Enterococcus and a unclassified Enterobacteriaceae (Figure 3). Seven genera, Bacillus, R1–R3, the first to third generation of C. medinalis fed rice; M1–M3, the first to third generation of C. medinalis fed maize. Empedobacter, Flavobacterium, Rhizobium, Rhodococcus, Frontiers in Microbiology | www.frontiersin.org 4 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 5 Yang et al. Gut Microbiota in C. medinalis FIGURE 1 | Relative abundance of the gut microbiota from rice-feeding C. medinalis for three generations at the phylum (A) and family (B) levels. R1–R3: the first to third-generation of C. medinalis fed on rice. FIGURE 2 | Relative abundance of the gut microbiota from maize-feeding C. medinalis for three generations at the phylum (A) and family (B) levels. M1–M3: the first to third generation of C. medinalis fed on maize. Frontiers in Microbiology | www.frontiersin.org 5 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 6 Yang et al. Gut Microbiota in C. medinalis 77.73 and 71.25% of the total OTUs of the first generation of C. medinalis fed rice or maize, were shared by C. medinalis fed rice or maize (Figure 6A). The second generation of C. medinalis fed on rice or maize shared 243 OTUs, which accounted for 85.56 and 86.17% of the total OTUs of the second generation of C. medinalis fed on rice or maize, respectively (Figure 6B). The third generation of C. medinalis fed on rice or maize shared 82 OTUs, which accounted for 40.39 and 64.57% of the total OTUs of the third generation of C. medinalis fed on rice or maize, respectively (Figure 6C). To find the biomarkers with significant differences between different groups, LDA effect size (LEfSe) was used to screen out different taxa at various levels (kingdom, phylum, class, order, family, genus, and species) between different groups based on a standard LDA value greater than two (Figure 7). Meanwhile, the cladogram from phylum to genus was graphed to fully understand the distribution of these different taxa at various taxonomic levels (Figure 8). In the third generation of C. medinalis fed maize (M3), the gut microbiota had the most of the different taxa (LDA > 2). There were 60 taxa FIGURE 3 | Relative abundance of the genera in the gut microbiotas found in mainly belonging to Firmicutes, Bacteroidota, Acidobacteriota, all samples of C. medinalis. R1–R3: the first to third generation of C. medinalis Proteobacteria, Actinobacteria, and Ignavibacteriae. A total of fed rice; M1–M3: the first to third generation of C. medinalis fed maize. six taxa belonging to Actinobacteria were in the gut microbiota of the first generation of C. medinalis fed rice (R1). A total of two taxa belonging to Proteobacteria and one taxon belonging Sphingobacterium, and unclassified Beutenbergiaceae, were stably to Bacteroidetes were in the gut microbiota of the second found in all samples of maize-feeding C. medinalis for three generation of C. medinalis fed rice (R2). A total of two taxa generations, whereas Tsukamurella and Ochrobactrum were belonging to Proteobacteria were in the gut microbiota of the stably found in all samples of rice-feeding C. medinalis for three third generation of C. medinalis fed rice (R3). A total of five generations (Supplementary Table 4). taxa belonging to Proteobacteria were in the gut microbiota of Principal coordinate analysis based on the Bray–Curtis the first generation of C. medinalis fed maize (M1). A total distance and weighted UniFrac distance was used to compare of three taxa belonging to Proteobacteria, five taxa exclusive to the community similarities between samples. The PCoA scatter Ignavibacteriae, five taxa belonging to Firmicutes, and four taxa plot showed that the abscissa and ordinate represent the two belonging to Actinobacteria were in the gut microbiota of the characteristic values that contribute to the largest differences second generation of C. medinalis fed maize (M2). A total of between the samples, and their influence degrees were 74.09 fourteen taxa belonging to Proteobacteria, seven taxa belonging and 14.73% based on weighted UniFrac distance (Figure 4A) to Actinobacteria, and six taxa belonging to Bacteroidetes were and 65.61 and 18.16% based on the Bray–Curtis (Figure 4B), in the gut microbiota of the third generation of C. medinalis respectively. PerMANOVA showed that there were significant fed on maize (M3). LEfSe was also used to find the biomarkers differences in the gut microbiota of rice- and maize-feeding with significant differences between samples fed different host C. medinalis (Table 3; PerMANOVA: R = 0.35, p = 0.001). Host plants (Supplementary Figure 1). A total of forty-seven taxa were plant  generation significantly affected the gut microbiota of identified as the biomarkers in the gut microbiota of C. medinalis C. medinalis (Table 3; PerMANOVA: R = 0.28, p = 0.004). No fed on different host plants (Supplementary Figure 2). A total significant differences were observed between the samples from of six taxa belonging to Actinobacteria and one taxon belonging different generations of C. medinalis (Table 3; PerMANOVA: to Proteobacteria were in the gut microbiota of C. medinalis fed R = 0.02, p = 0.751). rice. Nineteen taxa belonging to Proteobacteria, 10 taxa belonging Non-metric multidimensional scaling analysis revealed to Bacteroidetes, and 11 taxa belonging to Actinobacteria were in significant differences between the gut microbiota of rice- the gut microbiota of C. medinalis fed maize. and maize-feeding C. medinalis (Figure 5). ANOSIM showed that there were significant differences in the gut microbiota of Functional Prediction of the Gut rice- and maize-feeding C. medinalis (R = 0.5538, p = 0.001) (Supplementary Table 5). There were no significant differences Microbitota of Cnaphalocrocis medinalis in the gut microbiota of rice- and maize-feeding C. medinalis in To better understand the important role of the gut microbiota the same generations (Supplementary Table 5). of C. mednialis, we used R software with Tax4Fun2 to predict Venn diagrams showed overlapping OTUs of C. medinalis fed the function in samples based on 16S rDNA sequencing data on rice or maize from the first generation to the third generation and compared them with the Ref99NR database (Figure 9). (Figure 6). The results indicated that 171 OTUs, which comprised The results showed that the most functional prediction Frontiers in Microbiology | www.frontiersin.org 6 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 7 Yang et al. Gut Microbiota in C. medinalis FIGURE 4 | PCoA bacterial communities of C. medinalis fed on different host plants over generations based on weighted UniFrac (A) and Bray–Curtis (B) distances. M1–M3: the first to third generation of C. medinalis fed on maize; R1–R3: the first to third generation of C. medinalis fed on rice. categories were related to metabolism (70.12–71.18%) followed DISCUSSION by environmental information processing (16.17–17.08%), cellular processes (5.39–5.94%), and genetic information This study profiled the gut bacterial community in C. medinalis processing (3.87–4.06%). In the metabolism category, global fed on different host plants for three generations. Considering and overview maps had the highest abundance (34.18– the limited gut bacterial information isolated and cultured by 34.73%) followed by carbohydrate metabolism (14.93–15.75%), traditional methods (Yang, 2012), we obtained the bacterial amino acid metabolism (5.08–5.45%), energy metabolism information of C. medinalis by MiSeq sequencing. Recently, we (3.00–3.10%), metabolism of cofactors and vitamins (2.16– reported that the composition of the gut bacterial community 2.40%), nucleotide metabolism (2.18–2.26%), lipid metabolism changes across the life cycle of C. medinalis, and the phyla (2.02–2.17%), xenobiotics biodegradation and metabolism Proteobacteria and Firmicutes were the dominant bacterial (1.34–1.55%), biosynthesis of other secondary metabolites (1.31– taxa (Yang et al., 2020a). In the guts of both C. medinalis 1.35%), metabolism of other amino acids (1.12–1.28%), glycan fed rice and maize, the phyla Proteobacteria and Firmicutes biosynthesis and metabolism (1.08–1.14%), and metabolism of were also the dominant bacterial taxa. In this study, host terpenoids and polyketides (0.74–0.85%). In the environmental plants, generation, and their interaction did not significantly information processing category, membrane transport had affect the alpha diversity indices of the gut microbiota in the highest abundance (12.05–12.87%) followed by signal C. medinalis. Ace and choa1 values indicated that community transduction (4.08–4.26%). In the cellular processes category, richness did not differ among the different groups. Shannon the cellular community had the highest abundance (3.85–4.21%) and Simpson values indicated that community diversity did followed by cell motility (0.88–1.06%), cell growth and death not differ among the different groups. The experimental results (0.49–0.52%), and transport and catabolism (0.14–0.15%). In the provide a more comprehensive understanding of the relationship genetic information processing category, replication and repair between C. medinalis and its microbiota. Our results revealed had the highest abundance (1.55–1.64%), followed by translation the influence of host plants and insect generation on the gut (1.45–1.53%) and folding, sorting, and degradation (0.76–0.79%). bacterial community in C. medinalis and provide a foundation for investigating gut microbe C. medinalis–host plant interactions. Diet is one of the important factors for insect development (Karley et al., 2002; Qubaiová et al., 2021), and it also plays an important role in shaping insect phenotypes and gut microbial TABLE 3 | PERMANOVA of the bacterial communities of C. medinalis fed rice or maize for three generations. communities (Colman et al., 2012; Xu et al., 2019; Luo et al., 2021; Mason et al., 2021). Host diet could influence the diversity, Source df SS MS Pseudo-F R p-value structure, or composition of the gut in many insects (Strano Host plant 1.17 1.5924 1.5924 8.66 0.35 0.001 et al., 2018; Lü et al., 2019; Leite-Mondin et al., 2021; Yuan Generation 1.17 0.1086 0.1086 0.39 0.02 0.751 et al., 2021). Leite-Mondin et al. (2021) discovered that the gut Host plant  Generation 1.17 1.2699 1.2699 6.22 0.28 0.004 microbiota composition of Trichoplusia ni (Hubner) altered by diet may influence its polyphagous behavior. An imbalanced PERMANOVA was generated using 999 permutations, and the individual repeat was included in the model as a random effect. diet-altered variation in gut microbiota is detrimental to mirid Frontiers in Microbiology | www.frontiersin.org 7 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 8 Yang et al. Gut Microbiota in C. medinalis FIGURE 5 | NMDS analysis of the bacterial communities of C. medinalis fed maize and rice for three generations. NMDS plots were constructed using Bray–Curtis with the main groups of host plants (A) and generations (B). Stress values (stress = 0.079) indicate a good fit in two dimensions. M: samples from C. medinalis fed maize; R: samples from C. medinalis fed rice. FIGURE 6 | Venn diagram of bacterial community OTUs of C. medinalis fed different host plants for three generations. M1–M3: the first to third generation of C. medinalis fed on maize; R1–R3: the first to third generation of C. medinalis fed on rice. (A) M1 vs. R1; (B) M2 vs. R2; (C) M3 vs. R3. bugs, Adelphocoris suturalis Jakovlev (Luo et al., 2021). In fed rice or maize, whereas their relative abundances occupied this study, at the family and genus levels, the composition of more than 90% of the gut microbiota of C. medinalis fed rice the gut microbiota of C. medinalis differed between the host or maize. In addition, we found that two genera (Tsukamurella plants. Among the genera found in the gut of C. medinalis fed and Ochrobactrum) were stable in the gut of rice-feeding different host plants, only 21 genera were found in all samples C. medinalis, but unstable in the gut microbiota of maize- of three generations of rice-feeding C. medinalis, and only 26 feeding C. medinalis, and seven genera (Bacillus, Empedobacter, genera were found in all samples of three generations of maize- Flavobacterium, Rhizobium, Rhodococcus, Sphingobacterium, and feeding C. medinalis. These results indicated that most kinds unclassified Beutenbergiaceae) were stable in the gut of maize- of microbes are not stably colonized in the gut of C. medinalis feeding C. medinalis, but unstable in the gut of rice-feeding fed a particular host plant. Hammer et al. (2017) reported C. medinalis. For example, some genera that were stable in the that caterpillars lack a resident gut microbiome. Jones et al. gut of maize-feeding C. medinalis were found in some but not (2019) found high variability in gut bacterial composition and all samples of rice-feeding C. medinalis. The gut bacteria that abundance between the individuals of the same insect species were stable in the gut of C. medinalis for three generations may even fed on the same food source. The reports from other have an important role in shaping the microbiota community lepidopteran species showed that gut microbial assemblages in C. medinalis. Through LEfSe, 47 taxa were found to be the differed between individuals (Priya et al., 2012; Staudacher et al., biomarkers for the gut microbiota of C. medinalis fed different 2016). In this study, only 19 genera coexisted in C. medinalis host plants. Stable host-related bacteria may function to help Frontiers in Microbiology | www.frontiersin.org 8 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 9 Yang et al. Gut Microbiota in C. medinalis FIGURE 7 | Bacterial taxa with LDA scores greater than two in the gut microbiota of C. medinalis fed different host plants for three generations. Frontiers in Microbiology | www.frontiersin.org 9 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 10 Yang et al. Gut Microbiota in C. medinalis FIGURE 8 | Cladogram of bacterial biomarkers, from the phylum (innermost ring) to genus (outermost ring) level, with an LDA score > 2. Differential bacterial taxa are marked by lowercase letters. Each small circle at different taxonomic levels represents a taxon at that level, and the diameter of the circle is proportional to the relative abundance. The coloring principle is to color the species with no significant difference as yellow and the other different species as the group with the highest abundance of the species. Different colors represent different groups, and nodes with different colors represent the communities that play an important role in the group represented by the color. C. medinalis to adapt to host plants. In addition to diet, there major morphological changes with dietary transformation, could are many factors that influence the gut microbiota in insects. also have a strong impact on the gut microbiota composition Life stage and environment could shape the insect gut microbial (Voirol et al., 2018). However, certain taxa can persist throughout community combined with diets as drivers (Colman et al., 2012). all the stages of the insect (Hammer et al., 2014; Yang Host plant and population sources could drive the diversity of the et al., 2020a). In insects, the gut microbiota can promote gut microbial community in two polyphagous insects (Jones et al., homeostasis (Buchon et al., 2013), and core microbes in the gut 2019). Different host genotypes and microbial sources could microbiota may reach homeostasis by interacting with the factors influence the gut bacterial communities in lepidopterans (Mason in the environment. Gut microbes coexisting in all samples of et al., 2021). In this study, host plant  insect generation may rice- and maize-feeding C. medinalis may compose the core be a factor influencing the gut microbiota in C. medinalis. In the microbes in C. medinalis. colonization of gut microbes, the interaction of the host plant and The gut microbiota could play a crucial role in the generation may play an important role. A recent study indicated whole life of insects. The lepidopteran gut microbiota could that diet is not the primary driver of gut bacterial community function in digestion and nutrient acquisition, protection against structure in wood- and litter-feeding cockroaches (Lampert entomopathogens, and counteraction to anti-herbivore plant et al., 2019). The phyllosphere microbiome in host plants defenses (Voirol et al., 2018). Jing et al. (2020) found that contributes more than leaf phytochemicals to the variation in the most dominant role of gut bacteria is essential nutrient the gut microbiome structure in Agrilus planipennis (Mogouong provisioning, followed by digestion and detoxification. In this et al., 2021). In lepidopterans, metamorphosis, which entails study, functional prediction indicated that the most dominant Frontiers in Microbiology | www.frontiersin.org 10 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 11 Yang et al. Gut Microbiota in C. medinalis FIGURE 9 | Comparison of predicted GO functions of the gut bacteria of C. medinalis fed different host plants for three generations. Frontiers in Microbiology | www.frontiersin.org 11 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 12 Yang et al. Gut Microbiota in C. medinalis role of the gut microbiota in C. medinalis is metabolism, followed both rice- and maize-feeding C. medinalis for three generations by environmental information processing, cellular processes, may play an important role in the development of insects, and genetic information processing. Distinct antimicrobials while stably colonized bacteria in C. medinalis fed a particular could alter gut microbial communities as a result of different plant may function in host adaptation. The most dominant mortalities of P. xylostella (Lin et al., 2015). The gut microbiota role of the gut microbiota in C. medinalis is metabolism, involved in P. xylostella susceptibility to Bt Cry1Ac protoxin followed by environmental information processing, cellular is associated with the host immune response (Li et al., 2021). processes, and genetic information processing. Furthermore, In the guts of both C. medinalis fed rice and maize, the further experiments should be performed to reveal the function Proteobacteria and Firmicutes phyla were the dominant bacterial of these microbes, which may promote the identification of new taxa. Proteobacteria and Firmicutes have also been reported as targets for the management of C. medinalis. Our results provide a dominant taxa in many insects’ gut microbiota, especially in theoretical basis for the study of gut microbes in C. medinalis. Lepidoptera (Chen et al., 2020; Liu et al., 2020). They may function in carbohydrate metabolism, amino acid metabolism, DATA AVAILABILITY STATEMENT and membrane transport pathways of the host (Liu et al., 2020; Wang et al., 2020a; Chen et al., 2021). In particular, The datasets presented in this study can be found in online stably colonized gut bacteria may be crucial for insects to repositories. The names of the repository/repositories and adapt to host plants (Yang et al., 2020b). Global and overview accession number(s) can be found below: NCBI—PRJNA785679, maps, carbohydrate metabolism, membrane transport, amino SRR17106748–SRR17106753 acid metabolism, signal transduction, and cellular community were the top six pathways in the functions of the gut microbiota in C. medinalis. Enterococcus is an important flora that exists in AUTHOR CONTRIBUTIONS both rice- and maize-feeding C. medinalis for three generations, followed by the unclassified Enterobacteriaceae, Pectobacterium, YY, YL, and ZL contributed to conceptualization of the study. YY and Corynebacterium. Enterococcus has also been reported to be and ZL contributed to funding acquisition. XL investigated the stably maintained in many insects, and it can protect insects study. HX contributed to methodology. YL and ZL contributed against pathogens, fix toxic molecules from plants, increase host to supervision. YY and XL visualized the study. YY wrote the fitness, and tolerate toxic diets (Shao et al., 2011; Johnston original draft and contributed to writing, reviewing, and editing and Rolff, 2015; Vilanova et al., 2016; Shao et al., 2017). the manuscript. All authors have read and agreed to the published Enterobacteriaceae is one of the important dominant taxa in version of the manuscript. the gut microbiota of many insects (Wang et al., 2014; Yun et al., 2018; Raza et al., 2020). Enterobacteriaceae are involved in insect metabolism (Pers and Hansen, 2021; Zhou et al., 2021), FUNDING insect resistance or susceptibility to parasites, and pathogens This research was funded by the Zhejiang Provincial and insecticides (Oliver et al., 2003; Álvarez-Lagazzi et al., 2021; Polenogova et al., 2021) and play an important role in the host Natural Science Foundation of China (grant no. LY20C140004), the earmarked fund for the China Agriculture adaptability and reproduction of insects (Shi et al., 2012; Wang et al., 2020b). Pectobacterium, a clade of Enterobacteriaceae, is Research System (grant no. CARS-01-39), and the State Key Laboratory for Managing Biotic and Chemical Treats to known as a function of nitrogen fixation (Behar et al., 2005, 2008). In addition to fixing nitrogen, the gut microbiota may the Quality and Safety of Agro-products (grant nos. 2010DS700124-ZZ2007 and 2010DS700124-KF1908). help recycle nitrogenous waste products into usable compounds, such as uric acid and ammonia (Behar et al., 2005, 2008). Corynebacterium-related bacteria grow on a variety of sugars, ACKNOWLEDGMENTS organic acids, and alcohols as the single or combined carbon and energy sources as a workhorse for the large-scale production of We are thankful to Josie Lynn Catindig for her generous help with amino acids (Eikmanns and Blombach, 2014). The detailed actual manuscript editing. functions of these microbes in the gut of C. medinalis need to be proven and verified in further investigations. SUPPLEMENTARY MATERIAL CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. In conclusion, our results indicated that the alpha diversity 2022.824224/full#supplementary-material indices of gut microbes in C. medinalis could not be affected by the host plant, generation, or host plant  generation. Supplementary Figure 1 | Bacterial taxa with linear discriminant analysis (LDA) score >2 in the gut microbiota of C. medinalis fed on different host plants. PerMANOVA indicated that the gut bacteria of C. medinalis could be significantly affected by the host plant and host Supplementary Figure 2 | Cladogram of bacterial biomarkers, from the phylum plant  generation. Coexisting bacteria that were found in (innermost ring) to genus (outermost ring) level, with an LDA score >2. Differential Frontiers in Microbiology | www.frontiersin.org 12 April 2022 | Volume 13 | Article 824224 fmicb-13-824224 April 5, 2022 Time: 19:11 # 13 Yang et al. Gut Microbiota in C. medinalis bacterial taxa are marked by lowercase letters. Each small circle at different Supplementary Table 2 | ANOVA of Alpha diversity indices of gut bacterial taxonomic levels represents a taxon at that level, and the diameter of the circle is communities in Cnaphalocrocis medinalis fed rice or maize for three generations. proportional to the relative abundance. The coloring principle is to color the Supplementary Table 3 | Relative abundance (%) of genus in gut microbiota of species with no significant difference as yellow and the other different species as rice- or maize-feeding Cnaphalocrocis medinalis for three generations. the group with the highest abundance of the species. Different colors represent different groups, and nodes with different colors represent the communities that Supplementary Table 4 | List of genera of the gut microbiota only stable in all play an important role in the group represented by the color. samples of rice- or maize-feeding Cnaphalocrocis medinalis. Supplementary Table 1 | Sequencing statistics of gut microbiota from rice- or Supplementary Table 5 | Analysis of similarity (ANOSIM) between gut microbial maize-feeding Cnaphalocrocis medinalis for three generations. communities from within sample groups of Cnaphalocrocis medinalis. Chung, S. H., Rosa, C., Scully, E. D., Peiffer, M., Tooker, J. F., Hoover, K., et al. REFERENCES (2013). Herbivore exploits orally secreted bacteria to suppress plant defenses. Álvarez-Lagazzi, A. P., Cabrera, N., Francis, F., and Ramírez, C. C. (2021). Proc. Natl. Acad. Sci. 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Progresses in management technology of rice leaffolders in China. J. Plant Prot. Publisher’s Note: All claims expressed in this article are solely those of the authors 42, 691–701. and do not necessarily represent those of their affiliated organizations, or those of Yuan, X. Q., Zhang, X., Liu, X. Y., Dong, Y. L., Yan, Z. Z., Lv, D. B., et al. (2021). the publisher, the editors and the reviewers. Any product that may be evaluated in Comparison of gut bacterial communities of Grapholita molesta (Lepidoptera: this article, or claim that may be made by its manufacturer, is not guaranteed or Tortricidae) reared on different host plants. Int. J. Mol. Sci. 22:6843. doi: 10. endorsed by the publisher. 3390/ijms22136843 Yun, J. H., Jung, M. J., Kim, P. S., and Bae, J. W. (2018). Social status shapes Copyright © 2022 Yang, Liu, Xu, Liu and Lu. This is an open-access article distributed the bacterial and fungal gut communities of the honey bee. Sci. 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