Structure of gut and ovary, with associated microbiota across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae)

Structure of gut and ovary, with associated microbiota across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structure of gut and ovary, with associated microbiota across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae) Haiying Zhong, Juefeng Zhang, Fang Li, Kaili Yu, Jianming Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6652146/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The striped stem borer Chilo suppressalis (Lepidoptera: Crambidae) was one of the most serious pests of water-oat Zizania latifolia . Microbiota associated with gut, ovary and egg influenced a wide variety of their host traits (such as fitness and immunity), and understand the microbiota structure and its dynamics across C. suppressalis ’s life was a prerequisite for comprehending the symbiotic relationship between C. suppressalis and its microbiota as well as transmission pattern. Herein, we characterized the structure of the gut, ovary and associated microbiota of a striped stem borer C. suppressalis . The gut was structurally divided into foregut, midgut and hindgut, and its structure and ultrastructure were very different between adults and larvae, however, this was not documented in detailed before. Microbiota in gut, ovary and egg showed variation in relative abundance. Proteobacteria and Firmicutes became the predominant phyla, and Bacillaceae, Enterobacteriaceae, Enterococcaceae, Halomonadaceae, Moraxellaceae and Streptococcaceae were shared among the gut, ovary and egg of C. suppressalis , although they exhibited different relative abundance. The highest bacteria diversity was found in the larval midgut. The bacterial genera distribution showed great differences due to developmental stage, diet and gut compartments. Our results demonstrated that the developmental stage, diet and gut compartment had a considerable impact on gut microbiota of C. suppressalis . Genera Klebsiella , Enterococcus , Bacillus , Citrobacter and Lactococcus were core microbiota, which transferred from the gut to the ovaries and eggs. Our study provided an important insight into investigation of insect-bacteria symbioses as well as its transmission pattern, so as to perform effective biocontrol of this species. Biological sciences/Microbiology Biological sciences/Zoology Health sciences/Anatomy Gut Reproductive system Morphology Ultrastructure Microbiota Stem borer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction The insects’ gut is a tube opening from the mouth to the anus, and is divided into foregut, midgut and hindgut. The midgut and hindgut are major regions; useful materials (nutrients) are absorbed in the midgut, and partial nutrients and water are absorbed in the hindgut. Variations in guts among different species can be attributed to differences of feeding biology and food types. The solid-food feeding species usually possess relatively short, thick guts, whereas the fluid feeding species generally have long narrow guts with some modified structures 1 . The gut is a desirable, nutrient-rich ecological niche where multiple microbiota flourish and reproduce, of which the midgut is the main site habouring various microbiota; the anterior hindgut is the most densely symbiont-inhabited site, due to the available, partially digested food being from the midgut. Conversely, those microbiota may help their hosts in utilizing life-stage-specific resources by providing various functions related to nutrient supplementation 2 , 3 , 4 , detoxification 5 , 6 , reproduction 7 and population differentiation 8 , 9 , and this is especially obvious in phytophagous insects. Gut microbes can be transmitted from one generation of the host to the next, a process known as vertical transmission or inheritance 10 , 11 . Vertical transmission of microbiota provides a direct way to start a gut microbiome 12 , 13 . Yet, the gut microbiota may partly assemble from the food and local environments in each generation 14 . Zizania latifolia (Gramineae: Oryzeae) is an asexual aquatic vegetable with an edible swelling, plump fleshy stem formed by infection of fungus Ustilago esculenta to its meristems. The striped stem borer (SSB) Chilo suppressalis is one of the destructive generalists of rice Oryza sativa in Asia, southern Europe and northern Africa 15 , 16 , 17 . The inter-cropping pattern of rice O. sativa and Z. latifolia facilitates a transfer of C. suppressalis from O. sativa plant to Z. latifolia plant. In comparison, C. suppressalis feeding on water-oat fruit pulps possess higher survival rate, pupal weight and shorter developmental duration than those feeding on O. sativa plant 18 , 19 , thus leading C. suppressalis become a most serious pests of Z. latifolia . Larvae of this species bore into sheaths, stems and pulps of Z. latifolia , resulting in ‘dead hearts’, ‘dead sheaths’ and ‘boring pulps’ 20 , 21 , emphasizing the urgency for developing innovative, effective biological prevention strategies to this pest. Diet is a major factor in structuring microbiota across animal taxa 22 , and diet shift results in a microbiota variation across developmental stages of a single host. Larvae of C. suppressalis chew the solid parts of plants, while the adults suck nectar or fruit juice. Nutritional or chemical differences of the diets between larvae and adults might significantly effect on their microbiota resided at different stages. Characterization of microbiota structure is a prerequisite for understanding how a microbiota population functions in the gut ecosystem. To date, some documents focused on resistance affect on gut microbiota 23 , 24 or diets on larval gut microbiota of C. suppressalis 9 , 25 . However, structure of the gut and ovary, and comprehensive characterization of gut microbiota dynamics across life history of C. suppressalis is not yet available. In this study, we illustrated the gut and ovary, and systematically profiled the gut microbiota of different life stages (i.e., larvae, adults, ovary and eggs) of C. suppressalis , using light and electron microscopes, transmission electron microscope and high throughput pyrosequencing of microbiota 16S rRNA gene fragments. Our data will be informative to better understand the functional role of each organ and associated community shifts during life stage transitions and community structure associated with dietary regimes; thus provide ideas for controlling this pest based on the source and transmission of gut microbiota. Materials and methods Plant obtain Ten clusters of water-oat Zizania latifolia variety ‘Zhejiao No. 7’ were obtained from the field of the Jinhai Yao, a farmer who has long been cultivated Zizania latifolia and cooperated with our team in terms of technical guidance of Z. latifolia . The obtained ‘Zhejiao No. 7’ were cultivated in the plant land of our laboratory experimental field for the subsequent investigation. Thus a permission was not required to collect the plants. A good management such as water, fertilizer provision and weed removal was carried out in the field. During the laboratory experiment, pulps of Z. latifolia variety “Zhejiao No. 7” were collected from our laboratory experimental field, and the use of the plant parts in the present study complies with laboratory guidelines. Specimen collection and rearing Larvae of C. suppressalis were collected from the Z. latifolia field in Lishui, Zhejiang, where large areas of Z. latifolia are exclusively planted. Larval C. suppressalis were reared exclusively with fresh pulps of Z. latifolia “Zhejiao No. 7” at 28 ± 1°C, with a photoperiod of 16 h: 8 h (light/dark), and a relative humidity > 80%. All the larvae were reared in the laboratory for three continuous generations and the 4th instar individuals were sampled. Newly hatched larvae of C. suppressalis were introduced into canned bottles (diameter: 10.0 cm, height: 7.0 cm) where fresh pulps of Z. latifolia were provided. The bottles were incubated under conditions of 28 ± 1°C, with a photoperiod of 16 h: 8 h (light: dark), and a relative humidity > 85%. Replace water-oats residues by fresh ones every three days. Repeating this procedure until the larvae enter into pupae stage. Picking out the pupae and put them in clean petri dishes (8.8 cm in diameter), containing a small chunk of watery sponge. The petri dishes were kept at the same incubated condition. Female adults were emerged from the pupae after 6 days. Females were fed with 8.5% sucrose water and paired to males for oviposition. Light microscopy After three generations of rearing, the females and larvae of C. suppressalis were anesthetized for dissection. Under a Motic SMZ168 Stereoscopic Zoom Microscope, the whole guts of both females and larvae and reproductive systems of females were respectively dissected out in a phosphate buffered solution (PBS, 0.2 M, pH 7.2). The guts and reproductive systems were carefully plated into concave dishes separately, and were observed structurally. Related photographs were taken via a Scientific Digital Micrography System (SDMS) which was equipped with an Auto-montage imaging system and a high sensitive Qimaging Retiga 2000R digital camera. Transmission electron microscopy (TEM) The guts and ovaries were respectively fixed with 2.5% glutaraldehyde for 12 h at 4℃. Then they were rinsed three times in phosphate buffer (PBS, 0.1 M, pH 7.2), and were post-fixed in 1% osmium tetroxide (PBS, 0.1 M, pH 7.2). After three times’ rinse, the guts and ovaries were dehydrated in series of ethanols (30–100%, v/v), and were embedded in Epon 812 for incubation 1 . Ultra-thin sections were stained with uranyl acetate and lead citrate, and were observed by a transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan) at 80 kV. Gut, ovary and egg samples collection Healthy, uniformly developed eggs, adults and larvae of the same batch of C. suppressalis were collected, and were externally sterilized with 75% ethanol and rinsed 3 times with sterilized water. The sterilized eggs were directly placed in sterile microcentrifuge tubes. Individuals of the adults and larvae were anesthetized by placing on ice, and their guts and ovaries were dissected out with a sterilized fine-tip forcep. The guts and ovaries were carefully separated and placed in different sterile microcentrifuge tubes. They were immediately frozen in liquid nitrogen and stored at -80°C for DNA isolation. Six groups of samples (larval midguts, larval hindguts, adult midgut, adult hindguts, ovaries and eggs) were set; the midguts, hindguts and ovaries of 50 individuals, and 100 eggs were respectively collected as one group of sample, and three replicates were taken. DNA isolation, 16S rDNA amplification Total DNA was extracted from six groups of samples (guts, ovaries and eggs) using a Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) followed the manufacturer’s instructions. Purity and concentration of the DNA were measured by a NanoDrop 2000 spectrophotometer (Nano-drop Technologies, Wilmington, DE, USA) 9 . Variable V3 − V4 regions of the bacterial 16S rRNA were were amplified using two universal primers 341F (5’-CCTAYGGGRBGCASCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’). The Polymerase Chain Reaction (PCR) reaction solution was composed of 4.0 µL 5×FastPfu Buffer, 2.0 µL 2.5 mM dNTPs, 0.4 µL FastPfu Polymerase, 0.8 µL Primer (5.0 µM) and 10 ng template DNA 9 . The amplification procedure was 95°C for 2 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s. The product was kept at 72°C for 5 min to extent complately 9 . Illumina MiSeq sequencing Concentration of the PCR amplifications was examined using 2% agarose gel electrophoresis and quantified via NanoDrop 2000. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq6000 platform according to the instructions. Processing of sequencing data Raw sequencing reads were processed in Qiime (v1.9.1) for quality control, fltering, splicing, chimera removal, and sequence optimization. 9 . Reads being not assembled were discarded. Operational Units (OTUs) clustering was performed by UPARSE (v7.1 http://drive5.com/uparse/ ) and chimeric sequences were removed by UCHIME. RDP Classifer (v2.2) Bayesian algorithm and Qiime (v1.9.1) sofware were used for phylogenetic affiliation of each gene sequence and comparison annotation of species and . Results General structure of adult gut The adult gut was structurally divided into foregut, midgut and hindgut. The foregut contained a narrow oesophagus and a bag-like crop; the oesophagus proceeded posteriorly into the crop and extended into the anterior of the midgut; the pouch-shaped midgut was well-developed, which began from the crop and extended to the hindgut; the hindgut consisted of a long convoluted ileum and a well-developed rectal pouch. Malpighian tubules emerging from the junction of the midgut and the ileum, ran laterally along the midgut in the posterior direction. After reaching the anterior midgut they followed their own course in a posterior direction and gather near the rectum (Fig. 1 A). General structure of larval gut The gut of larvae was a continuous tube, which ran from the mouth to the anus. Structurally, the gut was divided into three distinct segment: foregut, midgut and hindgut. The foregut was slender and elongate, expanded posteriorly and constricted at its end. The midgut was well-developed and saclike in shape, which began from the end of the foregut and extended to the anterior of the hindgut. The hindgut is about one third the length of midgut, and narrower than the midgut. When the individual was freshly dissected out, the three segments were very different in their color and easy to be differentiated: the foregut was translucent, the midgut was opaque white, and the hindgut was yellowish-brown (Fig. 1 B). General structure of female reproductive system The female reproductive system was complex for consisting of the following characteristics (Fig. 1 C). An ovary (Ov) contained four ovarioles; pedicel of each ovariole connected directly with a lateral oviduct (LO) and finally connected with a common oviduct (CO). Basal region of the common oviduct entered into a gential chamber (GC), which laterally connected with a spermatotheca duct (SD). The spermatotheca consisted of a pricinpal spermatotheca (PSp) and an accessory spermatotheca (ASp); a convoluted spermatotheca gland (SpG) emerged from the former apex. A duct of accessory gland (DAG) emerged at the lower middle region of the gential chamber, and apically bifurcated to a pair of accessory gland reservoirs (AGR). A coiled accessory gland (AG) situated basally from the reservoir. An afferent duct (AD) emerged from the other sides of the gential chamber, and its distal area connected with a duetus bursae (DB) enlarging distally to a sac-like corpus bursae (CB). Ultrastructure of the midgut and hindgut of adult C. suppressalis The midgut consisted of two types of cells resting on a basal lamina (~ 0.5 µm). In the first type of cells, round or gourd-shaped nuclei with clumps of heterochromatin bounded by nuclear membrane were found adjacent to the middle region (Fig. 2 A). Narrow, long and well-developed basal infoldings (~ 2.5 µm long) associated with elongated mitochondria are observed at the basal region(Fig. 2 A, B). Densely packed microvilli (~ 8.5 µm ) were obvious at the apical area (Fig. 2 A). The mitochondria occupied the most part, arranging in a row below the microvilli and tending to orientated with their long axes in the direction of the microvilli (Fig. 2 B) The cytoplasm contained extensive rough endoplasmic reticulum, electron-dense secretory granules and abundant electron-lucent secretory vesicles (Fig. 2 C, D). These secretory vesicles vary in size and shape, and it appears that some small vesicles fused to become larger ones (Fig. 2 C, 3 B). Many electron-dense lysosome-like granules containing debris of organelles were also observed in the cytoplasm (Figs. 2 E, 3 A). In comparison, cells of the second type possessed very shallow basal labriths, though their apical border bears well-developed microvilli (Fig. 3 C). Heavy staining of lateral cell membranes were observed in the cytoplasm (Fig. 3 D). Desmosomes cannot be discerned due to the over-staining of the membranes by osmium. Large areas of well-developed rough endoplasmic reticulum were found in the cytoplasm (Fig. 4 A). Accumulations of glycogen granules (~ 50.0 nm in diameter), electron-lucent secretory vesicles were present in the cytoplasm (Fig. 4 A). These particles were spherical or possibly polyhedral. Electron-dense secretory granules, mitochondria with cristae scattered through the cytoplasm (Fig. 4 B). Small secretory granules seemed to fuse into bigger ones. Nuclei with small nucleoli were bounded by nuclear membrane (Fig. 4 B, C). The cytoplasm seemed to be ruptured due to the load of virus-like particles. Electron-dense secretory granules, mitochondria with cristae, and nuclei with small nucleoli were bounded by nuclear membrane (Fig. 4 B, C). Microorganisms (~ 2.4 µm long, 0.2 ~ 0.5 µm in diameter) scattered among the mitochondria (Fig. 4 A, D). The ileum was lined with a highly convoluted cuticle containing an electron-dense epicuticle (0.5 µm) and an electron-lucent endocuticle (~ 0.3 µm) facing the lumen (Fig. 5 A). Cells beneath the cuticle were featured by elaborated extensive apical leaflets formed by invagination of the apical plasma membrane. Abundant mitochondria packed the cytoplasm and were associated with the leaflets. The basal plasma membrane invaginated into a few extremely wide and very shallow infoldings associated with mitochondria. Large numbers of oval or spherical microorganisms with electron-dense membrane were observed in the lumen of this organ (Fig. 5 B). Within the membrane, electron-lucent vesicles and brush-like structures adjacent to the particles were also present (Fig. 5 B). Ultrastructure of the midgut and hindgut of larvae C. suppressalis Cell of the midgut rested on a basal lamina (~ 0.2 µm), and its basal region was equipped with elongated mitochondria with evident cristae (Fig. 6 A).Among the mitochondria, rough endoplasmic reticulum was scattered. Apical plasma membrane beared well-developed apical microvilli,(about 2.5 ~ 4.0 µm long), adjacent to which were some oval mitochondria (Fig. 6 B). The mitochondria and rough endoplasmic reticulum occupied the most part of the cytoplasm. Cells of the hindgut rested on a layer of very thin basal lamina (~ 0.05 µm) (Fig. 6 C). Basal plasma membrane invaginated into very wide infoldings associated with oval mitochondria and rough endoplasmic reticulum; whereas the apical plasma membrane was elaborated to well-developed apical leaflets (Fig. 6 D). Ultrastructure of ovary of female C. suppressalis The ovary contained numerous nurse cells; cylindrical follicle cells with evident nuclei enveloped entirely the oocyte (Fig. 7 A, B). Basal membrane of the follicle cell invaginated into long wide infoldings associated with elongated or oval mitochondria (Fig. 7 C). Well-developed microvilli were visible at the apical area of the follicle cells, of which the cytoplasm was filled with yolk granules and lipid droplets (Fig. 7 A, D, 8 A). These granules and droplets vary in size and electron-density, and some smaller ones fused into be larger ones (Fig. 8 A). Rough endoplasmic reticulum and virus-like particles scattered among the granules and droplets (Fig. 8 B). Abundant elongated or oval mitochondria of various size were visible throughout the cytoplasm. Sparse less-developed apical microvilli and numerous elongate mitochondria seemed to been taking part in the secretion (Fig. 8 C). Different from the oocyte, the nurse cell was filled with secretory granules of different size and electron-density (Fig. 8 D). Analysis of bacterial 16S rDNA gene sequences A total of 687,799 sequences clustering into 2524 operational taxonomic units (OTUs) were generated to analyze to microbiota (Table 1 ). Detected OTU numbers, Chao and Shannon were calculated as alpha diversity indicators. The relative bacterial abundance of 17 phyla differs significantly among the guts, egg and ovary ( Kruskal-Wallis test, p < 0.0001) (Table S1 ). Although the bacteria identified in the larval gut (JMG) were more diverse than those in the adult egg (JEG), both organs possessed the highest bacteria diversity and richness (Table 1 ). Table 1 Diversity of gut bacterial communities based on sequencing Developmental stages Diet type Sample Abbreviations Reads Bases (bp) OTUs Coverage Richness estimate Diversity index Chao1 S Shannon larvae/adults Pulps Midgut JMG1 40138 17434180 248 0.998729 303 40769 1.82 JMG2 30164 13022139 156 0.998110 225 30361 1.76 JMG3 38225 16566823 185 0.998483 242 38626 2.03 Hindgut JHG1 39198 17058995 154 0.998954 182 39778 1.78 JHG2 29999 13129341 97 0.999033 142 30626 1.84 JHG3 41964 18898628 105 0.999261 129 44068 1.45 No Midgut AMG1 29622 13457815 116 0.999122 134 31413 1.29 AMG2 AMG3 31377 14267149 119 0.999076 140 33296 1.31 Hindgut AHG1 42226 18171436 94 0.999124 145 42360 1.65 AHG2 40005 17192557 137 0.998700 178 40081 0.78 AHG3 36054 15537581 100 0.998835 151 36219 1.35 Ovary/Egg - Ovary JOV1 38043 16419021 60 0.999448 95 38220 0.86 JOV2 42406 18247431 77 0.999245 148 42501 1.00 JOV3 37306 16100611 66 0.999383 94 37483 0.89 Egg JEG1 30535 14170587 102 0.999247 115 33125 1.85 JEG2 41371 18295563 106 0.999202 150 42683 1.68 JEG3 35379 16197473 117 0.998954 159 37890 1.94 Note: S, Number of Sequences; WO, water-oat; RS, rice seedlings. Diversity of gut microbiota A total of 46 OTUs were shared by all of the samples (gut, ovary and egg) (Fig. 9 ), and the core OTUs belonged to the four phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria (S1 Fig). The OTUs detected from different samples grouped into distinct families with vary abundance: these coming from the midgut contained 85 families, and Enterobacteriaceae (35.93%~94.12%) and Enterococcaceae (3.86%~12.41%) were the most abundant families (Table S2 A; Table S3 ); these from the hindgut were classified into 71 families, with Enterobacteriaceae (67.25%~77.89) and Enterococcaceae (10.4%~11.4%) being predominant families (Table S2 B; Table S3 ); those identified from the egg were classified to 101 families, and the Bacillaceae (32.5%), Streptococcaceae (16.2%), Enterococcaceae (8.3%), Enterobacteriaceae (5.3%) and Flavobacteriaceae (5.1%) become previlent families (Table S3 ); these from the ovary were grouped into 96 families, with Enterobacteriaceae (40.7%), Enterococcaceae (28.8%) and Bacillaceae (13.1%) become the predominant families (Table S3 ). OTUs from the different gut compartments exhibited an great different within larvae or adult: these from the adult hindgut (AHG) were grouped into 45 families, and Enterobacteriaceae (78.4%), Enterococcaceae (11.5%) and Bacillaceae (4.0%) were the predominant ones (Table S3 ); those from the adult midgut (AMG) were classified as 35 families, of which Enterobacteriaceae (94.1%) was the dominance taxa (Table S3 ). In comparison, OTUs obtained from AHG were grouped into 67 families, with Enterobacteriaceae (66.7%), Bacillaceae (11.3%), Enterococcaceae (10.1%) and Streptococcaceae (5.9%) being previlent families (Table S3 ); these from the larval midgut (JMG) were classified into 84 families, and Enterobacteriaceae (35.9%), Bacillaceae (26.9%), Streptococcaceae (14.1%), Enterococcaceae (12.4%), and Halomonadaceae (4.9%) become the predominant families (Table S3 ). Taxonomic distribution of gut microbiota of C. suppressalis In terms of phylum composition, a homogeneous distribution was found in all samples (AHG, AMG, JMG, JEG, JOV) and larval hindgut (JHG): Proteobacteria (8.6–96.3%), Firmicutes (2.5–89.7%), respectively (Fig. S1 ; Table S4 ); and the two predominant phyla ‘Proteobacteria and Firmicutes’ changed significantly between the JEG and JMG (Figs. 10 , S1; Table S3 and Table S4 ). A total of 17 bacterial phyla grouped into 129 families, and the most predominant family was Enterobacteriaceae, followed by Bacillaceae and Enterococcaceae (Table S3 ). The families Bacillaceae, Enterobacteriaceae, Enterococcaceae, Halomonadaceae, Moraxellaceae, Streptococcaceae and Pseudomonadaceae were found across all samples (AHG, AMG, JHG, JMG, JEG, JOV), although they exhibited different relative abundance; but the Brucellaceae, Clostridiaceae, Lactobacillaceae, Microbacteriaceae and Planococcaceae were only found in the JEG, JOV, JHG, JMG, although it was low in relative abundance (Figs. 10 , 11 ; Table S3 ). Bacillaceae was enriched in the egg (32.5%), followed by the JMG (26.9%) and JOV (13.1%). Enterobacteriaceae was enriched in the AMG and AHG (94.1%, 78.4%) and JHG (66.7%), respectively. Enterococcaceae was predominant in the JEG (28.8%), JMG (12.41%) and AHG (11.5%). Halomonadaceae and Streptococcaceae were abundant in the JOV (5.8%, 6.4%), JEG (5.2%, 16.2%) and JMG (4.9%, 14.1%), respectively. Moraxellaceae was enriched in the JEG (4.4%), and presented with a lower abundance in JOV, AHG and JMG (0.4%, 0.2%, and 0.2%). Some families have a more lower abundance, for example, Brucellaceae in the JEG (0.4%) and JOV (0.2%), Clostridiaceae, Lactobacillaceae in the JMG (0.2%, 0.7%) and JEG (0.2%, 1.0%), Microbacteriaceae and Planococcaceae in the JEG (0.2%), and the Pseudomonadaceae in the JOV (0.4) and JMG (0.4%). These bacteria exhibited a variation of relative abundance associated with developmental stage, diet and gut compartment. Based on analysis of developmental stage and gut compartments, the bacterial genera from adults and larvae showed distinct distribution (Fig. 11 ; Table S5 ). Klebsiella was dominant in the AMG (64.3%) and AHG (75.6%), followed by Enterococcus in the AHG (11.4%) and Morganella in the AMG (12.4%). Citrobacter and Enterococcus in the AHG (5.1%, 11.4%) has a higher abundance than those in AMG (2.2%, 3.8%). In the JEG, Bacillus (32.5%) was prevailed, followed by Lactococcus (15.8%), Enterococcus (8.3%) and Halomonas ( 5.2%). Citrobacter was dominant in the JHG (40.81%), followed by Klebsiella (25.7%). In the midgut of larvae, Klebsiella (35.1%) was dominant in the JMG, followed by Bacillus , Lactococcus and Enterococcus (26.9%, 13.9%, 12.4% ). Halomonas was present in the JHG and JMG with a lower abundance (3.4%, 4.9%). Enterococcus was notably dominant in the JOV (28.4%), followed by Klebsiella (28.4%), Bacillus (13.1%) and Lactococcus (6.3%); Halomona and Morganella were presented with a similar predominance (5.8%). Providencia was only identified in the AMG, AHG and JOV (3.7%, 5.6%, 8.2%), respectively. Development-, compartment- and diet-related variations in the microbiota In C. Suppressalis , 99 bacterial genera were identified, and influence of compartment proved to be significant with a well-defined cluster formed within larvae and adults (i.e., JHG, JMG or AHG, AMG). Bacteria from the JOV and JEG were more heterogeneous for constituting clusters (Fig. 12 ). Enterobacteriaceae, Bacillaceae and Streptococcaceae exhibited a significant difference in abundance among the guts. Enterobacteriaceae was dominant in the AHG and AMG (78.4%, 94.1%), but it was decreased in the JHG, JMG (66.7%, 35.9%). In comparison, Bacillaceae was less abundant in the AHG and AMG (4.0%, 0.7%), whereas it was increased significantly in the two regions of the larvae (11.3%, 26.9%). Streptococcaceae and Halomonadaceae were less rich in the AHG (1.9%, 1.6%) and AMG (0.3%, 0.3%), but were increased in the JHG (5.9%, 3.4%) and JMG (14.1%, 4.9%) (Fig. 12 ; Table S3 ). The non-metric multidimensional scaling (NMDS) analysis revealed a clear separation of all samples in accordance to developmental stage and gut regions, and a closer association between JMG and JOV. Clusters were well-defined and the relatively great variable in the samples (i.e., JMG, JHG, AMG, AHG, EGG) excepting for JOV. The JEG cluster exhibited the most different microbiota, followed by the AHG and AMG clusters; the JHG cluster showed an intermediate composition. The JEG and JOV clusters exhibited a higher inter-sample variation; the JHG cluster showed an intermediate composition respecting to the AMG, AHG and JHG clusters. The JMG and JOV clusters were similarly homogeneous; the JEG was the most heterogeneous, followed by JOV (Fig. 13 ). Discussion Morphology and ultrastructure of gut in C. suppressalis In the present study, three types of basal infoldings were observed in the gut cells of C. suppressalis : 1) very narrow, long infoldings; 2) shallow, scattered infoldings; 3) extremely wide,very shallow infoldings. The first type of infolding were frequently reported in many insects, such as the filter chamber of leafhopper Cicadella viridis and midgut of midge Belgica antarctica 1 , 26 . The second type of infolding were morphologically similar to those previously found in the midgut of leafworm Alabama argillacea 27 , ileum of C. viridis 1 and rectum of hangingfly Bittacus cirratus 28 . Well-developed apical microvilli only observing in the midugt of C. suppressalis were similar to those reported in the midgut of katydids Gampsocleis gratiosa 29 and black soldier fly Hermetia illucens 30 . The microvilli and infoldings can enormously increased the surface area of the cell membrane and indicated a secretory function 31 . However, some investigators suggested that infoldings and microvilli provided “effective coupling of passive solvent movements to active salt transport during fluid” 32 , 33 . In our opinion, well-developed infoldings and microvilli, abundant mitochondria, extensive rough endoplasmic reticulum and many secretory vesicles existed in the cytoplasm were probably play a important role during ion transport of midgut cells in C. suppressalis The most striking character of ileum cells in C. suppressalis was well-developed apical leaflets associated with abundant mitochondria, resembling those found in the C. viridis and fruit fly Bactrocera dorsalis 1 , 34 . Such leaflets could greatly increase the contact surface with the luminal fluid 35 , and the “apical infoldings and their associated mitochondria formed mitochondrial pumps for ion transport from the gut lumen to the hemocoel” 36 . Thus the extensive apical leaflets in the hindgut cells of C. suppressalis might be play a vital role in efficient re-absorption of ions. Lysosome-like granules were observed in the midgut epithelium of C. suppressalis , resemble those in the midgut of cockroach Periplaneta americana 37 and G. gratiosa 29 . Such structure were probably involved in the ultrastructural changes in aging cells via the activity of phagocytes or autophagy of cells. The presence of such structures suggested the midgut cells of C. suppressalis were probably in an aging stage and their ultrastructure is undergoing an alteration. Morphology and ultrastructure of the ovary of C. suppressalis The female reproductive system of C. suppressalis was similar to other lepidopterans, such as Psilogramma menephron , Tirathaba rufivena , Holcocerus hippophaecolus , Opisina arenosella 38 , 39 , 40 , 41 , and they shared the following characters: 1) it consisted of paired ovaries, and each ovary contained three to five ovarioles; 2) two lateral oviducts fused into a common oviduct; 3) a gential chamber connected laterally with a long convoluted spermatotheca gland; 4) a spermatotheca was composed of a primary gland and an accessory gland. However, there were some differences among the species, such as moths Grapholita molesta 42 , Phauda flammans 43 , genus Palumbina 7 , Sphecodoptera sheni 44 and butterfly Tirumala limniace 45 . These differences were probably attributed to the species difference, but needs to be confirmed in the future. Follicle cells played an important in the formation of egg 46 , and margins of the follicular cells were blurred at the beginning. The distribution of abundant mitochondria and rough endoplasmic reticulum was very similar to that observed in the bug Lygus lineloaris 47 , probably suggesting that the follicle cells of C. suppressalis need more substances coming from the hemolymph to synthetize precursors of egg, in the early stage of egg shell formation, follicular cells began to secrete and form proteinaceous yolk membrane, as indicated previously by Simiczyjew (2003) 46 . With the gradual densification of the yolk membrane, follicular cells might secrete a dense inner egg shell. Subsequently, the secretions continued to accumulate on the inner egg shell to form the outer egg shell. Microbiota associated with gut, ovary and egg In the present study, we characterized the microbiota composition and relative abundance of C. suppressalis across its life stage. The phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria together constituted the most microbiota (90.7 ~ 99.9%), suggesting the microbiota was rather conserved in adults regardless of diet shift occurring after the metamorphosis. Bacterial diversity was notably greater in the larval midgut of C. suppressalis , comparing with other region or egg. Since midgut was the main region of food digestion, and its pH, physiological habitat and avaliable food facilitated the residence of abundant microbiota. The recurrent detection of Klebsiella , Enterococcus , Bacillus , Lactococcus and Citrobacter in ovary, egg and gut suggested that they were core microbiota, and may be influenced by compartment, diet, and developmental stage. Since die of the adult and larvae of C. suppressalis were very different in nutritional ingredient, and the pulps ingested by larvae contain some secondary compounds (i.e., oxalic acid and tannin); it was probable that the core bacteria identified in C. suppressalis gut was potential symbiont or beneficial bacteria, which might have evolved in closely related to hosts or and was simply transient in the gut from the food, and be able to colonize in different gut regions, as indicated by Negash et al. (2020) and Berg (1996) 48 , 49 , further research is necessary to elucidate their potential functions. The composition and richness of bacterial genera in C. suppressalis exhibited significant differences across different life stages (adult, larvae, egg). Klebsiella dominated in the AMG, AHG and JMG, were suggested to contribute to nitrogen requirements 26 , 50 . Citrobacter and Bacillus were only prevailed in the JHG and JEG, respectively; Providencia was found exclusively in the AMG, AHG and JOV, and their functions merits further investigation. Enterococcus was indicated to be associated with insecticide and pathogen resistances 23 , 51 , thus the distribution of this genus in all the organ of C. suppressalis may closely related to the immune of this pest. Diet and developmental stage modulated microbiota 52 , 53 , 54 , 55 , and some bacteria probably suppress other phyla in the same habitat 56 . Therefore, we inferred that the different bacteria dominance of C. suppressalis might be related to successful reproduction of some bacteria genus and suppression of others species in turn. Our findings showed that a great variation in different microbiota in the AMG, JMG and JEG. The divergence in the microbiota may reflect divergent functional roles in specific resource use. Diet and developmental stage presumably drives the remarkable difference in microbiota between pulp-feeding larvae and sucrose water-feeding adults of C. suppressalis . The difference in microbiome between the AMG and JMG was attributed to be a combination of metamorphosis and diet consumed, as holometabolous insects typically voided their gut contents and lost their gut microbiome during pupation, and the diet is different between larvae and adult. After emergence, adult feeding might stimulate the growth of persisted bacteria, or new bacterial taxa sourced from the diet restoring community richness. The well-defined clusters of gut regions of C. suppressalis suggested a stable microbiota profiles, and the bacteria variation could be attributed to acquire microorganisms from a greatly diverse environmental reservoir microflora randomly, as was indicated by Curtis and Sloan (2004) 57 . The present results illuminated the abundance of two dominant phyla (i.e., Proteobacteria and Firmicutes) and the difference of three families (Enterobacteriaceae, Bacillaceae and Enterococcaceae) in C. suppressalis . Proteobacteria was reported to be involved in nitrogen 58 and carbohydrate degradation, such as starches and hemicellulose 59 . Firmicutes was suggested to take part in energy absorption from the diet and may influence the development 60 . Enterobacteriaceae was known to be involved in pheromone production 61 . Our findings suggested that the rapid fluctuation of microbiota from larval to adult guts was probably influenced by the diet shift and microbiota recombination which were important factors in modulating the microbiota, as was documented for other Lepidopterans, Coleopterans and Isopterans 56 , 62 , 63 , 64 , 65 , 53 . The persistent of some bacteria of C. suppressalis throughout their life history suggested that these core bacteria could be transmitted to ovaries and eggs, and the bacteria carried by eggs can further spread to the next generation, and may be associated with host adaptation. However, further investigations are needed to examine their stability inside the eggs and in the gut of C. suppressalis after multiple generations. Conclusion Structure of gut and ovary, with associated bacteria across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae) We investigated the structure of gut and ovary and associated microbiota across life stages in C. suppressalis . The comparison of gut structure and ovary with associated microbiota supplied important information about gut structure and microbiota changes due to development-, compartment- and diet-alters of C. suppressalis . Analysis of microbiota provided an some vital important about the mechanisms underlying C. suppressalis adaptation to development and host plants at the bacterial level. The present study showed that the larval midgut had the highest bacteria diversity; the most previlent phyla were Proteobacteria and Firmicutes; and the dominent families were Enterobacteriaceae, followed by Bacillaceae and Enterococcaceae. The diet, development and compartment were probably contributed the highly diversed microbiota at the genera level adult and larvae. The bacteria change of microbiota parallels to those of own morphology. These findings not only provided an important insight into investigation of insect-bacteria symbioses but also supplied an effective control of lepidopterans from the aspect of microbiota. Abbreviations AHG: Hindgut of adult; AMG: Midgut of adult; JEG: Egg deposited by female; JHG: Hindgut of lalrvae; JMG: Midgut of larvae; JOV: Ovary of female; NCBI: National Center for Biotechnology Information; NMDS: Non-metric multidimensional scaling; OTUs: Operational Units; PCR: Polymerase chain reaction; SSB: Striped stem borer; SRA: Sequence Read Archive. Declarations Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: This work is financially supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY24C040001), Science and technology planning project of Huangyan District (Grant No. 2023031) and China Agriculture Research System of MOF and MARA (Grant No. CARS-24-G-07). Author Contribution H.Z. conducted experiments, analyzed data and wrote original draft. J.Z. conceived and designed the research. F.L. sampled the insect and conducted part of the experiments. K.Y. reared and sampled insect, and analyzed data. J.C. reviewed and revised the manuscript. All authors reviewed and approved the final version of the manuscript. Acknowledgement The authors sincerely thank editors for their critical review and providing valuable comments to this manuscript. The authors are grateful to the laboratory staff for technical assistance, especially for the microbiota and transcriptome analysis support by Mingke Biotechnology (Hangzhou) Co., LTD. We also thank for the laboratory staff Liao Zhenfeng of Transmission Electron Microscope for her technical assistance. Data Availability The raw reads were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the BioProject: PRJNA1142714. 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Supplementary Files S1table.xls S2ATable.xls S2BTable.xls S2CTable.xls S3table.xls S4table.xls S5table.xls S6table.xls FigureS1.tif SupplementaryInformationFigurelegendsandTabletitles6June2025.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6652146","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473104182,"identity":"0bc02ff8-5982-49bd-be77-ac8f5dcf0962","order_by":0,"name":"Haiying Zhong","email":"","orcid":"","institution":"ZheJiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haiying","middleName":"","lastName":"Zhong","suffix":""},{"id":473104183,"identity":"43de5e0c-60b5-4e1e-9be9-49effd0662b9","order_by":1,"name":"Juefeng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIie2PMQuCUBDHnwi6XLReGPkZ4oEF9WF6Sy7WEjSWIDTZHhT1FWppTXhQSzS3WtBSg0OEgUOKzeoY9H7DHQf/33FHiEDwi2BSgBDVjnuQjgUV8AiRZsUVkioyFLlLnzs3/1nlNVCn/N6Omv2yLfuXc4YiLfYNWgVOAU7dVm+CA/QUSq0MRcaOoSFwtkPLoD0bme2BomUpCpqvRBm7+sOgzQjZKk+BeHklAN4BBHqNN7B1noJoDTUCZt0Fy5CmE2QbnvOLPjO3lbfb0kE90iCMRmx5cPxrlpIgl9zvX+mYE0+QwjDNBgXCAoFA8Id8AOUJReBiAPDrAAAAAElFTkSuQmCC","orcid":"","institution":"ZheJiang Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Juefeng","middleName":"","lastName":"Zhang","suffix":""},{"id":473104184,"identity":"947abdad-d00d-40ff-a0f0-0a00214f845f","order_by":2,"name":"Fang Li","email":"","orcid":"","institution":"ZheJiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Li","suffix":""},{"id":473104185,"identity":"a2780ede-46c2-4702-99f5-d194e08021ac","order_by":3,"name":"Kaili Yu","email":"","orcid":"","institution":"ZheJiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kaili","middleName":"","lastName":"Yu","suffix":""},{"id":473104186,"identity":"1ae563c4-7d56-4326-a6b4-c9c31677d25a","order_by":4,"name":"Jianming Chen","email":"","orcid":"","institution":"ZheJiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jianming","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-13 06:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6652146/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6652146/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85078778,"identity":"539889c6-7ea1-46be-a5fe-e2d1730a5613","added_by":"auto","created_at":"2025-06-20 17:09:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":739023,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral structure of the alimentary canal and reproductive system of the striped stem borer \u003cem\u003eChilo suppressalis\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Adult. (\u003cstrong\u003eB\u003c/strong\u003e) Larvae. (\u003cstrong\u003eC\u003c/strong\u003e) Reproductive system of female. AD, afferent duct; AG, accessory gland; AGR, accessory gland reservoir; ASp, accessory spermatotheca; AT, anal tube; CB, corpus bursae; Co, ileum; CO, common oviduct; Cr, crop; DAG, duct of accessory gland; DB, duetus bursae; GC, gential chamber; LO, lateral oviduct; Mg, midgut; MT, Malpighian tubule; Oe, oesophagus; Ov, ovary; PSp, pricinpal spermatotheca; Rc, rectum; SD, spermatotheca duct; SpG, spermatotheca gland. Scale bars: A–B: 0.4 mm; C: 0.5 mm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/9fe6d72099e47a113c2d81bb.png"},{"id":85077859,"identity":"14fa3099-e2cd-40e9-8c4e-08286a67235f","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":963844,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the first midgut cells in adult \u003cem\u003eC. suppressalis\u003c/em\u003e.(\u003cstrong\u003eA\u003c/strong\u003e) Transverse section of the midgut. (\u003cstrong\u003eB\u003c/strong\u003e) High magnification of the basal region of midgut cells, showing well-developed basal infoldings contain abundant mitochondria and electron-lucent secretory granules. (\u003cstrong\u003eC-E\u003c/strong\u003e) High magnification of the middle region of midgut cells, showing scatterred nuclei and rough endoplasmic reticulum, secretory vesicles and mitochondria scatter in the cytoplasm. bl, basal lamina; if, basal infoldings; l, lumen; ly, lysosome-like structures; Mi, mitochondria; n, nucleoli; N, nuclei; rer, endoplasmic reticulum; sg, secretory granules; sv, secretory vesicles. Scale bars: A: 5.0 μm; B–E: 0.5 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/ab733c80509dd019a68acd25.png"},{"id":85078782,"identity":"ae2f2901-2ce8-4367-b2a0-a407fa35c3da","added_by":"auto","created_at":"2025-06-20 17:09:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1209828,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the midgut in \u003cem\u003eC. suppressalis\u003c/em\u003e. (\u003cstrong\u003eA-B\u003c/strong\u003e) High magnification of the middle region of the first midgut cell. (\u003cstrong\u003eC-D\u003c/strong\u003e) High magnification of the middle region of the second type of midgut cell, showing scatterred nuclei and rough endoplasmic reticulum, secretory vesicles and mitochondria scatter in the cytoplasm. bl, basal lamina; if, basal infoldings; lysosome-like structures; Mi, mitochondria; rer, endoplasmic reticulum; sd, septate desmosomes; sg, secretory granules; sv, secretory vesicles. Scale bars: A, B: 0.2 μm; C, D: 0.5 μm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/e447fa834955a7ebb2db1a15.png"},{"id":85078786,"identity":"98968c81-d419-47e3-aad6-f87760e07b99","added_by":"auto","created_at":"2025-06-20 17:09:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1138044,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the second midgut cells in \u003cem\u003eC. suppressalis\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Middle region of the first type of cell, showing well developed rough endoplasmic reticulum, accumulations of glycogen granules (asterisks), and electron-lucent secretory vesicles. Arrows indicate microorganisms. (\u003cstrong\u003eB-C\u003c/strong\u003e) Close-up of the nucleus, secretory granules and mitochondria. (\u003cstrong\u003eD\u003c/strong\u003e) Close-up of a microorganism. bl, basal lamina; Mi, mitochondria; n, nucleoli; N, nuclei; rer, rough endoplasmic reticulum; sg, secretory granules; sv, secretory vesicles. Nuclei are bounded by nuclear membrane (as arrows indicates). Scale bars: A–D: 0.2 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/e888b83749a7f56e5f7693be.png"},{"id":85078777,"identity":"cf7ba3f3-411f-4b29-8403-b0d7bb4f56e8","added_by":"auto","created_at":"2025-06-20 17:09:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":491394,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the hindgut in \u003cem\u003eC. suppressalis. \u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The ileum cells contain abundant mitochondria. al, apical leaflets; bl, basal lamina; en, endocuticle; ep, epicuticle; if, basal infoldings; Mi, mitochondria. Arrows indicate microorganisms in the lumen. (\u003cstrong\u003eB\u003c/strong\u003e) Close-up of microorganisms in the lumen. Scale bars: A: 2.0 μm; B: 0.2 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/26fd8dc73c3a1d61bf6ab16f.png"},{"id":85077848,"identity":"4b024a64-cfc4-484b-87cd-194f3a328cc2","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1201549,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of midgut cells of larvae of \u003cem\u003eC. suppressalis\u003c/em\u003e. Scale bars: A, C and D 0.2 μm; B 0.5 μm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/80ec725503a7357e322de413.png"},{"id":85079042,"identity":"2c4b6b8c-120f-442e-8467-a1f1f05c7deb","added_by":"auto","created_at":"2025-06-20 17:17:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":910848,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the ovary of female \u003cem\u003eC. suppressalis\u003c/em\u003e. FC, follicle cell; Fl, infoldings; LD, lipid droplets; Mt, mitochondria; Mu, muscles; N, nucleus; Nc, nurse cell; O,oocyte; VM, vitelline membrane; Y, yolk granules. Scale bars: C 2.0 μm; D 5.0 μm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/c4d771cc6ceb5f486158ecb2.png"},{"id":85078793,"identity":"34c5748e-25ce-4432-aad9-e320e694f3b2","added_by":"auto","created_at":"2025-06-20 17:09:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1187715,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of the ovary of female \u003cem\u003eC. suppressalis\u003c/em\u003e. Fl, infoldings; LD, lipid droplets; Mt, mitochondria; RER, rough endoplasmic reticulum; Se, secretions; Sg, secretory granules; Y, yolk granules. Scale bars: A and D 2.0 μm; B: 0.2 μm; C 0.5 μm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/0a97ae53f5dfcd8f023fa26f.png"},{"id":85077873,"identity":"97e573de-1b6f-4218-a8ed-06c3c55dc5f8","added_by":"auto","created_at":"2025-06-20 17:01:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":111917,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of OTUs from unique species owned by each sample and common species shared by two or more samples. AHG, hindguts of adult; AMG, midguts of adults; JHG, hindguts of larvae; JEG, eggslaied by females; JMG, midguts of larvae; JOV, ovary of females.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/289559de74c422bac7d72785.png"},{"id":85077868,"identity":"1dcd28ad-7715-4a90-89e2-94b040ec17d2","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":115984,"visible":true,"origin":"","legend":"\u003cp\u003eCompositions of gut microbiota at the family level across different developmental stages of \u003cem\u003eC. suppressalis\u003c/em\u003e. The Y-axis represents the proportion of each taxon. Abbreviations: AHG1–AHG3, hindguts of adults; AMG1–AMG3, midguts of adults; JEG1–JEG3, eggs oviposited by females; JHG1–JHG3, hindguts of larvae; JMG1–JMG3, midguts of larvae; JOV1–JOV3, ovary of females.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/bba2a5098dbcd9ca9158626f.png"},{"id":85078780,"identity":"7ac4284f-0d30-4e48-b5b5-f108f4ce4442","added_by":"auto","created_at":"2025-06-20 17:09:11","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":121064,"visible":true,"origin":"","legend":"\u003cp\u003eCompositions of gut microbiota at the genus level across different developmental stages of \u003cem\u003eC. suppressalis\u003c/em\u003e.. The composition of each sample was based on the taxonomic assignment of the 16S rDNA sequences. The Y-axis represented the proportion of each taxon. Abbreviations: AHG1–AHG3, hindguts of adults; AMG1–AMG3, midguts of adults; JEG1–JEG3, eggs oviposited by females; JHG1–JHG3, hindguts of larvae; JMG1–JMG3, midguts of larvae; JOV1–JOV3, ovary of females.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/53229d6efa400721ac9278ec.png"},{"id":85078794,"identity":"51c558cb-14d1-40d8-a616-96f802bd3d34","added_by":"auto","created_at":"2025-06-20 17:09:12","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":336593,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance and composition of gut microbiota of all samples. Heatmap represent the proportions of OTUs at the family level. The X-coordinate represents the sample of each population, and the Y-coordinate represents the taxon. The color code indicates relative abundance, ranging from blue (low abundance) to yellow to red (high abundance). Abbreviations: AHG1–AHG3, hindguts of adults; AMG1–AMG3, midguts of adults; JEG1–JEG3, eggs oviposited by females; JHG1–JHG3, hindguts of larvae; JMG1–JMG3, midguts of larvae; JOV1–JOV3, ovary of females.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/25c8f80ac892957a623e3d28.png"},{"id":85077850,"identity":"bbfce89a-0a43-4bfa-9acb-e958170252e7","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":124131,"visible":true,"origin":"","legend":"\u003cp\u003eNMDS of the gut, ovary and egg microbiota of \u003cem\u003eC. suppressalis\u003c/em\u003e. The samples were clustered by developmental stage, diet and compartment and represented with different colors: AHG (red, circles), AMG (blue, squares), JMG (purpl, inverted triangle), JHG (saffron yellow, triangle), JEG (green, rhombus) and JOV (yellow green, cross). The ellipses represent the standard error of the centroid for each group of samples with a confident limit of 95%. Abbreviations: AHG1–AHG3, hindguts of adults; AMG1–AMG3, midguts of adults; JEG1–JEG3, eggs oviposited by females; JHG1–JHG3, hindguts of larvae; JMG1–JMG3, midguts of larvae; JOV1–JOV3, ovary of females.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/fef56673e29dcfb72e9b1130.png"},{"id":87991474,"identity":"6ad132af-5783-4649-8428-41cb8c47a551","added_by":"auto","created_at":"2025-07-31 08:32:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11572800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/19197381-1f12-4184-83bf-eeb2c2568af2.pdf"},{"id":85077836,"identity":"3e8eacc2-0432-41c7-b785-b08fbe767d79","added_by":"auto","created_at":"2025-06-20 17:01:10","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1210,"visible":true,"origin":"","legend":"","description":"","filename":"S1table.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/b60a122fc7dc4497d3d21ad0.xls"},{"id":85077847,"identity":"5795bb0f-ef5d-422b-8ce5-c7325d608e62","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2524,"visible":true,"origin":"","legend":"","description":"","filename":"S2ATable.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/1493f335246353b63bf89a59.xls"},{"id":85077833,"identity":"bd08dd5c-df9e-4684-a466-5685e053bb97","added_by":"auto","created_at":"2025-06-20 17:01:10","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1597,"visible":true,"origin":"","legend":"","description":"","filename":"S2BTable.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/90327ccadb51d072f1e3bee5.xls"},{"id":85077842,"identity":"8f6a4ff9-e60f-4f6f-b91d-60f663007015","added_by":"auto","created_at":"2025-06-20 17:01:10","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3492,"visible":true,"origin":"","legend":"","description":"","filename":"S2CTable.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/0be7146d8b934ecb8935851f.xls"},{"id":85078802,"identity":"1a6b898e-89de-41a2-b61c-dc29ffbc51da","added_by":"auto","created_at":"2025-06-20 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17:01:10","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3151,"visible":true,"origin":"","legend":"","description":"","filename":"S5table.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/345ffb59de2b063f1df3a618.xls"},{"id":85077857,"identity":"797455a7-93ef-4819-a935-61a085ebd3c9","added_by":"auto","created_at":"2025-06-20 17:01:11","extension":"xls","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":42170,"visible":true,"origin":"","legend":"","description":"","filename":"S6table.xls","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/e5b8b78681b0c296eb966c24.xls"},{"id":85079044,"identity":"177b821a-fd44-424f-a5ce-9aa3c756023f","added_by":"auto","created_at":"2025-06-20 17:17:11","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":10343744,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/54cb2a5b1dc8a4b73a542647.tif"},{"id":85078787,"identity":"3b735fef-2dae-4537-8dd2-e2fb4bc65e0e","added_by":"auto","created_at":"2025-06-20 17:09:11","extension":"doc","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":19456,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFigurelegendsandTabletitles6June2025.doc","url":"https://assets-eu.researchsquare.com/files/rs-6652146/v1/30c6e6a568e7e6f957d32781.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eStructure of gut and ovary, with associated microbiota across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe insects\u0026rsquo; gut is a tube opening from the mouth to the anus, and is divided into foregut, midgut and hindgut. The midgut and hindgut are major regions; useful materials (nutrients) are absorbed in the midgut, and partial nutrients and water are absorbed in the hindgut. Variations in guts among different species can be attributed to differences of feeding biology and food types. The solid-food feeding species usually possess relatively short, thick guts, whereas the fluid feeding species generally have long narrow guts with some modified structures\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe gut is a desirable, nutrient-rich ecological niche where multiple microbiota flourish and reproduce, of which the midgut is the main site habouring various microbiota; the anterior hindgut is the most densely symbiont-inhabited site, due to the available, partially digested food being from the midgut. Conversely, those microbiota may help their hosts in utilizing life-stage-specific resources by providing various functions related to nutrient supplementation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, detoxification\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, reproduction\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and population differentiation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and this is especially obvious in phytophagous insects. Gut microbes can be transmitted from one generation of the host to the next, a process known as vertical transmission or inheritance\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Vertical transmission of microbiota provides a direct way to start a gut microbiome\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Yet, the gut microbiota may partly assemble from the food and local environments in each generation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eZizania latifolia\u003c/em\u003e (Gramineae: Oryzeae) is an asexual aquatic vegetable with an edible swelling, plump fleshy stem formed by infection of fungus \u003cem\u003eUstilago esculenta\u003c/em\u003e to its meristems. The striped stem borer (SSB) \u003cem\u003eChilo suppressalis\u003c/em\u003e is one of the destructive generalists of rice \u003cem\u003eOryza sativa\u003c/em\u003e in Asia, southern Europe and northern Africa\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The inter-cropping pattern of rice \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. latifolia\u003c/em\u003e facilitates a transfer of \u003cem\u003eC. suppressalis\u003c/em\u003e from \u003cem\u003eO. sativa\u003c/em\u003e plant to \u003cem\u003eZ. latifolia\u003c/em\u003e plant. In comparison, \u003cem\u003eC. suppressalis\u003c/em\u003e feeding on water-oat fruit pulps possess higher survival rate, pupal weight and shorter developmental duration than those feeding on \u003cem\u003eO. sativa\u003c/em\u003e plant\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, thus leading \u003cem\u003eC. suppressalis\u003c/em\u003e become a most serious pests of \u003cem\u003eZ. latifolia\u003c/em\u003e. Larvae of this species bore into sheaths, stems and pulps of \u003cem\u003eZ. latifolia\u003c/em\u003e, resulting in \u0026lsquo;dead hearts\u0026rsquo;, \u0026lsquo;dead sheaths\u0026rsquo; and \u0026lsquo;boring pulps\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, emphasizing the urgency for developing innovative, effective biological prevention strategies to this pest.\u003c/p\u003e \u003cp\u003eDiet is a major factor in structuring microbiota across animal taxa\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and diet shift results in a microbiota variation across developmental stages of a single host. Larvae of \u003cem\u003eC. suppressalis\u003c/em\u003e chew the solid parts of plants, while the adults suck nectar or fruit juice. Nutritional or chemical differences of the diets between larvae and adults might significantly effect on their microbiota resided at different stages. Characterization of microbiota structure is a prerequisite for understanding how a microbiota population functions in the gut ecosystem. To date, some documents focused on resistance affect on gut microbiota\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e or diets on larval gut microbiota of \u003cem\u003eC. suppressalis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, structure of the gut and ovary, and comprehensive characterization of gut microbiota dynamics across life history of \u003cem\u003eC. suppressalis\u003c/em\u003e is not yet available.\u003c/p\u003e \u003cp\u003eIn this study, we illustrated the gut and ovary, and systematically profiled the gut microbiota of different life stages (i.e., larvae, adults, ovary and eggs) of \u003cem\u003eC. suppressalis\u003c/em\u003e, using light and electron microscopes, transmission electron microscope and high throughput pyrosequencing of microbiota 16S rRNA gene fragments. Our data will be informative to better understand the functional role of each organ and associated community shifts during life stage transitions and community structure associated with dietary regimes; thus provide ideas for controlling this pest based on the source and transmission of gut microbiota.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant obtain\u003c/h2\u003e \u003cp\u003eTen clusters of water-oat \u003cem\u003eZizania latifolia\u003c/em\u003e variety \u0026lsquo;Zhejiao No. 7\u0026rsquo; were obtained from the field of the Jinhai Yao, a farmer who has long been cultivated \u003cem\u003eZizania latifolia\u003c/em\u003e and cooperated with our team in terms of technical guidance of \u003cem\u003eZ. latifolia\u003c/em\u003e. The obtained \u0026lsquo;Zhejiao No. 7\u0026rsquo; were cultivated in the plant land of our laboratory experimental field for the subsequent investigation. Thus a permission was not required to collect the plants. A good management such as water, fertilizer provision and weed removal was carried out in the field. During the laboratory experiment, pulps of \u003cem\u003eZ. latifolia\u003c/em\u003e variety \u0026ldquo;Zhejiao No. 7\u0026rdquo; were collected from our laboratory experimental field, and the use of the plant parts in the present study complies with laboratory guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpecimen collection and rearing\u003c/h3\u003e\n\u003cp\u003eLarvae of \u003cem\u003eC. suppressalis\u003c/em\u003e were collected from the \u003cem\u003eZ. latifolia\u003c/em\u003e field in Lishui, Zhejiang, where large areas of \u003cem\u003eZ. latifolia\u003c/em\u003e are exclusively planted.\u003c/p\u003e \u003cp\u003eLarval \u003cem\u003eC. suppressalis\u003c/em\u003e were reared exclusively with fresh pulps of \u003cem\u003eZ. latifolia\u003c/em\u003e \u0026ldquo;Zhejiao No. 7\u0026rdquo; at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, with a photoperiod of 16 h: 8 h (light/dark), and a relative humidity\u0026thinsp;\u0026gt;\u0026thinsp;80%. All the larvae were reared in the laboratory for three continuous generations and the 4th instar individuals were sampled.\u003c/p\u003e \u003cp\u003eNewly hatched larvae of \u003cem\u003eC. suppressalis\u003c/em\u003e were introduced into canned bottles (diameter: 10.0 cm, height: 7.0 cm) where fresh pulps of \u003cem\u003eZ. latifolia\u003c/em\u003e were provided. The bottles were incubated under conditions of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, with a photoperiod of 16 h: 8 h (light: dark), and a relative humidity\u0026thinsp;\u0026gt;\u0026thinsp;85%. Replace water-oats residues by fresh ones every three days. Repeating this procedure until the larvae enter into pupae stage. Picking out the pupae and put them in clean petri dishes (8.8 cm in diameter), containing a small chunk of watery sponge. The petri dishes were kept at the same incubated condition. Female adults were emerged from the pupae after 6 days. Females were fed with 8.5% sucrose water and paired to males for oviposition.\u003c/p\u003e\n\u003ch3\u003eLight microscopy\u003c/h3\u003e\n\u003cp\u003eAfter three generations of rearing, the females and larvae of \u003cem\u003eC. suppressalis\u003c/em\u003e were anesthetized for dissection. Under a Motic SMZ168 Stereoscopic Zoom Microscope, the whole guts of both females and larvae and reproductive systems of females were respectively dissected out in a phosphate buffered solution (PBS, 0.2 M, pH 7.2). The guts and reproductive systems were carefully plated into concave dishes separately, and were observed structurally. Related photographs were taken via a Scientific Digital Micrography System (SDMS) which was equipped with an Auto-montage imaging system and a high sensitive Qimaging Retiga 2000R digital camera.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eThe guts and ovaries were respectively fixed with 2.5% glutaraldehyde for 12 h at 4℃. Then they were rinsed three times in phosphate buffer (PBS, 0.1 M, pH 7.2), and were post-fixed in 1% osmium tetroxide (PBS, 0.1 M, pH 7.2). After three times\u0026rsquo; rinse, the guts and ovaries were dehydrated in series of ethanols (30\u0026ndash;100%, v/v), and were embedded in Epon 812 for incubation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Ultra-thin sections were stained with uranyl acetate and lead citrate, and were observed by a transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan) at 80 kV.\u003c/p\u003e\n\u003ch3\u003eGut, ovary and egg samples collection\u003c/h3\u003e\n\u003cp\u003eHealthy, uniformly developed eggs, adults and larvae of the same batch of \u003cem\u003eC. suppressalis\u003c/em\u003e were collected, and were externally sterilized with 75% ethanol and rinsed 3 times with sterilized water. The sterilized eggs were directly placed in sterile microcentrifuge tubes. Individuals of the adults and larvae were anesthetized by placing on ice, and their guts and ovaries were dissected out with a sterilized fine-tip forcep. The guts and ovaries were carefully separated and placed in different sterile microcentrifuge tubes. They were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for DNA isolation. Six groups of samples (larval midguts, larval hindguts, adult midgut, adult hindguts, ovaries and eggs) were set; the midguts, hindguts and ovaries of 50 individuals, and 100 eggs were respectively collected as one group of sample, and three replicates were taken.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA isolation, 16S rDNA amplification\u003c/h2\u003e \u003cp\u003eTotal DNA was extracted from six groups of samples (guts, ovaries and eggs) using a Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) followed the manufacturer\u0026rsquo;s instructions. Purity and concentration of the DNA were measured by a NanoDrop 2000 spectrophotometer (Nano-drop Technologies, Wilmington, DE, USA)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVariable V3\u0026thinsp;\u0026minus;\u0026thinsp;V4 regions of the bacterial 16S rRNA were were amplified using two universal primers 341F (5\u0026rsquo;-CCTAYGGGRBGCASCAG-3\u0026rsquo;) and 806R (5\u0026rsquo;-GGACTACNNGGGTATCTAAT-3\u0026rsquo;). The Polymerase Chain Reaction (PCR) reaction solution was composed of 4.0 \u0026micro;L 5\u0026times;FastPfu Buffer, 2.0 \u0026micro;L 2.5 mM dNTPs, 0.4 \u0026micro;L FastPfu Polymerase, 0.8 \u0026micro;L Primer (5.0 \u0026micro;M) and 10 ng template DNA\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The amplification procedure was 95\u0026deg;C for 2 min, followed by 25 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 50\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s. The product was kept at 72\u0026deg;C for 5 min to extent complately\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIllumina MiSeq sequencing\u003c/h3\u003e\n\u003cp\u003eConcentration of the PCR amplifications was examined using 2% agarose gel electrophoresis and quantified via NanoDrop 2000. Purified amplicons were pooled in equimolar and paired-end sequenced (2 \u0026times; 250) on an Illumina MiSeq6000 platform according to the instructions.\u003c/p\u003e\n\u003ch3\u003eProcessing of sequencing data\u003c/h3\u003e\n\u003cp\u003eRaw sequencing reads were processed in Qiime (v1.9.1) for quality control, fltering, splicing, chimera removal, and sequence optimization.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Reads being not assembled were discarded. Operational Units (OTUs) clustering was performed by UPARSE (v7.1 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://drive5.com/uparse/\u003c/span\u003e\u003cspan address=\"http://drive5.com/uparse/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and chimeric sequences were removed by UCHIME. RDP Classifer (v2.2) Bayesian algorithm and Qiime (v1.9.1) sofware were used for phylogenetic affiliation of each gene sequence and comparison annotation of species and .\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneral structure of adult gut\u003c/h2\u003e\n \u003cp\u003eThe adult gut was structurally divided into foregut, midgut and hindgut. The foregut contained a narrow oesophagus and a bag-like crop; the oesophagus proceeded posteriorly into the crop and extended into the anterior of the midgut; the pouch-shaped midgut was well-developed, which began from the crop and extended to the hindgut; the hindgut consisted of a long convoluted ileum and a well-developed rectal pouch. Malpighian tubules emerging from the junction of the midgut and the ileum, ran laterally along the midgut in the posterior direction. After reaching the anterior midgut they followed their own course in a posterior direction and gather near the rectum (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneral structure of larval gut\u003c/h2\u003e\n \u003cp\u003eThe gut of larvae was a continuous tube, which ran from the mouth to the anus. Structurally, the gut was divided into three distinct segment: foregut, midgut and hindgut. The foregut was slender and elongate, expanded posteriorly and constricted at its end. The midgut was well-developed and saclike in shape, which began from the end of the foregut and extended to the anterior of the hindgut. The hindgut is about one third the length of midgut, and narrower than the midgut. When the individual was freshly dissected out, the three segments were very different in their color and easy to be differentiated: the foregut was translucent, the midgut was opaque white, and the hindgut was yellowish-brown (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneral structure of female reproductive system\u003c/h2\u003e\n \u003cp\u003eThe female reproductive system was complex for consisting of the following characteristics (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). An ovary (Ov) contained four ovarioles; pedicel of each ovariole connected directly with a lateral oviduct (LO) and finally connected with a common oviduct (CO). Basal region of the common oviduct entered into a gential chamber (GC), which laterally connected with a spermatotheca duct (SD). The spermatotheca consisted of a pricinpal spermatotheca (PSp) and an accessory spermatotheca (ASp); a convoluted spermatotheca gland (SpG) emerged from the former apex. A duct of accessory gland (DAG) emerged at the lower middle region of the gential chamber, and apically bifurcated to a pair of accessory gland reservoirs (AGR). A coiled accessory gland (AG) situated basally from the reservoir. An afferent duct (AD) emerged from the other sides of the gential chamber, and its distal area connected with a duetus bursae (DB) enlarging distally to a sac-like corpus bursae (CB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eUltrastructure of the midgut and hindgut of adult \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe midgut consisted of two types of cells resting on a basal lamina (~\u0026thinsp;0.5 \u0026micro;m). In the first type of cells, round or gourd-shaped nuclei with clumps of heterochromatin bounded by nuclear membrane were found adjacent to the middle region (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Narrow, long and well-developed basal infoldings (~\u0026thinsp;2.5 \u0026micro;m long) associated with elongated mitochondria are observed at the basal region(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Densely packed microvilli (~\u0026thinsp;8.5 \u0026micro;m ) were obvious at the apical area (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The mitochondria occupied the most part, arranging in a row below the microvilli and tending to orientated with their long axes in the direction of the microvilli (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) The cytoplasm contained extensive rough endoplasmic reticulum, electron-dense secretory granules and abundant electron-lucent secretory vesicles (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). These secretory vesicles vary in size and shape, and it appears that some small vesicles fused to become larger ones (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Many electron-dense lysosome-like granules containing debris of organelles were also observed in the cytoplasm (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eIn comparison, cells of the second type possessed very shallow basal labriths, though their apical border bears well-developed microvilli (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Heavy staining of lateral cell membranes were observed in the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Desmosomes cannot be discerned due to the over-staining of the membranes by osmium. Large areas of well-developed rough endoplasmic reticulum were found in the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Accumulations of glycogen granules (~\u0026thinsp;50.0 nm in diameter), electron-lucent secretory vesicles were present in the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). These particles were spherical or possibly polyhedral. Electron-dense secretory granules, mitochondria with cristae scattered through the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Small secretory granules seemed to fuse into bigger ones. Nuclei with small nucleoli were bounded by nuclear membrane (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). The cytoplasm seemed to be ruptured due to the load of virus-like particles. Electron-dense secretory granules, mitochondria with cristae, and nuclei with small nucleoli were bounded by nuclear membrane (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Microorganisms (~\u0026thinsp;2.4 \u0026micro;m long, 0.2\u0026thinsp;~\u0026thinsp;0.5 \u0026micro;m in diameter) scattered among the mitochondria (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, D).\u003c/p\u003e\n \u003cp\u003eThe ileum was lined with a highly convoluted cuticle containing an electron-dense epicuticle (0.5 \u0026micro;m) and an electron-lucent endocuticle (~\u0026thinsp;0.3 \u0026micro;m) facing the lumen (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cells beneath the cuticle were featured by elaborated extensive apical leaflets formed by invagination of the apical plasma membrane. Abundant mitochondria packed the cytoplasm and were associated with the leaflets. The basal plasma membrane invaginated into a few extremely wide and very shallow infoldings associated with mitochondria. Large numbers of oval or spherical microorganisms with electron-dense membrane were observed in the lumen of this organ (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Within the membrane, electron-lucent vesicles and brush-like structures adjacent to the particles were also present (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eUltrastructure of the midgut and hindgut of larvae \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eCell of the midgut rested on a basal lamina (~\u0026thinsp;0.2 \u0026micro;m), and its basal region was equipped with elongated mitochondria with evident cristae (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA).Among the mitochondria, rough endoplasmic reticulum was scattered. Apical plasma membrane beared well-developed apical microvilli,(about 2.5\u0026thinsp;~\u0026thinsp;4.0 \u0026micro;m long), adjacent to which were some oval mitochondria (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). The mitochondria and rough endoplasmic reticulum occupied the most part of the cytoplasm.\u003c/p\u003e\n \u003cp\u003eCells of the hindgut rested on a layer of very thin basal lamina (~\u0026thinsp;0.05 \u0026micro;m) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Basal plasma membrane invaginated into very wide infoldings associated with oval mitochondria and rough endoplasmic reticulum; whereas the apical plasma membrane was elaborated to well-developed apical leaflets (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eUltrastructure of ovary of female \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe ovary contained numerous nurse cells; cylindrical follicle cells with evident nuclei enveloped entirely the oocyte (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Basal membrane of the follicle cell invaginated into long wide infoldings associated with elongated or oval mitochondria (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Well-developed microvilli were visible at the apical area of the follicle cells, of which the cytoplasm was filled with yolk granules and lipid droplets (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, D, \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). These granules and droplets vary in size and electron-density, and some smaller ones fused into be larger ones (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). Rough endoplasmic reticulum and virus-like particles scattered among the granules and droplets (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). Abundant elongated or oval mitochondria of various size were visible throughout the cytoplasm. Sparse less-developed apical microvilli and numerous elongate mitochondria seemed to been taking part in the secretion (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). Different from the oocyte, the nurse cell was filled with secretory granules of different size and electron-density (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of bacterial 16S rDNA gene sequences\u003c/h2\u003e\n \u003cp\u003eA total of 687,799 sequences clustering into 2524 operational taxonomic units (OTUs) were generated to analyze to microbiota (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Detected OTU numbers, Chao and Shannon were calculated as alpha diversity indicators. The relative bacterial abundance of 17 phyla differs significantly among the guts, egg and ovary (\u003cem\u003eKruskal-Wallis\u003c/em\u003e test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Although the bacteria identified in the larval gut (JMG) were more diverse than those in the adult egg (JEG), both organs possessed the highest bacteria diversity and richness (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDiversity of gut bacterial communities based on sequencing\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 10.0848%;\"\u003e\n \u003cp\u003eDevelopmental stages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 4.1793%;\"\u003e\n \u003cp\u003eDiet type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAbbreviations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003eReads\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003eBases (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003eOTUs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003eCoverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 8.8128%;\"\u003e\n \u003cp\u003eRichness estimate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 0.9176%;\"\u003e\n \u003cp\u003eDiversity index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003eChao1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003eShannon\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\" style=\"width: 10.0848%;\"\u003e\n \u003cp\u003e\u003cstrong\u003elarvae/adults\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\" style=\"width: 4.1793%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePulps\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMidgut\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJMG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e40138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e17434180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e40769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJMG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e30164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e13022139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e30361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJMG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e38225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e16566823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e38626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHindgut\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJHG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e39198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e17058995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e39778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJHG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e29999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e13129341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e30626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJHG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e41964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e18898628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e44068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\" style=\"width: 4.1793%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMidgut\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAMG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e29622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e13457815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e31413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAMG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAMG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e31377\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e14267149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e33296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHindgut\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAHG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e42226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e18171436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e42360\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAHG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e40005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e17192557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e40081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eAHG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e36054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e15537581\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e36219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\" style=\"width: 10.0848%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOvary/Egg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\" style=\"width: 4.1793%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOvary\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJOV1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e38043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e16419021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e38220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJOV2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e42406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e18247431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e42501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJOV3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e37306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e16100611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e37483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\" style=\"width: 5.9964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEgg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJEG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e30535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e14170587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e33125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJEG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e41371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e18295563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.999202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e42683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 9.358%;\"\u003e\n \u003cp\u003eJEG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e35379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.7232%;\"\u003e\n \u003cp\u003e16197473\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.2701%;\"\u003e\n \u003cp\u003e117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.4506%;\"\u003e\n \u003cp\u003e0.998954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.5427%;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 4.361%;\"\u003e\n \u003cp\u003e37890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.8141%;\"\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"11\" style=\"width: 67.9588%;\"\u003eNote: S, Number of Sequences; WO, water-oat; RS, rice seedlings.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eDiversity of gut microbiota\u003c/h2\u003e\n \u003cp\u003eA total of 46 OTUs were shared by all of the samples (gut, ovary and egg) (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), and the core OTUs belonged to the four phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria (S1 Fig). The OTUs detected from different samples grouped into distinct families with vary abundance: these coming from the midgut contained 85 families, and Enterobacteriaceae (35.93%~94.12%) and Enterococcaceae (3.86%~12.41%) were the most abundant families (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eA; Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e); these from the hindgut were classified into 71 families, with Enterobacteriaceae (67.25%~77.89) and Enterococcaceae (10.4%~11.4%) being predominant families (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eB; Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e); those identified from the egg were classified to 101 families, and the Bacillaceae (32.5%), Streptococcaceae (16.2%), Enterococcaceae (8.3%), Enterobacteriaceae (5.3%) and Flavobacteriaceae (5.1%) become previlent families (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e); these from the ovary were grouped into 96 families, with Enterobacteriaceae (40.7%), Enterococcaceae (28.8%) and Bacillaceae (13.1%) become the predominant families (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eOTUs from the different gut compartments exhibited an great different within larvae or adult: these from the adult hindgut (AHG) were grouped into 45 families, and Enterobacteriaceae (78.4%), Enterococcaceae (11.5%) and Bacillaceae (4.0%) were the predominant ones (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e); those from the adult midgut (AMG) were classified as 35 families, of which Enterobacteriaceae (94.1%) was the dominance taxa (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). In comparison, OTUs obtained from AHG were grouped into 67 families, with Enterobacteriaceae (66.7%), Bacillaceae (11.3%), Enterococcaceae (10.1%) and Streptococcaceae (5.9%) being previlent families (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e); these from the larval midgut (JMG) were classified into 84 families, and Enterobacteriaceae (35.9%), Bacillaceae (26.9%), Streptococcaceae (14.1%), Enterococcaceae (12.4%), and Halomonadaceae (4.9%) become the predominant families (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eTaxonomic distribution of gut microbiota of \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eIn terms of phylum composition, a homogeneous distribution was found in all samples (AHG, AMG, JMG, JEG, JOV) and larval hindgut (JHG): Proteobacteria (8.6\u0026ndash;96.3%), Firmicutes (2.5\u0026ndash;89.7%), respectively (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e; Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e); and the two predominant phyla \u0026lsquo;Proteobacteria and Firmicutes\u0026rsquo; changed significantly between the JEG and JMG (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, S1; Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). A total of 17 bacterial phyla grouped into 129 families, and the most predominant family was Enterobacteriaceae, followed by Bacillaceae and Enterococcaceae (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). The families Bacillaceae, Enterobacteriaceae, Enterococcaceae, Halomonadaceae, Moraxellaceae, Streptococcaceae and Pseudomonadaceae were found across all samples (AHG, AMG, JHG, JMG, JEG, JOV), although they exhibited different relative abundance; but the Brucellaceae, Clostridiaceae, Lactobacillaceae, Microbacteriaceae and Planococcaceae were only found in the JEG, JOV, JHG, JMG, although it was low in relative abundance (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e; Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). Bacillaceae was enriched in the egg (32.5%), followed by the JMG (26.9%) and JOV (13.1%). Enterobacteriaceae was enriched in the AMG and AHG (94.1%, 78.4%) and JHG (66.7%), respectively. Enterococcaceae was predominant in the JEG (28.8%), JMG (12.41%) and AHG (11.5%). Halomonadaceae and Streptococcaceae were abundant in the JOV (5.8%, 6.4%), JEG (5.2%, 16.2%) and JMG (4.9%, 14.1%), respectively. Moraxellaceae was enriched in the JEG (4.4%), and presented with a lower abundance in JOV, AHG and JMG (0.4%, 0.2%, and 0.2%). Some families have a more lower abundance, for example, Brucellaceae in the JEG (0.4%) and JOV (0.2%), Clostridiaceae, Lactobacillaceae in the JMG (0.2%, 0.7%) and JEG (0.2%, 1.0%), Microbacteriaceae and Planococcaceae in the JEG (0.2%), and the Pseudomonadaceae in the JOV (0.4) and JMG (0.4%). These bacteria exhibited a variation of relative abundance associated with developmental stage, diet and gut compartment.\u003c/p\u003e\n \u003cp\u003eBased on analysis of developmental stage and gut compartments, the bacterial genera from adults and larvae showed distinct distribution (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e; Table \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e). \u003cem\u003eKlebsiella\u003c/em\u003e was dominant in the AMG (64.3%) and AHG (75.6%), followed by \u003cem\u003eEnterococcus\u003c/em\u003e in the AHG (11.4%) and \u003cem\u003eMorganella\u003c/em\u003e in the AMG (12.4%). \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e in the AHG (5.1%, 11.4%) has a higher abundance than those in AMG (2.2%, 3.8%). In the JEG, \u003cem\u003eBacillus\u003c/em\u003e (32.5%) was prevailed, followed by \u003cem\u003eLactococcus\u003c/em\u003e (15.8%), \u003cem\u003eEnterococcus\u003c/em\u003e (8.3%) and \u003cem\u003eHalomonas (\u003c/em\u003e5.2%). \u003cem\u003eCitrobacter\u003c/em\u003e was dominant in the JHG (40.81%), followed by \u003cem\u003eKlebsiella\u003c/em\u003e (25.7%). In the midgut of larvae, \u003cem\u003eKlebsiella\u003c/em\u003e (35.1%) was dominant in the JMG, followed by \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e (26.9%, 13.9%, 12.4% ). \u003cem\u003eHalomonas\u003c/em\u003e was present in the JHG and JMG with a lower abundance (3.4%, 4.9%). \u003cem\u003eEnterococcus\u003c/em\u003e was notably dominant in the JOV (28.4%), followed by \u003cem\u003eKlebsiella\u003c/em\u003e (28.4%), \u003cem\u003eBacillus\u003c/em\u003e (13.1%) and \u003cem\u003eLactococcus\u003c/em\u003e (6.3%); \u003cem\u003eHalomona\u003c/em\u003e and \u003cem\u003eMorganella\u003c/em\u003e were presented with a similar predominance (5.8%). \u003cem\u003eProvidencia\u003c/em\u003e was only identified in the AMG, AHG and JOV (3.7%, 5.6%, 8.2%), respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eDevelopment-, compartment- and diet-related variations in the microbiota\u003c/h2\u003e\n \u003cp\u003eIn \u003cem\u003eC. Suppressalis\u003c/em\u003e, 99 bacterial genera were identified, and influence of compartment proved to be significant with a well-defined cluster formed within larvae and adults (i.e., JHG, JMG or AHG, AMG). Bacteria from the JOV and JEG were more heterogeneous for constituting clusters (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e). Enterobacteriaceae, Bacillaceae and Streptococcaceae exhibited a significant difference in abundance among the guts. Enterobacteriaceae was dominant in the AHG and AMG (78.4%, 94.1%), but it was decreased in the JHG, JMG (66.7%, 35.9%). In comparison, Bacillaceae was less abundant in the AHG and AMG (4.0%, 0.7%), whereas it was increased significantly in the two regions of the larvae (11.3%, 26.9%). Streptococcaceae and Halomonadaceae were less rich in the AHG (1.9%, 1.6%) and AMG (0.3%, 0.3%), but were increased in the JHG (5.9%, 3.4%) and JMG (14.1%, 4.9%) (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e; Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe non-metric multidimensional scaling (NMDS) analysis revealed a clear separation of all samples in accordance to developmental stage and gut regions, and a closer association between JMG and JOV. Clusters were well-defined and the relatively great variable in the samples (i.e., JMG, JHG, AMG, AHG, EGG) excepting for JOV. The JEG cluster exhibited the most different microbiota, followed by the AHG and AMG clusters; the JHG cluster showed an intermediate composition. The JEG and JOV clusters exhibited a higher inter-sample variation; the JHG cluster showed an intermediate composition respecting to the AMG, AHG and JHG clusters. The JMG and JOV clusters were similarly homogeneous; the JEG was the most heterogeneous, followed by JOV (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eMorphology and ultrastructure of gut in \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eIn the present study, three types of basal infoldings were observed in the gut cells of \u003cem\u003eC. suppressalis\u003c/em\u003e: 1) very narrow, long infoldings; 2) shallow, scattered infoldings; 3) extremely wide,very shallow infoldings. The first type of infolding were frequently reported in many insects, such as the filter chamber of leafhopper \u003cem\u003eCicadella viridis\u003c/em\u003e and midgut of midge \u003cem\u003eBelgica antarctica\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The second type of infolding were morphologically similar to those previously found in the midgut of leafworm \u003cem\u003eAlabama argillacea\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, ileum of \u003cem\u003eC. viridis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and rectum of hangingfly \u003cem\u003eBittacus cirratus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Well-developed apical microvilli only observing in the midugt of \u003cem\u003eC. suppressalis\u003c/em\u003e were similar to those reported in the midgut of katydids \u003cem\u003eGampsocleis gratiosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and black soldier fly \u003cem\u003eHermetia illucens\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The microvilli and infoldings can enormously increased the surface area of the cell membrane and indicated a secretory function\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, some investigators suggested that infoldings and microvilli provided \u0026ldquo;effective coupling of passive solvent movements to active salt transport during fluid\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In our opinion, well-developed infoldings and microvilli, abundant mitochondria, extensive rough endoplasmic reticulum and many secretory vesicles existed in the cytoplasm were probably play a important role during ion transport of midgut cells in \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe most striking character of ileum cells in \u003cem\u003eC. suppressalis\u003c/em\u003e was well-developed apical leaflets associated with abundant mitochondria, resembling those found in the \u003cem\u003eC. viridis\u003c/em\u003e and fruit fly \u003cem\u003eBactrocera dorsalis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Such leaflets could greatly increase the contact surface with the luminal fluid\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and the \u0026ldquo;apical infoldings and their associated mitochondria formed mitochondrial pumps for ion transport from the gut lumen to the hemocoel\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Thus the extensive apical leaflets in the hindgut cells of \u003cem\u003eC. suppressalis\u003c/em\u003e might be play a vital role in efficient re-absorption of ions.\u003c/p\u003e \u003cp\u003eLysosome-like granules were observed in the midgut epithelium of \u003cem\u003eC. suppressalis\u003c/em\u003e, resemble those in the midgut of cockroach \u003cem\u003ePeriplaneta americana\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eG. gratiosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Such structure were probably involved in the ultrastructural changes in aging cells via the activity of phagocytes or autophagy of cells. The presence of such structures suggested the midgut cells of \u003cem\u003eC. suppressalis\u003c/em\u003e were probably in an aging stage and their ultrastructure is undergoing an alteration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMorphology and ultrastructure of the ovary of \u003cem\u003eC. suppressalis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe female reproductive system of \u003cem\u003eC. suppressalis\u003c/em\u003e was similar to other lepidopterans, such as \u003cem\u003ePsilogramma menephron\u003c/em\u003e, \u003cem\u003eTirathaba rufivena\u003c/em\u003e, \u003cem\u003eHolcocerus hippophaecolus\u003c/em\u003e, \u003cem\u003eOpisina arenosella\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and they shared the following characters: 1) it consisted of paired ovaries, and each ovary contained three to five ovarioles; 2) two lateral oviducts fused into a common oviduct; 3) a gential chamber connected laterally with a long convoluted spermatotheca gland; 4) a spermatotheca was composed of a primary gland and an accessory gland. However, there were some differences among the species, such as moths \u003cem\u003eGrapholita molesta\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePhauda flammans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, genus \u003cem\u003ePalumbina\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eSphecodoptera sheni\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and butterfly \u003cem\u003eTirumala limniace\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. These differences were probably attributed to the species difference, but needs to be confirmed in the future.\u003c/p\u003e \u003cp\u003eFollicle cells played an important in the formation of egg\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and margins of the follicular cells were blurred at the beginning. The distribution of abundant mitochondria and rough endoplasmic reticulum was very similar to that observed in the bug \u003cem\u003eLygus lineloaris\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, probably suggesting that the follicle cells of \u003cem\u003eC. suppressalis\u003c/em\u003e need more substances coming from the hemolymph to synthetize precursors of egg, in the early stage of egg shell formation, follicular cells began to secrete and form proteinaceous yolk membrane, as indicated previously by Simiczyjew (2003)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. With the gradual densification of the yolk membrane, follicular cells might secrete a dense inner egg shell. Subsequently, the secretions continued to accumulate on the inner egg shell to form the outer egg shell.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMicrobiota associated with gut, ovary and egg\u003c/h2\u003e \u003cp\u003eIn the present study, we characterized the microbiota composition and relative abundance of \u003cem\u003eC. suppressalis\u003c/em\u003e across its life stage. The phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria together constituted the most microbiota (90.7\u0026thinsp;~\u0026thinsp;99.9%), suggesting the microbiota was rather conserved in adults regardless of diet shift occurring after the metamorphosis. Bacterial diversity was notably greater in the larval midgut of \u003cem\u003eC. suppressalis\u003c/em\u003e, comparing with other region or egg. Since midgut was the main region of food digestion, and its pH, physiological habitat and avaliable food facilitated the residence of abundant microbiota. The recurrent detection of \u003cem\u003eKlebsiella\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e and \u003cem\u003eCitrobacter\u003c/em\u003e in ovary, egg and gut suggested that they were core microbiota, and may be influenced by compartment, diet, and developmental stage. Since die of the adult and larvae of \u003cem\u003eC. suppressalis\u003c/em\u003e were very different in nutritional ingredient, and the pulps ingested by larvae contain some secondary compounds (i.e., oxalic acid and tannin); it was probable that the core bacteria identified in \u003cem\u003eC. suppressalis\u003c/em\u003e gut was potential symbiont or beneficial bacteria, which might have evolved in closely related to hosts or and was simply transient in the gut from the food, and be able to colonize in different gut regions, as indicated by Negash et al. (2020) and Berg (1996)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, further research is necessary to elucidate their potential functions.\u003c/p\u003e \u003cp\u003eThe composition and richness of bacterial genera in \u003cem\u003eC. suppressalis\u003c/em\u003e exhibited significant differences across different life stages (adult, larvae, egg). \u003cem\u003eKlebsiella\u003c/em\u003e dominated in the AMG, AHG and JMG, were suggested to contribute to nitrogen requirements\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e were only prevailed in the JHG and JEG, respectively; \u003cem\u003eProvidencia\u003c/em\u003e was found exclusively in the AMG, AHG and JOV, and their functions merits further investigation. \u003cem\u003eEnterococcus\u003c/em\u003e was indicated to be associated with insecticide and pathogen resistances\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, thus the distribution of this genus in all the organ of \u003cem\u003eC. suppressalis\u003c/em\u003e may closely related to the immune of this pest. Diet and developmental stage modulated microbiota\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, and some bacteria probably suppress other phyla in the same habitat\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Therefore, we inferred that the different bacteria dominance of \u003cem\u003eC. suppressalis\u003c/em\u003e might be related to successful reproduction of some bacteria genus and suppression of others species in turn.\u003c/p\u003e \u003cp\u003eOur findings showed that a great variation in different microbiota in the AMG, JMG and JEG. The divergence in the microbiota may reflect divergent functional roles in specific resource use. Diet and developmental stage presumably drives the remarkable difference in microbiota between pulp-feeding larvae and sucrose water-feeding adults of \u003cem\u003eC. suppressalis\u003c/em\u003e. The difference in microbiome between the AMG and JMG was attributed to be a combination of metamorphosis and diet consumed, as holometabolous insects typically voided their gut contents and lost their gut microbiome during pupation, and the diet is different between larvae and adult. After emergence, adult feeding might stimulate the growth of persisted bacteria, or new bacterial taxa sourced from the diet restoring community richness. The well-defined clusters of gut regions of \u003cem\u003eC. suppressalis\u003c/em\u003e suggested a stable microbiota profiles, and the bacteria variation could be attributed to acquire microorganisms from a greatly diverse environmental reservoir microflora randomly, as was indicated by Curtis and Sloan (2004)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present results illuminated the abundance of two dominant phyla (i.e., Proteobacteria and Firmicutes) and the difference of three families (Enterobacteriaceae, Bacillaceae and Enterococcaceae) in \u003cem\u003eC. suppressalis\u003c/em\u003e. Proteobacteria was reported to be involved in nitrogen\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and carbohydrate degradation, such as starches and hemicellulose\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Firmicutes was suggested to take part in energy absorption from the diet and may influence the development\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Enterobacteriaceae was known to be involved in pheromone production\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Our findings suggested that the rapid fluctuation of microbiota from larval to adult guts was probably influenced by the diet shift and microbiota recombination which were important factors in modulating the microbiota, as was documented for other Lepidopterans, Coleopterans and Isopterans\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The persistent of some bacteria of \u003cem\u003eC. suppressalis\u003c/em\u003e throughout their life history suggested that these core bacteria could be transmitted to ovaries and eggs, and the bacteria carried by eggs can further spread to the next generation, and may be associated with host adaptation. However, further investigations are needed to examine their stability inside the eggs and in the gut of \u003cem\u003eC. suppressalis\u003c/em\u003e after multiple generations.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eStructure of gut and ovary, with associated bacteria across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eWe investigated the structure of gut and ovary and associated microbiota across life stages in \u003cem\u003eC. suppressalis\u003c/em\u003e. The comparison of gut structure and ovary with associated microbiota supplied important information about gut structure and microbiota changes due to development-, compartment- and diet-alters of \u003cem\u003eC. suppressalis\u003c/em\u003e. Analysis of microbiota provided an some vital important about the mechanisms underlying \u003cem\u003eC. suppressalis\u003c/em\u003e adaptation to development and host plants at the bacterial level. The present study showed that the larval midgut had the highest bacteria diversity; the most previlent phyla were Proteobacteria and Firmicutes; and the dominent families were Enterobacteriaceae, followed by Bacillaceae and Enterococcaceae. The diet, development and compartment were probably contributed the highly diversed microbiota at the genera level adult and larvae. The bacteria change of microbiota parallels to those of own morphology. These findings not only provided an important insight into investigation of insect-bacteria symbioses but also supplied an effective control of lepidopterans from the aspect of microbiota.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAHG: Hindgut of adult; AMG: Midgut of adult; JEG: Egg deposited by female; JHG: Hindgut of lalrvae; JMG: Midgut of larvae; JOV: Ovary of female; NCBI: National Center for Biotechnology Information; NMDS: Non-metric multidimensional scaling; OTUs: Operational Units; PCR: Polymerase chain reaction; SSB: Striped stem borer; SRA: Sequence Read Archive.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work is financially supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY24C040001), Science and technology planning project of Huangyan District (Grant No. 2023031) and China Agriculture Research System of MOF and MARA (Grant No. CARS-24-G-07).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.Z. conducted experiments, analyzed data and wrote original draft. J.Z. conceived and designed the research. F.L. sampled the insect and conducted part of the experiments. K.Y. reared and sampled insect, and analyzed data. J.C. reviewed and revised the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors sincerely thank editors for their critical review and providing valuable comments to this manuscript. The authors are grateful to the laboratory staff for technical assistance, especially for the microbiota and transcriptome analysis support by Mingke Biotechnology (Hangzhou) Co., LTD. We also thank for the laboratory staff Liao Zhenfeng of Transmission Electron Microscope for her technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw reads were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the BioProject: PRJNA1142714. Because the Release date set is 2026-12-31, the deposited data is not accessible until 2026-12-31. https://dataview.ncbi.nlm.nih.gov/object/PRJNA1142714?archive=sra\u003c/p\u003e\n\u003ch2\u003eSupplementary information: \u003c/h2\u003e\n\u003cp\u003eAccompanies this paper at https://submission.springernature.com/new/submission/cf30f102-4062-4a67-aae3-ea6e1ebd4acb/upload-files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhong, H., Zhang, Y. \u0026amp; Wei, C. Morphology of the alimentary canal of the leafhopper, \u003cem\u003eCicadella viridis\u003c/em\u003e (Hemiptera: Cicadellidae). \u003cem\u003eAnn. Entomol. Soc. AM.\u003c/em\u003e \u003cb\u003e108\u003c/b\u003e, 57\u0026ndash;69 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, B. et al. 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Rep.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 2792 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gut, Reproductive system, Morphology, Ultrastructure, Microbiota, Stem borer","lastPublishedDoi":"10.21203/rs.3.rs-6652146/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6652146/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe striped stem borer \u003cem\u003eChilo suppressalis\u003c/em\u003e (Lepidoptera: Crambidae) was one of the most serious pests of water-oat \u003cem\u003eZizania latifolia\u003c/em\u003e. Microbiota associated with gut, ovary and egg influenced a wide variety of their host traits (such as fitness and immunity), and understand the microbiota structure and its dynamics across \u003cem\u003eC. suppressalis\u003c/em\u003e\u0026rsquo;s life was a prerequisite for comprehending the symbiotic relationship between \u003cem\u003eC. suppressalis\u003c/em\u003e and its microbiota as well as transmission pattern. Herein, we characterized the structure of the gut, ovary and associated microbiota of a striped stem borer \u003cem\u003eC. suppressalis\u003c/em\u003e. The gut was structurally divided into foregut, midgut and hindgut, and its structure and ultrastructure were very different between adults and larvae, however, this was not documented in detailed before. Microbiota in gut, ovary and egg showed variation in relative abundance. Proteobacteria and Firmicutes became the predominant phyla, and Bacillaceae, Enterobacteriaceae, Enterococcaceae, Halomonadaceae, Moraxellaceae and Streptococcaceae were shared among the gut, ovary and egg of \u003cem\u003eC. suppressalis\u003c/em\u003e, although they exhibited different relative abundance. The highest bacteria diversity was found in the larval midgut. The bacterial genera distribution showed great differences due to developmental stage, diet and gut compartments. Our results demonstrated that the developmental stage, diet and gut compartment had a considerable impact on gut microbiota of \u003cem\u003eC. suppressalis\u003c/em\u003e. Genera \u003cem\u003eKlebsiella\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eLactococcus\u003c/em\u003e were core microbiota, which transferred from the gut to the ovaries and eggs. Our study provided an important insight into investigation of insect-bacteria symbioses as well as its transmission pattern, so as to perform effective biocontrol of this species.\u003c/p\u003e","manuscriptTitle":"Structure of gut and ovary, with associated microbiota across life stages in the striped stem borer Chilo suppressalis (Lepidoptera: Crambidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 17:01:05","doi":"10.21203/rs.3.rs-6652146/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9822a304-1bc2-45ab-9a10-29d50532a908","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50365598,"name":"Biological sciences/Microbiology"},{"id":50365599,"name":"Biological sciences/Zoology"},{"id":50365600,"name":"Health sciences/Anatomy"}],"tags":[],"updatedAt":"2025-07-31T08:24:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 17:01:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6652146","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6652146","identity":"rs-6652146","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}