Analysis of gut microbial diversity and function in Hyblaea puera (Lepidoptera: Hyblaeidae)

Analysis of gut microbial diversity and function in Hyblaea puera (Lepidoptera: Hyblaeidae) | 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 Research Article Analysis of gut microbial diversity and function in Hyblaea puera (Lepidoptera: Hyblaeidae) Wen Zhang, Qiaoling Zhao, Xiaoya Zhang, Yuehua Liu, Gaole Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8081780/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 Symbiotic microorganisms play a significant role in the physiology and biochemistry of insects. Hyblaea puera (Lepidoptera: Hyblaeidae), is an emerging invasive pest seriously damaging the mangrove ecosystem (mainly Avicennia marina ) along the coast of China. This study systematically investigated the community structure, diversity and potential functions of the gut bacteria of instar larvae of H. puera under field collection, indoor rearing and starvation treatment. The results revealed that laboratory rearing significantly altered the gut microbial composition. The gut bacteria of laboratory-reared larvae were dominated by the phylum Firmicutes-D, genus Enterococcus and Ligilactobacillus , whereas field-collected larvae exhibited a bacterial community primarily composed of the phyla Bacteroidota and Proteobacteria, genera JC017 and Fulvimarina . Starvation treatment significantly reduced alpha diversity and amplicon sequence variant (ASV) richness of the gut bacteria, and the alpha diversity was significantly lower in laboratory-reared larvae compared to field-collected ones. After starvation, the relative abundance of Enterococcus in laboratory-reared larvae increased markedly to 83.36%, resulting in a mono-dominant microbial structure. In contrast, starved field-collected larvae maintained a more diverse and tolerant community, predominantly consisting of Burkholderia and Acinetobacter . Functional prediction indicated that the gut bacteria was extensively involved in metabolic pathways such as amino acid and vitamin biosynthesis, as well as degradation of aromatic compounds, suggesting its potential role in helping the host overcome mangrove chemical defenses and compensate for nutritional challenges. This study provides a theoretical foundation for understanding the ecological adaptation mechanisms of H. puera and developing microbiome-based strategies for environmentally friendly pest control. Hyblaea puera high-throughput sequencing gut bacteria community structure function prediction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hyblaea puera (Lepidoptera: Hyblaeidae) is characterized by its high reproductive capacity, rapid dispersal, broad adaptability, and wide host range. Originating from Southeast Asia, it is the most serious defoliating pest in teak plantations. Following the introduction of teak, it has now spread to Latin America, where it continues to threaten teak production (Chandrasekhar et al. 2008 ). In recent years, H. puera has emerged as a major invasive pest damaging mangrove plants, particularly Avicennia marina , along the coast of China. (Hu et al. 2016 ). During the larval stage, H. puera feeds on the tender leaves of A. marina . Initially, the larvae consume approximately half of a leaf blade and then secrete silk-like saliva to roll the leaf, concealing themselves within the curled structure to continue feeding. Older instar larvae are capable of consuming entire leaves, leaving only bare branches. Pupation occurs inside the rolled leaves, and adults lay large numbers of eggs on the foliage, facilitating rapid population outbreaks. It has been documented that this pest has infested over 300 hectares of mangrove forests in Guangxi, China, posing a serious threat to mangrove ecosystem health and coastal conservation (Hu et al. 2016 , Yang et al. 2020 ). Current research on H. puera primarily focuses on the following aspects. Wei et al. ( 2019 ) systematically described its damage characteristics and proposed an integrated monitoring approach combining remote sensing and manual surveys to establish a cross-sectoral pest monitoring network (Wei et al. 2019 ). Chang et al. (2023) investigated the morphology and antennal sensilla of male and female adults, developing a rapid identification method that supports the application of sex pheromone-based control techniques (Chang 2023 ). In terms of biological control, the baculovirus HpNPV has been confirmed to be transmitted via feces and can effectively suppress H. puera populations (Biji et al. 2006 ), while Metarhizium spp. have demonstrated strong pathogenicity against the H. puera (Velavan et al. 2022 ). At the molecular level, the cloning of chitin metabolism-related genes ( HpCHS1 and HpChi-h ) has laid the groundwork for RNAi-based targeted control strategies (Kottaipalayam-Somasundaram et al. 2022 ), and plant-derived insecticidal proteins such as WsMBP1 have shown significant toxicity (George et al. 2018 ). Population genetics studies using RAGEP markers have revealed seasonal migration patterns (Chandrasekhar et al. 2005 ), and mitochondrial genome analyses have provided new insights into its phylogenetic classification (Shah et al. 2022 ). These findings have enhanced the understanding of H. puera and facilitated the development of integrated sustainable management strategies incorporating remote sensing, biological, and molecular technologies. The gut serves as a vital organ for food digestion and absorption in insects, harboring a vast array of symbiotic bacteria. These microbial symbionts play crucial roles in multiple physiological processes, including food digestion and nutrient assimilation (Warnecke et al. 2007 ), growth and reproduction (Sharon et al. 2010 ), immune development and regulation (Hernández-Martínez et al. 2010 ), pathogen resistance (Dillon et al. 2005 ), and pesticide detoxification (Broderick et al. 2006 ). The host insect and its gut microbiota interact through complex mechanisms of mutual dependence, reciprocal regulation, and co-adaptation. For instance, the composition and metabolic activity of gut bacteria can significantly influence host physiology and behavior, while the microenvironment of the insect gut also shapes—and often determines—the structure and assembly of the bacterial community (Jia et al. 2021 , Yao et al. 2022 ). Given these multifaceted interactions, symbiotic bacteria are increasingly regarded as a “multifunctional organ” in pest management strategies, driving research that targets insect-microbe interactions to develop novel control techniques. However, the diversity and functional roles of the gut microbiota in H. puera remain largely unexplored. The present study investigated the gut bacterial diversity, community structure, taxonomic composition, and biological functions of fifth-instar larvae of H. puera —a major invasive pest damaging mangrove ecosystems—using 16S rRNA high-throughput amplicon sequencing and bioinformatic approaches. Comparisons were made among field-collected populations, laboratory-reared populations, and both groups subjected to starvation treatment. The findings provide a foundation for further elucidating the biological roles of gut bacteria in the host insect and support the development of novel integrated pest management strategies based on insect-microbe interactions. This study also offers important theoretical and practical insights for the conservation of mangrove ecosystems. Materials and methods Sample Collection and Rearing Field-collected larvae of H. puera were obtained from A. marina of mangrove in Zhanjiang, Guangdong Province, China (10°54′21″N, 108°54′21″E) in April 2024. For the laboratory population, field-collected individuals were reared for more than ten generations in a controlled greenhouse environment using an artificial diet. The rearing conditions were maintained as follows: temperature at 28 ± 2°C, photoperiod of 16 h light : 8 h dark, and relative humidity of 60% ± 10%. Sample Preparation and 16S rRNA High-Throughput Sequencing The fifth-instar larvae of H. puera from field-collected, laboratory-reared, and 24 h starved groups were dissected under sterile conditions using forceps, scissors, and dissection needles to isolate intact gut tissues. The extracted guts were immediately flash-frozen in liquid nitrogen and stored at -80°C until further processing. Each gut was considered an independent biological replicate, with six replicates per group. All samples were sent to Nanjing Personalgene Technology Co., Ltd. for 16S rRNA amplicon sequencing. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified with the universal primers 338F (5ʼ-ACTCCTACGGGAGGCAGCA-3ʼ) and 806R (5ʼ-GGACTACHVGGGTWTCTAAT-3ʼ), with unique barcodes incorporated into the forward primer. The 25 µL PCR reaction mixture consisted of 0.25 µL of Q5 High-Fidelity DNA Polymerase, 5 µL of 5× Reaction Buffer, 5 µL of 5× High GC Buffer, 2 µL of dNTPs (10 mM), 2 µL of template DNA, 1 µL of each forward and reverse primer (10 µM), and 8.75 µL of ddH₂O. The thermal cycling protocol included an initial denaturation at 98°C for 5 min; 25 cycles of denaturation at 98°C for 10 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s; followed by a final extension at 72°C for 5 min. Amplification products were electrophoresed on a 2% agarose gel, and target bands were excised and purified using the AxyPrep DNA Gel Extraction Kit. Purified amplicons were quantified with the Quant-iT™ PicoGreen™ dsDNA Assay Kit on a Microplate Reader (BioTek, FLx800), pooled in equimolar amounts according to the required sequencing depth, and subjected to sequencing on the Illumina platform. Bioinformatic Analysis and Functional Prediction Raw sequencing data were processed using QIIME2 (version 2019.4; https://github.com/QIIME2/q2-feature-classifier ) (Bolyen et al. 2019 ). Demultiplexing was performed with the demux plugin, followed by primer trimming using cutadapt. Sequences were subsequently quality-filtered, denoised, merged (skipped for single-end reads), and checked for chimeras with the DADA2 plugin (Callahan et al. 2016 ). Amplicon sequence variants (ASV) were generated by clustering sequences at 100% similarity, producing an ASV abundance table. Taxonomic assignment of representative ASV sequences was conducted by alignment against the Greengenes database (Release 13.8; http://greengenes.secondgenome.com ) within QIIME2 (DeSantis et al. 2006 ). Alpha and beta diversity analyses were performed using the q2-diversity plugin in QIIME2. Rarefaction curves were generated and visualized with R software (version 4.5.1; https://www.r-project.org/ ). Functional potential of the gut microbiota was predicted from the 16S rRNA data using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; https://github.com/picrust/picrust2/wiki ) (Douglas et al. 2020 ). Results Taxonomic Annotation and Evaluation of Gut Bacteria in H. puera Larvae A total of 2,336,149 high-quality sequences were obtained from 24 gut samples of fifth-instar H. puera larvae after high-throughput sequencing. Basic information of the 16S rRNA amplicon sequencing is summarized in Table 1 . After denoising and clustering using the DADA2 pipeline, a total of 27 phyla, 46 classes, 106 orders, 204 families, 451 genera, and 761 species were identified. The laboratory-reared group exhibited a significantly higher number of bacterial ASVs compared to the field-collected group. Following starvation treatment, both groups showed a significant reduction in gut bacterial ASV richness. The rarefaction curves flattened as sequencing depth increased (Fig. 1 A), indicating that sufficient sequencing coverage was achieved and that additional reads would not substantially increase ASV detection. This confirms the high quality of the sequencing data and supports subsequent analyses. Venn diagram analysis revealed 42 ASVs shared across all four experimental groups. Specifically, 1782, 411, 2574, and 469 unique ASVs were identified in the field-collected non-starved (HAN), field-collected starved (HAS), laboratory-reared non-starved (HFN), and laboratory-reared starved (HAS) groups, respectively (Fig. 1 B). These results suggest that laboratory rearing with an artificial diet increased ASV richness in the larval gut bacteria, while subsequent starvation significantly reduced gut bacterial ASV richness in both populations. Table 1 Basic information of 16S rRNA amplicon sequencing of gut microbiota in fifth-instar larvae of Hyblaea puera Group phylum class order family genus species ASV HAN 20 29 39 71 125 195 1974 HAS 18 27 30 43 60 70 557 HFN 21 30 46 77 142 225 3027 HFS 17 25 29 43 57 74 839 Total 27 46 106 204 451 761 5278 HAN, field-collected larvae; HAS, field-collected larvae starved for 24 h; HFN, laboratory-reared larvae fed artificial diet; HFS, laboratory-reared larvae starved for 24 h. Analysis of Gut Bacterial Diversity in H. puera Larvae Alpha diversity of the gut bacteria in fifth-instar H. puera larvae was evaluated using multiple indices, including Chao1, Goods_coverage, Simpson, Pielou_e, Faith_pd, Shannon, and Observed_species (Fig. 2 ; Table S1 ). The results showed that field-collected larvae exhibited lower values in Chao1, Simpson, Pielou_e, Faith_pd, Shannon, and Observed_species compared to laboratory-reared larvae, though these differences were not statistically significant ( P > 0.05). After starvation treatment, field-collected and laboratory-reared groups showed reductions in Chao1, Faith_pd, and Observed_species relative to their non-starved counterparts, but again these changes were not significant. In contrast, significant decreases in Simpson, Pielou_e, and Shannon indices were observed in starved laboratory-reared larvae compared to the non-starved group ( P < 0.01). Furthermore, starved field-collected larvae displayed significantly higher Simpson and Pielou_e values than starved laboratory-reared larvae ( P < 0.05). These findings indicate that starvation significantly reduced the alpha diversity of the gut bacteria in laboratory-reared H. puera larvae. Moreover, under starvation conditions, laboratory-reared larvae showed significantly lower alpha diversity compared to field-collected larvae. Principal coordinates analysis (PCoA) was performed using the Unweighted Unifrac distance metric. Eigenvalues and eigenvectors were computed, and the axes with the highest contribution rates were selected for visualization. Greater distances between samples in the ordination plot reflect larger dissimilarities in microbial community structure. As shown in Fig. 3 A, slight differences were observed in the gut bacterial composition between starved groups of field-collected and laboratory-reared larvae. In contrast, pronounced separation was detected between non-starved field-collected and laboratory-reared groups, indicating significant structural divergence. Non-metric multidimensional scaling (NMDS) was further applied to rank inter-sample distances based on ordinal relationships rather than absolute values, providing more robust visualization for complex ecological data (Fig. 3 B). The stress value for the NMDS ordination was 0.138, which is below the 0.2 threshold, confirming the reliability of the configuration. Confidence intervals partially overlapped between starved field-collected and laboratory-reared groups, whereas no overlap was observed between non-starved field-collected and laboratory-reared groups. These results demonstrate that the gut microbial community structure and composition differ significantly between field-collected and laboratory-reared larvae under non-starved conditions. Gut Bacterial Community Composition in H. puera Larvae At the phylum level (Table S1 ), the top five bacterial phyla in field-collected fifth-instar larvae were Bacteroidota, Proteobacteria, Firmicutes-A, Actinobacteriota, and Firmicutes-D. After 24 hours of starvation, the dominant phyla in field-collected larvae shifted to Proteobacteria, Bacteroidota, Actinobacteriota, Firmicutes-D, and Cyanobacteria. In contrast, laboratory-reared larvae were dominated by Firmicutes-D, Proteobacteria, Firmicutes-A, Actinobacteriota, and Bacteroidota. Starvation of laboratory-reared larvae resulted in a predominance of Firmicutes-D, Proteobacteria, Actinobacteriota, Methylomirabilota, and Firmicutes-A. At the genus level (Fig. 4 A–B, Table S2), the gut bacteria of non-starved laboratory-reared larvae was dominated by Enterococcus (33.32%), Ligilactobacillus (18.39%), Stenotrophomonas (9.01%), Bacillus (4.35%), Levilactobacillus (3.80%), Lactiplantibacillus (2.90%), Acinetobacter (1.31%), Faecalibacterium (1.25%), Collinsella (1.08%), and Liquorilactobacillus (0.88%). After 24 hours of starvation, the community shifted markedly, with Enterococcus becoming overwhelmingly dominant (83.36%), followed by Acinetobacter (2.86%), Bacillus (1.73%), Ligilactobacillus (1.60%), Burkholderia (0.76%), Lactiplantibacillus (0.43%), Liquorilactobacillus (0.38%), Levilactobacillus (0.37%), Stenotrophomonas (0.34%), and DSHD01 (0.11%). In field-collected non-starved larvae, the dominant genera were JC017 (38.03%), Fulvimarina (14.82%), Stenotrophomonas (10.47%), Pseudokineococcus (5.76%), Alistipes (1.72%), Burkholderia (1.71%), Faecalibacterium (1.60%), Acinetobacter (1.53%), Barnesiella (1.17%), and Prevotella (1.00%). After starvation, the microbial composition changed significantly, with Burkholderia (34.48%) and Acinetobacter (20.64%) becoming the most abundant taxa, followed by JC017 (8.20%), Brevundimonas (3.63%), Pseudomonas (3.18%), Methylobacterium (2.70%), Actinotalea (1.91%), Fulvimarina (1.89%), Nocardioides (1.55%), and Actinomyces (1.46%). Functional Prediction of Gut Bacteria Based on PICRUSt2 analysis, a statistical functional assessment of metabolic pathways was performed (Fig. 5 ). At Level 1 pathway categories, the gut bacteria of H. puera was predominantly enriched in six major functional classes: Biosynthesis, Degradation/ Utilization/Assimilation, Detoxification, Generation of Precursor Metabolites and Energy, Glycan Pathways, and Metabolic Clusters. Predicted Level 2 pathway annotations revealed that gut symbiotic bacteria are involved in diverse physiological and biochemical processes, with significant enrichment in multiple metabolic pathways. These include amino acid biosynthesis; biosynthesis of cofactors, prosthetic groups, electron carriers, and vitamins; carbohydrate biosynthesis; fatty acid and lipid biosynthesis; amino acid degradation; aromatic compound degradation; carbohydrate degradation; carboxylate degradation; and nucleoside and nucleotide degradation. These results indicate that the gut bacteria of H. puera participates in a wide range of host physiological processes and may play crucial roles in supporting development and reproduction. Discussion Symbiotic microbes are closely associated with various aspects of insect physiology, including nutrition, reproduction, defense, immunity, and stress resistance (Han et al. 2024 ), and directly or indirectly influence insect growth, development, metabolism, and environmental adaptability (Yao et al. 2023 , Liu et al. 2024 ). Hyblaea puera has recently emerged as a major invasive pest damaging mangrove plants, particularly A. marina . However, the diversity and functional roles of the gut microbiota in H. puera remain largely unexplored. In this study, we employed 16S rRNA high-throughput sequencing to systematically investigate the diversity, community structure, and potential functions of the gut bacteria in fifth-instar larvae of H. puera under field-collected, laboratory-reared, and starvation-treated conditions. The gut microbiota of insects is largely acquired through oral intake, and microorganisms carried by food significantly shape the microbial community, indicating that dietary sources play a decisive role in structuring the gut microbiota (Douglas 2015 ). For example, in silkworm larvae (4th–5th instar) fed with lettuce leaves, the gut microbial composition differed markedly from that of conventionally reared silkworms. Lettuce-fed larvae exhibited substantial abundances of Acinetobacter and Anaerofilum -genera that are nearly absent in normal silkworm larvae (Liang et al. 2014 ). Our results indicated that the gut bacteria of laboratory- reared H. puera larvae is dominated by the phylum Firmicutes-D, with Enterococcus and Ligilactobacillus being the predominant genera. Such a community structure is characteristic of insects reared on artificial diets rich in sugars and starches (Chen et al. 2018 ). As facultative anaerobes, lactobacilli efficiently ferment carbohydrates to produce lactate, which not only helps establish an acidic microenvironment that inhibits pathogens but may also enhance the host’s energy acquisition efficiency from the artificial diet (Engel and Moran 2013 ). In stark contrast, the gut bacteria of field-collected H. puera larvae is dominated by the phylum level by Bacteroidota and Proteobacteria, and at the genus level by JC017 , Fulvimarina , and Stenotrophomonas . Bacteroidota are well-known for their robust capacity to degrade complex polysaccharides, aiding the host in breaking down plant cell wall components such as cellulose and pectin (Hammer and Bowers 2015 ). The presence of Fulvimarina , a genus commonly associated with marine environments, strongly suggests that the larvae acquire habitat-specific microorganisms through feeding on A. marina (Yang et al. 2023 ). This highly specialized microbial structure likely results from long-term co-evolution between the larvae and their natural host plants, potentially facilitating the digestion of host-specific plant compounds and enhancing adaptation to their ecological niche. Interestingly, although metrics such as Observed_species were slightly lower in the field group compared to the laboratory group, the difference was not statistically significant. This may be attributed to the uniform composition of artificial diet, which provides a stable environment conducive to the proliferation of generalist bacterial species. In contrast, the complex and varied nature of natural food sources may promote competitive dominance of more specialized taxa, thereby reducing the total number of microbial species (ASV richness) and diversity in the larval gut. Starvation treatment significantly reduced the number of ASVs in field-collected and laboratory-reared H. puera larvae, indicating that nutrient deprivation acts as a strong ecological filter that eliminates microbial taxa incapable of surviving independently under resource-limited conditions, thereby leading to a simplified gut microbiota (Akami et al. 2019 ). Crucially, our study revealed that the initial structure of the microbiota predetermined the stress response pattern. In starved laboratory-reared larvae (HAS), the relative abundance of Enterococcus increased dramatically from 33.32% to 83.36%, resulting in a near-monodominant community. Enterococcus is well-known for its high environmental adaptability, including tolerance to pH shifts, nutrient stress, and a strong capacity for biofilm formation, enabling it to survive and proliferate rapidly under harsh conditions (Zhang et al. 2023 ). In contrast, the response to starvation in field-collected larvae (HAS) demonstrated greater functional redundancy and ecological resilience. Instead of dominance by a single taxon, the microbial community shifted to a structure co-dominated by Burkholderia (34.48%) and Acinetobacter (20.64%). Species within Burkholderia are known not only for their ability to degrade various aromatic pollutants-such as phenols, polycyclic aromatic hydrocarbons, and pesticides—but also for performing coupled denitrification, producing siderophores, and potentially helping maintain gut ecological balance (Jang et al. 2024 ). Acinetobacter , on the other hand, exhibits remarkable metabolic plasticity, enabling it to utilize diverse carbon and nitrogen sources, degrade hydrocarbons and lipids, and form biofilms under adverse conditions, underscoring its ecological versatility and potential role in gut homeostasis (Zhang et al. 2022 ). These taxa may aid the host through nutrient supplementation or detoxification during starvation. This diversified response strategy likely stems from the inherently higher complexity of the field-acquired microbiota, offering more functional options to cope with environmental stress. The functional prediction analysis using PICRUSt2 provides critical insights into the ecological implications of the observed microbial community structure. The results indicate that the gut microbiota of H. puera possesses extensive metabolic potential, being heavily involved in two major functional modules: biosynthesis-including amino acids, vitamins, cofactors, and fatty acids-and degradation/utilization-such as carbohydrates, aromatic compounds, and carboxylates. This metabolic versatility is crucial for an insect feeding on mangrove plants, which are well-known for their potent chemical defenses. Mangrove plants typically contain abundant secondary metabolites, such as tannins and phenolics (Lang et al. 2025 ). The significant enrichment of pathways related to "aromatic compound degradation" and "detoxification" strongly suggests that these symbiotic bacteria play a key role in assisting the larvae in counteracting plant chemical defenses (Hammer and Bowers 2015 ). Concurrently, pathways involved in "vitamin and amino acid biosynthesis" imply that the gut microbiota may act as an external nutrient reservoir, supplementing essential nutrients that are scarce in the host’s diet, thereby supporting normal growth, development, and reproduction (Berg et al. 2025 ). This strategy of "microbial facilitation"-gaining dual capabilities in toxin degradation and nutritional supplementation through symbiotic bacteria—may represent a core ecological mechanism enabling H. puera to successfully adapt to its specialized diet and emerge as a major pest. In summary, our findings indicated that laboratory rearing significantly altered the gut microbial composition. The gut bacteria of laboratory-reared larvae were dominated by the phylum Firmicutes-D, genus Enterococcus , and Ligilactobacillus , whereas field-collected larvae exhibited a bacterial community primarily composed of the phyla Bacteroidota and Proteobacteria, with JC017 and Fulvimarina as the dominant genera. Starvation treatment significantly reduced alpha diversity and ASV richness of the gut bacteria, and the alpha diversity was significantly lower in laboratory-reared larvae compared to field-collected ones. After starvation, the relative abundance of Enterococcus in laboratory-reared larvae increased markedly to 83.36%, resulting in a mono-dominant microbial structure. In contrast, starved field-collected larvae maintained a more diverse and tolerant community, predominantly consisting of Burkholderia and Acinetobacter . Functional prediction indicated that the gut bacteria was extensively involved in metabolic pathways such as amino acid and vitamin biosynthesis, as well as degradation of aromatic compounds, suggesting its potential role in helping the host overcome mangrove chemical defenses and compensate for nutritional challenges. This study provides a theoretical foundation for understanding the ecological adaptation of H. puera and supports the development of microbiome-based strategies for eco-friendly pest control. Future research should focus on the isolation and culture of gut bacteria, combined with metagenomic and metabolomic approaches, to precisely elucidate the functional genes and metabolites of key taxa (e.g., Enterococcus and Burkholderia ) and validate their specific roles in nutrient provision and detoxification. Declarations Supplementary material The online version contains supplementary material available at Author Contribution Statement Conceptualization, Liangjian Qu, Hao Yu. Formal analysis, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Funding acquisition, Liangjian Qu, Hao Yu. Investigation, Wen Zhang, Yuehua Liu, Gaole Chen, Yi Yang, Junli Jiang. Project administration, Liangjian Qu, Hao Yu. Methodology, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Resources, Wen Zhang, Yuehua Liu, Liangjian Qu. Supervision, Gaole Chen, Yi Yang, Junli Jiang. Validation, Wen Zhang, Yuehua Liu, Gaole Chen. Visualization, Yi Yang, Junli Jiang. Writing—original draft, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Writing—review & editing, Liangjian Qu, Hao Yu. Funding This study was supported by the National Key R&D Program of China (2023YFC260480 1), the Key R&D programs of Henan Province (No. 241111311900), the Undergraduate Innovation Training Program Project of Henan Province (10467CXCY2025032) Data Availability The datasets and analyses of the current study are available from the corresponding author on reasonable request. Ethics Approval Not applicable. Conflict of interest The authors declare no competing interests. References Akami M, Njintang NY, Gbaye OA, Andongma AA, Rashid MA, Niu C-Y, Nukenine EN (2019) Gut bacteria of the cowpea beetle mediate its resistance to dichlorvos and susceptibility to Lippia adoensis essential oil. Sci Rep 9:6435. https://doi.org/10.1038/s41598-019-42843-1 Berg G, Toledo GV, Schierstaedt J, Hyöty H, Wicaksono WA (2025) Linking the edible plant microbiome and human gut microbiome. Gut Microbes 17:2551113. https://doi.org/10.1080/19490976.2025.2551113 Biji CP, Sudheendrakumar VV, Sajeev TV (2006) Quantitative estimation of Hyblaea puera NPV production in three larval stages of the teak defoliator, Hyblaea puera (Cramer). J Virol Methods 136:78–82. https://doi.org/10.1016/j.jviromet.2006.04.001 Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852–857. https://doi.org/10.1038/s41587-019-0209-9 Broderick NA, Raffa KF, Handelsman J (2006) Midgut bacteria required for Bacillus thuringiensis insecticidal activity. PNAS 103:15916–15919. https://doi.org/10.1073/pnas.0604865103 Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869 Chandrasekhar N, Sajeev TV, Sudheendrakumar VV, Banerjee M (2005) Population dynamics of the teak defoliator ( Hyblaea puera Cramer) in Nilambur teak plantations using Randomly Amplified Gene Encoding Primers (RAGEP). BMC Ecol 5:1. https://doi.org/10.1186/1472-6785-5-1 Chandrasekhar N, Neetha N, Vaidyan L, Banerjee M (2008) Deciphering the Molecular Phylogenetics of Family Hyblaeidae and Inferring the Phylogeographical Relationships Using DNA Barcoding. J Genet Mol Biology 19:158–167. https://doi.org/10.30047/JGMB.200809.0004 Chang M (2023) Identification of morphological characters and antennal sensilla of themoth Hyblaea puera Cramer. Biol Disaster Sci 46:342–347. https://doi.org/10.3969/j.issn.2095-3704.2023.03.51 Chen B, Du K, Sun C, Vimalanathan A, Liang X, Li Y, Wang B, Lu X, Li L, Shao Y (2018) Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives. ISME J 12:2252–2262. https://doi.org/10.1038/s41396-018-0174-1 DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072. https://doi.org/10.1128/aem.03006-05 Dillon RJ, Vennard CT, Buckling A, Charnley AK (2005) Diversity of locust gut bacteria protects against pathogen invasion. Ecol Lett 8:1291–1298. https://doi.org/10.1111/j.1461-0248.2005.00828.x Douglas AE (2015) Multiorganismal insects: diversity and function of resident microorganisms. Annu Rev Entomol 60:17–34. https://doi.org/10.1146/annurev-ento-010814-020822 Douglas GM, Maffei VJ, Zaneveld JR, Yurgel SN, Brown JR, Taylor CM, Huttenhower C, Langille MGI (2020) PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 38:685–688. https://doi.org/10.1038/s41587-020-0548-6 Engel P, Moran NA (2013) The gut microbiota of insects-diversity in structure and function. FEMS Microbiol Rev 37:699–735. https://doi.org/10.1111/1574-6976.12025 George BS, Silambarasan S, Senthil K, Jacob JP, Dasgupta MG (2018) Characterization of an insecticidal protein from withania somnifera against lepidopteran and hemipteran pest. Mol Biotechnol 60:290–301. https://doi.org/10.1007/s12033-018-0070-y Hammer TJ, Bowers MD (2015) Gut microbes may facilitate insect herbivory of chemically defended plants. Oecologia 179:1–14. https://doi.org/10.1007/s00442-015-3327-1 Han S, Akhtar MR, Xia X (2024) Functions and regulations of insect gut bacteria. Pest Manag Sci 80:4828–4840. https://doi.org/10.1002/ps.8261 Hernández-Martínez P, Naseri B, Navarro-Cerrillo G, Escriche B, Ferré J, Herrero S (2010) Increase in midgut microbiota load induces an apparent immune priming and increases tolerance to Bacillus thuringiensis . Environ Microbiol 12:2730–2737. https://doi.org/10.1111/j.1462-2920.2010.02241.x Hu R, Chen H, Yang K, Cai B, Li J (2016) Review of a new pest, Hyblaea puera (Cramer), in mangrove forest in China. For Pest Disease 35:34–37 Jang S, Ishigami K, Mergaert P, Kikuchi Y (2024) Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect. PNAS 121:e2315540121. https://doi.org/10.1073/pnas.2315540121 Jia Y, Jin S, Hu K, Geng L, Han C, Kang R, Pang Y, Ling E, Tan EK, Pan Y et al (2021) Gut microbiome modulates drosophila aggression through octopamine signaling. Nat Commun 12:2698. https://doi.org/10.1038/s41467-021-23041-y Kottaipalayam-Somasundaram SR, Jacob JP, Aiyar B, Merzendorfer H, Nambiar-Veetil M (2022) Chitin metabolism as a potential target for RNAi-based control of the forestry pest Hyblaea puera Cramer (Lepidoptera: Hyblaeidae). Pest Manag Sci 78:296–303. https://doi.org/10.1002/ps.6634 Epub 2021 Sep 27 Lang T, Hussain M, Li M, Tam NF-Y, Min Pan 5 FW-FL, Xu SJ-L, Jiang M, Wang Y, Mu L, Zhou H (2025) Unlocking the structure-activity relationship of mangrove condensed and hydrolysable tannins: Unveiling their potential ecological significance in antioxidant and antibacterial functions. Int J Biol Macromol 307: 141918. https://doi.org/10.1016/j.ijbiomac.2025.141918 Liang X, Fu Y, Tong L, Liu H (2014) Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding. Appl Microbiol Biotechnol 98:3769–3776. https://doi.org/10.1007/s00253-014-5532-y Liu BQ, Bao XY, Yan JY, Zhang D, Sun X, Li CQ, Chen ZB, Luan JB (2024) Rickettsia symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating. Proc. Natl. Acad. Sci. U. S. A 121: e2406788121. https://doi.org/10.1073/pnas.2406788121 Shah RA, Riyaz M, Ignacimuthu S, Sivasankaran K (2022) Characterization of four mitochondrial genomes from superfamilies Noctuoidea and Hyblaeoidea with their phylogenetic implications. Sci Rep 12:18926. https://doi.org/10.1038/s41598-022-21502-y Sharon G, Segal D, Ringo JM, Hefetz A, Zilber-Rosenberg I, Rosenberg E (2010) Commensal bacteria play a role in mating preference of Drosophila melanogaster . PNAS 107:20051–20056. https://doi.org/10.1073/pnas.1009906107 Velavan V, Dhanapa R, Ramkumar G, Karthi S, Senthil-Nathan S, Ndomba OA, Kweka EJ (2022) Characterization and Evaluation of Metarhizium spp. (Metsch.) Sorokin Isolates for Their Temperature Tolerance. Journal of Fungi 8: 68. https://doi.org/10.3390/jof8010068 Warnecke F, Luginbühl P, Ivanova N, Ghassemian M, Richardson TH, Stege JT, Cayouette M, McHardy AC, Djordjevic G, Aboushadi N et al (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450:560–565. https://doi.org/10.1038/nature06269 Wei J, Liu W, Huang Q, Chen Q, Su B (2019) Investigation of main insect pests in shankou national mangrove reserve of Guangxi In recent 15 years. FuJian Agric Sci Technol 46:66–69. https://doi.org/10.13428/j.cnki.fjlk.2019.04.015 Yang S, Peng J, Xue Y, Zheng Z, Zhou H (2020) Species and integrated control of pests in mangrove community in China. For pest disease 39:32–41. https://doi.org/10.19688/j.cnki.issn1671-0886.20190031 Yang SR, Liu YY, Ge XF, Liu WZ (2023) Caldimonas mangrovi sp. nov Isolated Mangrove Soil Microbiol 92:500–507. https://doi.org/10.1134/S0026261723600477 Yao YL, Ma XY, Wang TY, Yan JY, Chen NF, Hong JS, Liu BQ, Xu ZQ, Zhang N, Lv C et al (2023) A bacteriocyte symbiont determines whitefly sex ratio by regulating mitochondrial function. Cell Rep 42:112102. https://doi.org/10.1016/j.celrep.2023.112102 Yao Z, Cai Z, Ma Q, Bai S, Wang Y, Zhang P, Guo Q, Gu J, Lemaitre B, Zhang H (2022) Compartmentalized PGRP expression along the dipteran Bactrocera dorsalis gut forms a zone of protection for symbiotic bacteria. 41:111523. https://doi.org/10.1016/j.celrep.2022.111523 Zhang L, Liu J, Deng M, Chen X, Jiang L, Zhang J, Tao L, Yu W, Qiu Y (2023) Enterococcus faecalis promotes the progression of colorectal cancer via its metabolite: biliverdin. J Translational Med 21. https://doi.org/10.1186/s12967-023-03929-7 Zhang S, Li Z, Shu J, Xue H, Guo K, Zhou X (2022) Soil-derived bacteria endow Camellia weevil with more ability to resist plant chemical defense. Microbiome 10:97. https://doi.org/10.1186/s40168-022-01290-3 Supplementary Files Supplementarymaterialstables.docx 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-8081780","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":555842749,"identity":"84a35ef5-cfee-4878-9f66-17d197c056b5","order_by":0,"name":"Wen Zhang","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhang","suffix":""},{"id":555842750,"identity":"8e21e787-e88c-4b56-af1c-b97236db03f7","order_by":1,"name":"Qiaoling Zhao","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qiaoling","middleName":"","lastName":"Zhao","suffix":""},{"id":555842751,"identity":"d01d95c7-4596-4ade-8e74-fff2a5ca79d7","order_by":2,"name":"Xiaoya Zhang","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoya","middleName":"","lastName":"Zhang","suffix":""},{"id":555842752,"identity":"c4216b4a-d742-4f44-945a-4d9f9dbb4c3f","order_by":3,"name":"Yuehua Liu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuehua","middleName":"","lastName":"Liu","suffix":""},{"id":555842753,"identity":"162f2512-a460-4be5-bb1b-de51e31fa4bc","order_by":4,"name":"Gaole Chen","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Gaole","middleName":"","lastName":"Chen","suffix":""},{"id":555842754,"identity":"e10236ae-b6a2-4c71-9024-79fa63f9d0ac","order_by":5,"name":"Yi Yang","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""},{"id":555842755,"identity":"12fc90e6-be26-4819-a2c4-88c196d0d77c","order_by":6,"name":"Junli Jiang","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Junli","middleName":"","lastName":"Jiang","suffix":""},{"id":555842756,"identity":"9a229e62-a645-4e2b-8e64-3aa87e38c3dc","order_by":7,"name":"Xinan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIie3PoQoCQRCA4ZGVsYxuXYvPsCJc8mFGBJtgEoOgINwFUau+hcl8OmA6u8GgxS6CYBEv2BT3bIb9wk7Zn90B8Lw/VFIAcTorqNT6yL26O8FXUtMFbNpj0sqQvGZjMaOgfAo3GZICVaXTP+SWQkGPMQYdjdnxMWSZb8/KCnb3TAcwyW7pSFQshIJW1GrP5gzWtF1Jbij0ELICQYfTM0OSvlIMxZRH+QCYMyXpLsWpWK2waThukXMXrZPalW4yCLWsL/dHvaKjyffkDf123fM8z/voCXsORUt7oGzVAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9080-9350","institution":"Henan Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Xinan","middleName":"","lastName":"Li","suffix":""},{"id":555842757,"identity":"b135a278-3799-4dce-8302-1363d986927e","order_by":8,"name":"Liangjian Qu","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Liangjian","middleName":"","lastName":"Qu","suffix":""},{"id":555842758,"identity":"5fff4dfd-842d-4042-a121-c74e4d1e50e4","order_by":9,"name":"Hao Yu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-11-11 03:05:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8081780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8081780/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97813157,"identity":"dfb7cb87-36b3-4c3e-93eb-f673354d2690","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3245264,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/c5165080408b98f797ca8bf5.tif"},{"id":97898938,"identity":"f48a2658-ee00-411b-86ec-33aa6d26b926","added_by":"auto","created_at":"2025-12-10 15:40:14","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7318432,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/8cbfa901b67e994f6c94d22d.tif"},{"id":97898616,"identity":"d9a427c1-0c1a-4ab6-ba1a-e405c7f52eb9","added_by":"auto","created_at":"2025-12-10 15:39:22","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2224558,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/d8e96113f9185af4bcdd591d.tif"},{"id":97813158,"identity":"b4f5f6ec-482f-4884-8332-ff8353513c5b","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3344636,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/4bd8210bac87fe581c01075d.tif"},{"id":97898502,"identity":"79a27bb3-2b15-49de-9aa4-02147dd35788","added_by":"auto","created_at":"2025-12-10 15:39:13","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4225026,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/c1c96fa9ffe62608af8bfe4d.tif"},{"id":97897559,"identity":"e7bc6389-11ca-40df-b9a5-a7fa9fa6fe4d","added_by":"auto","created_at":"2025-12-10 15:37:57","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13401,"visible":true,"origin":"","legend":"","description":"","filename":"nentNENTD2500523.xml","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/5c7e8760acad95dfbe7b15b6.xml"},{"id":97813176,"identity":"fda1269c-b7aa-4bd5-9018-91fcf7c80e6d","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1153,"visible":true,"origin":"","legend":"","description":"","filename":"NENTD250052311947.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/e37267a4304443a743925829.xml"},{"id":97896310,"identity":"2d6a28be-8896-435e-ad4b-20f0620ef0dd","added_by":"auto","created_at":"2025-12-10 15:36:19","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":860,"visible":true,"origin":"","legend":"","description":"","filename":"NENTD2500523Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/3b01fbfff07f24cdfd0e8dbc.xml"},{"id":97898189,"identity":"8b5ac101-0898-4bac-a960-777790c35fd3","added_by":"auto","created_at":"2025-12-10 15:38:47","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110209,"visible":true,"origin":"","legend":"","description":"","filename":"NENTD25005230enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/0aef451c6f09ba488193d4bd.xml"},{"id":97898877,"identity":"78d8346f-d7f9-4dd5-8c2f-0c88b037bc05","added_by":"auto","created_at":"2025-12-10 15:39:57","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3245264,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/4b2e15f21145df9c7420551d.tif"},{"id":97813168,"identity":"1c46fe32-578a-43fa-b7ac-a755da1bfa35","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7318432,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/067e59758649feb8bece2e73.tif"},{"id":97898509,"identity":"7c9730fa-4c31-48f4-9526-fd02b18c469e","added_by":"auto","created_at":"2025-12-10 15:39:14","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2224558,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/b959a2c07c4c1eb3140227a9.tif"},{"id":97813172,"identity":"9d7b3c95-aa15-44ce-83ac-cc7cd0dd68ee","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3344636,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/23f5255a8f10c300c3ff4c02.tif"},{"id":97813165,"identity":"babb748a-5355-424a-a923-8415b19f63c4","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4225026,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/ac39ff1bdf8be05f603c76c8.tif"},{"id":97898758,"identity":"b2f94636-cc19-4ca0-a186-02089dfa23bf","added_by":"auto","created_at":"2025-12-10 15:39:34","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":249097,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/9fceb78f92dc9cc704578139.png"},{"id":97898269,"identity":"d5c5df87-2699-468a-b446-2f2d0e514c44","added_by":"auto","created_at":"2025-12-10 15:38:55","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":650306,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/1fb82ea9c2fa251391b80b7d.png"},{"id":97813164,"identity":"5c1e4da1-0023-4480-8dbc-3da8d011e5c8","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":236482,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/2fe1e47520f9c7aff07a8955.png"},{"id":97896590,"identity":"f8a854f2-f436-4dde-a6c7-6e86429c9701","added_by":"auto","created_at":"2025-12-10 15:36:48","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":254614,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/811c36a001020131ccda34b7.png"},{"id":97813173,"identity":"e243dbe9-8186-4974-8fe3-898d7a722b46","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":383693,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/4c0f4d181163eed2e941fbdb.png"},{"id":97813174,"identity":"a84e4a55-39d4-4ef9-9d21-e3a5177982f5","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109912,"visible":true,"origin":"","legend":"","description":"","filename":"NENTD25005230structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/4c8ef6de2e50d8f1ec8bc27a.xml"},{"id":97897540,"identity":"a5514291-a8bd-44fa-8405-73f5cbbc1d98","added_by":"auto","created_at":"2025-12-10 15:37:56","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117960,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/2cd79fa791ab639a0fee3cab.html"},{"id":97898507,"identity":"6a8d677c-5201-46c4-8b82-7991ed261151","added_by":"auto","created_at":"2025-12-10 15:39:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1709088,"visible":true,"origin":"","legend":"\u003cp\u003eRarefaction curves (A) and venn diagram (B) of gut bacteria in \u003cem\u003eHyblaea puera Larvae\u003c/em\u003e. HAN, field-collected larvae; HAS, field-collected larvae starved for 24 h; HFN, laboratory-reared larvae fed an artificial diet; HFS, laboratory-reared larvae starved for 24 h.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/05c372b6dc4cbdbd2eda6a23.png"},{"id":97897768,"identity":"19602f65-4ad1-459c-b454-7da7b532d435","added_by":"auto","created_at":"2025-12-10 15:38:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2861288,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity of gut bacteria in fifth-instar larvae of \u003cem\u003eHyblaea puera\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e* and **, above the bars denote highly significant differences between groups based on paired bootstrap tests (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, respectively). HAN, field-collected larvae; HAS, field-collected larvae starved for 24 h; HFN, laboratory-reared larvae fed an artificial diet; HFS, laboratory-reared larvae starved for 24 h.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/e8f0cbe187f862da53b1532d.png"},{"id":97813151,"identity":"9217fe34-6b54-4b74-b145-9491324a8f49","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1147832,"visible":true,"origin":"","legend":"\u003cp\u003eBeta diversity of gut bacteria in fifth-instar larvae of \u003cem\u003eHyblaea puera\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eHAN, field-collected larvae; HAS, field-collected larvae starved for 24 h; HFN, laboratory-reared larvae fed an artificial diet; HFS, laboratory-reared larvae starved for 24 h.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/ca38d001372bc77f4021a208.png"},{"id":97813153,"identity":"e9beb9ec-6109-44e0-adde-e438648925b6","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1586235,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of gut bacteria at genus level in fifth-instar larvae of \u003cem\u003eHyblaea puera\u003c/em\u003e. A, HFN, laboratory-reared larvae fed an artificial diet; B, HFS, laboratory-reared larvae starved for 24 h；C, HAN, field-collected larvae; D, HAS, field-collected larvae starved for 24 h.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/e4c499aa4a897dbccacf8e70.png"},{"id":97813154,"identity":"382ca46a-e3c3-4acb-bf76-e409351990cc","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2598019,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical plot of metabolic pathways of gut bacteria in fifth-instar larvae of \u003cem\u003eHyblaea puera\u003c/em\u003e. X-axis, abundance (in units of KO/PWY/COG per million) or count of functional pathways/categories; Y-axis: functional pathways/categories at the second hierarchical level of KEGG/MetaCyc/COG classification; Rightmost annotation, first-level pathway/category to which the corresponding pathway belongs.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/8564e82d1b3a4d18b634da56.png"},{"id":105565265,"identity":"675d4b34-c9ed-48a6-bd1d-4cb81c664129","added_by":"auto","created_at":"2026-03-27 12:52:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11400472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/0c95224c-4617-4a37-a6ca-298856dba8c3.pdf"},{"id":97813160,"identity":"e5ccb98e-fcad-4281-a76c-df5166081057","added_by":"auto","created_at":"2025-12-09 16:16:37","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":21120,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialstables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8081780/v1/89f0b7ebe7371d80a3bfa51b.docx"}],"financialInterests":"","formattedTitle":"Analysis of gut microbial diversity and function in Hyblaea puera (Lepidoptera: Hyblaeidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eHyblaea puera\u003c/em\u003e (Lepidoptera: Hyblaeidae) is characterized by its high reproductive capacity, rapid dispersal, broad adaptability, and wide host range. Originating from Southeast Asia, it is the most serious defoliating pest in teak plantations. Following the introduction of teak, it has now spread to Latin America, where it continues to threaten teak production (Chandrasekhar et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In recent years, \u003cem\u003eH. puera\u003c/em\u003e has emerged as a major invasive pest damaging mangrove plants, particularly \u003cem\u003eAvicennia marina\u003c/em\u003e, along the coast of China. (Hu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During the larval stage, \u003cem\u003eH. puera\u003c/em\u003e feeds on the tender leaves of \u003cem\u003eA. marina\u003c/em\u003e. Initially, the larvae consume approximately half of a leaf blade and then secrete silk-like saliva to roll the leaf, concealing themselves within the curled structure to continue feeding. Older instar larvae are capable of consuming entire leaves, leaving only bare branches. Pupation occurs inside the rolled leaves, and adults lay large numbers of eggs on the foliage, facilitating rapid population outbreaks. It has been documented that this pest has infested over 300 hectares of mangrove forests in Guangxi, China, posing a serious threat to mangrove ecosystem health and coastal conservation (Hu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrent research on \u003cem\u003eH. puera\u003c/em\u003e primarily focuses on the following aspects. Wei et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) systematically described its damage characteristics and proposed an integrated monitoring approach combining remote sensing and manual surveys to establish a cross-sectoral pest monitoring network (Wei et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chang et al. (2023) investigated the morphology and antennal sensilla of male and female adults, developing a rapid identification method that supports the application of sex pheromone-based control techniques (Chang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In terms of biological control, the baculovirus HpNPV has been confirmed to be transmitted via feces and can effectively suppress \u003cem\u003eH. puera\u003c/em\u003e populations (Biji et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), while \u003cem\u003eMetarhizium\u003c/em\u003e spp. have demonstrated strong pathogenicity against the \u003cem\u003eH. puera\u003c/em\u003e (Velavan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). At the molecular level, the cloning of chitin metabolism-related genes (\u003cem\u003eHpCHS1\u003c/em\u003e and \u003cem\u003eHpChi-h\u003c/em\u003e) has laid the groundwork for RNAi-based targeted control strategies (Kottaipalayam-Somasundaram et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and plant-derived insecticidal proteins such as WsMBP1 have shown significant toxicity (George et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Population genetics studies using RAGEP markers have revealed seasonal migration patterns (Chandrasekhar et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and mitochondrial genome analyses have provided new insights into its phylogenetic classification (Shah et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings have enhanced the understanding of \u003cem\u003eH. puera\u003c/em\u003e and facilitated the development of integrated sustainable management strategies incorporating remote sensing, biological, and molecular technologies.\u003c/p\u003e\u003cp\u003eThe gut serves as a vital organ for food digestion and absorption in insects, harboring a vast array of symbiotic bacteria. These microbial symbionts play crucial roles in multiple physiological processes, including food digestion and nutrient assimilation (Warnecke et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), growth and reproduction (Sharon et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), immune development and regulation (Hern\u0026aacute;ndez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), pathogen resistance (Dillon et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and pesticide detoxification (Broderick et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The host insect and its gut microbiota interact through complex mechanisms of mutual dependence, reciprocal regulation, and co-adaptation. For instance, the composition and metabolic activity of gut bacteria can significantly influence host physiology and behavior, while the microenvironment of the insect gut also shapes\u0026mdash;and often determines\u0026mdash;the structure and assembly of the bacterial community (Jia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Yao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given these multifaceted interactions, symbiotic bacteria are increasingly regarded as a \u0026ldquo;multifunctional organ\u0026rdquo; in pest management strategies, driving research that targets insect-microbe interactions to develop novel control techniques. However, the diversity and functional roles of the gut microbiota in \u003cem\u003eH. puera\u003c/em\u003e remain largely unexplored.\u003c/p\u003e\u003cp\u003eThe present study investigated the gut bacterial diversity, community structure, taxonomic composition, and biological functions of fifth-instar larvae of \u003cem\u003eH. puera\u003c/em\u003e\u0026mdash;a major invasive pest damaging mangrove ecosystems\u0026mdash;using 16S rRNA high-throughput amplicon sequencing and bioinformatic approaches. Comparisons were made among field-collected populations, laboratory-reared populations, and both groups subjected to starvation treatment. The findings provide a foundation for further elucidating the biological roles of gut bacteria in the host insect and support the development of novel integrated pest management strategies based on insect-microbe interactions. This study also offers important theoretical and practical insights for the conservation of mangrove ecosystems.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample Collection and Rearing\u003c/h2\u003e\u003cp\u003eField-collected larvae of \u003cem\u003eH. puera\u003c/em\u003e were obtained from \u003cem\u003eA. marina\u003c/em\u003e of mangrove in Zhanjiang, Guangdong Province, China (10\u0026deg;54\u0026prime;21\u0026Prime;N, 108\u0026deg;54\u0026prime;21\u0026Prime;E) in April 2024. For the laboratory population, field-collected individuals were reared for more than ten generations in a controlled greenhouse environment using an artificial diet. The rearing conditions were maintained as follows: temperature at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, photoperiod of 16 h light : 8 h dark, and relative humidity of 60% \u0026plusmn; 10%.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample Preparation and 16S rRNA High-Throughput Sequencing\u003c/h3\u003e\n\u003cp\u003eThe fifth-instar larvae of \u003cem\u003eH. puera\u003c/em\u003e from field-collected, laboratory-reared, and 24 h starved groups were dissected under sterile conditions using forceps, scissors, and dissection needles to isolate intact gut tissues. The extracted guts were immediately flash-frozen in liquid nitrogen and stored at -80\u0026deg;C until further processing. Each gut was considered an independent biological replicate, with six replicates per group. All samples were sent to Nanjing Personalgene Technology Co., Ltd. for 16S rRNA amplicon sequencing.\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. The V3\u0026ndash;V4 hypervariable region of the bacterial 16S rRNA gene was amplified with the universal primers 338F (5ʼ-ACTCCTACGGGAGGCAGCA-3ʼ) and 806R (5ʼ-GGACTACHVGGGTWTCTAAT-3ʼ), with unique barcodes incorporated into the forward primer. The 25 \u0026micro;L PCR reaction mixture consisted of 0.25 \u0026micro;L of Q5 High-Fidelity DNA Polymerase, 5 \u0026micro;L of 5\u0026times; Reaction Buffer, 5 \u0026micro;L of 5\u0026times; High GC Buffer, 2 \u0026micro;L of dNTPs (10 mM), 2 \u0026micro;L of template DNA, 1 \u0026micro;L of each forward and reverse primer (10 \u0026micro;M), and 8.75 \u0026micro;L of ddH₂O. The thermal cycling protocol included an initial denaturation at 98\u0026deg;C for 5 min; 25 cycles of denaturation at 98\u0026deg;C for 10 s, annealing at 53\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s; followed by a final extension at 72\u0026deg;C for 5 min. Amplification products were electrophoresed on a 2% agarose gel, and target bands were excised and purified using the AxyPrep DNA Gel Extraction Kit. Purified amplicons were quantified with the Quant-iT\u0026trade; PicoGreen\u0026trade; dsDNA Assay Kit on a Microplate Reader (BioTek, FLx800), pooled in equimolar amounts according to the required sequencing depth, and subjected to sequencing on the Illumina platform.\u003c/p\u003e\n\u003ch3\u003eBioinformatic Analysis and Functional Prediction\u003c/h3\u003e\n\u003cp\u003eRaw sequencing data were processed using QIIME2 (version 2019.4; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/QIIME2/q2-feature-classifier\u003c/span\u003e\u003cspan address=\"https://github.com/QIIME2/q2-feature-classifier\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Bolyen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Demultiplexing was performed with the demux plugin, followed by primer trimming using cutadapt. Sequences were subsequently quality-filtered, denoised, merged (skipped for single-end reads), and checked for chimeras with the DADA2 plugin (Callahan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Amplicon sequence variants (ASV) were generated by clustering sequences at 100% similarity, producing an ASV abundance table. Taxonomic assignment of representative ASV sequences was conducted by alignment against the Greengenes database (Release 13.8; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://greengenes.secondgenome.com\u003c/span\u003e\u003cspan address=\"http://greengenes.secondgenome.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) within QIIME2 (DeSantis et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Alpha and beta diversity analyses were performed using the q2-diversity plugin in QIIME2. Rarefaction curves were generated and visualized with R software (version 4.5.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org/\u003c/span\u003e\u003cspan address=\"https://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Functional potential of the gut microbiota was predicted from the 16S rRNA data using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/picrust/picrust2/wiki\u003c/span\u003e\u003cspan address=\"https://github.com/picrust/picrust2/wiki\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Douglas et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eTaxonomic Annotation and Evaluation of Gut Bacteria in\u003c/b\u003e \u003cb\u003eH. puera\u003c/b\u003e \u003cb\u003eLarvae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 2,336,149 high-quality sequences were obtained from 24 gut samples of fifth-instar \u003cem\u003eH. puera\u003c/em\u003e larvae after high-throughput sequencing. Basic information of the 16S rRNA amplicon sequencing is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After denoising and clustering using the DADA2 pipeline, a total of 27 phyla, 46 classes, 106 orders, 204 families, 451 genera, and 761 species were identified. The laboratory-reared group exhibited a significantly higher number of bacterial ASVs compared to the field-collected group. Following starvation treatment, both groups showed a significant reduction in gut bacterial ASV richness. The rarefaction curves flattened as sequencing depth increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating that sufficient sequencing coverage was achieved and that additional reads would not substantially increase ASV detection. This confirms the high quality of the sequencing data and supports subsequent analyses. Venn diagram analysis revealed 42 ASVs shared across all four experimental groups. Specifically, 1782, 411, 2574, and 469 unique ASVs were identified in the field-collected non-starved (HAN), field-collected starved (HAS), laboratory-reared non-starved (HFN), and laboratory-reared starved (HAS) groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results suggest that laboratory rearing with an artificial diet increased ASV richness in the larval gut bacteria, while subsequent starvation significantly reduced gut bacterial ASV richness in both populations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic information of 16S rRNA amplicon sequencing of gut microbiota in fifth-instar larvae of \u003cem\u003eHyblaea puera\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ephylum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eclass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eorder\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003efamily\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003egenus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003especies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eASV\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHAN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e195\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1974\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHAS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e557\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHFN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3027\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHFS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e839\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e761\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e5278\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eHAN, field-collected larvae; HAS, field-collected larvae starved for 24 h; HFN, laboratory-reared larvae fed artificial diet; HFS, laboratory-reared larvae starved for 24 h.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of Gut Bacterial Diversity in\u003c/b\u003e \u003cb\u003eH. puera\u003c/b\u003e \u003cb\u003eLarvae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlpha diversity of the gut bacteria in fifth-instar \u003cem\u003eH. puera\u003c/em\u003e larvae was evaluated using multiple indices, including Chao1, Goods_coverage, Simpson, Pielou_e, Faith_pd, Shannon, and Observed_species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The results showed that field-collected larvae exhibited lower values in Chao1, Simpson, Pielou_e, Faith_pd, Shannon, and Observed_species compared to laboratory-reared larvae, though these differences were not statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After starvation treatment, field-collected and laboratory-reared groups showed reductions in Chao1, Faith_pd, and Observed_species relative to their non-starved counterparts, but again these changes were not significant. In contrast, significant decreases in Simpson, Pielou_e, and Shannon indices were observed in starved laboratory-reared larvae compared to the non-starved group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, starved field-collected larvae displayed significantly higher Simpson and Pielou_e values than starved laboratory-reared larvae (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that starvation significantly reduced the alpha diversity of the gut bacteria in laboratory-reared \u003cem\u003eH. puera\u003c/em\u003e larvae. Moreover, under starvation conditions, laboratory-reared larvae showed significantly lower alpha diversity compared to field-collected larvae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrincipal coordinates analysis (PCoA) was performed using the Unweighted Unifrac distance metric. Eigenvalues and eigenvectors were computed, and the axes with the highest contribution rates were selected for visualization. Greater distances between samples in the ordination plot reflect larger dissimilarities in microbial community structure. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, slight differences were observed in the gut bacterial composition between starved groups of field-collected and laboratory-reared larvae. In contrast, pronounced separation was detected between non-starved field-collected and laboratory-reared groups, indicating significant structural divergence. Non-metric multidimensional scaling (NMDS) was further applied to rank inter-sample distances based on ordinal relationships rather than absolute values, providing more robust visualization for complex ecological data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The stress value for the NMDS ordination was 0.138, which is below the 0.2 threshold, confirming the reliability of the configuration. Confidence intervals partially overlapped between starved field-collected and laboratory-reared groups, whereas no overlap was observed between non-starved field-collected and laboratory-reared groups. These results demonstrate that the gut microbial community structure and composition differ significantly between field-collected and laboratory-reared larvae under non-starved conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGut Bacterial Community Composition in\u003c/b\u003e \u003cb\u003eH. puera\u003c/b\u003e \u003cb\u003eLarvae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the phylum level (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), the top five bacterial phyla in field-collected fifth-instar larvae were Bacteroidota, Proteobacteria, Firmicutes-A, Actinobacteriota, and Firmicutes-D. After 24 hours of starvation, the dominant phyla in field-collected larvae shifted to Proteobacteria, Bacteroidota, Actinobacteriota, Firmicutes-D, and Cyanobacteria. In contrast, laboratory-reared larvae were dominated by Firmicutes-D, Proteobacteria, Firmicutes-A, Actinobacteriota, and Bacteroidota. Starvation of laboratory-reared larvae resulted in a predominance of Firmicutes-D, Proteobacteria, Actinobacteriota, Methylomirabilota, and Firmicutes-A.\u003c/p\u003e\u003cp\u003eAt the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B, Table S2), the gut bacteria of non-starved laboratory-reared larvae was dominated by \u003cem\u003eEnterococcus\u003c/em\u003e (33.32%), \u003cem\u003eLigilactobacillus\u003c/em\u003e (18.39%), \u003cem\u003eStenotrophomonas\u003c/em\u003e (9.01%), \u003cem\u003eBacillus\u003c/em\u003e (4.35%), \u003cem\u003eLevilactobacillus\u003c/em\u003e (3.80%), \u003cem\u003eLactiplantibacillus\u003c/em\u003e (2.90%), \u003cem\u003eAcinetobacter\u003c/em\u003e (1.31%), \u003cem\u003eFaecalibacterium\u003c/em\u003e (1.25%), \u003cem\u003eCollinsella\u003c/em\u003e (1.08%), and \u003cem\u003eLiquorilactobacillus\u003c/em\u003e (0.88%). After 24 hours of starvation, the community shifted markedly, with \u003cem\u003eEnterococcus\u003c/em\u003e becoming overwhelmingly dominant (83.36%), followed by \u003cem\u003eAcinetobacter\u003c/em\u003e (2.86%), \u003cem\u003eBacillus\u003c/em\u003e (1.73%), \u003cem\u003eLigilactobacillus\u003c/em\u003e (1.60%), \u003cem\u003eBurkholderia\u003c/em\u003e (0.76%), \u003cem\u003eLactiplantibacillus\u003c/em\u003e (0.43%), \u003cem\u003eLiquorilactobacillus\u003c/em\u003e (0.38%), \u003cem\u003eLevilactobacillus\u003c/em\u003e (0.37%), \u003cem\u003eStenotrophomonas\u003c/em\u003e (0.34%), and \u003cem\u003eDSHD01\u003c/em\u003e (0.11%). In field-collected non-starved larvae, the dominant genera were \u003cem\u003eJC017\u003c/em\u003e (38.03%), \u003cem\u003eFulvimarina\u003c/em\u003e (14.82%), \u003cem\u003eStenotrophomonas\u003c/em\u003e (10.47%), \u003cem\u003ePseudokineococcus\u003c/em\u003e (5.76%), \u003cem\u003eAlistipes\u003c/em\u003e (1.72%), \u003cem\u003eBurkholderia\u003c/em\u003e (1.71%), \u003cem\u003eFaecalibacterium\u003c/em\u003e (1.60%), \u003cem\u003eAcinetobacter\u003c/em\u003e (1.53%), \u003cem\u003eBarnesiella\u003c/em\u003e (1.17%), and \u003cem\u003ePrevotella\u003c/em\u003e (1.00%). After starvation, the microbial composition changed significantly, with \u003cem\u003eBurkholderia\u003c/em\u003e (34.48%) and \u003cem\u003eAcinetobacter\u003c/em\u003e (20.64%) becoming the most abundant taxa, followed by \u003cem\u003eJC017\u003c/em\u003e (8.20%), \u003cem\u003eBrevundimonas\u003c/em\u003e (3.63%), \u003cem\u003ePseudomonas\u003c/em\u003e (3.18%), \u003cem\u003eMethylobacterium\u003c/em\u003e (2.70%), \u003cem\u003eActinotalea\u003c/em\u003e (1.91%), \u003cem\u003eFulvimarina\u003c/em\u003e (1.89%), \u003cem\u003eNocardioides\u003c/em\u003e (1.55%), and \u003cem\u003eActinomyces\u003c/em\u003e (1.46%).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFunctional Prediction of Gut Bacteria\u003c/h3\u003e\n\u003cp\u003eBased on PICRUSt2 analysis, a statistical functional assessment of metabolic pathways was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At Level 1 pathway categories, the gut bacteria of \u003cem\u003eH. puera\u003c/em\u003e was predominantly enriched in six major functional classes: Biosynthesis, Degradation/ Utilization/Assimilation, Detoxification, Generation of Precursor Metabolites and Energy, Glycan Pathways, and Metabolic Clusters. Predicted Level 2 pathway annotations revealed that gut symbiotic bacteria are involved in diverse physiological and biochemical processes, with significant enrichment in multiple metabolic pathways. These include amino acid biosynthesis; biosynthesis of cofactors, prosthetic groups, electron carriers, and vitamins; carbohydrate biosynthesis; fatty acid and lipid biosynthesis; amino acid degradation; aromatic compound degradation; carbohydrate degradation; carboxylate degradation; and nucleoside and nucleotide degradation. These results indicate that the gut bacteria of \u003cem\u003eH. puera\u003c/em\u003e participates in a wide range of host physiological processes and may play crucial roles in supporting development and reproduction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSymbiotic microbes are closely associated with various aspects of insect physiology, including nutrition, reproduction, defense, immunity, and stress resistance (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and directly or indirectly influence insect growth, development, metabolism, and environmental adaptability (Yao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eHyblaea puera\u003c/em\u003e has recently emerged as a major invasive pest damaging mangrove plants, particularly \u003cem\u003eA. marina\u003c/em\u003e. However, the diversity and functional roles of the gut microbiota in \u003cem\u003eH. puera\u003c/em\u003e remain largely unexplored. In this study, we employed 16S rRNA high-throughput sequencing to systematically investigate the diversity, community structure, and potential functions of the gut bacteria in fifth-instar larvae of \u003cem\u003eH. puera\u003c/em\u003e under field-collected, laboratory-reared, and starvation-treated conditions.\u003c/p\u003e\u003cp\u003eThe gut microbiota of insects is largely acquired through oral intake, and microorganisms carried by food significantly shape the microbial community, indicating that dietary sources play a decisive role in structuring the gut microbiota (Douglas \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For example, in silkworm larvae (4th\u0026ndash;5th instar) fed with lettuce leaves, the gut microbial composition differed markedly from that of conventionally reared silkworms. Lettuce-fed larvae exhibited substantial abundances of \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003eAnaerofilum\u003c/em\u003e-genera that are nearly absent in normal silkworm larvae (Liang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our results indicated that the gut bacteria of laboratory- reared \u003cem\u003eH. puera\u003c/em\u003e larvae is dominated by the phylum Firmicutes-D, with \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eLigilactobacillus\u003c/em\u003e being the predominant genera. Such a community structure is characteristic of insects reared on artificial diets rich in sugars and starches (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As facultative anaerobes, lactobacilli efficiently ferment carbohydrates to produce lactate, which not only helps establish an acidic microenvironment that inhibits pathogens but may also enhance the host\u0026rsquo;s energy acquisition efficiency from the artificial diet (Engel and Moran \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn stark contrast, the gut bacteria of field-collected \u003cem\u003eH. puera\u003c/em\u003e larvae is dominated by the phylum level by Bacteroidota and Proteobacteria, and at the genus level by \u003cem\u003eJC017\u003c/em\u003e, \u003cem\u003eFulvimarina\u003c/em\u003e, and \u003cem\u003eStenotrophomonas\u003c/em\u003e. Bacteroidota are well-known for their robust capacity to degrade complex polysaccharides, aiding the host in breaking down plant cell wall components such as cellulose and pectin (Hammer and Bowers \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The presence of \u003cem\u003eFulvimarina\u003c/em\u003e, a genus commonly associated with marine environments, strongly suggests that the larvae acquire habitat-specific microorganisms through feeding on \u003cem\u003eA. marina\u003c/em\u003e (Yang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This highly specialized microbial structure likely results from long-term co-evolution between the larvae and their natural host plants, potentially facilitating the digestion of host-specific plant compounds and enhancing adaptation to their ecological niche. Interestingly, although metrics such as Observed_species were slightly lower in the field group compared to the laboratory group, the difference was not statistically significant. This may be attributed to the uniform composition of artificial diet, which provides a stable environment conducive to the proliferation of generalist bacterial species. In contrast, the complex and varied nature of natural food sources may promote competitive dominance of more specialized taxa, thereby reducing the total number of microbial species (ASV richness) and diversity in the larval gut.\u003c/p\u003e\u003cp\u003eStarvation treatment significantly reduced the number of ASVs in field-collected and laboratory-reared \u003cem\u003eH. puera\u003c/em\u003e larvae, indicating that nutrient deprivation acts as a strong ecological filter that eliminates microbial taxa incapable of surviving independently under resource-limited conditions, thereby leading to a simplified gut microbiota (Akami et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Crucially, our study revealed that the initial structure of the microbiota predetermined the stress response pattern. In starved laboratory-reared larvae (HAS), the relative abundance of \u003cem\u003eEnterococcus\u003c/em\u003e increased dramatically from 33.32% to 83.36%, resulting in a near-monodominant community. \u003cem\u003eEnterococcus\u003c/em\u003e is well-known for its high environmental adaptability, including tolerance to pH shifts, nutrient stress, and a strong capacity for biofilm formation, enabling it to survive and proliferate rapidly under harsh conditions (Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, the response to starvation in field-collected larvae (HAS) demonstrated greater functional redundancy and ecological resilience. Instead of dominance by a single taxon, the microbial community shifted to a structure co-dominated by \u003cem\u003eBurkholderia\u003c/em\u003e (34.48%) and \u003cem\u003eAcinetobacter\u003c/em\u003e (20.64%). Species within \u003cem\u003eBurkholderia\u003c/em\u003e are known not only for their ability to degrade various aromatic pollutants-such as phenols, polycyclic aromatic hydrocarbons, and pesticides\u0026mdash;but also for performing coupled denitrification, producing siderophores, and potentially helping maintain gut ecological balance (Jang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eAcinetobacter\u003c/em\u003e, on the other hand, exhibits remarkable metabolic plasticity, enabling it to utilize diverse carbon and nitrogen sources, degrade hydrocarbons and lipids, and form biofilms under adverse conditions, underscoring its ecological versatility and potential role in gut homeostasis (Zhang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These taxa may aid the host through nutrient supplementation or detoxification during starvation. This diversified response strategy likely stems from the inherently higher complexity of the field-acquired microbiota, offering more functional options to cope with environmental stress.\u003c/p\u003e\u003cp\u003eThe functional prediction analysis using PICRUSt2 provides critical insights into the ecological implications of the observed microbial community structure. The results indicate that the gut microbiota of \u003cem\u003eH. puera\u003c/em\u003e possesses extensive metabolic potential, being heavily involved in two major functional modules: biosynthesis-including amino acids, vitamins, cofactors, and fatty acids-and degradation/utilization-such as carbohydrates, aromatic compounds, and carboxylates. This metabolic versatility is crucial for an insect feeding on mangrove plants, which are well-known for their potent chemical defenses. Mangrove plants typically contain abundant secondary metabolites, such as tannins and phenolics (Lang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The significant enrichment of pathways related to \"aromatic compound degradation\" and \"detoxification\" strongly suggests that these symbiotic bacteria play a key role in assisting the larvae in counteracting plant chemical defenses (Hammer and Bowers \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Concurrently, pathways involved in \"vitamin and amino acid biosynthesis\" imply that the gut microbiota may act as an external nutrient reservoir, supplementing essential nutrients that are scarce in the host\u0026rsquo;s diet, thereby supporting normal growth, development, and reproduction (Berg et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This strategy of \"microbial facilitation\"-gaining dual capabilities in toxin degradation and nutritional supplementation through symbiotic bacteria\u0026mdash;may represent a core ecological mechanism enabling \u003cem\u003eH. puera\u003c/em\u003e to successfully adapt to its specialized diet and emerge as a major pest.\u003c/p\u003e\u003cp\u003eIn summary, our findings indicated that laboratory rearing significantly altered the gut microbial composition. The gut bacteria of laboratory-reared larvae were dominated by the phylum Firmicutes-D, genus \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eLigilactobacillus\u003c/em\u003e, whereas field-collected larvae exhibited a bacterial community primarily composed of the phyla Bacteroidota and Proteobacteria, with \u003cem\u003eJC017\u003c/em\u003e and \u003cem\u003eFulvimarina\u003c/em\u003e as the dominant genera. Starvation treatment significantly reduced alpha diversity and ASV richness of the gut bacteria, and the alpha diversity was significantly lower in laboratory-reared larvae compared to field-collected ones. After starvation, the relative abundance of \u003cem\u003eEnterococcus\u003c/em\u003e in laboratory-reared larvae increased markedly to 83.36%, resulting in a mono-dominant microbial structure. In contrast, starved field-collected larvae maintained a more diverse and tolerant community, predominantly consisting of \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e. Functional prediction indicated that the gut bacteria was extensively involved in metabolic pathways such as amino acid and vitamin biosynthesis, as well as degradation of aromatic compounds, suggesting its potential role in helping the host overcome mangrove chemical defenses and compensate for nutritional challenges. This study provides a theoretical foundation for understanding the ecological adaptation of \u003cem\u003eH. puera\u003c/em\u003e and supports the development of microbiome-based strategies for eco-friendly pest control. Future research should focus on the isolation and culture of gut bacteria, combined with metagenomic and metabolomic approaches, to precisely elucidate the functional genes and metabolites of key taxa (e.g., \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eBurkholderia\u003c/em\u003e) and validate their specific roles in nutrient provision and detoxification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eSupplementary material\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003eAuthor Contribution Statement\u003c/p\u003e\n\u003cp\u003eConceptualization, Liangjian Qu, Hao Yu. Formal analysis, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Funding acquisition, Liangjian Qu, Hao Yu. Investigation, Wen Zhang, Yuehua Liu, Gaole Chen, Yi Yang, Junli Jiang. Project administration, Liangjian Qu, Hao Yu. Methodology, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Resources, Wen Zhang, Yuehua Liu, Liangjian Qu. Supervision, Gaole Chen, Yi Yang, Junli Jiang. Validation, Wen Zhang, Yuehua Liu, Gaole Chen. Visualization, Yi Yang, Junli Jiang. Writing\u0026mdash;original draft, Xinan Li, Qiaoling Zhao, Xiaoya Zhang. Writing\u0026mdash;review \u0026amp; editing, Liangjian Qu, Hao Yu.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key R\u0026amp;D Program of China (2023YFC260480\u003c/p\u003e\n\u003cp\u003e1), the Key R\u0026amp;D programs of Henan Province (No. 241111311900), the Undergraduate Innovation Training Program Project of Henan Province (10467CXCY2025032)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets and analyses of the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkami M, Njintang NY, Gbaye OA, Andongma AA, Rashid MA, Niu C-Y, Nukenine EN (2019) Gut bacteria of the cowpea beetle mediate its resistance to dichlorvos and susceptibility to \u003cem\u003eLippia adoensis\u003c/em\u003e essential oil. Sci Rep 9:6435. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-42843-1\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-42843-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerg G, Toledo GV, Schierstaedt J, Hy\u0026ouml;ty H, Wicaksono WA (2025) Linking the edible plant microbiome and human gut microbiome. Gut Microbes 17:2551113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/19490976.2025.2551113\u003c/span\u003e\u003cspan address=\"10.1080/19490976.2025.2551113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiji CP, Sudheendrakumar VV, Sajeev TV (2006) Quantitative estimation of \u003cem\u003eHyblaea puera\u003c/em\u003e NPV production in three larval stages of the teak defoliator, \u003cem\u003eHyblaea puera\u003c/em\u003e (Cramer). J Virol Methods 136:78\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jviromet.2006.04.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jviromet.2006.04.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852\u0026ndash;857. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-019-0209-9\u003c/span\u003e\u003cspan address=\"10.1038/s41587-019-0209-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroderick NA, Raffa KF, Handelsman J (2006) Midgut bacteria required for \u003cem\u003eBacillus thuringiensis\u003c/em\u003e insecticidal activity. PNAS 103:15916\u0026ndash;15919. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0604865103\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0604865103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCallahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581\u0026ndash;583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.3869\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.3869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandrasekhar N, Sajeev TV, Sudheendrakumar VV, Banerjee M (2005) Population dynamics of the teak defoliator (\u003cem\u003eHyblaea puera\u003c/em\u003e Cramer) in Nilambur teak plantations using Randomly Amplified Gene Encoding Primers (RAGEP). BMC Ecol 5:1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1472-6785-5-1\u003c/span\u003e\u003cspan address=\"10.1186/1472-6785-5-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandrasekhar N, Neetha N, Vaidyan L, Banerjee M (2008) Deciphering the Molecular Phylogenetics of Family Hyblaeidae and Inferring the Phylogeographical Relationships Using DNA Barcoding. J Genet Mol Biology 19:158\u0026ndash;167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.30047/JGMB.200809.0004\u003c/span\u003e\u003cspan address=\"10.30047/JGMB.200809.0004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang M (2023) Identification of morphological characters and antennal sensilla of themoth \u003cem\u003eHyblaea puera\u003c/em\u003e Cramer. Biol Disaster Sci 46:342\u0026ndash;347. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3969/j.issn.2095-3704.2023.03.51\u003c/span\u003e\u003cspan address=\"10.3969/j.issn.2095-3704.2023.03.51\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen B, Du K, Sun C, Vimalanathan A, Liang X, Li Y, Wang B, Lu X, Li L, Shao Y (2018) Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives. ISME J 12:2252\u0026ndash;2262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-018-0174-1\u003c/span\u003e\u003cspan address=\"10.1038/s41396-018-0174-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069\u0026ndash;5072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.03006-05\u003c/span\u003e\u003cspan address=\"10.1128/aem.03006-05\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDillon RJ, Vennard CT, Buckling A, Charnley AK (2005) Diversity of locust gut bacteria protects against pathogen invasion. Ecol Lett 8:1291\u0026ndash;1298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1461-0248.2005.00828.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1461-0248.2005.00828.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDouglas AE (2015) Multiorganismal insects: diversity and function of resident microorganisms. Annu Rev Entomol 60:17\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-ento-010814-020822\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-010814-020822\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDouglas GM, Maffei VJ, Zaneveld JR, Yurgel SN, Brown JR, Taylor CM, Huttenhower C, Langille MGI (2020) PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 38:685\u0026ndash;688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-020-0548-6\u003c/span\u003e\u003cspan address=\"10.1038/s41587-020-0548-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEngel P, Moran NA (2013) The gut microbiota of insects-diversity in structure and function. FEMS Microbiol Rev 37:699\u0026ndash;735. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1574-6976.12025\u003c/span\u003e\u003cspan address=\"10.1111/1574-6976.12025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGeorge BS, Silambarasan S, Senthil K, Jacob JP, Dasgupta MG (2018) Characterization of an insecticidal protein from withania somnifera against lepidopteran and hemipteran pest. Mol Biotechnol 60:290\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12033-018-0070-y\u003c/span\u003e\u003cspan address=\"10.1007/s12033-018-0070-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHammer TJ, Bowers MD (2015) Gut microbes may facilitate insect herbivory of chemically defended plants. Oecologia 179:1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00442-015-3327-1\u003c/span\u003e\u003cspan address=\"10.1007/s00442-015-3327-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan S, Akhtar MR, Xia X (2024) Functions and regulations of insect gut bacteria. Pest Manag Sci 80:4828\u0026ndash;4840. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.8261\u003c/span\u003e\u003cspan address=\"10.1002/ps.8261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Mart\u0026iacute;nez P, Naseri B, Navarro-Cerrillo G, Escriche B, Ferr\u0026eacute; J, Herrero S (2010) Increase in midgut microbiota load induces an apparent immune priming and increases tolerance to \u003cem\u003eBacillus thuringiensis\u003c/em\u003e. Environ Microbiol 12:2730\u0026ndash;2737. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1462-2920.2010.02241.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-2920.2010.02241.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu R, Chen H, Yang K, Cai B, Li J (2016) Review of a new pest, \u003cem\u003eHyblaea puera\u003c/em\u003e (Cramer), in mangrove forest in China. For Pest Disease 35:34\u0026ndash;37\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJang S, Ishigami K, Mergaert P, Kikuchi Y (2024) Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect. PNAS 121:e2315540121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.2315540121\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2315540121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia Y, Jin S, Hu K, Geng L, Han C, Kang R, Pang Y, Ling E, Tan EK, Pan Y et al (2021) Gut microbiome modulates \u003cem\u003edrosophila\u003c/em\u003e aggression through octopamine signaling. Nat Commun 12:2698. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-021-23041-y\u003c/span\u003e\u003cspan address=\"10.1038/s41467-021-23041-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKottaipalayam-Somasundaram SR, Jacob JP, Aiyar B, Merzendorfer H, Nambiar-Veetil M (2022) Chitin metabolism as a potential target for RNAi-based control of the forestry pest \u003cem\u003eHyblaea puera\u003c/em\u003e Cramer (Lepidoptera: Hyblaeidae). Pest Manag Sci 78:296\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ps.6634\u003c/span\u003e\u003cspan address=\"10.1002/ps.6634\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2021 Sep 27\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLang T, Hussain M, Li M, Tam NF-Y, Min Pan 5 FW-FL, Xu SJ-L, Jiang M, Wang Y, Mu L, Zhou H (2025) Unlocking the structure-activity relationship of mangrove condensed and hydrolysable tannins: Unveiling their potential ecological significance in antioxidant and antibacterial functions. Int J Biol Macromol 307: 141918. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2025.141918\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2025.141918\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang X, Fu Y, Tong L, Liu H (2014) Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding. Appl Microbiol Biotechnol 98:3769\u0026ndash;3776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-014-5532-y\u003c/span\u003e\u003cspan address=\"10.1007/s00253-014-5532-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu BQ, Bao XY, Yan JY, Zhang D, Sun X, Li CQ, Chen ZB, Luan JB (2024) \u003cem\u003eRickettsia\u003c/em\u003e symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating. Proc. Natl. Acad. Sci. U. S. A 121: e2406788121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.2406788121\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2406788121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShah RA, Riyaz M, Ignacimuthu S, Sivasankaran K (2022) Characterization of four mitochondrial genomes from superfamilies Noctuoidea and Hyblaeoidea with their phylogenetic implications. Sci Rep 12:18926. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-21502-y\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-21502-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharon G, Segal D, Ringo JM, Hefetz A, Zilber-Rosenberg I, Rosenberg E (2010) Commensal bacteria play a role in mating preference of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. PNAS 107:20051\u0026ndash;20056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1009906107\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1009906107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVelavan V, Dhanapa R, Ramkumar G, Karthi S, Senthil-Nathan S, Ndomba OA, Kweka EJ (2022) Characterization and Evaluation of \u003cem\u003eMetarhizium\u003c/em\u003e spp. (Metsch.) Sorokin Isolates for Their Temperature Tolerance. Journal of Fungi 8: 68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof8010068\u003c/span\u003e\u003cspan address=\"10.3390/jof8010068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarnecke F, Luginb\u0026uuml;hl P, Ivanova N, Ghassemian M, Richardson TH, Stege JT, Cayouette M, McHardy AC, Djordjevic G, Aboushadi N et al (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450:560\u0026ndash;565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature06269\u003c/span\u003e\u003cspan address=\"10.1038/nature06269\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei J, Liu W, Huang Q, Chen Q, Su B (2019) Investigation of main insect pests in shankou national mangrove reserve of Guangxi In recent 15 years. FuJian Agric Sci Technol 46:66\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13428/j.cnki.fjlk.2019.04.015\u003c/span\u003e\u003cspan address=\"10.13428/j.cnki.fjlk.2019.04.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang S, Peng J, Xue Y, Zheng Z, Zhou H (2020) Species and integrated control of pests in mangrove community in China. For pest disease 39:32\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19688/j.cnki.issn1671-0886.20190031\u003c/span\u003e\u003cspan address=\"10.19688/j.cnki.issn1671-0886.20190031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang SR, Liu YY, Ge XF, Liu WZ (2023) \u003cem\u003eCaldimonas mangrovi\u003c/em\u003e sp. nov Isolated Mangrove Soil Microbiol 92:500\u0026ndash;507. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S0026261723600477\u003c/span\u003e\u003cspan address=\"10.1134/S0026261723600477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYao YL, Ma XY, Wang TY, Yan JY, Chen NF, Hong JS, Liu BQ, Xu ZQ, Zhang N, Lv C et al (2023) A bacteriocyte symbiont determines whitefly sex ratio by regulating mitochondrial function. Cell Rep 42:112102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2023.112102\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2023.112102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYao Z, Cai Z, Ma Q, Bai S, Wang Y, Zhang P, Guo Q, Gu J, Lemaitre B, Zhang H (2022) Compartmentalized PGRP expression along the dipteran \u003cem\u003eBactrocera dorsalis\u003c/em\u003e gut forms a zone of protection for symbiotic bacteria. 41:111523. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2022.111523\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2022.111523\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Liu J, Deng M, Chen X, Jiang L, Zhang J, Tao L, Yu W, Qiu Y (2023) Enterococcus faecalis promotes the progression of colorectal cancer via its metabolite: biliverdin. J Translational Med 21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12967-023-03929-7\u003c/span\u003e\u003cspan address=\"10.1186/s12967-023-03929-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang S, Li Z, Shu J, Xue H, Guo K, Zhou X (2022) Soil-derived bacteria endow \u003cem\u003eCamellia weevil\u003c/em\u003e with more ability to resist plant chemical defense. Microbiome 10:97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-022-01290-3\u003c/span\u003e\u003cspan address=\"10.1186/s40168-022-01290-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"Hyblaea puera, high-throughput sequencing, gut bacteria, community structure, function prediction","lastPublishedDoi":"10.21203/rs.3.rs-8081780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8081780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSymbiotic microorganisms play a significant role in the physiology and biochemistry of insects. \u003cem\u003eHyblaea puera\u003c/em\u003e (Lepidoptera: Hyblaeidae), is an emerging invasive pest seriously damaging the mangrove ecosystem (mainly \u003cem\u003eAvicennia marina\u003c/em\u003e) along the coast of China. This study systematically investigated the community structure, diversity and potential functions of the gut bacteria of instar larvae of \u003cem\u003eH. puera\u003c/em\u003e under field collection, indoor rearing and starvation treatment. The results revealed that laboratory rearing significantly altered the gut microbial composition. The gut bacteria of laboratory-reared larvae were dominated by the phylum Firmicutes-D, genus \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eLigilactobacillus\u003c/em\u003e, whereas field-collected larvae exhibited a bacterial community primarily composed of the phyla Bacteroidota and Proteobacteria, genera \u003cem\u003eJC017\u003c/em\u003e and \u003cem\u003eFulvimarina\u003c/em\u003e. Starvation treatment significantly reduced alpha diversity and amplicon sequence variant (ASV) richness of the gut bacteria, and the alpha diversity was significantly lower in laboratory-reared larvae compared to field-collected ones. After starvation, the relative abundance of \u003cem\u003eEnterococcus\u003c/em\u003e in laboratory-reared larvae increased markedly to 83.36%, resulting in a mono-dominant microbial structure. In contrast, starved field-collected larvae maintained a more diverse and tolerant community, predominantly consisting of \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e. Functional prediction indicated that the gut bacteria was extensively involved in metabolic pathways such as amino acid and vitamin biosynthesis, as well as degradation of aromatic compounds, suggesting its potential role in helping the host overcome mangrove chemical defenses and compensate for nutritional challenges. This study provides a theoretical foundation for understanding the ecological adaptation mechanisms of \u003cem\u003eH. puera\u003c/em\u003e and developing microbiome-based strategies for environmentally friendly pest control.\u003c/p\u003e","manuscriptTitle":"Analysis of gut microbial diversity and function in Hyblaea puera (Lepidoptera: Hyblaeidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 16:16:32","doi":"10.21203/rs.3.rs-8081780/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":"a449ee03-01b3-44e2-8e19-2e6080fb8568","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T22:39:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-09 16:16:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8081780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8081780","identity":"rs-8081780","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}