Gut microbiomes of four Australian stink bug species associated with citrus, and fitness effects of a dominant Pantoea-like symbiont in Biprorulus bibax

Gut microbiomes of four Australian stink bug species associated with citrus, and fitness effects of a dominant Pantoea-like symbiont in Biprorulus bibax | 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 Gut microbiomes of four Australian stink bug species associated with citrus, and fitness effects of a dominant Pantoea-like symbiont in Biprorulus bibax Alihan Katlav, Juntao Wang, Piotr Trębicki, Amir Tourani, Jennifer Morrow, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8890409/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Gut microbiomes are fundamental to animal biology, evolution and health. True bugs (Hemiptera) maintain heritable microbial gut symbionts and are research models for host-microbe interactions. However, current knowledge is mostly limited to the bacterial symbionts in the Holarctic-Oriental bug fauna, leaving the microbial symbioses of the Australasian diversity unknown. Using high-throughput amplicon sequencing, we characterized the bacterial and fungal communities of four Australian stink bug species associated with citrus: Biprorulus bibax , Poecilometis strigatus (Pentatomidae), Lyramorpha rosea and Musgraveia sulciventris (Tessaratomidae). Across all species, bacterial communities were low in diversity, with each species harbouring a dominant and distinct gammaproteobacterial symbiont within the Pantoea-Erwinia complex. However, L. rosea and P. strigatus contained more diverse assemblages including low-abundance secondary taxa. Furthermore, each host species harboured a differentiated fungal consortium that was diverse across hosts and dominated by taxa including Cladosporium , Eremothecium and Malassezia . Although the dominant bacterial symbionts were host-specific, their phylogeny was incongruent with the host phylogeny, probably indicating host switches and decoupled host-symbiont evolutionary histories. We also found evidence that in B. bibax , the Pantoea -like symbiont was vertically transmitted via egg smearing. Egg surface sterilisation resulted in aposymbiotic offspring with delayed development, reduced longevity and lower fecundity, demonstrating a symbiont contribution to host fitness. Overall, our findings of the first comparative gut microbiome analysis of Australian stink bugs support globally conserved association patterns with Pantoea -like symbionts alongside species-specific microbial community structure and advanced the understanding of host-microbe evolution in these insects. Pantoea Gammaproteobacteria fungal microbiome spined citrus bug gumtree shield bug lychee stink bug bronze orange bug Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Animal guts host diverse microbial communities, including bacteria and fungi that play key roles in host biology and ecology [ 1 ]. These microbes often improve nutrition by helping digest and metabolise complex or otherwise inaccessible dietary components in gut [ 2 ], and can protect hosts by producing antimicrobial compounds or enhancing tolerance to stressors such as heat and chemical agents [ 3 , 4 ]. In insects, especially those feeding on nutritionally restrictive diets (e.g., plant sap, blood, woody tissue), these factors have driven tight symbioses with specialised microbes that occupy distinct gut niches or are housed in bacteriocytes/mycetocytes, where they help overcome nutritional constraints and shape ecological interactions [ 2 , 5 , 6 ]. Gut microbial associations are well documented in Hemiptera, particularly Pentatomoidea (stink bugs and relatives; ~8,000 species) [ 7 , 8 ]. In many species, the posterior midgut (M4) contains crypts packed with extracellular symbionts, usually dominated by a single, often host-specific Enterobacteriaceae lineage [ 9 , 10 ] that remains poorly characterised across numerous taxa. These bacteria can supplement limiting nutrients, contribute to detoxification, and facilitate niche expansion and diversification [ 11 – 13 ]. Stink bug gut symbionts are typically maternally transmitted via a few main routes [ 14 ]. Most commonly, females smear symbionts onto eggs during oviposition, and hatchlings acquire them by feeding on the eggshells [ 15 ]. In some species, females deposit symbiont-filled capsules beside the eggs that nymphs consume [ 16 ]. Less commonly, symbionts are acquired via maternal excretions or from environmental reservoirs contaminated with the mother’s faeces/rectal secretions, rather than strict vertical transmission [ 17 ]. Fungal communities (mycobiomes) in Hemiptera, including yeast-like symbionts (YLS), remain far less studied than bacterial microbiota [ 18 , 19 ]. However, YLS can strongly influence host physiology and fitness [ 20 ]; for example, in the brown planthopper Nilaparvata lugens they are essential for development and digestion and provide key nutrients (e.g., sterols and B vitamins) [ 21 ]. In stink bugs, fungal diversity is still poorly characterised, though surveys often recover Cladosporium [ 22 ]. Some species also vector the plant-pathogenic yeast Eremothecium coryli, which causes yeast spot/fruit rot (stigmatomycosis) [ 23 ]. Australia hosts > 400 pentatomoid species, many endemic; Pentatomidae and Tessaratomidae comprise > 65% of this fauna and include many key sap-sucking pests in Australian agroecosystems (Australian Faunal Directory). Australia is also vulnerable to incursions by exotic pentatomoids such as the brown marmorated stink bug ( Halyomorpha halys ), a highly invasive polyphagous species reported from wide range of host plants; fairly recently, a post-border incursion in New South Wales during summer 2017-18 was declared eradicated in August 2018 [ 24 ]. Despite this ecological, economic and biosecurity significance, the microbial symbionts of Australia’s native stink bugs remain unstudied; there is, to our knowledge no detailed information on their bacterial or fungal communities. This is a major gap because gut symbionts are often essential for stink bug development and survival, and their removal in many non-Australian species causes delayed growth, elevated mortality, reduced fecundity, and morphological abnormalities [ 25 – 27 ]. Here we conducted a comparative analysis of gut microbial diversity and structure across four Australian stink bug species of two Pentatomoidea families collected from citrus trees: the spined citrus bug (SCB) Biprorulus bibax (Pentatomidae; citrus specialist), the gumtree shield bug (GSB) Poecilometis strigatus (Pentatomidae; primarily Eucalyptus -associated but also on citrus), the lychee stink bug (LSB) Lyramorpha rosea (Tessaratomidae; broadly polyphagous on fruit trees), and the bronze orange bug (BOB) Musgraveia sulciventris (Tessaratomidae; major exclusive citrus pest) [ 28 – 31 ]. We dissected the posterior midgut (M4) of adult bugs (Fig. 1 A–D) and used high-throughput sequencing of the bacterial 16S rRNA gene and fungal ITS region to profile both core and transient gut communities. We hypothesised that gut microbial diversity and community composition would differ among species and between families, reflecting host phylogeny and ecological strategy, with specialist species harbouring more stable and less diverse microbiomes than generalists. In addition, we hypothesised that dominant vertically transmitted symbionts are functionally essential for host fitness. To test these predictions, we assessed microbiome diversity differences among the four species. To assess functional importance, we established a laboratory culture of B. bibax (Fig. 1 E–H) and used egg-surface sterilisation to disrupt vertical symbiont acquisition, which markedly impaired nymphal development and reduced adult longevity and fecundity. Together, these results provide the first baseline data on bacterial and fungal gut microbiomes in Australian stink bugs and highlight their importance for host fitness, ecology, and evolution. MATERIAL & METHODS Gut dissection and sample preparations We characterised gut bacterial and fungal communities using high-throughput amplicon sequencing of total genomic DNA from 32 individuals representing four stink bug species across two families: B. bibax (n = 10) and P. strigatus (n = 5) (Pentatomidae), and L. rosea (n = 9) and M. sulciventris (n = 8 for 16S rRNA; n = 6 for fungal ITS) (Tessaratomidae). Adult males and females were collected in 2021 from citrus trees in an orchard at the Hawkesbury Campus, Western Sydney University (33°36′36″S, 150°44′47″E; Supplementary Table S1 ). Sampling was spread across multiple spatially distant trees (one individual per tree) within ~ 1000 m² to minimise close relatedness and direct contact among individuals. Specimens were freeze-killed at − 20°C for 24 h, surface-sterilised (2% sodium hypochlorite for 2 minutes, Milli-Q rinse, brief ethanol dip, air-dried), and dissected under sterile conditions in Ringer’s solution using sterilised tools. The posterior midgut M4 (crypt region) was excised and transferred individually to sterile 1.5 mL tubes for DNA extraction. DNA extraction from gut samples and library preparation for 16S rRNA gene and ITS amplicon sequencing The midgut M4 samples were homogenized using a TissueLyser (Qiagen, Hilden, Germany) with one 2-mm stainless steel ball per tube, operated at a frequency of 25 Hz for 4 minutes. Subsequently, DNA extractions were carried out using the Qiagen DNeasy Blood and Tissue kit as per the manufacturer's instructions, followed by an elution with 100 µl of nuclease-free water. Extracted genomic DNA was quantified using fluorometry (Qubit, Thermofisher Scientific) and spectrophotometry (Nanodrop 2000, Thermofisher Scientific, Waltham, USA), and its integrity was verified by agarose electrophoresis. As an additional quality check, and to obtain host barcodes, we PCR-amplified the mitochondrial cytochrome c oxidase I (COI) gene from each DNA extract using the universal primers LCO1490 and HCO2198 [ 32 ]. The resulting PCR amplicons were Sanger sequenced, and the COI sequences generated for all four stink bug species were used for host COI barcoding and downstream host phylogenetic analyses. DNA extracts were stored at − 20°C until library preparation and sequencing. DNA extracts were submitted to the Western Sydney University Next Generation Sequencing facility for high-throughput amplicon sequencing was using an Illumina MiSeq platform (paired-end 2 × 301 bp). For bacteria, the V3–V4 region of the 16S rRNA gene was amplified using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). For fungi, the ITS2 region was amplified using primers fITS7 (5′-GTGARTCATCGAATCTTTG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). A bacterial mock community (ZymoBIOMICS Microbial Community DNA Standard) provided by the facility was included as a positive control. Bioinformatics and phylogenetic analysis Raw sequencing reads were demultiplexed, and adapter sequences were removed with quality filtering based on Phred33 scores. The 16S rRNA gene and ITS amplicon datasets were analysed using USEARCH [ 33 ] combined with the Quantitative Insights Into Microbial Ecology (QIIME2) framework [ 34 ]. The quality filtering (trimming the low-quality bases, Q value > 20) of raw reads was performed in USEARCH, and chimeric sequences were removed before denoising (error-correction) of amplicon reads to generate the zOTU (zero-radius operational taxonomic unit) using UNOISE [ 35 ]. The resultant zOTUs were assigned with taxonomic information in QIIME2 (version 2023.7) using a Naïve Bayes classifier trained on the SILVA 16S rRNA gene database for bacterial 16S rRNA gene sequences and the UNITE v8.0 database for fungal ITS sequences [ 36 , 37 ]. Any bug-associated mitochondrial, plant-associated and chloroplast sequences were removed prior to downstream analyses. To assess whether the sequencing depth was sufficient, rarefaction curves were generated by plotting the number of observed microbial zOTUs against a range of sampling depths, averaged over 20 iterations. The curves reached a plateau for all samples, indicating that the sequencing depth was adequate and that the samples captured the majority of microbial diversity present (Supplementary Fig. S1 ). Alpha diversity was estimated among the four bug species using Shannon diversity index, and Simpson and Chao estimator. Values were compared using one-way ANOVA (LSD test; P ≤ 0.05 was considered statistically significant), or the nonparametric Kruskal–Wallis test when the data were not normally distributed. The analyses were performed using SPSS software (v.27; IBM Corp, Chicago, USA). Beta diversity was assessed using weighted UniFrac distance (phylogenetic relationships and relative abundance), unweighted UniFrac distance (phylogenetic relationships by presence/absence) and Bray–Curtis distance (relative abundance) to determine the microbial community difference in the four bug species. The effect of stinkbug species and host range on gut microbial diversity and community composition were investigated using permutational multivariate analysis of variance (PERMANOVA, permutation = 9999), and visualised with principal component analysis (PCA) using the Vegan package (v.2.5–6) [ 38 ] in R (v.4.0.3; R Core Team 2021, https://www.R- project.org/). We further used ggplot2 package (version 3.3.3) in R to produce the stacked bar plots of gut bacteria and fungi at class and OTU level. For the analysis of the core microbiome (core OTUs), we used an online microbiome analysis tool (MicrobiomeAnalyst, https://www.microbiomeanalyst.ca/ ) following recommended parameters [ 39 ]. Here, we computed the core microbiomes of the four targeted stink bug species first using a 20% threshold, and subsequently we extended our analyses using a 40% and 60% threshold aiming to enhance the possibility of the detected gut microbiome to be biologically relevant to their hosts. We identified core bacterial populations (OTUs) as those present in at least half of the individuals within each host species or with a relative abundance greater than 1%. For phylogenetic analyses of the dominant bacterium in each host, we further PCR-amplified the close to full-length bacterial 16S rRNA gene (~ 1,500 bp; V1–V9) using primers 16SA1 (5′-AGAGTTTGATCMTGGCTCAG-3′) and 16SB1 (5′-TACGGYTACCTTGTTACGACTT-3′) from the genomic DNA extracts of dissected guts of three individuals per stink bug species. Individuals were selected from those in which a single bacterial OTU dominated the community (70–98% relative abundance in the OTU table). These PCR amplicons were then directly Sanger-sequenced without bacterial culturing. High-quality electropherograms showed single, well-resolved peaks, confirming read accuracy; therefore, no additional bacterial culturing or molecular cloning was necessary. When V3–V4 reads were aligned to the matching region of the V1–V9 16S rRNA gene sequences, the overlap exhibited 100% nucleotide identity across samples, indicating specific, single-template amplification. The V1–V9 16S rRNA gene sequences were aligned using ClustalX [ 40 ]. Phylogenetic inference was conducted on a trimmed 1,350 bp alignment together with reference sequences of gut-associated stink bug symbionts retrieved from NCBI, which robustly resolved the placement of the identified core symbionts. Molecular phylogenetic analyses based on Maximum Likelihood (ML) and Neighbor Joining (NJ) algorithms were performed in MEGA v11.0.13; ML used the GTR + G+I substitution model (selected as the best fit for the 16S rRNA gene). Node support was assessed with 1,000 bootstrap replicates for both methods. Phylogenetic trees were rooted with Pseudomonas aeruginosa (Pseudomonadales: Gammaproteobacteria) as a non-Enterobacteriaceae outgroup. To assess whether the phylogeny of the bacterial symbionts mirrored that of their stink bug hosts, we compared host phylogenies inferred from mitochondrial COI sequences (615 bp) with corresponding symbiont phylogenies based on 16S rRNA gene sequences using a tanglegram approach. Maximum Likelihood (ML) trees for both host and symbiont datasets were reconstructed and visualized in Dendroscope software (version 3.8.10) [ 41 ]. A tanglegram was generated to illustrate and evaluate topological correspondences between host and symbiont trees. The degree of phylogenetic congruence was quantified using the I cong index, which tests whether the observed similarity between trees exceeds that expected by random association. Symbiont elimination and analysis of fitness parameters of B. bibax A laboratory culture of B. bibax was established from adults (20 males and 20 females) collected from the same citrus orchard during austral winter (June 2024). Insects were maintained in mesh-lidded containers [ 42 ] at 25 ± 1°C, 60 ± 5% RH and a 16:8 h light:dark photoperiod, and were provided with fresh oranges. Because adults collected in winter were reproductively dormant, females did not oviposit immediately; dormancy ended after 20–23 days. After oviposition commenced, > 10 newly laid egg batches were collected. Egg masses were surface-sterilized in 70% ethanol for 10 min, immersed in 10% sodium hypochlorite for 15 s, and rinsed twice with sterile water, whereas control egg masses were rinsed twice with sterile water only [ 26 ]. DNA-based PCR assays of a subset of eggs (5 eggs per treatment) confirmed complete elimination of the dominant bacterial gut symbiont in treated eggs but its presence in control eggs (not surface-sterilized) based on 16S rRNA gene PCR using the V1-V9 primers. All egg masses in both treatments hatched successfully, with no detectable difference in hatching rate between sterilized and control groups. Hatchlings from each treatment were reared under the same laboratory conditions, and individual nymphs were monitored daily. This involved the assessment of development time to adult, adult longevity (days from adult eclosion to death) and fecundity. To quantify fecundity, newly emerged adults were separated by sex, and individual females were paired with individual males (Fig. 1 E). Each pair was maintained in an individual container supplied with a fresh orange. Containers were inspected daily and all egg masses were collected and counted, with total fecundity calculated as the cumulative number of eggs produced per female over her lifespan. Fitness traits (development time, adult longevity, and fecundity) were compared between treatments using Kruskal–Wallis and Mood’s median tests in SPSS. Data availability The 16S rRNA gene and ITS amplicon sequencing datasets generated in this study are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1420412. Associated metadata and raw data from the symbiont elimination experiment (fitness parameters of Biprorulus bibax ) are available via Figshare at: https://doi.org/10.6084/m9.figshare.31341679 Please note Cytochrome oxidase I (COI) Sanger sequences and bacterial 16S rRNA gene sequences (V1–V9) of the symbiotic bacteria have been submitted to the NCBI GenBank database and are currently being processed; accession numbers are pending and will be added to a revised version, should the manuscript proceed. RESULTS Sequencing overview Using high-throughput amplicon sequencing, we characterized the gut bacterial and fungal communities of four Australian native stink bug species. After quality control and filtering of the bacterial 16S rRNA gene amplicons, we obtained a total of 1,349,660 sequence reads (mean 42,177 reads per sample; range 2,211–75,951), representing 476 OTUs clustered at 97% sequence similarity. For fungal ITS amplicons, we obtained 2,274,402 sequence reads (mean 22,519 reads per sample) and identified 824 fungal OTUs; reads were rarefied to 110 reads per sample prior to downstream analyses to retain samples with low sequencing depth. Gut bacterial community composition across stinkbug species At both the class and OTU levels, gut bacterial communities showed low overall diversity across all four host species but clear host-associated differences in composition (R2=, P=) (Fig. 2 ). Biprorulus bibax . The bacterial community was dominated by Gammaproteobacteria (72–100% relative abundance), except in SCB2 where Gammaproteobacteria was only ~ 44% and Alphaproteobacteria was ~ 56% (Fig. 2 A). At the OTU level, B. bibax was dominated by a Pantoea -like symbiont (OTU2), which represents a clear core member and accounted for 90–99% of reads in most individuals, but was only ~ 43–50% in SCB1 and SCB2 (Fig. 2 B). Other taxa were generally low to moderate, including Commensalibacter (OTU7; ~0.8–2.5%) and an unclassified Gammaproteobacteria (OTU291; ~2.5–55%). In SCB1, additional low-abundance taxa were detected, including Serratia (OTU35 ~ 2.3%, OTU373 ~ 4.5%), Enterobacter (OTU238 ~ 6.5%) and another Commensalibacter (OTU180 ~ 1.2%) (Fig. 2 B). Poecilometis strigatus . Gut communities were dominated by Gammaproteobacteria (~ 60–100%), with Alphaproteobacteria (20–40%) detected mainly in GSB1 and GSB2 (Fig. 2 A). The dominant OTU was a Pantoea -like symbiont (OTU3; 46.3–75.2%). An unassigned Gammaproteobacteria (OTU12) was consistently present at moderate levels (10.7–24.1%), while Commensalibacter (OTU11) was prominent (~ 18–38%) only in GSB1 and GSB2. Other taxa occurred at low abundance (generally 0.4–1.4%) (Fig. 2 B). Lyramorpha rosea. This species exhibited the highest bacterial diversity among the four stink bug species, although Gammaproteobacteria still dominated (~ 70.2–99.8%), except in LSB6 where it was only ~ 45% (Fig. 2 A). Other consistently detected classes included Actinomycetes (2.1–37.2%), Clostridia (2.3–16.6%), Bacilli (0.2–8.1%), and a range of unclassified taxa (0.1–2.9%) (Fig. 2 A). At the OTU level, a Pantoea -like symbiont (OTU1) dominated (46.3–75.2%). Additional abundant taxa included Frankia (OTU9; ~0.9–34.2%) and an unassigned bacterium (OTU14; ~1.5–12.5%). Several genera (e.g., Lactococcus , Weissella , Kosakonia , Commensalibacter , Erwinia ) occurred sporadically and at trace levels (Fig. 2 B). Musgraveia sulciventris . The bacterial community was low-diversity and strongly dominated by Gammaproteobacteria, primarily a Pantoea -like symbiont (OTU5) accounting for 67.7–99.9% of reads (Fig. 2 A). Other gammaproteobacterial taxa occurred sporadically, including Kosakonia (0.2–8.5%), Erwinia (0.03–9.4%), Sodalis (1.55–9.4%), and Frankia (0.1–0.6%). Other bacteria were detected only at trace levels (< 0.01%) (Fig. 2 B). Gut bacterial community diversity and structure across host species Alpha diversity differed significantly among host species based on observed species, Chao1 richness, and Shannon diversity indices (Fig. 3 A–C). The bacterial community of L. rosea exhibited significantly higher observed species and Chao1 estimates than in B. bibax and P. strigatus (observed species: χ² = 8.72, df = 3, p = 0.027; Chao1: χ² = 9.15, df = 3, p = 0.033). Comparisons of Chao1 between L. rosea and M. sulciventris were marginally non-significant but suggested a trend toward higher richness in L. rosea (observed species: p = 0.067; Chao1: p = 0.091). Shannon diversity also differed significantly among species (χ² = 10.64, df = 3, p = 0.014), with L. rosea and P. strigatus showing similarly higher diversity than B. bibax and M. sulciventris (Fig. 3 A–C). Beta diversity analyses (Bray–Curtis, weighted and unweighted UniFrac) showed clear host-range clustering and significant differences in bacterial community composition among stinkbug species (Fig. 3 D–F). PERMANOVA tests (999 permutations; Table 1 ) confirmed that bacterial communities differed significantly among all bug species, as well as between species within Pentatomidae and Tessaratomidae (Table 1 ). Significant differences were also detected between the two families and between host-range groups (broad vs narrow host plant specificity) (Table 1 ), indicating that both host phylogeny and ecological traits contribute to gut bacterial community structure. Table 1 Summary of PERMANOVA results evaluating pairwise differences in beta diversity of gut bacterial (based on 16S rRNA gene sequence data) and fungal (based on ITS sequence data) communities among stink bug species using weighted UniFrac, Bray–Curtis, and unweighted UniFrac distance metrics. Comparisons include: (i) among all four stink bug species; (ii) between pentatomid species ( Biprorulus bibax and Poecilometis strigatus ); (iii) between tessaratomid species ( Lyramorpha rosea and Musgraveia sulciventris ); (iv) between families (Pentatomidae vs Tessaratomidae); and (v) between host range groups (narrow host range: M. sulciventris & B. bibax vs broad host range: L. rosea & P. strigatus ). Gene PERMANOVA N Permutations Weighted UniFrac Bray–Curtis Unweighted UniFrac F P F P F P 16S rRNA All stinkbug species 32 999 11.11 < 0.001 32.14 < 0.001 6.24 < 0.001 Between species in Pentatomidae 15 999 8.49 0.004 34.28 0.002 8.73 < 0.001 Between species in Tessaratomidae 17 999 12.32 0.003 29.06 < 0.001 4.51 0.004 Between families 32 999 7.24 < 0.001 11.51 < 0.001 4.39 < 0.001 Between groups with different host range 32 999 9.25 < 0.001 10.88 < 0.001 2.96 0.008 ITS All stinkbug species 38 999 4.01 0.01 2.53 < 0.001 3.26 < 0.001 Between species in Pentatomidae 21 999 3.57 0.069 2.35 < 0.001 2.51 0.013 Between species in Tessaratomidae 17 999 6.01 0.013 2.97 < 0.001 4.61 < 0.001 Between families 38 999 3.43 0.042 2.13 < 0.001 2.94 0.003 Between groups with different host range 38 999 0.88 0.337 2.82 < 0.001 3.11 < 0.001 Phylogenetic placement of the dominant bacterial symbionts and their cophylogeny with hosts All symbionts formed distinct lineages within Gammaproteobacteria but occupied different positions within the broader Pantoea – Erwinia symbiont complex (Fig. 4 A). The B. bibax symbiont clustered closely with Pantoea septica (98.7% identity) and Erwinia amylovora (98.2%). The P. strigatus symbiont formed a more divergent lineage, branching separately and forming a sister lineage to symbionts of Eurydema species (97.3%) and Niphe elongata (97.1%) (Pentatomidae). The M. sulciventris symbiont clustered more basally relative to the B. bibax symbiont and grouped within an ancestral clade including the symbiont of Nezara viridula (Pentatomidae). The L. rosea symbiont grouped within a moderately supported clade of pentatomoid-associated symbionts including Graphosoma lineatum (97.9%) and Erthesina fullo (97.6%) (Pentatomidae) (Fig. 4 A). Cophylogenetic analyses using host mitochondrial COI and symbiont 16S rRNA gene sequences showed no significant congruence between host and symbiont lineages (Fig. 4 B). Multiple mismatched branching patterns suggested largely independent evolutionary histories. The I cong congruence index confirmed that the topologies were not more similar than expected by chance ( P = 0.85), which may indicate host switching and/or symbiont replacement during diversification. Gut fungal community composition across stinkbug species Gut fungal composition showed high within-species variability, yet each stink bug species displayed a distinct overall fungal community profile (Fig. 5 ). Biprorulus bibax . The fungal community showed consistently low diversity but substantial individual variation, typically dominated by an unclassified Ascomycota (~ 70–90%) (Fig. 5 A). In SC1, SC2 and SC5 its proportion dropped below 30%, with Saccharomycetes becoming more prominent, while SCF10 was dominated by Tremellomycetes (~ 95%), representing a clear outlier (Fig. 5 A). At the OTU level, core members included an unassigned Ascomycota (OTU127) and Eremothecium spp. (OTU12, OTU13), together contributing up to ~ 70% across individuals (Fig. 5 B). Other taxa (e.g., Naganishia sp. OTU5, dominant in SCB10 > 80%; Malassezia sp. OTU3; unassigned Chytridiomycota OTU106) were sporadic and did not form a consistent core (Fig. 5 B). Poecilometis strigatus. The fungal community was primarily dominated by Dothideomycetes (10–95%), with Sordariomycetes contributing substantially in some individuals (up to 40%). Other fungal classes (e.g., Cystobasidiomycetes, Tremellomycetes, other Ascomycota) occurred sporadically and at lower abundance (Fig. 5 A). At the OTU level, Cladosporium sp. (OTU7) was consistently present (10–95%) and appeared as a dominant and potential core member, while Aureobasidium sp. (OTU10) reached up to 40% in some individuals (Fig. 5 B). Additional taxa (e.g., Diaporthe sp. OTU17, Paraconiothyrium sp. (II) OTU37, Meyerozyma sp. OTU25) occurred at low abundance in subsets of samples (Fig. 5 B). Lyramorpha rosea. The fungal community varied across individuals but were typically dominated by Dothideomycetes and an unclassified Ascomycota, together comprising ~ 20% to > 90% of abundance. Saccharomycetes also occurred at moderate to high levels in approximately half of individuals (~ 20% to > 70%). Other classes (e.g., Sordariomycetes, Cystobasidiomycetes, Tremellomycetes, Eurotiomycetes) were sporadic and generally low abundance (Fig. 5 A). At the OTU level, Eremothecium sp. (I) (OTU2) dominated (up to ~ 90%), with Paraconiothyrium sp. (I) (OTU26; ~4–48%) and additional taxa (e.g., unassigned Mycosphaerellaceae OTU23 ~ 9%; Malasseziaceae OTU14 ~ 2.6%) detected in some individuals (Fig. 5 B). Musgraveia sulciventris. The gut fungal community varied among individuals but was typically dominated by Malasseziomycetes, with highly variable relative abundance (~ 10–80%) (Fig. 5 A). Sordariomycetes (0–50%) and unclassified Ascomycota (0–40%) also contributed substantially across samples (Fig. 5 A). Other classes (e.g., Exobasidiomycetes, Tremellomycetes, Dothideomycetes) occurred sporadically and generally at low to moderate abundance (Fig. 5 A). At the OTU level, Malassezia sp. (I) (OTU3) was detected in all individuals, while Malassezia sp. (II) (OTU35) occurred only in BOB3 and BOB4 (Fig. 5 B). Additional taxa, including Hyphopichia sp. (OTU90) and Cladosporium sp. (OTU7), were present at low abundance in some individuals (Fig. 5 B). Gut fungal community diversity and structure across host species Alpha diversity analyses showed no significant differences in fungal community richness or diversity among the four stink bug species (Fig. 6 A–C): observed species (χ² = 4.26, df = 3, p = 0.235), Chao1 richness (χ² = 3.84, df = 3, p = 0.279), and Shannon diversity (χ² = 5.19, df = 3, p = 0.158). Nevertheless, within-species variation was substantial, with M. sulciventris showing the lowest variability (observed species = 10.4 ± 3.3; Chao1 = 13.1 ± 4.8; Shannon = 1.7 ± 0.4), indicating a consistently depauperate and uniform fungal community, whereas P. strigatus showed the highest variability in Chao1 and Shannon (Chao1 = 30.7 ± 12.2; Shannon = 3.3 ± 1.0). Beta diversity analyses (Bray–Curtis, weighted and unweighted UniFrac) revealed significant differences in fungal community composition among host species (Fig. 6 D–F; Table 1 ). PERMANOVA tests (999 permutations) supported overall host-associated structuring. Fungal assemblages differed more strongly among Tessaratomidae species (F = 2.97–6.01, p ≤ 0.013) than among Pentatomidae species (F = 2.35–3.57, p = 0.013–0.069), while between-family and host-range comparisons were also significant for most metrics (Table 1 ). Together, these results indicate that gut fungal communities vary with host taxonomy and ecological traits, with stronger divergence within Tessaratomidae and among species differing in plant host-range breadth. Gut symbiont elimination negatively impacted B. bibax fitness Egg-surface sterilization resulted in the elimination of the Pantoea-like gut symbiont from B. bibax eggs, thereby markedly affecting the biological performance of the offspring. Aposymbiotic individuals exhibited a markedly prolonged egg-to-adult development time compared with the control group ( χ² ₍₁,₄₂₎ = 19.99, p < 0.001; Fig. 7 A), taking on average 51.3 ± 2.8 days versus 43.2 ± 1.7 days in controls. Adult longevity was also substantially reduced in aposymbiotic insects ( χ² ₍₁,₄₂₎ = 16.72, p < 0.001; Fig. 7 B), averaging 29.4 ± 3.1 days compared with 48.1 ± 2.5 days in symbiotic controls. Likewise, total fecundity (number of eggs per female during life time) was significantly lower in aposymbiotic females ( χ² ₍₁,₂₁₎ = 13.20, p < 0.001; Fig. 7 C), with a mean of 44.6 ± 6.2 eggs compared with 77.8 ± 7.1 eggs in controls. DISCUSSION Low-diversity gut bacterial communities dominated by host-specific Pantoea -like symbionts in four Australian stink bug species Our study provides the first comprehensive characterization of gut microbial communities in four Australian native stinkbug species, B. bibax and P. strigatus (Pentatomidae), and L. rosea and M. sulciventris (Tessaratomidae) (Fig. 1 A–D). Across all species, M4 gut bacterial diversity was consistently low and dominated by Gammaproteobacteria, driven in each host by a single host-specific OTU affiliated with the Pantoea – Erwinia complex (Enterobacteriaceae), as is typical of pentatomoid midgut-crypt symbioses [ 14 , 25 ]. Communities were most streamlined in the citrus specialists B. bibax and M. sulciventris , where the dominant Pantoea -like OTUs (OTU2 and OTU5, respectively) comprised > 90% of reads. In contrast, L. rosea showed greater inter-individual variation and measurable contributions from non-proteobacterial classes (Actinomycetes and Bacilli), including Frankia and sporadic low-abundance genera (e.g., Lactococcus , Weissella , Kosakonia , Commensalibacter and Erwinia ). Similarly, P. strigatus consistently harboured a second, unassigned core gammaproteobacterial OTU (OTU12) at moderate abundance alongside the dominant Pantoea-like OTU and other low-abundance taxa (e.g., Commensalibacter ). These compositional patterns are reflected in alpha diversity metrics: L. rosea showed significantly higher richness (observed species and Chao1) than B. bibax and M. sulciventris , and Shannon diversity was higher in L. rosea and P. strigatus , indicating more even communities. Beta diversity ordinations separated the four species into distinct clusters, and PERMANOVA confirmed significant differences among species, between families, and between host-range groups (narrow vs broad). Together, this suggests that while a conserved Pantoea - Erwinia complex symbiosis underpins community structure across these pentatomoid bugs, variation in richness and composition is further shaped by host ecology via secondary (environmental) bacteria. This aligns with the broader host plant ranges of P. strigatus (Rutaceae, Myrtaceae, Proteaceae, Rosaceae, Oleaceae) [ 28 ] and L. rosea (Sapindaceae and Rutaceae) [ 31 ], compared with the more citrus-restricted B. bibax and M. sulciventris (James, 1989; Cant et al., 1996). Broader host use likely increases exposure to diverse microenvironments, diets, and plant compounds (including antimicrobials), and may increase opportunities for horizontal acquisition, collectively promoting higher secondary microbial diversity [ 2 , 43 – 45 ]. Phylogenetic placement of gut bacterial symbionts and host–symbiont coevolution Bacterial symbionts of pentatomoids are phylogenetically grouped with plant-associated Erwinia and Pantoea bacteria [ 25 ], although without clear patterns of host symbiont co-evolution. One hypothesis for these patterns is that, over evolutionary times, the symbionts may be replaced with taxonomically similar bacteria through host switches and that different bug species may have different levels of dependency on this symbiotic relationship. Our phylogenetic analysis of near full-length 16S rRNA gene clearly show that the dominant gut symbionts of all four Australian stink bug species, despite belonging to four stink bug genera of two families (Pentatomidae vs Tessaratomidae), fall within the same broad gammaproteobacterial clade of the Pantoea – Erwinia complex. More specifically, the gut symbiont of B. bibax clustered closely with Pantoea septica and Erwinia amylovora , whereas the symbiont of P. strigatus formed a more divergent lineage and grouped as sister to symbionts reported from the pentatomoids Eurydema spp. and Niphe elongata . The L. rosea symbiont fell within a clade of pentatomoid-associated symbionts including those from Graphosoma lineatum and Erthesina fullo , and the M. sulciventris symbiont clustered more basally, near to the symbiont of Nezara viridula (Pentatomidae). This supports the idea that pentatomoid stink bugs generally share a conserved bacterial symbiosis type, in which an Enterobacteriaceae lineage dominates the crypt-bearing posterior midgut, but have diversified into host-specific lineages across evolutionary time [ 25 , 46 ]. The cophylogenetic analysis further supports this interpretation by showing that host and symbiont evolutionary histories are not tightly coupled. The host COI phylogeny and the symbiont 16S rRNA gene phylogeny showed no significant congruence (Fig. 3 B), and the multiple mismatched branching patterns indicate that host–symbiont associations have not been maintained through long-term strict cospeciation. This fits well with the view that pentatomoid gut symbionts are often monosymbiotic but polyphyletic within Enterobacteriaceae, reflecting evolutionary turnover and/or occasional replacement among closely related lineages that are pre-adapted to life in the stink bug midgut crypt environment [ 47 ]. Such turnover could be facilitated by transmission biology: gut symbionts are extracellular and commonly acquired early in development from egg-associated inocula, which may allow occasional replacement while still maintaining the same functional niche [ 47 , 48 ]. Gut fungal communities are highly variable and dominated by yeast-like and plant-associated fungi Across the four Australian stink bugs, the gut mycobiome was far more variable than the bacterial community and lacked a single dominant symbiont. This is unsurprising because yeast-like and other plant-associated fungi often form flexible, environmentally driven associations with insects, ranging from transient passengers to ecologically important partners [ 49 , 50 ]. Fungal profiles differed strongly among individuals within species, consistent with frequent renewal from diet and plant surfaces rather than strict vertical transmission. Nevertheless, recurrent taxa suggest ecologically meaningful links: Eremothecium OTUs (notably in B. bibax and L. rosea ) are well known from piercing–sucking Hemiptera and have been associated with stink bug–mediated transmission and yeast-spot plant diseases [ 23 , 51 ] Scarpari et al., 2018; Hojjati et al., 2023). Similarly, Dothideomycetes-dominated profiles (e.g., Cladosporium and Aureobasidium in P. strigatus and L. rosea ) match the common occurrence of these fungi in the phyllosphere and as endophytes that insects likely ingest during feeding [ 52 ]. Although fungal alpha diversity did not differ significantly among species, within-species variation was high and beta diversity showed clear host-associated structuring (including stronger divergence within Tessaratomidae and host-range effects). This combination, similar overall richness but different composition, is consistent with a scenario where gut fungi are shaped by host taxonomy and ecology (diet breadth, feeding microhabitat, exposure to plant-associated fungi), but do not form a uniformly conserved “core” across individuals in the way that midgut-crypt bacteria often do [ 49 ]. Functionally, these patterns motivate targeted follow-up work (e.g., culture-based assays, metagenomics/metatranscriptomics, and controlled diet experiments) to distinguish resident, beneficial yeasts from diet-derived transient fungi and to test whether dominant taxa (e.g., Eremothecium , Cladosporium , Aureobasidium ) contribute to nutrition, detoxification or interactions of host plants. Fitness role of gut symbiont in B. bibax In B. bibax (Fig. 1 E), newly hatched nymphs remained aggregated around the eggshell for ~ 2 days (Fig. 1 G–H), repeatedly probing the egg surface, likely to acquire the maternally deposited Pantoea -like symbiont. Egg-surface sterilisation eliminated this symbiont and produced aposymbiotic offspring, confirming vertical transmission via egg smearing—a common mechanism in stink bugs with midgut-crypt symbionts, where disruption typically reduces host performance [ 53 ]. Consistent with this, aposymbiotic B. bibax developed more slowly (~ 8-day delay), had reduced adult longevity (~ 19 days shorter), and females laid ~ 40% fewer eggs than controls. Similar fitness costs have been reported after symbiont-disruption in other stink bugs, including Graphosoma lineatum , Eurygaster integriceps , and Halyomorpha halys (26, 54–56). These effects are plausibly driven by nutritional constraints in sap-feeding bugs, where symbionts can supplement limiting nutrients; in H. halys , genomic evidence supports such roles for Candidatus Pantoea carbekii [ 56 ]. Overall, our results place B. bibax among pentatomids with strong dependence on extracellular gut symbionts, consistent with variation in symbiont reliance across stink bug lineages [ 53 ]. Future work should test fitness effects in the other hosts, resolve symbiont function using genome- and/or metabolite-based approaches, and determine transmission routes by tracking symbionts across life stages. In several Tessaratomidae species, females guard egg masses and first-instar nymphs, and the newly hatched nymphs are extremely flattened and reportedly non-feeding at the first instar [ 31 ]. It will therefore be important to test whether maternal care behaviours facilitate vertical transmission of gut symbionts (e.g., by maintaining close contact with eggs/nymphs surfaces or maternal secretions) in L. rosea and M. sulciventris . Conclusion Our study shows that four Australian native stink bugs harbour low-diversity gut bacterial communities dominated by host-specific Pantoea - Erwinia complex symbionts. Phylogenetic and cophylogenetic analyses revealed host-specific lineages but no strict host–symbiont cospeciation, consistent with symbiont turnover over evolutionary time. In contrast, secondary gut bacterial taxa and gut fungi were more variable, with richness and composition differing among species in ways broadly aligned with host ecology, particularly host plant range. In B. bibax , disrupting maternal transmission produced aposymbiotic offspring with reduced fitness, demonstrating functional importance of the dominant bacterial symbiont. Broader sampling across regions, seasons, host plants and life stages will be needed to test robustness and move beyond amplicon snapshots toward a more complete understanding of gut microbiome dynamics in Australian true bugs. Declarations Supplementary Information The supplementary material (Fig. S1 and Table S1 ) attached to this submission. Competing Interests The authors declare no competing interests. Funding This project was conducted as part of the Australian Postgraduate Research Intern (APR.Intern) program (Project Reference: INT-1050), in partnership with Agriculture Victoria Research (industry partner), with Western Sydney University as the student institution. The project was also supported by the 2021 Western Sydney University World Microbiome Day Student Grant, awarded to Alihan Katlav. Author Contribution AK, MR and PT conceived the study. AK and AT performed the experiments; AK, JW and JLM conducted data analysis; AK conducted field sampling, the insect material preparations and directions; AK, JW and JM prepared figures; MR and PT supervised the work; AK, MR and JM wrote main manuscript text with input from all authors. Acknowledgement Acknowledgements We acknowledge the APR.Intern team for their support of this project, with special thanks to Zak Blayney (APR.Intern CRM & Project Officer), Justin Mabbutt (APR.Intern Business Development Manager, Victoria & Tasmania), and James Krahe from Food Innovation Australia Ltd (FIAL). We also thank Dr Eleonora Egidi for arranging the Western Sydney University World Microbiome Day Award, Carl Ramirez for assisting with insect collections, and all members of the Microbial Ecology and Environmental Sustainability group at the Hawkesbury Institute for the Environment (HIE), Western Sydney University, for their support. Data Availability The 16S rRNA gene and ITS amplicon sequencing datasets generated in this study are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1420412. 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Supplementary Files SupplFig.S1.docx SupplTableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviews received at journal 20 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 22 Feb, 2026 Reviewers agreed at journal 20 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Editor assigned by journal 16 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 16 Feb, 2026 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. 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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-8890409","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595362858,"identity":"d24d7643-874d-456b-b986-1194298b3880","order_by":0,"name":"Alihan Katlav","email":"data:image/png;base64,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","orcid":"","institution":"Western Sydney University","correspondingAuthor":true,"prefix":"","firstName":"Alihan","middleName":"","lastName":"Katlav","suffix":""},{"id":595362859,"identity":"78fb4707-8d17-467d-9921-65619ec91333","order_by":1,"name":"Juntao Wang","email":"","orcid":"","institution":"Western Sydney University","correspondingAuthor":false,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Wang","suffix":""},{"id":595362860,"identity":"7ca0074f-7039-4568-b4a7-c87accc90b7c","order_by":2,"name":"Piotr Trębicki","email":"","orcid":"","institution":"Macquarie University","correspondingAuthor":false,"prefix":"","firstName":"Piotr","middleName":"","lastName":"Trębicki","suffix":""},{"id":595362861,"identity":"c7a6e930-79c2-40d7-97bb-40909fa8f7c8","order_by":3,"name":"Amir Tourani","email":"","orcid":"","institution":"Western Sydney University","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"","lastName":"Tourani","suffix":""},{"id":595362862,"identity":"8fb9287b-71b9-4555-b7b3-49c72ec9c16e","order_by":4,"name":"Jennifer Morrow","email":"","orcid":"","institution":"Western Sydney University","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Morrow","suffix":""},{"id":595362863,"identity":"c20c52c3-86e6-46e9-944e-3e5013eb77d9","order_by":5,"name":"Hongwei Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Liu","suffix":""},{"id":595362864,"identity":"d27859e2-05f2-4aa6-91e9-ee7573614e01","order_by":6,"name":"Markus Riegler","email":"","orcid":"","institution":"Western Sydney University","correspondingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"Riegler","suffix":""}],"badges":[],"createdAt":"2026-02-16 06:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8890409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8890409/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103300306,"identity":"088294b5-3cde-47b9-8fbe-2276f7d0c167","added_by":"auto","created_at":"2026-02-24 07:57:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5584037,"visible":true,"origin":"","legend":"\u003cp\u003eStudy species, dissected gut regions and spined citrus bug symbiont-disruption assays. \u003cstrong\u003e(A–D)\u003c/strong\u003eAdult habitus (left) and dissected alimentary tract (right) of the four Australian stink bug species examined: \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eBiprorulus bibax\u003c/em\u003e(spined citrus bug, SCB), \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003ePoecilometis strigatus\u003c/em\u003e (gumtree shield bug, GSB), \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eLyramorpha rosea\u003c/em\u003e (lychee stink bug, LSB), and \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eMusgraveia sulciventris\u003c/em\u003e (bronze orange bug, BOB). Midgut regions are indicated as M1–M3 (anterior midgut), M4B (M4 bulb, where present), and M4 (posterior, crypt-bearing midgut), followed by the hindgut (H). The M4 (crypt) region was excised for microbiome profiling. \u003cstrong\u003e(E–H)\u003c/strong\u003e Biology of \u003cem\u003eB. bibax\u003c/em\u003e relevant to vertical symbiont transmission: \u003cstrong\u003e(E)\u003c/strong\u003e mating pair, \u003cstrong\u003e(F) \u003c/strong\u003eegg mass, \u003cstrong\u003e(G)\u003c/strong\u003e aggregation of newly hatched first-instar nymphs around the egg mass, and \u003cstrong\u003e(H)\u003c/strong\u003e close-up of a first-instar nymph showing typical probing behaviour on the eggshell surface.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/e624b5bfe9b9227fda7e3fba.jpg"},{"id":103300318,"identity":"b098b37a-9f00-4781-a04c-be768402cdab","added_by":"auto","created_at":"2026-02-24 07:57:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1793961,"visible":true,"origin":"","legend":"\u003cp\u003eGut bacterial community composition (M4 crypt region) of four Australian stink bugs species. \u003cstrong\u003e(A)\u003c/strong\u003eStacked bar plots showing the relative abundance (%) of bacterial classes in individual bugs. \u003cstrong\u003e(B)\u003c/strong\u003e Heatmap of the dominant and prevalent bacterial OTUs (97% similarity OTUs) across the same individuals. Colour intensity indicates relative abundance (%) per sample.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/17855420e1cf298426a33846.jpg"},{"id":103300209,"identity":"c0f392e4-439c-44ae-bc52-d1450ff5b881","added_by":"auto","created_at":"2026-02-24 07:57:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1054279,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial alpha and beta diversity of M4 gut communities across four stink bug species. \u003cstrong\u003e(A–C)\u003c/strong\u003eAlpha diversity metrics for individual bugs: \u003cstrong\u003e(A)\u003c/strong\u003e observed species richness, \u003cstrong\u003e(B)\u003c/strong\u003e Chao1 estimated richness, and \u003cstrong\u003e(C)\u003c/strong\u003e Shannon diversity index. Boxplots show median (line), interquartile range (box), and range (whiskers), with outliers indicated as points. Different letters denote significant pairwise differences among host species (Kruskal–Wallis with post hoc comparisons; P ≤ 0.05). \u003cstrong\u003e(D–F)\u003c/strong\u003e Principal coordinate analysis ordinations of bacterial community composition based on \u003cstrong\u003e(D)\u003c/strong\u003e Bray–Curtis dissimilarity, \u003cstrong\u003e(E)\u003c/strong\u003e weighted UniFrac distance, and \u003cstrong\u003e(F)\u003c/strong\u003e unweighted UniFrac distance. Points represent individual bugs coloured by host species; axis labels indicate the percentage of variation explained by each principal coordinate.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/fe1bf063366e6ffe75e468c6.jpg"},{"id":103300230,"identity":"d40464d6-e58c-4b0c-9512-6b36ab3d84c4","added_by":"auto","created_at":"2026-02-24 07:57:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1625473,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic placement of dominant gut symbionts and host–symbiont cophylogeny. \u003cstrong\u003e(A)\u003c/strong\u003e Phylogeny based on near full-length 16S rRNA gene sequences (~1,350 bp) showing the placement of the dominant gut symbionts (GS) from four stink bug species within the \u003cem\u003ePantoea\u003c/em\u003e–\u003cem\u003eErwinia\u003c/em\u003e complex. Numbers at nodes indicate Maximum Likelihood/Neighbor Joining bootstrap support (1,000 replicates), and scale bar shows substitutions per site. \u003cstrong\u003e(B)\u003c/strong\u003e Tanglegram comparing host cytochrome c oxidase I phylogeny (left) and symbiont 16S rRNA gene phylogeny (right). Dotted lines link hosts to their GS; crossings indicate topological incongruence (no strict host–symbiont cospeciation).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/82f168c2fbe83c26bd19f5d6.jpg"},{"id":103300225,"identity":"dbcda26e-33fb-439c-a944-2436d7760890","added_by":"auto","created_at":"2026-02-24 07:57:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1514807,"visible":true,"origin":"","legend":"\u003cp\u003eGut fungal community composition (M4 crypt region) of four Australian stink bug species. \u003cstrong\u003e(A)\u003c/strong\u003eStacked bar plots showing the relative abundance (%) of fungal classes in individual bugs. \u003cstrong\u003e(B)\u003c/strong\u003e Heatmap showing the dominant and prevalent fungal operational taxonomic units (clustered at 97% sequence similarity) across the same individuals. Colour intensity indicates the relative abundance (%) of each operational taxonomic unit per sample.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/b6ec4495c106269780efe5fe.jpg"},{"id":103300232,"identity":"1cd90a07-0c4b-473f-be8e-12f6e6138fde","added_by":"auto","created_at":"2026-02-24 07:57:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1015925,"visible":true,"origin":"","legend":"\u003cp\u003eFungal alpha and beta diversity of M4 gut communities across four stink bug species. \u003cstrong\u003e(A–C)\u003c/strong\u003eAlpha diversity metrics for individual bugs: \u003cstrong\u003e(A)\u003c/strong\u003e observed species richness, \u003cstrong\u003e(B)\u003c/strong\u003e Chao1 estimated richness, and \u003cstrong\u003e(C)\u003c/strong\u003e Shannon diversity index. Boxplots show median (line), interquartile range (box), and range (whiskers), with outliers shown as points. Letters indicate results of post hoc comparisons; no significant differences were detected among host species (Kruskal–Wallis test; P \u0026gt; 0.05). \u003cstrong\u003e(D–F)\u003c/strong\u003e Principal coordinate analysis ordinations of fungal community composition based on \u003cstrong\u003e(D)\u003c/strong\u003e Bray–Curtis dissimilarity, \u003cstrong\u003e(E)\u003c/strong\u003e weighted UniFrac distance, and \u003cstrong\u003e(F)\u003c/strong\u003e unweighted UniFrac distance. Points represent individual bugs coloured by host species; axis labels indicate the percentage of variation explained by each principal coordinate.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/e3cf349ae7e1bb84ee66740f.jpg"},{"id":103300227,"identity":"0261e7d6-d6e4-46ec-bc85-9a71954e4c97","added_by":"auto","created_at":"2026-02-24 07:57:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":553168,"visible":true,"origin":"","legend":"\u003cp\u003eFitness effects of elimination of the dominant gut symbiont in \u003cem\u003eBiprorulus bibax\u003c/em\u003e. Boxplots compare control (symbiotic) and aposymbiotic individuals produced by egg-surface sterilisation. \u003cstrong\u003e(A)\u003c/strong\u003e Egg-to-adult development time (days). \u003cstrong\u003e(B)\u003c/strong\u003eAdult longevity (days). \u003cstrong\u003e(C)\u003c/strong\u003e Total lifetime fecundity (total number of eggs laid per female). Boxes show the median (centre line) and interquartile range, whiskers show the range, and open circles indicate outliers. Asterisks denote significant differences between treatments (*** P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/c36132d9ab76cca41e2e18a0.jpg"},{"id":103506430,"identity":"7ea7a065-de65-4d18-8bd9-6d77d47def2c","added_by":"auto","created_at":"2026-02-26 13:36:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14536232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/e5ac4bcc-8229-4b16-ada1-ce34b0a86d07.pdf"},{"id":103300229,"identity":"04b41d57-573e-465d-89a0-9ec654cd1fce","added_by":"auto","created_at":"2026-02-24 07:57:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":124722,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFig.S1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/f7313a1426704a81e95ba41e.docx"},{"id":103300307,"identity":"3b26a855-be12-4841-bbec-eb349fdcff43","added_by":"auto","created_at":"2026-02-24 07:57:40","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22436,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8890409/v1/5132b2550fb272a07f60b507.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gut microbiomes of four Australian stink bug species associated with citrus, and fitness effects of a dominant Pantoea-like symbiont in Biprorulus bibax","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAnimal guts host diverse microbial communities, including bacteria and fungi that play key roles in host biology and ecology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These microbes often improve nutrition by helping digest and metabolise complex or otherwise inaccessible dietary components in gut [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and can protect hosts by producing antimicrobial compounds or enhancing tolerance to stressors such as heat and chemical agents [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In insects, especially those feeding on nutritionally restrictive diets (e.g., plant sap, blood, woody tissue), these factors have driven tight symbioses with specialised microbes that occupy distinct gut niches or are housed in bacteriocytes/mycetocytes, where they help overcome nutritional constraints and shape ecological interactions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGut microbial associations are well documented in Hemiptera, particularly Pentatomoidea (stink bugs and relatives; ~8,000 species) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In many species, the posterior midgut (M4) contains crypts packed with extracellular symbionts, usually dominated by a single, often host-specific Enterobacteriaceae lineage [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] that remains poorly characterised across numerous taxa. These bacteria can supplement limiting nutrients, contribute to detoxification, and facilitate niche expansion and diversification [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Stink bug gut symbionts are typically maternally transmitted via a few main routes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Most commonly, females smear symbionts onto eggs during oviposition, and hatchlings acquire them by feeding on the eggshells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In some species, females deposit symbiont-filled capsules beside the eggs that nymphs consume [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Less commonly, symbionts are acquired via maternal excretions or from environmental reservoirs contaminated with the mother\u0026rsquo;s faeces/rectal secretions, rather than strict vertical transmission [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFungal communities (mycobiomes) in Hemiptera, including yeast-like symbionts (YLS), remain far less studied than bacterial microbiota [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, YLS can strongly influence host physiology and fitness [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; for example, in the brown planthopper Nilaparvata lugens they are essential for development and digestion and provide key nutrients (e.g., sterols and B vitamins) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In stink bugs, fungal diversity is still poorly characterised, though surveys often recover Cladosporium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Some species also vector the plant-pathogenic yeast Eremothecium coryli, which causes yeast spot/fruit rot (stigmatomycosis) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAustralia hosts\u0026thinsp;\u0026gt;\u0026thinsp;400 pentatomoid species, many endemic; Pentatomidae and Tessaratomidae comprise\u0026thinsp;\u0026gt;\u0026thinsp;65% of this fauna and include many key sap-sucking pests in Australian agroecosystems (Australian Faunal Directory). Australia is also vulnerable to incursions by exotic pentatomoids such as the brown marmorated stink bug (\u003cem\u003eHalyomorpha halys\u003c/em\u003e), a highly invasive polyphagous species reported from wide range of host plants; fairly recently, a post-border incursion in New South Wales during summer 2017-18 was declared eradicated in August 2018 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Despite this ecological, economic and biosecurity significance, the microbial symbionts of Australia\u0026rsquo;s native stink bugs remain unstudied; there is, to our knowledge no detailed information on their bacterial or fungal communities. This is a major gap because gut symbionts are often essential for stink bug development and survival, and their removal in many non-Australian species causes delayed growth, elevated mortality, reduced fecundity, and morphological abnormalities [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we conducted a comparative analysis of gut microbial diversity and structure across four Australian stink bug species of two Pentatomoidea families collected from citrus trees: the spined citrus bug (SCB) \u003cem\u003eBiprorulus bibax\u003c/em\u003e (Pentatomidae; citrus specialist), the gumtree shield bug (GSB) \u003cem\u003ePoecilometis strigatus\u003c/em\u003e (Pentatomidae; primarily \u003cem\u003eEucalyptus\u003c/em\u003e-associated but also on citrus), the lychee stink bug (LSB) \u003cem\u003eLyramorpha rosea\u003c/em\u003e (Tessaratomidae; broadly polyphagous on fruit trees), and the bronze orange bug (BOB) \u003cem\u003eMusgraveia sulciventris\u003c/em\u003e (Tessaratomidae; major exclusive citrus pest) [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We dissected the posterior midgut (M4) of adult bugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D) and used high-throughput sequencing of the bacterial 16S rRNA gene and fungal ITS region to profile both core and transient gut communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe hypothesised that gut microbial diversity and community composition would differ among species and between families, reflecting host phylogeny and ecological strategy, with specialist species harbouring more stable and less diverse microbiomes than generalists. In addition, we hypothesised that dominant vertically transmitted symbionts are functionally essential for host fitness. To test these predictions, we assessed microbiome diversity differences among the four species. To assess functional importance, we established a laboratory culture of \u003cem\u003eB. bibax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;H) and used egg-surface sterilisation to disrupt vertical symbiont acquisition, which markedly impaired nymphal development and reduced adult longevity and fecundity. Together, these results provide the first baseline data on bacterial and fungal gut microbiomes in Australian stink bugs and highlight their importance for host fitness, ecology, and evolution.\u003c/p\u003e"},{"header":"MATERIAL \u0026 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGut dissection and sample preparations\u003c/h2\u003e \u003cp\u003eWe characterised gut bacterial and fungal communities using high-throughput amplicon sequencing of total genomic DNA from 32 individuals representing four stink bug species across two families: \u003cem\u003eB. bibax\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;10) and \u003cem\u003eP. strigatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5) (Pentatomidae), and \u003cem\u003eL. rosea\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;9) and \u003cem\u003eM. sulciventris\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8 for 16S rRNA; n\u0026thinsp;=\u0026thinsp;6 for fungal ITS) (Tessaratomidae). Adult males and females were collected in 2021 from citrus trees in an orchard at the Hawkesbury Campus, Western Sydney University (33\u0026deg;36\u0026prime;36\u0026Prime;S, 150\u0026deg;44\u0026prime;47\u0026Prime;E; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Sampling was spread across multiple spatially distant trees (one individual per tree) within ~\u0026thinsp;1000 m\u0026sup2; to minimise close relatedness and direct contact among individuals. Specimens were freeze-killed at \u0026minus;\u0026thinsp;20\u0026deg;C for 24 h, surface-sterilised (2% sodium hypochlorite for 2 minutes, Milli-Q rinse, brief ethanol dip, air-dried), and dissected under sterile conditions in Ringer\u0026rsquo;s solution using sterilised tools. The posterior midgut M4 (crypt region) was excised and transferred individually to sterile 1.5 mL tubes for DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction from gut samples and library preparation for 16S rRNA gene and ITS amplicon sequencing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe midgut M4 samples were homogenized using a TissueLyser (Qiagen, Hilden, Germany) with one 2-mm stainless steel ball per tube, operated at a frequency of 25 Hz for 4 minutes. Subsequently, DNA extractions were carried out using the Qiagen DNeasy Blood and Tissue kit as per the manufacturer's instructions, followed by an elution with 100 \u0026micro;l of nuclease-free water. Extracted genomic DNA was quantified using fluorometry (Qubit, Thermofisher Scientific) and spectrophotometry (Nanodrop 2000, Thermofisher Scientific, Waltham, USA), and its integrity was verified by agarose electrophoresis. As an additional quality check, and to obtain host barcodes, we PCR-amplified the mitochondrial cytochrome c oxidase I (COI) gene from each DNA extract using the universal primers LCO1490 and HCO2198 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The resulting PCR amplicons were Sanger sequenced, and the COI sequences generated for all four stink bug species were used for host COI barcoding and downstream host phylogenetic analyses. DNA extracts were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until library preparation and sequencing. DNA extracts were submitted to the Western Sydney University Next Generation Sequencing facility for high-throughput amplicon sequencing was using an Illumina MiSeq platform (paired-end 2 \u0026times; 301 bp). For bacteria, the V3\u0026ndash;V4 region of the 16S rRNA gene was amplified using primers 341F (5\u0026prime;-CCTACGGGNGGCWGCAG-3\u0026prime;) and 805R (5\u0026prime;-GACTACHVGGGTATCTAATCC-3\u0026prime;). For fungi, the ITS2 region was amplified using primers fITS7 (5\u0026prime;-GTGARTCATCGAATCTTTG-3\u0026prime;) and ITS4 (5\u0026prime;-TCCTCCGCTTATTGATATGC-3\u0026prime;). A bacterial mock community (ZymoBIOMICS Microbial Community DNA Standard) provided by the facility was included as a positive control.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatics and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eRaw sequencing reads were demultiplexed, and adapter sequences were removed with quality filtering based on Phred33 scores. The 16S rRNA gene and ITS amplicon datasets were analysed using USEARCH [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] combined with the Quantitative Insights Into Microbial Ecology (QIIME2) framework [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The quality filtering (trimming the low-quality bases, Q value\u0026thinsp;\u0026gt;\u0026thinsp;20) of raw reads was performed in USEARCH, and chimeric sequences were removed before denoising (error-correction) of amplicon reads to generate the zOTU (zero-radius operational taxonomic unit) using UNOISE [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The resultant zOTUs were assigned with taxonomic information in QIIME2 (version 2023.7) using a Na\u0026iuml;ve Bayes classifier trained on the SILVA 16S rRNA gene database for bacterial 16S rRNA gene sequences and the UNITE v8.0 database for fungal ITS sequences [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Any bug-associated mitochondrial, plant-associated and chloroplast sequences were removed prior to downstream analyses.\u003c/p\u003e \u003cp\u003eTo assess whether the sequencing depth was sufficient, rarefaction curves were generated by plotting the number of observed microbial zOTUs against a range of sampling depths, averaged over 20 iterations. The curves reached a plateau for all samples, indicating that the sequencing depth was adequate and that the samples captured the majority of microbial diversity present (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Alpha diversity was estimated among the four bug species using Shannon diversity index, and Simpson and Chao estimator. Values were compared using one-way ANOVA (LSD test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant), or the nonparametric Kruskal\u0026ndash;Wallis test when the data were not normally distributed. The analyses were performed using SPSS software (v.27; IBM Corp, Chicago, USA).\u003c/p\u003e \u003cp\u003eBeta diversity was assessed using weighted UniFrac distance (phylogenetic relationships and relative abundance), unweighted UniFrac distance (phylogenetic relationships by presence/absence) and Bray\u0026ndash;Curtis distance (relative abundance) to determine the microbial community difference in the four bug species. The effect of stinkbug species and host range on gut microbial diversity and community composition were investigated using permutational multivariate analysis of variance (PERMANOVA, permutation\u0026thinsp;=\u0026thinsp;9999), and visualised with principal component analysis (PCA) using the Vegan package (v.2.5\u0026ndash;6) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] in R (v.4.0.3; R Core Team 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-\u003c/span\u003e\u003cspan address=\"https://www.R-\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e project.org/). We further used ggplot2 package (version 3.3.3) in R to produce the stacked bar plots of gut bacteria and fungi at class and OTU level.\u003c/p\u003e \u003cp\u003eFor the analysis of the core microbiome (core OTUs), we used an online microbiome analysis tool (MicrobiomeAnalyst, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.microbiomeanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://www.microbiomeanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) following recommended parameters [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Here, we computed the core microbiomes of the four targeted stink bug species first using a 20% threshold, and subsequently we extended our analyses using a 40% and 60% threshold aiming to enhance the possibility of the detected gut microbiome to be biologically relevant to their hosts. We identified core bacterial populations (OTUs) as those present in at least half of the individuals within each host species or with a relative abundance greater than 1%.\u003c/p\u003e \u003cp\u003eFor phylogenetic analyses of the dominant bacterium in each host, we further PCR-amplified the close to full-length bacterial 16S rRNA gene (~\u0026thinsp;1,500 bp; V1\u0026ndash;V9) using primers 16SA1 (5\u0026prime;-AGAGTTTGATCMTGGCTCAG-3\u0026prime;) and 16SB1 (5\u0026prime;-TACGGYTACCTTGTTACGACTT-3\u0026prime;) from the genomic DNA extracts of dissected guts of three individuals per stink bug species. Individuals were selected from those in which a single bacterial OTU dominated the community (70\u0026ndash;98% relative abundance in the OTU table). These PCR amplicons were then directly Sanger-sequenced without bacterial culturing. High-quality electropherograms showed single, well-resolved peaks, confirming read accuracy; therefore, no additional bacterial culturing or molecular cloning was necessary. When V3\u0026ndash;V4 reads were aligned to the matching region of the V1\u0026ndash;V9 16S rRNA gene sequences, the overlap exhibited 100% nucleotide identity across samples, indicating specific, single-template amplification. The V1\u0026ndash;V9 16S rRNA gene sequences were aligned using ClustalX [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Phylogenetic inference was conducted on a trimmed 1,350 bp alignment together with reference sequences of gut-associated stink bug symbionts retrieved from NCBI, which robustly resolved the placement of the identified core symbionts. Molecular phylogenetic analyses based on Maximum Likelihood (ML) and Neighbor Joining (NJ) algorithms were performed in MEGA v11.0.13; ML used the GTR\u0026thinsp;+\u0026thinsp;G+I substitution model (selected as the best fit for the 16S rRNA gene). Node support was assessed with 1,000 bootstrap replicates for both methods. Phylogenetic trees were rooted with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (Pseudomonadales: Gammaproteobacteria) as a non-Enterobacteriaceae outgroup.\u003c/p\u003e \u003cp\u003eTo assess whether the phylogeny of the bacterial symbionts mirrored that of their stink bug hosts, we compared host phylogenies inferred from mitochondrial COI sequences (615 bp) with corresponding symbiont phylogenies based on 16S rRNA gene sequences using a tanglegram approach. Maximum Likelihood (ML) trees for both host and symbiont datasets were reconstructed and visualized in Dendroscope software (version 3.8.10) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A tanglegram was generated to illustrate and evaluate topological correspondences between host and symbiont trees. The degree of phylogenetic congruence was quantified using the I\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026gt;cong\u0026lt;/sub\u0026gt; index, which tests whether the observed similarity between trees exceeds that expected by random association.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSymbiont elimination and analysis of fitness parameters of\u003c/b\u003e \u003cb\u003eB. bibax\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA laboratory culture of \u003cem\u003eB. bibax\u003c/em\u003e was established from adults (20 males and 20 females) collected from the same citrus orchard during austral winter (June 2024). Insects were maintained in mesh-lidded containers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH and a 16:8 h light:dark photoperiod, and were provided with fresh oranges. Because adults collected in winter were reproductively dormant, females did not oviposit immediately; dormancy ended after 20\u0026ndash;23 days. After oviposition commenced, \u0026gt;\u0026thinsp;10 newly laid egg batches were collected. Egg masses were surface-sterilized in 70% ethanol for 10 min, immersed in 10% sodium hypochlorite for 15 s, and rinsed twice with sterile water, whereas control egg masses were rinsed twice with sterile water only [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. DNA-based PCR assays of a subset of eggs (5 eggs per treatment) confirmed complete elimination of the dominant bacterial gut symbiont in treated eggs but its presence in control eggs (not surface-sterilized) based on 16S rRNA gene PCR using the V1-V9 primers. All egg masses in both treatments hatched successfully, with no detectable difference in hatching rate between sterilized and control groups. Hatchlings from each treatment were reared under the same laboratory conditions, and individual nymphs were monitored daily. This involved the assessment of development time to adult, adult longevity (days from adult eclosion to death) and fecundity. To quantify fecundity, newly emerged adults were separated by sex, and individual females were paired with individual males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Each pair was maintained in an individual container supplied with a fresh orange. Containers were inspected daily and all egg masses were collected and counted, with total fecundity calculated as the cumulative number of eggs produced per female over her lifespan. Fitness traits (development time, adult longevity, and fecundity) were compared between treatments using Kruskal\u0026ndash;Wallis and Mood\u0026rsquo;s median tests in SPSS.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eThe 16S rRNA gene and ITS amplicon sequencing datasets generated in this study are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1420412. Associated metadata and raw data from the symbiont elimination experiment (fitness parameters of \u003cem\u003eBiprorulus bibax\u003c/em\u003e) are available via Figshare at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.6084/m9.figshare.31341679\u003c/span\u003e\u003cspan address=\"10.6084/m9.figshare.31341679\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePlease note\u003c/strong\u003e \u003cp\u003eCytochrome oxidase I (COI) Sanger sequences and bacterial 16S rRNA gene sequences (V1\u0026ndash;V9) of the symbiotic bacteria have been submitted to the NCBI GenBank database and are currently being processed; accession numbers are pending and will be added to a revised version, should the manuscript proceed.\u003c/p\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSequencing overview\u003c/h2\u003e \u003cp\u003eUsing high-throughput amplicon sequencing, we characterized the gut bacterial and fungal communities of four Australian native stink bug species. After quality control and filtering of the bacterial 16S rRNA gene amplicons, we obtained a total of 1,349,660 sequence reads (mean 42,177 reads per sample; range 2,211\u0026ndash;75,951), representing 476 OTUs clustered at 97% sequence similarity. For fungal ITS amplicons, we obtained 2,274,402 sequence reads (mean 22,519 reads per sample) and identified 824 fungal OTUs; reads were rarefied to 110 reads per sample prior to downstream analyses to retain samples with low sequencing depth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGut bacterial community composition across stinkbug species\u003c/h2\u003e \u003cp\u003eAt both the class and OTU levels, gut bacterial communities showed low overall diversity across all four host species but clear host-associated differences in composition (R2=, P=) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiprorulus bibax\u003c/b\u003e. The bacterial community was dominated by Gammaproteobacteria (72\u0026ndash;100% relative abundance), except in SCB2 where Gammaproteobacteria was only\u0026thinsp;~\u0026thinsp;44% and Alphaproteobacteria was ~\u0026thinsp;56% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At the OTU level, \u003cem\u003eB. bibax\u003c/em\u003e was dominated by a \u003cem\u003ePantoea\u003c/em\u003e-like symbiont (OTU2), which represents a clear core member and accounted for 90\u0026ndash;99% of reads in most individuals, but was only\u0026thinsp;~\u0026thinsp;43\u0026ndash;50% in SCB1 and SCB2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Other taxa were generally low to moderate, including \u003cem\u003eCommensalibacter\u003c/em\u003e (OTU7; ~0.8\u0026ndash;2.5%) and an unclassified Gammaproteobacteria (OTU291; ~2.5\u0026ndash;55%). In SCB1, additional low-abundance taxa were detected, including \u003cem\u003eSerratia\u003c/em\u003e (OTU35\u0026thinsp;~\u0026thinsp;2.3%, OTU373\u0026thinsp;~\u0026thinsp;4.5%), \u003cem\u003eEnterobacter\u003c/em\u003e (OTU238\u0026thinsp;~\u0026thinsp;6.5%) and another \u003cem\u003eCommensalibacter\u003c/em\u003e (OTU180\u0026thinsp;~\u0026thinsp;1.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePoecilometis strigatus\u003c/b\u003e. Gut communities were dominated by Gammaproteobacteria (~\u0026thinsp;60\u0026ndash;100%), with Alphaproteobacteria (20\u0026ndash;40%) detected mainly in GSB1 and GSB2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The dominant OTU was a \u003cem\u003ePantoea\u003c/em\u003e-like symbiont (OTU3; 46.3\u0026ndash;75.2%). An unassigned Gammaproteobacteria (OTU12) was consistently present at moderate levels (10.7\u0026ndash;24.1%), while \u003cem\u003eCommensalibacter\u003c/em\u003e (OTU11) was prominent (~\u0026thinsp;18\u0026ndash;38%) only in GSB1 and GSB2. Other taxa occurred at low abundance (generally 0.4\u0026ndash;1.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyramorpha rosea.\u003c/b\u003e This species exhibited the highest bacterial diversity among the four stink bug species, although Gammaproteobacteria still dominated (~\u0026thinsp;70.2\u0026ndash;99.8%), except in LSB6 where it was only\u0026thinsp;~\u0026thinsp;45% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Other consistently detected classes included Actinomycetes (2.1\u0026ndash;37.2%), Clostridia (2.3\u0026ndash;16.6%), Bacilli (0.2\u0026ndash;8.1%), and a range of unclassified taxa (0.1\u0026ndash;2.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At the OTU level, a \u003cem\u003ePantoea\u003c/em\u003e-like symbiont (OTU1) dominated (46.3\u0026ndash;75.2%). Additional abundant taxa included \u003cem\u003eFrankia\u003c/em\u003e (OTU9; ~0.9\u0026ndash;34.2%) and an unassigned bacterium (OTU14; ~1.5\u0026ndash;12.5%). Several genera (e.g., \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eWeissella\u003c/em\u003e, \u003cem\u003eKosakonia\u003c/em\u003e, \u003cem\u003eCommensalibacter\u003c/em\u003e, \u003cem\u003eErwinia\u003c/em\u003e) occurred sporadically and at trace levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMusgraveia sulciventris\u003c/b\u003e. The bacterial community was low-diversity and strongly dominated by Gammaproteobacteria, primarily a \u003cem\u003ePantoea\u003c/em\u003e-like symbiont (OTU5) accounting for 67.7\u0026ndash;99.9% of reads (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Other gammaproteobacterial taxa occurred sporadically, including \u003cem\u003eKosakonia\u003c/em\u003e (0.2\u0026ndash;8.5%), \u003cem\u003eErwinia\u003c/em\u003e (0.03\u0026ndash;9.4%), \u003cem\u003eSodalis\u003c/em\u003e (1.55\u0026ndash;9.4%), and \u003cem\u003eFrankia\u003c/em\u003e (0.1\u0026ndash;0.6%). Other bacteria were detected only at trace levels (\u0026lt;\u0026thinsp;0.01%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGut bacterial community diversity and structure across host species\u003c/h3\u003e\n\u003cp\u003eAlpha diversity differed significantly among host species based on observed species, Chao1 richness, and Shannon diversity indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). The bacterial community of \u003cem\u003eL. rosea\u003c/em\u003e exhibited significantly higher observed species and Chao1 estimates than in \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eP. strigatus\u003c/em\u003e (observed species: χ\u0026sup2; = 8.72, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.027; Chao1: χ\u0026sup2; = 9.15, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.033). Comparisons of Chao1 between \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e were marginally non-significant but suggested a trend toward higher richness in \u003cem\u003eL. rosea\u003c/em\u003e (observed species: p\u0026thinsp;=\u0026thinsp;0.067; Chao1: p\u0026thinsp;=\u0026thinsp;0.091). Shannon diversity also differed significantly among species (χ\u0026sup2; = 10.64, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.014), with \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eP. strigatus\u003c/em\u003e showing similarly higher diversity than \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeta diversity analyses (Bray\u0026ndash;Curtis, weighted and unweighted UniFrac) showed clear host-range clustering and significant differences in bacterial community composition among stinkbug species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F). PERMANOVA tests (999 permutations; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) confirmed that bacterial communities differed significantly among all bug species, as well as between species within Pentatomidae and Tessaratomidae (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Significant differences were also detected between the two families and between host-range groups (broad vs narrow host plant specificity) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that both host phylogeny and ecological traits contribute to gut bacterial community structure.\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\u003eSummary of PERMANOVA results evaluating pairwise differences in beta diversity of gut bacterial (based on 16S rRNA gene sequence data) and fungal (based on ITS sequence data) communities among stink bug species using weighted UniFrac, Bray\u0026ndash;Curtis, and unweighted UniFrac distance metrics. Comparisons include: (i) among all four stink bug species; (ii) between pentatomid species (\u003cem\u003eBiprorulus bibax\u003c/em\u003e and \u003cem\u003ePoecilometis strigatus\u003c/em\u003e); (iii) between tessaratomid species (\u003cem\u003eLyramorpha rosea\u003c/em\u003e and \u003cem\u003eMusgraveia sulciventris\u003c/em\u003e); (iv) between families (Pentatomidae vs Tessaratomidae); and (v) between host range groups (narrow host range: \u003cem\u003eM. sulciventris\u003c/em\u003e \u0026amp; \u003cem\u003eB. bibax\u003c/em\u003e vs broad host range: \u003cem\u003eL. rosea\u003c/em\u003e \u0026amp; \u003cem\u003eP. strigatus\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePERMANOVA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eWeighted UniFrac\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eBray\u0026ndash;Curtis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eUnweighted UniFrac\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003e16S rRNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll stinkbug species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e6.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween species in Pentatomidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween species in Tessaratomidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e29.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween families\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween groups with different host range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eITS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll stinkbug species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt; 0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween species in Pentatomidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.013\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween species in Tessaratomidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.013\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween families\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.042\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026nbsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetween groups with different host range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt; 0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePhylogenetic placement of the dominant bacterial symbionts and their cophylogeny with hosts\u003c/h3\u003e\n\u003cp\u003eAll symbionts formed distinct lineages within Gammaproteobacteria but occupied different positions within the broader \u003cem\u003ePantoea\u003c/em\u003e\u0026ndash;\u003cem\u003eErwinia\u003c/em\u003e symbiont complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The \u003cem\u003eB. bibax\u003c/em\u003e symbiont clustered closely with \u003cem\u003ePantoea septica\u003c/em\u003e (98.7% identity) and \u003cem\u003eErwinia amylovora\u003c/em\u003e (98.2%). The \u003cem\u003eP. strigatus\u003c/em\u003e symbiont formed a more divergent lineage, branching separately and forming a sister lineage to symbionts of \u003cem\u003eEurydema\u003c/em\u003e species (97.3%) and \u003cem\u003eNiphe elongata\u003c/em\u003e (97.1%) (Pentatomidae). The \u003cem\u003eM. sulciventris\u003c/em\u003e symbiont clustered more basally relative to the \u003cem\u003eB. bibax\u003c/em\u003e symbiont and grouped within an ancestral clade including the symbiont of \u003cem\u003eNezara viridula\u003c/em\u003e (Pentatomidae). The \u003cem\u003eL. rosea\u003c/em\u003e symbiont grouped within a moderately supported clade of pentatomoid-associated symbionts including \u003cem\u003eGraphosoma lineatum\u003c/em\u003e (97.9%) and \u003cem\u003eErthesina fullo\u003c/em\u003e (97.6%) (Pentatomidae) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cophylogenetic analyses using host mitochondrial COI and symbiont 16S rRNA gene sequences showed no significant congruence between host and symbiont lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Multiple mismatched branching patterns suggested largely independent evolutionary histories. The I\u0026thinsp;\u0026lt;\u0026thinsp;sub\u0026gt;cong\u0026lt;/sub\u0026gt; congruence index confirmed that the topologies were not more similar than expected by chance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.85), which may indicate host switching and/or symbiont replacement during diversification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGut fungal community composition across stinkbug species\u003c/h2\u003e \u003cp\u003eGut fungal composition showed high within-species variability, yet each stink bug species displayed a distinct overall fungal community profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiprorulus bibax\u003c/b\u003e. The fungal community showed consistently low diversity but substantial individual variation, typically dominated by an unclassified Ascomycota (~\u0026thinsp;70\u0026ndash;90%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In SC1, SC2 and SC5 its proportion dropped below 30%, with Saccharomycetes becoming more prominent, while SCF10 was dominated by Tremellomycetes (~\u0026thinsp;95%), representing a clear outlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the OTU level, core members included an unassigned Ascomycota (OTU127) and \u003cem\u003eEremothecium\u003c/em\u003e spp. (OTU12, OTU13), together contributing up to ~\u0026thinsp;70% across individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Other taxa (e.g., \u003cem\u003eNaganishia\u003c/em\u003e sp. OTU5, dominant in SCB10\u0026thinsp;\u0026gt;\u0026thinsp;80%; \u003cem\u003eMalassezia\u003c/em\u003e sp. OTU3; unassigned Chytridiomycota OTU106) were sporadic and did not form a consistent core (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePoecilometis strigatus.\u003c/b\u003e The fungal community was primarily dominated by Dothideomycetes (10\u0026ndash;95%), with Sordariomycetes contributing substantially in some individuals (up to 40%). Other fungal classes (e.g., Cystobasidiomycetes, Tremellomycetes, other Ascomycota) occurred sporadically and at lower abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the OTU level, \u003cem\u003eCladosporium\u003c/em\u003e sp. (OTU7) was consistently present (10\u0026ndash;95%) and appeared as a dominant and potential core member, while \u003cem\u003eAureobasidium\u003c/em\u003e sp. (OTU10) reached up to 40% in some individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additional taxa (e.g., \u003cem\u003eDiaporthe\u003c/em\u003e sp. OTU17, \u003cem\u003eParaconiothyrium\u003c/em\u003e sp. (II) OTU37, \u003cem\u003eMeyerozyma\u003c/em\u003e sp. OTU25) occurred at low abundance in subsets of samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyramorpha rosea.\u003c/b\u003e The fungal community varied across individuals but were typically dominated by Dothideomycetes and an unclassified Ascomycota, together comprising\u0026thinsp;~\u0026thinsp;20% to \u0026gt;\u0026thinsp;90% of abundance. Saccharomycetes also occurred at moderate to high levels in approximately half of individuals (~\u0026thinsp;20% to \u0026gt;\u0026thinsp;70%). Other classes (e.g., Sordariomycetes, Cystobasidiomycetes, Tremellomycetes, Eurotiomycetes) were sporadic and generally low abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the OTU level, \u003cem\u003eEremothecium\u003c/em\u003e sp. (I) (OTU2) dominated (up to ~\u0026thinsp;90%), with \u003cem\u003eParaconiothyrium\u003c/em\u003e sp. (I) (OTU26; ~4\u0026ndash;48%) and additional taxa (e.g., unassigned Mycosphaerellaceae OTU23\u0026thinsp;~\u0026thinsp;9%; Malasseziaceae OTU14\u0026thinsp;~\u0026thinsp;2.6%) detected in some individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMusgraveia sulciventris.\u003c/b\u003e The gut fungal community varied among individuals but was typically dominated by Malasseziomycetes, with highly variable relative abundance (~\u0026thinsp;10\u0026ndash;80%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Sordariomycetes (0\u0026ndash;50%) and unclassified Ascomycota (0\u0026ndash;40%) also contributed substantially across samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Other classes (e.g., Exobasidiomycetes, Tremellomycetes, Dothideomycetes) occurred sporadically and generally at low to moderate abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the OTU level, \u003cem\u003eMalassezia\u003c/em\u003e sp. (I) (OTU3) was detected in all individuals, while \u003cem\u003eMalassezia\u003c/em\u003e sp. (II) (OTU35) occurred only in BOB3 and BOB4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additional taxa, including \u003cem\u003eHyphopichia\u003c/em\u003e sp. (OTU90) and \u003cem\u003eCladosporium\u003c/em\u003e sp. (OTU7), were present at low abundance in some individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGut fungal community diversity and structure across host species\u003c/h2\u003e \u003cp\u003eAlpha diversity analyses showed no significant differences in fungal community richness or diversity among the four stink bug species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C): observed species (χ\u0026sup2; = 4.26, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.235), Chao1 richness (χ\u0026sup2; = 3.84, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.279), and Shannon diversity (χ\u0026sup2; = 5.19, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.158). Nevertheless, within-species variation was substantial, with \u003cem\u003eM. sulciventris\u003c/em\u003e showing the lowest variability (observed species\u0026thinsp;=\u0026thinsp;10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3; Chao1\u0026thinsp;=\u0026thinsp;13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8; Shannon\u0026thinsp;=\u0026thinsp;1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4), indicating a consistently depauperate and uniform fungal community, whereas \u003cem\u003eP. strigatus\u003c/em\u003e showed the highest variability in Chao1 and Shannon (Chao1\u0026thinsp;=\u0026thinsp;30.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2; Shannon\u0026thinsp;=\u0026thinsp;3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeta diversity analyses (Bray\u0026ndash;Curtis, weighted and unweighted UniFrac) revealed significant differences in fungal community composition among host species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;F; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PERMANOVA tests (999 permutations) supported overall host-associated structuring. Fungal assemblages differed more strongly among Tessaratomidae species (F\u0026thinsp;=\u0026thinsp;2.97\u0026ndash;6.01, p\u0026thinsp;\u0026le;\u0026thinsp;0.013) than among Pentatomidae species (F\u0026thinsp;=\u0026thinsp;2.35\u0026ndash;3.57, p\u0026thinsp;=\u0026thinsp;0.013\u0026ndash;0.069), while between-family and host-range comparisons were also significant for most metrics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Together, these results indicate that gut fungal communities vary with host taxonomy and ecological traits, with stronger divergence within Tessaratomidae and among species differing in plant host-range breadth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGut symbiont elimination negatively impacted\u003c/b\u003e \u003cb\u003eB. bibax\u003c/b\u003e \u003cb\u003efitness\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEgg-surface sterilization resulted in the elimination of the \u003cem\u003ePantoea-like\u003c/em\u003e gut symbiont from \u003cem\u003eB. bibax\u003c/em\u003e eggs, thereby markedly affecting the biological performance of the offspring. Aposymbiotic individuals exhibited a markedly prolonged egg-to-adult development time compared with the control group (\u003cem\u003eχ\u0026sup2;\u003c/em\u003e₍₁,₄₂₎ = 19.99, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), taking on average 51.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 days versus 43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 days in controls. Adult longevity was also substantially reduced in aposymbiotic insects (\u003cem\u003eχ\u0026sup2;\u003c/em\u003e₍₁,₄₂₎ = 16.72, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), averaging 29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 days compared with 48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 days in symbiotic controls. Likewise, total fecundity (number of eggs per female during life time) was significantly lower in aposymbiotic females (\u003cem\u003eχ\u0026sup2;\u003c/em\u003e₍₁,₂₁₎ = 13.20, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), with a mean of 44.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 eggs compared with 77.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 eggs in controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cb\u003eLow-diversity gut bacterial communities dominated by host-specific\u003c/b\u003e \u003cb\u003ePantoea\u003c/b\u003e\u003cb\u003e-like symbionts in four Australian stink bug species\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur study provides the first comprehensive characterization of gut microbial communities in four Australian native stinkbug species, \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eP. strigatus\u003c/em\u003e (Pentatomidae), and \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e (Tessaratomidae) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). Across all species, M4 gut bacterial diversity was consistently low and dominated by Gammaproteobacteria, driven in each host by a single host-specific OTU affiliated with the \u003cem\u003ePantoea\u003c/em\u003e\u0026ndash;\u003cem\u003eErwinia\u003c/em\u003e complex (Enterobacteriaceae), as is typical of pentatomoid midgut-crypt symbioses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Communities were most streamlined in the citrus specialists \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e, where the dominant \u003cem\u003ePantoea\u003c/em\u003e-like OTUs (OTU2 and OTU5, respectively) comprised\u0026thinsp;\u0026gt;\u0026thinsp;90% of reads. In contrast, \u003cem\u003eL. rosea\u003c/em\u003e showed greater inter-individual variation and measurable contributions from non-proteobacterial classes (Actinomycetes and Bacilli), including \u003cem\u003eFrankia\u003c/em\u003e and sporadic low-abundance genera (e.g., \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eWeissella\u003c/em\u003e, \u003cem\u003eKosakonia\u003c/em\u003e, \u003cem\u003eCommensalibacter\u003c/em\u003e and \u003cem\u003eErwinia\u003c/em\u003e). Similarly, \u003cem\u003eP. strigatus\u003c/em\u003e consistently harboured a second, unassigned core gammaproteobacterial OTU (OTU12) at moderate abundance alongside the dominant Pantoea-like OTU and other low-abundance taxa (e.g., \u003cem\u003eCommensalibacter\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThese compositional patterns are reflected in alpha diversity metrics: \u003cem\u003eL. rosea\u003c/em\u003e showed significantly higher richness (observed species and Chao1) than \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e, and Shannon diversity was higher in \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eP. strigatus\u003c/em\u003e, indicating more even communities. Beta diversity ordinations separated the four species into distinct clusters, and PERMANOVA confirmed significant differences among species, between families, and between host-range groups (narrow \u003cem\u003evs\u003c/em\u003e broad). Together, this suggests that while a conserved \u003cem\u003ePantoea\u003c/em\u003e-\u003cem\u003eErwinia\u003c/em\u003e complex symbiosis underpins community structure across these pentatomoid bugs, variation in richness and composition is further shaped by host ecology via secondary (environmental) bacteria. This aligns with the broader host plant ranges of \u003cem\u003eP. strigatus\u003c/em\u003e (Rutaceae, Myrtaceae, Proteaceae, Rosaceae, Oleaceae) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and \u003cem\u003eL. rosea\u003c/em\u003e (Sapindaceae and Rutaceae) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], compared with the more citrus-restricted \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e (James, 1989; Cant et al., 1996). Broader host use likely increases exposure to diverse microenvironments, diets, and plant compounds (including antimicrobials), and may increase opportunities for horizontal acquisition, collectively promoting higher secondary microbial diversity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic placement of gut bacterial symbionts and host\u0026ndash;symbiont coevolution\u003c/h2\u003e \u003cp\u003eBacterial symbionts of pentatomoids are phylogenetically grouped with plant-associated \u003cem\u003eErwinia\u003c/em\u003e and \u003cem\u003ePantoea\u003c/em\u003e bacteria [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], although without clear patterns of host symbiont co-evolution. One hypothesis for these patterns is that, over evolutionary times, the symbionts may be replaced with taxonomically similar bacteria through host switches and that different bug species may have different levels of dependency on this symbiotic relationship. Our phylogenetic analysis of near full-length 16S rRNA gene clearly show that the dominant gut symbionts of all four Australian stink bug species, despite belonging to four stink bug genera of two families (Pentatomidae \u003cem\u003evs\u003c/em\u003e Tessaratomidae), fall within the same broad gammaproteobacterial clade of the \u003cem\u003ePantoea\u003c/em\u003e\u0026ndash;\u003cem\u003eErwinia\u003c/em\u003e complex. More specifically, the gut symbiont of \u003cem\u003eB. bibax\u003c/em\u003e clustered closely with \u003cem\u003ePantoea septica\u003c/em\u003e and \u003cem\u003eErwinia amylovora\u003c/em\u003e, whereas the symbiont of \u003cem\u003eP. strigatus\u003c/em\u003e formed a more divergent lineage and grouped as sister to symbionts reported from the pentatomoids \u003cem\u003eEurydema\u003c/em\u003e spp. and \u003cem\u003eNiphe elongata\u003c/em\u003e. The \u003cem\u003eL. rosea\u003c/em\u003e symbiont fell within a clade of pentatomoid-associated symbionts including those from \u003cem\u003eGraphosoma lineatum\u003c/em\u003e and \u003cem\u003eErthesina fullo\u003c/em\u003e, and the \u003cem\u003eM. sulciventris\u003c/em\u003e symbiont clustered more basally, near to the symbiont of \u003cem\u003eNezara viridula\u003c/em\u003e (Pentatomidae). This supports the idea that pentatomoid stink bugs generally share a conserved bacterial symbiosis type, in which an Enterobacteriaceae lineage dominates the crypt-bearing posterior midgut, but have diversified into host-specific lineages across evolutionary time [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cophylogenetic analysis further supports this interpretation by showing that host and symbiont evolutionary histories are not tightly coupled. The host COI phylogeny and the symbiont 16S rRNA gene phylogeny showed no significant congruence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and the multiple mismatched branching patterns indicate that host\u0026ndash;symbiont associations have not been maintained through long-term strict cospeciation. This fits well with the view that pentatomoid gut symbionts are often monosymbiotic but polyphyletic within Enterobacteriaceae, reflecting evolutionary turnover and/or occasional replacement among closely related lineages that are pre-adapted to life in the stink bug midgut crypt environment [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Such turnover could be facilitated by transmission biology: gut symbionts are extracellular and commonly acquired early in development from egg-associated inocula, which may allow occasional replacement while still maintaining the same functional niche [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGut fungal communities are highly variable and dominated by yeast-like and plant-associated fungi\u003c/h2\u003e \u003cp\u003eAcross the four Australian stink bugs, the gut mycobiome was far more variable than the bacterial community and lacked a single dominant symbiont. This is unsurprising because yeast-like and other plant-associated fungi often form flexible, environmentally driven associations with insects, ranging from transient passengers to ecologically important partners [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Fungal profiles differed strongly among individuals within species, consistent with frequent renewal from diet and plant surfaces rather than strict vertical transmission. Nevertheless, recurrent taxa suggest ecologically meaningful links: \u003cem\u003eEremothecium\u003c/em\u003e OTUs (notably in \u003cem\u003eB. bibax\u003c/em\u003e and \u003cem\u003eL. rosea\u003c/em\u003e) are well known from piercing\u0026ndash;sucking Hemiptera and have been associated with stink bug\u0026ndash;mediated transmission and yeast-spot plant diseases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] Scarpari et al., 2018; Hojjati et al., 2023). Similarly, Dothideomycetes-dominated profiles (e.g., \u003cem\u003eCladosporium\u003c/em\u003e and \u003cem\u003eAureobasidium\u003c/em\u003e in \u003cem\u003eP. strigatus\u003c/em\u003e and \u003cem\u003eL. rosea\u003c/em\u003e) match the common occurrence of these fungi in the phyllosphere and as endophytes that insects likely ingest during feeding [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough fungal alpha diversity did not differ significantly among species, within-species variation was high and beta diversity showed clear host-associated structuring (including stronger divergence within Tessaratomidae and host-range effects). This combination, similar overall richness but different composition, is consistent with a scenario where gut fungi are shaped by host taxonomy and ecology (diet breadth, feeding microhabitat, exposure to plant-associated fungi), but do not form a uniformly conserved \u0026ldquo;core\u0026rdquo; across individuals in the way that midgut-crypt bacteria often do [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Functionally, these patterns motivate targeted follow-up work (e.g., culture-based assays, metagenomics/metatranscriptomics, and controlled diet experiments) to distinguish resident, beneficial yeasts from diet-derived transient fungi and to test whether dominant taxa (e.g., \u003cem\u003eEremothecium\u003c/em\u003e, \u003cem\u003eCladosporium\u003c/em\u003e, \u003cem\u003eAureobasidium\u003c/em\u003e) contribute to nutrition, detoxification or interactions of host plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFitness role of gut symbiont in\u003c/b\u003e \u003cb\u003eB. bibax\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eB. bibax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), newly hatched nymphs remained aggregated around the eggshell for ~\u0026thinsp;2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026ndash;H), repeatedly probing the egg surface, likely to acquire the maternally deposited \u003cem\u003ePantoea\u003c/em\u003e-like symbiont. Egg-surface sterilisation eliminated this symbiont and produced aposymbiotic offspring, confirming vertical transmission via egg smearing\u0026mdash;a common mechanism in stink bugs with midgut-crypt symbionts, where disruption typically reduces host performance [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Consistent with this, aposymbiotic \u003cem\u003eB. bibax\u003c/em\u003e developed more slowly (~\u0026thinsp;8-day delay), had reduced adult longevity (~\u0026thinsp;19 days shorter), and females laid\u0026thinsp;~\u0026thinsp;40% fewer eggs than controls. Similar fitness costs have been reported after symbiont-disruption in other stink bugs, including \u003cem\u003eGraphosoma lineatum\u003c/em\u003e, \u003cem\u003eEurygaster integriceps\u003c/em\u003e, and \u003cem\u003eHalyomorpha halys\u003c/em\u003e (26, 54\u0026ndash;56). These effects are plausibly driven by nutritional constraints in sap-feeding bugs, where symbionts can supplement limiting nutrients; in \u003cem\u003eH. halys\u003c/em\u003e, genomic evidence supports such roles for \u003cem\u003eCandidatus Pantoea carbekii\u003c/em\u003e [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Overall, our results place \u003cem\u003eB. bibax\u003c/em\u003e among pentatomids with strong dependence on extracellular gut symbionts, consistent with variation in symbiont reliance across stink bug lineages [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Future work should test fitness effects in the other hosts, resolve symbiont function using genome- and/or metabolite-based approaches, and determine transmission routes by tracking symbionts across life stages. In several Tessaratomidae species, females guard egg masses and first-instar nymphs, and the newly hatched nymphs are extremely flattened and reportedly non-feeding at the first instar [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It will therefore be important to test whether maternal care behaviours facilitate vertical transmission of gut symbionts (e.g., by maintaining close contact with eggs/nymphs surfaces or maternal secretions) in \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eM. sulciventris\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study shows that four Australian native stink bugs harbour low-diversity gut bacterial communities dominated by host-specific \u003cem\u003ePantoea\u003c/em\u003e-\u003cem\u003eErwinia\u003c/em\u003e complex symbionts. Phylogenetic and cophylogenetic analyses revealed host-specific lineages but no strict host\u0026ndash;symbiont cospeciation, consistent with symbiont turnover over evolutionary time. In contrast, secondary gut bacterial taxa and gut fungi were more variable, with richness and composition differing among species in ways broadly aligned with host ecology, particularly host plant range. In \u003cem\u003eB. bibax\u003c/em\u003e, disrupting maternal transmission produced aposymbiotic offspring with reduced fitness, demonstrating functional importance of the dominant bacterial symbiont. Broader sampling across regions, seasons, host plants and life stages will be needed to test robustness and move beyond amplicon snapshots toward a more complete understanding of gut microbiome dynamics in Australian true bugs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupplementary Information\u003c/h2\u003e \u003cp\u003eThe supplementary material (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) attached to this submission.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project was conducted as part of the Australian Postgraduate Research Intern (APR.Intern) program (Project Reference: INT-1050), in partnership with Agriculture Victoria Research (industry partner), with Western Sydney University as the student institution. The project was also supported by the 2021 Western Sydney University World Microbiome Day Student Grant, awarded to Alihan Katlav.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAK, MR and PT conceived the study. AK and AT performed the experiments; AK, JW and JLM conducted data analysis; AK conducted field sampling, the insect material preparations and directions; AK, JW and JM prepared figures; MR and PT supervised the work; AK, MR and JM wrote main manuscript text with input from all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgements We acknowledge the APR.Intern team for their support of this project, with special thanks to Zak Blayney (APR.Intern CRM \u0026amp; Project Officer), Justin Mabbutt (APR.Intern Business Development Manager, Victoria \u0026amp; Tasmania), and James Krahe from Food Innovation Australia Ltd (FIAL). We also thank Dr Eleonora Egidi for arranging the Western Sydney University World Microbiome Day Award, Carl Ramirez for assisting with insect collections, and all members of the Microbial Ecology and Environmental Sustainability group at the Hawkesbury Institute for the Environment (HIE), Western Sydney University, for their support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe 16S rRNA gene and ITS amplicon sequencing datasets generated in this study are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1420412. Associated metadata and raw data from the symbiont elimination experiment (fitness parameters of *Biprorulus bibax* ) are available via Figshare at: [https://doi.org/10.6084/m9.figshare.31341679](https:/doi.org/10.6084/m9.figshare.31341679)Please note : Cytochrome oxidase I (COI) Sanger sequences and bacterial 16S rRNA gene sequences (V1\u0026ndash;V9) of the symbiotic bacteria have been submitted to the NCBI GenBank database and are currently being processed; accession numbers are pending and will be added to a revised version, should the manuscript proceed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCornwallis CK, van\u0026rsquo;t Padje A, Ellers J, Klein M, Jackson R, Kiers ET, West SA, Henry LM (2023) Symbioses shape feeding niches and diversification across insects. 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Zool Lett 2:24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan H, Wu W, Sun Z, Chen J, Li H (2021) The gut microbiota of the insect infraorder Pentatomomorpha (Hemiptera: Heteroptera) in the light of ecology and evolution. Microorganisms 9:464\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStefanini I (2018) Yeast\u0026ndash;insect associations: it takes guts. Yeast 35:315\u0026ndash;330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalassign\u0026eacute; S, Minard G, Vallon L, Martin E, Valiente Moro C, Luis P (2021) Diversity and functions of yeast communities associated with insects. Microorganisms 9:1552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimura S, Tokumaru S, Kuge K (2008) \u003cem\u003eEremothecium ashbyi\u003c/em\u003e causes soybean yeast-spot and is associated with stink bug, \u003cem\u003eRiptortus clavatus\u003c/em\u003e. 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J Insect Sci 13:99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor CM, Coffey PL, DeLay BD, Dively GP (2014) The importance of gut symbionts in the development of the brown marmorated stink bug, \u003cem\u003eHalyomorpha halys\u003c/em\u003e. PLoS ONE 9:e90312\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenyon LJ, Meulia T, Sabree ZL (2015) Habitat visualization and genomic analysis of \u003cem\u003eCandidatus Pantoea carbekii\u003c/em\u003e, the primary symbiont of the brown marmorated stink bug. Genome Biol Evol 7:620\u0026ndash;635\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pantoea, Gammaproteobacteria, fungal microbiome, spined citrus bug, gumtree shield bug, lychee stink bug, bronze orange bug","lastPublishedDoi":"10.21203/rs.3.rs-8890409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8890409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGut microbiomes are fundamental to animal biology, evolution and health. True bugs (Hemiptera) maintain heritable microbial gut symbionts and are research models for host-microbe interactions. However, current knowledge is mostly limited to the bacterial symbionts in the Holarctic-Oriental bug fauna, leaving the microbial symbioses of the Australasian diversity unknown. Using high-throughput amplicon sequencing, we characterized the bacterial and fungal communities of four Australian stink bug species associated with citrus: \u003cem\u003eBiprorulus bibax\u003c/em\u003e, \u003cem\u003ePoecilometis strigatus\u003c/em\u003e (Pentatomidae), \u003cem\u003eLyramorpha rosea\u003c/em\u003e and \u003cem\u003eMusgraveia sulciventris\u003c/em\u003e (Tessaratomidae). Across all species, bacterial communities were low in diversity, with each species harbouring a dominant and distinct gammaproteobacterial symbiont within the \u003cem\u003ePantoea-Erwinia\u003c/em\u003e complex. However, \u003cem\u003eL. rosea\u003c/em\u003e and \u003cem\u003eP. strigatus\u003c/em\u003e contained more diverse assemblages including low-abundance secondary taxa. Furthermore, each host species harboured a differentiated fungal consortium that was diverse across hosts and dominated by taxa including \u003cem\u003eCladosporium\u003c/em\u003e, \u003cem\u003eEremothecium\u003c/em\u003e and \u003cem\u003eMalassezia\u003c/em\u003e. Although the dominant bacterial symbionts were host-specific, their phylogeny was incongruent with the host phylogeny, probably indicating host switches and decoupled host-symbiont evolutionary histories. We also found evidence that in \u003cem\u003eB. bibax\u003c/em\u003e, the \u003cem\u003ePantoea\u003c/em\u003e-like symbiont was vertically transmitted via egg smearing. Egg surface sterilisation resulted in aposymbiotic offspring with delayed development, reduced longevity and lower fecundity, demonstrating a symbiont contribution to host fitness. Overall, our findings of the first comparative gut microbiome analysis of Australian stink bugs support globally conserved association patterns with \u003cem\u003ePantoea\u003c/em\u003e-like symbionts alongside species-specific microbial community structure and advanced the understanding of host-microbe evolution in these insects.\u003c/p\u003e","manuscriptTitle":"Gut microbiomes of four Australian stink bug species associated with citrus, and fitness effects of a dominant Pantoea-like symbiont in Biprorulus bibax","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 07:56:57","doi":"10.21203/rs.3.rs-8890409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T16:32:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T07:05:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T05:23:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T06:15:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78777580239588671120433155632110325386","date":"2026-02-23T04:58:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106323966227942596815662639729207949399","date":"2026-02-20T09:57:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283923348871327542782531940521756855471","date":"2026-02-20T04:38:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T16:12:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T04:45:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-17T04:43:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2026-02-16T06:44:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"65de04b4-7429-4014-9b17-c1273d347b6f","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T18:53:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 07:56:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8890409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8890409","identity":"rs-8890409","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}