Dominant Egg Surface Bacteria of Holotrichia Oblita (Coleoptera: Scarabaeidae) Inhibit The Multiplication of Bacillus Thuringiensis and Beauveria Bassiana

Dominant Egg Surface Bacteria of Holotrichia Oblita (Coleoptera: Scarabaeidae) Inhibit The Multiplication of Bacillus Thuringiensis and Beauveria Bassiana | 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 Dominant Egg Surface Bacteria of Holotrichia Oblita (Coleoptera: Scarabaeidae) Inhibit The Multiplication of Bacillus Thuringiensis and Beauveria Bassiana Kui Wang, Qi Liu, Chunqin Liu, Lili Geng, Guirong Wang, Jie Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-145611/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Many species of scarabs (Coleoptera: Scarabaeidae) are important pests. Bacillus thuringiensis (Bt) and Beauveria bassiana (Bb) are entomopathogens that have been used as biocontrol agents of various pests. However, neither provides adequate control of scarab pests in the field. In this study, we used Holotrichia oblita , an important scarab pest in China, as a model to explore the reason of difficult to control. Compared to other scarabs, H. oblita could form a unique egg cocoon structure, in which the young larvae complete the initial development process. We analyzed the microbiome of eggs surface, egg cocoon and bulk soil respectively, and investigated the role of these microorganisms in pathogen infection. 16S rRNA gene analysis revealed low bacterial richness in egg surface with Proteobacteria , Firmicutes , Bacteroidetes and Fusobacteria dominating in terms of OTUs number, and indicated that the microbial community structure varied among egg and soil samples. Some dominant species of the egg surface have been reported to exhibit antimicrobial activity, which may contribute to the low susceptibility of young larvae to entomopathogens. The further confrontation culture analysis verified this hypothesis. We found that 11 of 28 cultivable isolates possessed antimicrobial activity against both Bt and Bb, and all isolates from egg surface samples showed antagonistic ability. Four isolates belonging to Pseudomonas showed strong Bt- and weak Bb-antagonistic ability, and this genus significantly increased in egg surface samples. This study demonstrated that H. oblita egg has a unique microbial community structure, containing microorganisms with antimicrobial activity against entomopathogens. These microorganisms protect the newly hatched larvae from pathogen infection. General Microbiology pathogen infection entomopathogens Pseudomonas Firmicutes Bacteroidetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Many species of Scarabaeidae (Coleoptera) are plant pests that cause significant economic losses in agriculture, horticulture, and forestry 1 , 2 . The adults typically feed on aerial plant structures, while the larvae, known as white grubs, feed on plant roots. Some root-feeding species are major pests. Application of chemical insecticides is the main method used to control grubs. However, the extensive use of chemicals poses a threat to the environment and public health. Environmentally friendly microbial agents that could control scarabs would be a safer alternative. Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces one or more insecticidal crystal proteins (Cry, Cyt) or vegetative insecticidal proteins (Vip), which exhibit specific toxicity against many insects, especially Lepidoptera and Coleoptera 3 , 4 . Following the ingestion of the endotoxin crystals by susceptible insects, the crystals are solubilized by the alkaline conditions in the midgut and are subsequently proteolytically converted into a toxic core fragment. The activated toxin binds to midgut receptors and forms ionic pores that kill midgut cells, or induces a cell death signaling pathway, finally leading to insect death 5 , 6 . Different Bt strains, such as HBF-1, HBF-18, Bt185, and B-JJX, have insecticidal activity against scarab pests 7-10 . Fungi are also used as biological insecticides for the control of insect pests. Compared to bacteria and viruses, fungi can directly penetrate through the insect cuticle and multiply in the hemocoel. This makes them useful biological control agents of white grubs 11 , 12 . Beauveria bassiana (Bb) is the most widely distributed entomopathogenic fungus in the world. It infects a variety of insect species and causes white muscardine disease. Several Bb-based products are commercially available for the control of the coffee borer beetle ( Hypothenemus hampei ) and various species of Curculionidae 13-15 . Both Bt and Bb are effective against scarab larvae in lab experiments, but, under field conditions, they are often not as effective. In China, Holotrichia oblita , H. parallela , and Anomala corpulenta are the most important grub pest species. They damage many plants, such as sweet potatoes, soybeans, peanuts, and the Siberian elm 16 . Exposed to the same Bt or Bb agents, H. oblita was less susceptible than H. parallela or A. corpulenta 3 , 9 , 10 , 17 . H. oblita was able to survive using different defense mechanisms. Our previous study indicated that the larval gut bacteria may potentially affect the susceptibility of scarabs to Bt strains. The proportion of gut isolates with antibacterial activity against Bt strains was 90% in H. oblita , 75% in H. parallela and 80% in A. corpulenta 18 . Scarab adults usually oviposit in the soil near their preferred host plants. Compared to H. parallela and A. corpulenta , H. oblita forms a unique egg cocoon structure by mixing its eggs with the surrounding soil. There is a variety of microbes in the soil, some of which can produce compounds adversely affecting the growth of pathogens 19-21 . Biocontrol agents generally target young larvae; such as 3 to 5-day-old emerging larvae in lab experiments 9 , 10 , however, under natural conditions, the grubs can often resist pathogen invasion and hatch normally. This unique egg cocoon structure may contribute to the relatively low susceptibility of young larvae to entomopathogens. To study the role of egg bacteria in pathogen infection, we collected 20 samples from the H. oblita egg surface (E), egg cocoon (C), and bulk soil (B), and analyzed the microbial community structure using 16S rRNA gene sequencing. We tested the antimicrobial potential of 28 cultivable isolates against scarab-specific Bt and Bb strains using confrontation culture analysis. The results suggest that dominant egg surface bacteria can inhibit the multiplication of Bt and Bb strains. Methods Sampling and DNA extraction . H. oblita was collected from a field in Cangzhou, Hebei Province, China. The adults were reared in plastic boxes (66 cm by 41 cm by 18 cm) filled with soil containing willow leaves at a temperature of 25℃ until they laid eggs. Egg cocoons were collected and peeled off, then the eggs were transferred to a sterile 2 ml plastic centrifuge tube containing 1 ml sterile water and sonicated for 5 min in an ultrasonic cleaning bath (Bransonic 32) to dislodge bacteria. After centrifugation at 10,000×g for 5 min, the microorganisms in the wash buffer were collected and defined as the egg surface (E) sample, and resuspended with 1 ml sterile water. The egg cocoon soils (1 g) and soils (1 g) 10 cm away from the egg cocoons were suspended in 5 ml sterile water and centrifuged at 10,000×g for 5 min, and the pellets were defined as the egg cocoon (C) sample and the bulk soil (B) sample (Figure 7) and resuspended with 1 ml sterile water. A total of 20 samples were collected, including 6 E samples, 7 C samples, and 7 B samples. For egg surface (E) samples, 1 μl of the microorganism suspension was directly used as a template for PCR. For soil samples (B and C), a 900 μl suspension was used to extract genomic DNA, using a PowerSoil® DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA), and 1 μl of DNA was used as a template for PCR amplification. The remaining 100 μl suspension of 20 samples was kept for conventional culture using solid Luria Bertani (LB) agar medium, and single colonies were picked from the plates and repeatedly grown on solid agar plates until pure cultures were obtained. A total of 28 cultivable isolates were collected, including 3 isolates from E samples, 7 isolates from C samples, and 18 isolates from B samples. Genomic DNA of each isolate was extracted as previously described 49 . 16S rRNA gene sequencing and bioinformatic analysis. The V3-V4 region of microbial 16S rRNA genes of 20 samples were amplified by PCR using the specific primers, 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-'3). PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). The TruSeq ® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) was used for 16S rRNA gene amplicon library construction. The Qubit @ 2.0 Fluorometer (Thermo Scientific, Rockford, IL, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA) were used for library quality assessment. Finally, the library was sequenced on the Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA), and 250 bp paired-end reads were generated. Raw Data were trimmed using Trimmomatic (version 0.36) 50 with default parameters. Then the clean paired-end reads were assembled into raw tags using Usearch (version 9.2.64) 51 . The primer sequences in the raw tags were trimmed, and effective tags were obtained. The 16S rRNA sequence data of 20 samples were deposited in Sequence Read Archive (SRA) database under BioProject ID PRJNA637400, with accession number SRR11931252–SRR11931271. The operational taxonomic units (OTUs) were clustered at 97% identity cutoff with a Usearch UPARSE algorithm 52 . Then the chimera sequences were removed on the basis of the UPARSE pipeline analysis. The OTU annotation was performed using the Usearch SINTAX algorithm 53 , against RDP training set (version 16) 16S rRNA Database with a confidence threshold of 0.8. OTUs annotated as chloroplast or mitochondria or OTUs not annotated to the kingdom level were abandoned. QIIME (version 1.7.0) pyNAST algorithm 54 was used for species annotation against the GreenGene Database 55 . Usearch (version 9.2.64) 51 was used to calculate Alpha diversity metrics, including the indexes (shannon, chao1) reflecting the sample community richness, and indexes (simpson, dominance, and equitability) reflecting the sample community evenness. QIIME (version 1.7.0) 56 was used to calculate Beta diversity to estimate variation between samples. Principal Component Analysis (PCA) and Non-Metric Multi-Dimensional Scaling (NMDS) analysis were performed using R package (https://www.r-project.org/) to visualize complex relationships between samples. Lda Effective Size (LEfSe) test for variability of microbiota was calculated using lefse (Version 1.0.7) (http://huttenhower.sph.harvard.edu/galaxy/). For 28 cultivable isolates, the 16S rRNA genes of each isolate were amplified using the specific primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-'3). Then the 16S rRNA sequences were identified through aligning against NCBI 16S rRNA sequence (Bacteria and Archaea) database with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Confrontation culture analysis. Four entomopathogen strains were used: 3 scarab-specific Bt strains (HBF-1, HBF-18, Bt185) 7-9 , and one Bb strain BBNS-J9-16 (preservation number: CGMCC No.5288). The dual culture tests for antagonistic ability of 28 cultivable isolates against Bt strains were processed using the cup-plate confrontation culture method, as previously described 18 . Sterile water was used as a negative control. The observable inhibition zones were used as indicators of the antibacterial activity of the 28 isolates against Bt strains. For the antagonistic ability analysis of the 28 isolates against Bb strain, the isolates were cultured at 30℃ with shaking at 220 rpm. The Bb strain was cultured on PDA for 2–4 d. Subsequently, fungal culture plugs were placed in the middle of LB agar plates. Five dishes of sterile blotter paper (6 mm diam.) were placed on the surface of the plate and inoculated with 10 μl of the cultured bacterial suspension. The amphotericin-B and sterile water were used as positive and negative controls, respectively. The plates were incubated at 28℃ for 5 d, and inhibition zones induced by 28 isolates against Bb strain were recorded. Genome sequencing and secondary metabolite analysis. Four cultivable isolates with strong antagonistic ability against Bt strains and weak antagonistic ability against Bb strain were selected for draft genome sequencing, using the Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). The produced reads were cleaned by removing reads with Ns or more than 20% low-quality bases, and 1 Gb 2×100 bp pair-end clean reads for each isolate were obtained. The SPAdes 57 was used for genome assembly, and GeneMark 58 was used for gene prediction. The antiSMASH 2.0 pipeline 59 was used for secondary metabolite analysis of these four isolates. Phylogenetic analysis. All the 16S rRNA sequences of 28 cultivable isolates were analyzed using MEGA (version 7) 60 and an online tool iTOL: Interactive Tree of Life (http://itol.embl.de/) 61 . The analysis included bootstrapping values with 1000 replications. For the phylogenetic analysis of four genome sequenced isolates, we constructed the whole-genome-based tree using CVTree 26 with k-string = 6, and PHYLIP 62 . The iTOL was used to annotate the tree 61 . Results Microbial diversity comparisons between egg and soil samples. The bacterial composition of different samples from H. oblita egg surface (E), egg cocoon (C), and bulk soil (B) was determined by sequencing analysis of the 16S rRNA gene. A total of 2,748,824 raw reads were generated from 20 samples, including 667,427 raw reads from six E samples, 924,724 raw reads from seven C samples, and 1,156,673 raw reads from seven B samples. After removing the short reads and trimming the low-quality regions, a total of 1,894,784 effective tags were identified, with an average length of 415 bp. All the effective sequences were clustered into 49,503 OTUs at 97% DNA sequence similarity, including 1,863 OTUs from E samples, 22,827 OTUs from C samples, and 24,813 OTUs from B samples (Tables S1 and S2). Alpha diversity analysis was then performed to assess the diversity and evenness of the microbial population from different samples. The alpha diversity patterns were variable across the soil samples (B and C) and egg surface samples (E) (Figure 1). The number of observed OTUs and alpha diversity analysis based on Shannon and Chao1 indexes indicated that soil samples had more microbial diversity than the egg surface samples (Figure 1A and Table S2). A previous study in our laboratory showed that the microorganism collection method could affect the community structure, where the phyllosphere community diversity was lower for samples subjected to DNA extraction than for those subjected to direct PCR 22 . In the present study, we performed direct PCR for E samples and added a DNA extraction process before PCR for B and C samples. The results confirmed that the community diversity of soil samples was much higher than the egg surface samples. The results of Simpson, dominance, and equitability indexes indicated that, compared to the B and C samples, the evenness of sample E decreased (Figure 1B and Table S2). The rarefaction curve based on the Shannon index showed that all samples reached a plateau, suggesting that our sampling effort was sufficient to obtain a full estimate of OTU richness (Figure S1). Among all samples, 26 phyla, 143 families, and 300 genera were identified. Proteobacteria was the dominant phylum and comprised the majority of all detected microorganisms (approximately 44.63%) (Figure 2A). This is typically observed in other soil libraries 23-25 . Actinobacteria , Acidobacteria , and Bacteroidetes were also abundant in egg cocoon (C) samples and bulk soil (B) samples. In E samples, Firmicutes , Bacteroidetes , and Fusobacteria were the most abundant phyla (Figure 2A). The community structure varied markedly among different samples, outlined by the Lefse LDA results (Figure 3A). Compared with bulk soil samples, the composition of Firmicutes , Bacteroidetes , and Fusobacteria and the composition of Actinobacteria and Acidobacteria significantly increased in egg surface samples and egg cocoon samples, respectively (Figure 3A). Bray-Curtis tree and PCA analysis also indicated that microbiota in different samples were clearly separated at the phylum level (Figure 2A and Figure 3B). PC1 and PC2 explained 73.8% and 14.8% of the global variation, respectively (Figure 3B). Similar results were observed in the NMDS analysis based on Weighted UniFrac distances (Figure S2). At the family level, Sphingomonadaceae and Xanthomonadaceae in phylum Proteobacteria and Chitinophagaceae in phylum Bacteroidetes were enriched in B samples. Rhodospirillaceae in phylum Proteobacteria and Micrococcaceae in phylum Actinobacteria were enriched in C samples. The families Enterobacteriaceae, Moraxellaceae, and Desulfovibrionaceae in phylum Proteobacteria , Porphyromonadaceae in phylum Bacteroidetes , Leptotrichiaceae in phylum Fusobacteria , and Ruminococcaceae and Lachnospiraceae in phylum Firmicutes were enriched in E samples (Figure 2B). Differences were also observed at the class, order, and genus level (Figure S3). From 20 samples, we isolated 28 strains with different colony morphology and found the number of cultivable isolates from bulk soil samples (18 strains from B) was much higher than egg cocoon samples (seven strains from C) and egg surface samples (3 strains from E). Then we performed 16S rRNA gene sequencing to identify these 28 isolated strains. All the sequences were aligned against the NCBI database using BLAST, and the results showed that these 28 isolates belonged to two phyla, Proteobacteria and Firmicutes . Phylogenetic analysis on the basis of the 16S rRNA sequences revealed that these 28 isolates clustered into four major groups at the family level, i.e., Alcaligenaceae, Pseudomonadaceae, Enterobacteriaceae, and Bacillaceae (Figure 4). Alcaligenaceae, Enterobacteriaceae, and Pseudomonadaceae belonged to the Proteobacteria phylum, which constituted the largest group (23 isolates). The other five Bacillaceae strains belonged to the Firmicutes phylum (Table S3). The 18 isolates from bulk soil samples were composed of 7 different genera, Alcaligenes , Citrobacter , Bacillus , Pseudomonas , Klebsiella , Enterobacte , and Serratia . The seven isolates from egg cocoon samples were composed of four genera, Alcaligenes , Bacillus , Citrobacter , and Klebsiella . The three isolates from egg surface samples were composed of two genera, Alcaligenes and Pseudomonas (Table S3). The effects of cultivable isolates against pathogens. We assessed the antimicrobial activity of the 28 cultivable isolates against scarab-specific Bt and Bb strains. The confrontation culture analysis showed that strains (LD01, LD9) from H. oblita egg surface (E) samples and strains (T03, T162) from bulk soil (B) samples had strong antagonistic ability against all three scarab-specific Bt strains and weak antagonistic ability against the Bb strain. All of these four strains were Pseudomonas. Strain LD02 from E samples, strain L05 from C samples, and strains (T10, T16, T101, T161, T164) from B samples showed weak antagonistic ability against all three Bt strains but showed strong antagonistic ability against the Bb strain. These seven strains belonged to Alcaligenes. The remaining 17 strains showed no antagonistic ability against the Bt and Bb strains, including 12 Proteobacteria strains and 5 Firmicutes strains (Figure 5 and Table S3). All the three isolates from E samples showed antagonistic ability (100%, N=3) against pathogens, where the proportions of antimicrobial isolates in B and C samples were 38.89% (N=18) and 14.29% (N=7), respectively. Genome sequencing and secondary metabolite analysis of strains with antimicrobial activity. The four strains (LD01, LD9, T03, and T162) with strong Bt-antagonistic ability and weak Bb-antagonistic ability were genome sequenced using the Illumina platform. The 16S rRNA gene sequence identification showed that these four strains belonged to the genus Pseudomonas and had the highest similarity with P. aeruginosa strain DSM50071 (99.51%–99.79%). Therefore, we collected 20 additional Pseudomonas strain genomes from the NCBI GeneBank database (http://www.ncbi.nlm.nih.gov/), including 11 P. aeruginosa strains, 7 P. mendocina strains, 1 P. denitrificans strain, and 1 P. reidholzensis strain (Figure 6 and Table S4). The whole-genome-based phylogenetic tree was constructed using CVTree and PHYLIP, with Bt kurstaki strain HD73 as an outgroup. The CVTree is an alignment-free method where each organism is represented by a Composition Vector (CV) derived from all proteins present in its genome. CVTree has been effectively used in several phylogenetic studies of microorganisms including archaea, prokaryotes, and fungi 26-28 . The results showed that these four strains were clustered with P. aeruginosa strains, indicating they belonged to P. aeruginosa. The blue-green coloration produced during culture verified this result. Phylogenetic analysis also showed high genome similarity among these four P. aeruginosa strains, suggesting that they might be the same strain. As an opportunistic human pathogen, P. aeruginosa can be isolated from various sources, including humans, animals, hospitals, swimming pools, soil, rhizosphere, and plants 29 . P. aeruginosa is also a promising biocontrol agent for plant pathogens and pests such as Pythium sp. and the root-knot nematode (Meloidogyne incognita) 30 , 31 . Nga et al (2013) found that P. aeruginosa isolated from the rhizosphere of a watermelon plant showed high antagonistic ability against both bacterial and fungal pathogens on rice, watermelon, and cabbage 32 . Our study showed that P. aeruginosa also had antagonistic ability against entomopathogenic Bt and Bb strains. Then we used antiSMASH 2.0 pipeline to identify and annotate the putative secondary metabolite biosynthesis gene clusters in the four strains. A total of 62 gene clusters were identified, including 18 NRPS (non-ribosomal peptide synthetase cluster), 9 NRPS-like fragments, 8 hserlactone (homoserine lactone cluster), 7 bacteriocin, 8 phenazine, 4 CDPS (tRNA-dependent cyclodipeptide synthases), 4 NAGGN (N-acetyl-glutaminyl-glutamine-amide), and 4 thiopeptides (Table S5), some of which belonged to antimicrobial compounds. For example, phenazines, redox-active small molecules, have antibiotic properties toward many bacteria and fungi and can damage mammalian cells 33-35 . Thiopeptide antibiotics are a prominent class of antimicrobials with potent activity against gram-positive bacteria and many drug-resistant pathogens 36 . In this study, we also found 7 Alcaligenes strains showed strong Bb-antagonistic ability and weak Bt-antagonistic ability. Alcaligenes is known among bacteria having antibacterial and antifungal activity, for example, active on Microcrocystis spp. and Fusarium oxysporum 37 , 38 . These 7 Alcaligenes strains in the present study may also possess some antimicrobial compounds. Discussion H. oblita is one of the main soil-dwelling scarab pests in China. Biocontrol agents Bt and Bb usually target young larvae, such as 3 to 5-day-old emerging larvae in lab experiments 9 , 10 . However, the control effect by these agents is not as efficient as expected under field conditions. Compared to other scarab pests, H. oblita could form a unique egg cocoon structure, and the young larva completes the initial development process inside this structure. Microorganisms can confer various benefits on hosts, such as supplying nutrients, or protect against pathogen invasion 39 . The microbiota of different scarab insects exhibits some similarities, meanwhile can be affected by factors such as insect species, host developmental stage, habitats, pH, temperature, even different tissues of the host 40 , 41 . For example, significant difference of community structure was observed in the midgut and hindgut of one scarab larvae, Protaetia brevitarsis 42 . We previously demonstrated that H. oblita larvae gut isolates contributed to the relatively low susceptibility of young larvae to entomopathogens. 18 . However, as an important part of H. oblita developmental stage, the microbiota of egg and egg cocoon has not been investigated before, and the role of the unique egg cocoon structure in pathogen infection remains poorly understood. To this end, we analyzed the microbiome of egg surface, egg cocoon and bulk soil in this work respectively. The average numbers of OTUs in egg surface (310.50 ± 46.97) significantly decreased than egg cocoon (3261.00 ± 310.52) and bulk soil (3544.71 ± 124.14), indicating the microbial diversity of egg surface was less than soil. And the average Simpson indexes of egg surface, egg cocoon and bulk soil were 0.105 (± 0.024), 0.008 (± 0.002), and 0.008 (± 0.001) respectively, indicating the community unevenness of egg surface increased. The further microbial composition analysis demonstrated the community of egg surface was selective. At the genus level, the composition of Clostridium from Firmicutes , Pseudomonas , Acinetobacter, Desulfovibrio, Enhydrobacter, Delftia, Sphingobium from Proteobacteria , Dysgonomonas , Emticicia from Bacteroidetes , Sebaldella from Fusobacteria , and Deinococcus from Deinococcus-Thermus significantly increased in egg surface samples (Figure S4), some of which have been reported to exhibit antimicrobial activity. For example, the genus Clostridium mainly occurs in soil, and C. butyricum of this genus has been shown to have antagonistic interaction against Candida albicans , Escherichia coli , Klebsiella spp., Salmonella spp., Vibrio spp. and Helicobacter pylori , as well as some fish pathogens 43 , 44 . Acinetobacter is an emerging nosocomial pathogen, with a growing prevalence worldwide 45 . We previously reported that two Acinetobacter strains isolated from the grub gut exhibited strong antibacterial activity against Bt strains 18 . Many species of genus Pseudomonas have been proved synthesize a variety of compounds with antagonistic activity 32 , 46 . Species of Sphingobium and Delftia are also producing antimicrobial compounds, such as delftibactin A, which inhibit the growth of some common pathogenic microbes 47 , 48 . These species may play an important role in the defense process of H. oblita larvae against biocontrol agents. To further verify this hypothesis, we isolated 28 strains from egg and soil samples, and assessed the antimicrobial potential against Bt and Bb. All the four Pseudomonas isolates and seven Alcaligenes isolates showed antimicrobial activity against both Bt and Bb. Scarab larvae gut isolates from H. oblita , H. parallela , and A. corpulenta belonged to four phlya, Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidetes . Most of these gut isolates (81.25%, N=32) exhibited antibacterial activity against Bt strains. And Proteobacteria constituted the largest group (62.07%, N=29) 18 , which was also the dominant group among all samples in the current study (approximately 44.63%). Firmicute and Bacteroidetes were the most dominant phyla in egg surface, and Actinobacteria was the dominant phylum in egg cocoon. Under natural conditions, the newly hatched larvae will eat the egg shells and feed on the soil around the eggs. During this process, the larvae may feed on these specific microorganisms, which could help them build a stable intestinal microbiota, and protect them from pathogen infection. Declarations Acknowledgements This research was funded by the National Key Research and Development Program of China (Grant: 2017YFD0201200), and the National Natural Science Foundation of China (No. 32070511). Author Contributions C.S., G.W. and J.Z. designed the study, C.L. and L.G. provided the insect and collected samples, Q.L. carried out the experimental work, K.W. did the data analysis, K.W. and C.S. wrote the manuscript. All authors read and approved the final version of the manuscript. Additional information Conflicts of Interest: The authors declare no conflict of interest. References Harrison, J. D. G. & Wingfield, M. J. A taxonomic review of white grubs and leaf chafers (Coleoptera: Scarabaeidae: Melolonthinae) recorded from forestry and agricultural crops in Sub-Saharan Africa. Entomol. 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Characterization of cry9Da4 , cry9Eb2 , and cry9Ee1 genes from Bacillus thuringiensis strain T03B001. Microbiol. Biotechnol. 97 , 9705-9713 (2013). Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 , 2114-2120 (2014). Alloui, T., Boussebough, I., Chaoui, A., Nouar, A. Z. & Chettah, M. C. Usearch: a meta search engine based on a new result merging strategy. International Joint Conference on Knowledge Discovery 1, 531-536 (2015). Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Methods 10 , 996-998 (2013). Edgar, R. C. SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv , 074161 (2016). Caporaso, J. G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26 , 266-267 (2010). DeSantis, T. Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Environ. 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Res. 44 , 242-245 (2016). Felsenstein, J. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladistics-the International Journal of the Willi Hennig Society 5 , 164-166 (1989). Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 04 Mar, 2021 Reviews received at journal 19 Jan, 2021 Reviewers agreed at journal 14 Jan, 2021 Reviewers invited by journal 13 Jan, 2021 Editor assigned by journal 12 Jan, 2021 Editor invited by journal 12 Jan, 2021 Submission checks completed at journal 12 Jan, 2021 First submitted to journal 12 Jan, 2021 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-145611","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":8037924,"identity":"75455d6b-a451-45fa-bba7-bb33e9eccc35","order_by":0,"name":"Kui Wang","email":"","orcid":"","institution":"Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kui","middleName":"","lastName":"Wang","suffix":""},{"id":8037925,"identity":"0dbe9943-b4d9-4de3-a328-c7d8e4493908","order_by":1,"name":"Qi Liu","email":"","orcid":"","institution":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Liu","suffix":""},{"id":8037926,"identity":"6aac59b1-4e6e-4efd-906f-3e1f11206de0","order_by":2,"name":"Chunqin Liu","email":"","orcid":"","institution":"Cangzhou Academy of Agricultural and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunqin","middleName":"","lastName":"Liu","suffix":""},{"id":8037927,"identity":"1295454c-297f-43fd-859a-5d172d45a9a8","order_by":3,"name":"Lili Geng","email":"","orcid":"","institution":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Geng","suffix":""},{"id":8037928,"identity":"23a4e938-85aa-4f6f-b2bf-0b3e78ed9848","order_by":4,"name":"Guirong Wang","email":"","orcid":"","institution":"Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guirong","middleName":"","lastName":"Wang","suffix":""},{"id":8037929,"identity":"b3d5c76a-8a46-41a2-b0af-0f5acee53137","order_by":5,"name":"Jie Zhang","email":"","orcid":"","institution":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhang","suffix":""},{"id":8037930,"identity":"4d2c345e-5007-4c0c-a7a4-03a8b2564080","order_by":6,"name":"Changlong Shu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYDACdgY2BoYKCVK0MIO0nCFZC2MbKTr4m9mfPeadZ5Fnzn744ccfDHZ5BLVIHOYxN+bdJlFs2ZNmLM3DkFxMUIsBMw+bNFBL4oYbDAbSDAwHEhsIa2F/Js07B6SF/fPPH8RpYTCT5m0AaeExk+AhRgvQL2aSc46B/JJTZs1jkExYC397+zOJNzV1wBA7vvnmjwo7wlpgIMEA4k5i1SO0jIJRMApGwSjAAgC0cjM9c6vn5wAAAABJRU5ErkJggg==","orcid":"","institution":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Changlong","middleName":"","lastName":"Shu","suffix":""}],"badges":[],"createdAt":"2021-01-12 07:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-145611/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-145611/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5063139,"identity":"9058bbb2-6552-4d16-8996-fa2c055e986f","added_by":"auto","created_at":"2021-01-18 20:53:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27541,"visible":true,"origin":"","legend":"The distribution of alpha diversity indexes of OTUs from bulk soil (B), egg cocoon (C) and egg surface (E) samples. Shannon index (A) reflects the richness of the sample community, and Simpson index (B) reflects the evenness of the sample community.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/3866bfbcabc6016f5115f824.jpg"},{"id":5063189,"identity":"b7b8f7ee-d14b-44ee-9989-93b734e25525","added_by":"auto","created_at":"2021-01-18 20:56:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1887938,"visible":true,"origin":"","legend":"Taxonomic composition of microbial communities in bulk soil (B), egg cocoon (C) and egg surface (E) samples. Taxonomic resolution is at (A) phylum level and (B) family level respectively. The sample variation in the community structure is also highlighted in the Bray-Curtis tree (A) and cluster tree (B).","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/c3df0c78a74a2e45f6b02ef1.jpg"},{"id":5063231,"identity":"3f614730-c5a0-4edf-9f91-31ab474d5137","added_by":"auto","created_at":"2021-01-18 20:59:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1552456,"visible":true,"origin":"","legend":"Comparison of microbiota using Lefse (LDA) test at the phylum level (A). LDA scores represent the significant microbial difference among different samples. PCA analysis for microbiota variability at the phylum level (B). Each symbol represents a sample. The variance explained by the PCs is indicated on the axes.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/d52506ed12b02a3156a2c3fd.jpg"},{"id":5063191,"identity":"97c9700b-3378-4a80-ac75-171d981340e7","added_by":"auto","created_at":"2021-01-18 20:56:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":843685,"visible":true,"origin":"","legend":"Phylogenetic analysis of 28 cultivable isolates. The tree is constructed based on the 16S rRNA gene sequences of each strain. Bootstrap values over 0.5 (1000 replications) are indicated using filled purple circles on the branch. Strain ID prefixed with “LD” indicates egg surface (E) isolates, strain ID prefixed with “L” indicates egg cocoon (C) isolates, and strain ID prefixed with “T” indicates bulk soil (B) isolates. Different label background colors represent different clades at the family level. The antimicrobial activity against “Bt” or “Bb” strains is indicated using pentagram symbols, filled black pentagrams represent strong antimicrobial activity, partially filled pentagrams represent weak antimicrobial activity, and open pentagrams represent no antimicrobial activity.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/c4d3fe86670ddbc68d8eabd4.jpg"},{"id":5063142,"identity":"9b65f8dc-1ed6-4d21-8f49-0bbd11f5427e","added_by":"auto","created_at":"2021-01-18 20:53:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1419695,"visible":true,"origin":"","legend":"Effect of 28 cultivable isolates against the scarab-specific Bt strain Bt185 (A), HBF-18 (B), HBF-1 (C), and Bb strain (D). CK in (A), (B), and (C) and symbol “–” in (D) signifies negative control. The symbol “+” in (D) signifies positive control. Strain ID prefixed with “LD” indicates egg surface isolates, strain ID prefixed with “L” indicates egg cocoon isolates, and strain ID prefixed with “T” indicates bulk soil isolates.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/4376f2c3bdac6a398a26490c.jpg"},{"id":5063188,"identity":"c7b8c2cf-7bb0-458c-a2e4-a1367938f32e","added_by":"auto","created_at":"2021-01-18 20:56:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":301193,"visible":true,"origin":"","legend":"Phylogenetic analysis of four Pseudomonas strains (LD01, LD9, T03, and T162). This tree is constructed based on the whole genome of each strain. Different label background colors represent different clades at the species level. Bt strain Bacillus_thuringiensis_HD73 is used as the outgroup.","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/b8dd931b50ad324ea334e459.jpg"},{"id":5063144,"identity":"8f8ff75c-1de2-4d77-a5a3-dc122b76364d","added_by":"auto","created_at":"2021-01-18 20:53:48","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104024,"visible":true,"origin":"","legend":"Egg cocoon (C) samples and egg surface (E) samples were collected as shown in the figures above, and bulk soil (B) samples were collected from the soil about 10 cm away from the egg surface.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/f12869720e459a55ca3b080e.jpg"},{"id":13647695,"identity":"0567546b-ccdb-4f0b-868d-a556b4ba5476","added_by":"auto","created_at":"2021-09-17 09:29:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1432605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/9348134c-a1b4-4897-b776-18cf9cc39425.pdf"},{"id":5063232,"identity":"c5ae4306-9391-4874-bd78-c35631f7115e","added_by":"auto","created_at":"2021-01-18 20:59:48","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2585281,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-145611/v1/6cc19399c820751fba84f243.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDominant Egg Surface Bacteria of Holotrichia Oblita (Coleoptera: Scarabaeidae) Inhibit The Multiplication of Bacillus Thuringiensis and Beauveria Bassiana\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany species of Scarabaeidae (Coleoptera) are plant pests that cause significant economic losses in agriculture, horticulture, and forestry \u003ca href=\"#_ENREF_1\"\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_2\"\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/a\u003e. The adults typically feed on aerial plant structures, while the larvae, known as white grubs, feed on plant roots. Some root-feeding species are major pests.\u003c/p\u003e\n\u003cp\u003eApplication of chemical insecticides is the main method used to control grubs. However, the extensive use of chemicals poses a threat to the environment and public health. Environmentally friendly microbial agents that could control scarabs would be a safer alternative. \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) is a gram-positive bacterium that produces one or more insecticidal crystal proteins (Cry, Cyt) or vegetative insecticidal proteins (Vip), which exhibit specific toxicity against many insects, especially Lepidoptera and Coleoptera \u003ca href=\"#_ENREF_3\"\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_4\"\u003e\u003csup\u003e4\u003c/sup\u003e\u003c/a\u003e. Following the ingestion of the endotoxin crystals by susceptible insects, the crystals are solubilized by the alkaline conditions in the midgut and are subsequently proteolytically converted into a toxic core fragment. The activated toxin binds to midgut receptors and forms ionic pores that kill midgut cells, or induces a cell death signaling pathway, finally leading to insect death \u003ca href=\"#_ENREF_5\"\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_6\"\u003e\u003csup\u003e6\u003c/sup\u003e\u003c/a\u003e. Different Bt strains, such as HBF-1, HBF-18, Bt185, and B-JJX, have insecticidal activity against scarab pests \u003ca href=\"#_ENREF_7\"\u003e\u003csup\u003e7-10\u003c/sup\u003e\u003c/a\u003e. Fungi are also used as biological insecticides for the control of insect pests. Compared to bacteria and viruses, fungi can directly penetrate through the insect cuticle and multiply in the hemocoel. This makes them useful biological control agents of white grubs \u003ca href=\"#_ENREF_11\"\u003e\u003csup\u003e11\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_12\"\u003e\u003csup\u003e12\u003c/sup\u003e\u003c/a\u003e. \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Bb) is the most widely distributed entomopathogenic fungus in the world. It infects a variety of insect species and causes white muscardine disease. Several Bb-based products are commercially available for the control of the coffee borer beetle (\u003cem\u003eHypothenemus hampei\u003c/em\u003e) and various species of Curculionidae \u003ca href=\"#_ENREF_13\"\u003e\u003csup\u003e13-15\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003eBoth Bt and Bb are effective against scarab larvae in lab experiments, but, under field conditions, they are often not as effective. In China,\u003cem\u003e Holotrichia oblita\u003c/em\u003e, \u003cem\u003eH. parallela\u003c/em\u003e, and \u003cem\u003eAnomala corpulenta\u003c/em\u003e are the most important grub pest species. They damage many plants, such as sweet potatoes, soybeans, peanuts, and the Siberian elm \u003ca href=\"#_ENREF_16\"\u003e\u003csup\u003e16\u003c/sup\u003e\u003c/a\u003e. Exposed to the same Bt or Bb agents, \u003cem\u003eH. oblita\u003c/em\u003e was less susceptible than \u003cem\u003eH. parallela\u003c/em\u003e or \u003cem\u003eA. corpulenta\u003c/em\u003e\u003ca href=\"#_ENREF_3\"\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_9\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_10\"\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_17\"\u003e\u003csup\u003e17\u003c/sup\u003e\u003c/a\u003e. \u003cem\u003eH. oblita\u003c/em\u003e was able to survive using different defense mechanisms. Our previous study indicated that the larval gut bacteria may potentially affect the susceptibility of scarabs to Bt strains. The proportion of gut isolates with antibacterial activity against Bt strains was 90% in \u003cem\u003eH. oblita\u003c/em\u003e, 75% in \u003cem\u003eH. parallela\u003c/em\u003e and 80% in \u003cem\u003eA. corpulenta\u003c/em\u003e\u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003eScarab adults usually oviposit in the soil near their preferred host plants. Compared to \u003cem\u003eH. parallela\u003c/em\u003e and \u003cem\u003eA. corpulenta\u003c/em\u003e, \u003cem\u003eH. oblita \u003c/em\u003eforms a unique egg cocoon structure by mixing its eggs with the surrounding soil. There is a variety of microbes in the soil, some of which can produce compounds adversely affecting the growth of pathogens \u003ca href=\"#_ENREF_19\"\u003e\u003csup\u003e19-21\u003c/sup\u003e\u003c/a\u003e. Biocontrol agents generally target young larvae; such as 3 to 5-day-old emerging larvae in lab experiments \u003ca href=\"#_ENREF_9\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_10\"\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/a\u003e, however, under natural conditions, the grubs can often resist pathogen invasion and hatch normally. This unique egg cocoon structure may contribute to the relatively low susceptibility of young larvae to entomopathogens.\u003c/p\u003e\n\u003cp\u003eTo study the role of egg bacteria in pathogen infection, we collected 20 samples from the \u003cem\u003eH. oblita\u003c/em\u003e egg surface (E), egg cocoon (C), and bulk soil (B), and analyzed the microbial community structure using 16S rRNA gene sequencing. We tested the antimicrobial potential of 28 cultivable isolates against scarab-specific Bt and Bb strains using confrontation culture analysis. The results suggest that dominant egg surface bacteria can inhibit the multiplication of Bt and Bb strains.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSampling and DNA extraction\u003c/strong\u003e\u003cstrong\u003e. \u003c/strong\u003eH. oblita was collected from a field in Cangzhou, Hebei Province, China. The adults were reared in plastic boxes (66 cm by 41 cm by 18 cm) filled with soil containing willow leaves at a temperature of 25℃ until they laid eggs.\u003c/p\u003e\n\u003cp\u003eEgg cocoons were collected and peeled off, then the eggs were transferred to a sterile 2 ml plastic centrifuge tube containing 1 ml sterile water and sonicated for 5 min in an ultrasonic cleaning bath (Bransonic 32) to dislodge bacteria. After centrifugation at 10,000\u0026times;g for 5 min, the microorganisms in the wash buffer were collected and defined as the egg surface (E) sample, and resuspended with 1 ml sterile water. The egg cocoon soils (1 g) and soils (1 g) 10 cm away from the egg cocoons were suspended in 5\u0026thinsp;ml sterile water and centrifuged at 10,000\u0026times;g for 5 min, and the pellets were defined as the egg cocoon (C) sample and the bulk soil (B) sample (Figure 7) and resuspended with 1 ml sterile water. A total of 20 samples were collected, including 6 E samples, 7 C samples, and 7 B samples.\u003c/p\u003e\n\u003cp\u003eFor egg surface (E) samples, 1 \u0026mu;l of the microorganism suspension was directly used as a template for PCR. For soil samples (B and C), a 900 \u0026mu;l suspension was used to extract genomic DNA, using a PowerSoil\u0026reg; DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA), and 1 \u0026mu;l of DNA was used as a template for PCR amplification. The remaining 100 \u0026mu;l suspension of 20 samples was kept for conventional culture using solid Luria Bertani (LB) agar medium, and single colonies were picked from the plates and repeatedly grown on solid agar plates until pure cultures were obtained. A total of 28 cultivable isolates were collected, including 3 isolates from E samples, 7 isolates from C samples, and 18 isolates from B samples. Genomic DNA of each isolate was extracted as previously described \u003ca href=\"#_ENREF_49\"\u003e\u003csup\u003e49\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16S rRNA gene sequencing and bioinformatic analysis. \u003c/strong\u003eThe V3-V4 region of microbial 16S rRNA genes of 20 samples were amplified by PCR using the specific primers, 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-'3). PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). The TruSeq \u0026reg; DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) was used for 16S rRNA gene amplicon library construction. The Qubit \u003csup\u003e@\u003c/sup\u003e 2.0 Fluorometer (Thermo Scientific, Rockford, IL, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA) were used for library quality assessment. Finally, the library was sequenced on the Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA), and 250 bp paired-end reads were generated. Raw Data were trimmed using Trimmomatic (version 0.36) \u003ca href=\"#_ENREF_50\"\u003e\u003csup\u003e50\u003c/sup\u003e\u003c/a\u003e with default parameters. Then the clean paired-end reads were assembled into raw tags using Usearch (version 9.2.64) \u003ca href=\"#_ENREF_51\"\u003e\u003csup\u003e51\u003c/sup\u003e\u003c/a\u003e. The primer sequences in the raw tags were trimmed, and effective tags were obtained. The 16S rRNA sequence data of 20 samples were deposited in Sequence Read Archive (SRA) database under BioProject ID PRJNA637400, with accession number SRR11931252\u0026ndash;SRR11931271.\u003c/p\u003e\n\u003cp\u003eThe operational taxonomic units (OTUs) were clustered at 97% identity cutoff with a Usearch UPARSE algorithm \u003ca href=\"#_ENREF_52\"\u003e\u003csup\u003e52\u003c/sup\u003e\u003c/a\u003e. Then the chimera sequences were removed on the basis of the UPARSE pipeline analysis. The OTU annotation was performed using the Usearch SINTAX algorithm \u003ca href=\"#_ENREF_53\"\u003e\u003csup\u003e53\u003c/sup\u003e\u003c/a\u003e, against RDP training set (version 16) 16S rRNA Database with a confidence threshold of 0.8. OTUs annotated as chloroplast or mitochondria or OTUs not annotated to the kingdom level were abandoned. QIIME (version 1.7.0) pyNAST algorithm \u003ca href=\"#_ENREF_54\"\u003e\u003csup\u003e54\u003c/sup\u003e\u003c/a\u003e was used for species annotation against the GreenGene Database\u003ca href=\"#_ENREF_55\"\u003e\u003csup\u003e55\u003c/sup\u003e\u003c/a\u003e. Usearch (version 9.2.64) \u003ca href=\"#_ENREF_51\"\u003e\u003csup\u003e51\u003c/sup\u003e\u003c/a\u003e was used to calculate Alpha diversity metrics, including the indexes (shannon, chao1) reflecting the sample community richness, and indexes (simpson, dominance, and equitability) reflecting the sample community evenness. QIIME (version 1.7.0) \u003ca href=\"#_ENREF_56\"\u003e\u003csup\u003e56\u003c/sup\u003e\u003c/a\u003e was used to calculate Beta diversity to estimate variation between samples. Principal Component Analysis (PCA) and Non-Metric Multi-Dimensional Scaling (NMDS) analysis were performed using R package (https://www.r-project.org/) to visualize complex relationships between samples. Lda Effective Size (LEfSe) test for variability of microbiota was calculated using lefse (Version 1.0.7) (http://huttenhower.sph.harvard.edu/galaxy/).\u003c/p\u003e\n\u003cp\u003eFor 28 cultivable isolates, the 16S rRNA genes of each isolate were amplified using the specific primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-'3). Then the 16S rRNA sequences were identified through aligning against NCBI 16S rRNA sequence (Bacteria and Archaea) database with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfrontation culture analysis. \u003c/strong\u003eFour entomopathogen strains were used: 3 scarab-specific Bt strains (HBF-1, HBF-18, Bt185)\u003ca href=\"#_ENREF_7\"\u003e\u003csup\u003e7-9\u003c/sup\u003e\u003c/a\u003e, and one Bb strain BBNS-J9-16 (preservation number: CGMCC No.5288).\u003c/p\u003e\n\u003cp\u003eThe dual culture tests for antagonistic ability of 28 cultivable isolates against Bt strains were processed using the cup-plate confrontation culture method, as previously described \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e. Sterile water was used as a negative control. The observable inhibition zones were used as indicators of the antibacterial activity of the 28 isolates against Bt strains.\u003c/p\u003e\n\u003cp\u003eFor the antagonistic ability analysis of the 28 isolates against Bb strain, the isolates were cultured at 30℃ with shaking at 220 rpm. The Bb strain was cultured on PDA for 2\u0026ndash;4 d. Subsequently, fungal culture plugs were placed in the middle of LB agar plates. Five dishes of sterile blotter paper (6 mm diam.) were placed on the surface of the plate and inoculated with 10 \u0026mu;l of the cultured bacterial suspension. The amphotericin-B and sterile water were used as positive and negative controls, respectively. The plates were incubated at 28℃ for 5 d, and inhibition zones induced by 28 isolates against Bb strain were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing and secondary metabolite analysis. \u003c/strong\u003eFour cultivable isolates with strong antagonistic ability against Bt strains and weak antagonistic ability against Bb strain were selected for draft genome sequencing, using the Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). The produced reads were cleaned by removing reads with Ns or more than 20% low-quality bases, and 1 Gb 2\u0026times;100 bp pair-end clean reads for each isolate were obtained. The SPAdes \u003ca href=\"#_ENREF_57\"\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/a\u003e was used for genome assembly, and GeneMark \u003ca href=\"#_ENREF_58\"\u003e\u003csup\u003e58\u003c/sup\u003e\u003c/a\u003e was used for gene prediction. The antiSMASH 2.0 pipeline \u003ca href=\"#_ENREF_59\"\u003e\u003csup\u003e59\u003c/sup\u003e\u003c/a\u003e was used for secondary metabolite analysis of these four isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis. \u003c/strong\u003eAll the 16S rRNA sequences of 28 cultivable isolates were analyzed using MEGA (version 7) \u003csup\u003e60\u003c/sup\u003e and an online tool iTOL: Interactive Tree of Life (http://itol.embl.de/) \u003ca href=\"#_ENREF_61\"\u003e\u003csup\u003e61\u003c/sup\u003e\u003c/a\u003e. The analysis included bootstrapping values with 1000 replications.\u003c/p\u003e\n\u003cp\u003eFor the phylogenetic analysis of four genome sequenced isolates, we constructed the whole-genome-based tree using CVTree \u003ca href=\"#_ENREF_26\"\u003e\u003csup\u003e26\u003c/sup\u003e\u003c/a\u003e with \u003cem\u003ek-string\u003c/em\u003e = 6, and PHYLIP \u003ca href=\"#_ENREF_62\"\u003e\u003csup\u003e62\u003c/sup\u003e\u003c/a\u003e. The iTOL was used to annotate the tree \u003ca href=\"#_ENREF_61\"\u003e\u003csup\u003e61\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMicrobial diversity comparisons between egg and soil samples. \u003c/strong\u003eThe bacterial composition of different samples from H. oblita egg surface (E), egg cocoon (C), and bulk soil (B) was determined by sequencing analysis of the 16S rRNA gene. A total of 2,748,824 raw reads were generated from 20 samples, including 667,427 raw reads from six E samples, 924,724 raw reads from seven C samples, and 1,156,673 raw reads from seven B samples. After removing the short reads and trimming the low-quality regions, a total of 1,894,784 effective tags were identified, with an average length of 415 bp. All the effective sequences were clustered into 49,503 OTUs at 97% DNA sequence similarity, including 1,863 OTUs from E samples, 22,827 OTUs from C samples, and 24,813 OTUs from B samples (Tables S1 and S2).\u003c/p\u003e\n\u003cp\u003eAlpha diversity analysis was then performed to assess the diversity and evenness of the microbial population from different samples. The alpha diversity patterns were variable across the soil samples (B and C) and egg surface samples (E) (Figure 1). The number of observed OTUs and alpha diversity analysis based on Shannon and Chao1 indexes indicated that soil samples had more microbial diversity than the egg surface samples (Figure 1A and Table S2). A previous study in our laboratory showed that the microorganism collection method could affect the community structure, where the phyllosphere community diversity was lower for samples subjected to DNA extraction than for those subjected to direct PCR \u003ca href=\"#_ENREF_22\"\u003e\u003csup\u003e22\u003c/sup\u003e\u003c/a\u003e. In the present study, we performed direct PCR for E samples and added a DNA extraction process before PCR for B and C samples. The results confirmed that the community diversity of soil samples was much higher than the egg surface samples. The results of Simpson, dominance, and equitability indexes indicated that, compared to the B and C samples, the evenness of sample E decreased (Figure 1B and Table S2). The rarefaction curve based on the Shannon index showed that all samples reached a plateau, suggesting that our sampling effort was sufficient to obtain a full estimate of OTU richness (Figure S1).\u003c/p\u003e\n\u003cp\u003eAmong all samples, 26 phyla, 143 families, and 300 genera were identified. \u003cem\u003eProteobacteria\u003c/em\u003e was the dominant phylum and comprised the majority of all detected microorganisms (approximately 44.63%) (Figure 2A). This is typically observed in other soil libraries \u003ca href=\"#_ENREF_23\"\u003e\u003csup\u003e23-25\u003c/sup\u003e\u003c/a\u003e. \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eAcidobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e were also abundant in egg cocoon (C) samples and bulk soil (B) samples. In E samples, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, and \u003cem\u003eFusobacteria\u003c/em\u003e were the most abundant phyla (Figure 2A). The community structure varied markedly among different samples, outlined by the Lefse LDA results (Figure 3A). Compared with bulk soil samples, the composition of \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, and \u003cem\u003eFusobacteria\u003c/em\u003e and the composition of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eAcidobacteria\u003c/em\u003e significantly increased in egg surface samples and egg cocoon samples, respectively (Figure 3A). Bray-Curtis tree and PCA analysis also indicated that microbiota in different samples were clearly separated at the phylum level (Figure 2A and Figure 3B). PC1 and PC2 explained 73.8% and 14.8% of the global variation, respectively (Figure 3B). Similar results were observed in the NMDS analysis based on Weighted UniFrac distances (Figure S2).\u003c/p\u003e\n\u003cp\u003eAt the family level, Sphingomonadaceae and Xanthomonadaceae in phylum \u003cem\u003eProteobacteria\u003c/em\u003e and Chitinophagaceae in phylum \u003cem\u003eBacteroidetes\u003c/em\u003e were enriched in B samples. Rhodospirillaceae in phylum \u003cem\u003eProteobacteria\u003c/em\u003e and Micrococcaceae in phylum \u003cem\u003eActinobacteria\u003c/em\u003e were enriched in C samples. The families Enterobacteriaceae, Moraxellaceae, and Desulfovibrionaceae in phylum \u003cem\u003eProteobacteria\u003c/em\u003e, Porphyromonadaceae in phylum \u003cem\u003eBacteroidetes\u003c/em\u003e, Leptotrichiaceae in phylum \u003cem\u003eFusobacteria\u003c/em\u003e, and Ruminococcaceae and Lachnospiraceae in phylum \u003cem\u003eFirmicutes \u003c/em\u003ewere enriched in E samples (Figure 2B). Differences were also observed at the class, order, and genus level (Figure S3).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom 20 samples, we isolated 28 strains with different colony morphology and found the number of cultivable isolates from bulk soil samples (18 strains from B) was much higher than egg cocoon samples (seven strains from C) and egg surface samples (3 strains from E). Then we performed 16S rRNA gene sequencing to identify these 28 isolated strains. All the sequences were aligned against the NCBI database using BLAST, and the results showed that these 28 isolates belonged to two phyla, \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e. Phylogenetic analysis on the basis of the 16S rRNA sequences revealed that these 28 isolates clustered into four major groups at the family level, i.e., Alcaligenaceae, Pseudomonadaceae, Enterobacteriaceae, and Bacillaceae (Figure 4). Alcaligenaceae, Enterobacteriaceae, and Pseudomonadaceae belonged to the \u003cem\u003eProteobacteria\u003c/em\u003e phylum, which constituted the largest group (23 isolates). The other five Bacillaceae strains belonged to the \u003cem\u003eFirmicutes\u003c/em\u003e phylum (Table S3).\u003c/p\u003e\n\u003cp\u003eThe 18 isolates from bulk soil samples were composed of 7 different genera, \u003cem\u003eAlcaligenes\u003c/em\u003e, \u003cem\u003eCitrobacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e, \u003cem\u003eEnterobacte\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e. The seven isolates from egg cocoon samples were composed of four genera,\u003cem\u003e Alcaligenes\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eCitrobacter\u003c/em\u003e, and \u003cem\u003eKlebsiella\u003c/em\u003e. The three isolates from egg surface samples were composed of two genera, \u003cem\u003eAlcaligenes\u003c/em\u003e and\u003cem\u003e Pseudomonas\u003c/em\u003e (Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effects of cultivable isolates against pathogens. \u003c/strong\u003eWe assessed the antimicrobial activity of the 28 cultivable isolates against scarab-specific Bt and Bb strains. The confrontation culture analysis showed that strains (LD01, LD9) from H. oblita egg surface (E) samples and strains (T03, T162) from bulk soil (B) samples had strong antagonistic ability against all three scarab-specific Bt strains and weak antagonistic ability against the Bb strain. All of these four strains were Pseudomonas. Strain LD02 from E samples, strain L05 from C samples, and strains (T10, T16, T101, T161, T164) from B samples showed weak antagonistic ability against all three Bt strains but showed strong antagonistic ability against the Bb strain. These seven strains belonged to Alcaligenes. The remaining 17 strains showed no antagonistic ability against the Bt and Bb strains, including 12 Proteobacteria strains and 5 Firmicutes strains (Figure 5 and Table S3).\u003c/p\u003e\n\u003cp\u003eAll the three isolates from E samples showed antagonistic ability (100%, N=3) against pathogens, where the proportions of antimicrobial isolates in B and C samples were 38.89% (N=18) and 14.29% (N=7), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing and secondary metabolite analysis of strains with antimicrobial activity. \u003c/strong\u003eThe four strains (LD01, LD9, T03, and T162) with strong Bt-antagonistic ability and weak Bb-antagonistic ability were genome sequenced using the Illumina platform. The 16S rRNA gene sequence identification showed that these four strains belonged to the genus Pseudomonas and had the highest similarity with P. aeruginosa strain DSM50071 (99.51%\u0026ndash;99.79%). Therefore, we collected 20 additional Pseudomonas strain genomes from the NCBI GeneBank database (http://www.ncbi.nlm.nih.gov/), including 11 P. aeruginosa strains, 7 P. mendocina strains, 1 P. denitrificans strain, and 1 P. reidholzensis strain (Figure 6 and Table S4). The whole-genome-based phylogenetic tree was constructed using CVTree and PHYLIP, with Bt kurstaki strain HD73 as an outgroup. The CVTree is an alignment-free method where each organism is represented by a Composition Vector (CV) derived from all proteins present in its genome. CVTree has been effectively used in several phylogenetic studies of microorganisms including archaea, prokaryotes, and fungi \u003ca href=\"#_ENREF_26\"\u003e\u003csup\u003e26-28\u003c/sup\u003e\u003c/a\u003e. The results showed that these four strains were clustered with P. aeruginosa strains, indicating they belonged to P. aeruginosa. The blue-green coloration produced during culture verified this result. Phylogenetic analysis also showed high genome similarity among these four P. aeruginosa strains, suggesting that they might be the same strain. As an opportunistic human pathogen, P. aeruginosa can be isolated from various sources, including humans, animals, hospitals, swimming pools, soil, rhizosphere, and plants \u003ca href=\"#_ENREF_29\"\u003e\u003csup\u003e29\u003c/sup\u003e\u003c/a\u003e. P. aeruginosa is also a promising biocontrol agent for plant pathogens and pests such as Pythium sp. and the root-knot nematode (Meloidogyne incognita) \u003ca href=\"#_ENREF_30\"\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_31\"\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/a\u003e. Nga et al (2013) found that P. aeruginosa isolated from the rhizosphere of a watermelon plant showed high antagonistic ability against both bacterial and fungal pathogens on rice, watermelon, and cabbage \u003ca href=\"#_ENREF_32\"\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/a\u003e. Our study showed that P. aeruginosa also had antagonistic ability against entomopathogenic Bt and Bb strains.\u003c/p\u003e\n\u003cp\u003eThen we used antiSMASH 2.0 pipeline to identify and annotate the putative secondary metabolite biosynthesis gene clusters in the four strains. A total of 62 gene clusters were identified, including 18 NRPS (non-ribosomal peptide synthetase cluster), 9 NRPS-like fragments, 8 hserlactone (homoserine lactone cluster), 7 bacteriocin, 8 phenazine, 4 CDPS (tRNA-dependent cyclodipeptide synthases), 4 NAGGN (N-acetyl-glutaminyl-glutamine-amide), and 4 thiopeptides (Table S5), some of which belonged to antimicrobial compounds. For example, phenazines, redox-active small molecules, have antibiotic properties toward many bacteria and fungi and can damage mammalian cells \u003ca href=\"#_ENREF_33\"\u003e\u003csup\u003e33-35\u003c/sup\u003e\u003c/a\u003e. Thiopeptide antibiotics are a prominent class of antimicrobials with potent activity against gram-positive bacteria and many drug-resistant pathogens \u003ca href=\"#_ENREF_36\"\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, we also found 7 \u003cem\u003eAlcaligenes\u003c/em\u003e strains showed strong Bb-antagonistic ability and weak Bt-antagonistic ability. \u003cem\u003eAlcaligenes\u003c/em\u003e is known among bacteria having antibacterial and antifungal activity, for example, active on \u003cem\u003eMicrocrocystis\u003c/em\u003e spp. and \u003cem\u003eFusarium oxysporum\u003c/em\u003e \u003ca href=\"#_ENREF_37\"\u003e\u003csup\u003e37\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_38\"\u003e\u003csup\u003e38\u003c/sup\u003e\u003c/a\u003e. These 7 \u003cem\u003eAlcaligenes\u003c/em\u003e strains in the present study may also possess some antimicrobial compounds.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eH. oblita\u003c/em\u003e is one of the main soil-dwelling scarab pests in China. Biocontrol agents Bt and Bb usually target young larvae, such as 3 to 5-day-old emerging larvae in lab experiments \u003ca href=\"#_ENREF_9\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_10\"\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/a\u003e. However, the control effect by these agents is not as efficient as expected under field conditions. Compared to other scarab pests, \u003cem\u003eH. oblita\u003c/em\u003e could form a unique egg cocoon structure, and the young larva completes the initial development process inside this structure.\u003c/p\u003e\n\u003cp\u003eMicroorganisms can confer various benefits on hosts, such as supplying nutrients, or protect against pathogen invasion \u003ca href=\"#_ENREF_39\"\u003e\u003csup\u003e39\u003c/sup\u003e\u003c/a\u003e. The microbiota of different scarab insects exhibits some similarities, meanwhile can be affected by factors such as insect species, host developmental stage, habitats, pH, temperature, even different tissues of the host \u003ca href=\"#_ENREF_40\"\u003e\u003csup\u003e40\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_41\"\u003e\u003csup\u003e41\u003c/sup\u003e\u003c/a\u003e. For example, significant difference of community structure was observed in the midgut and hindgut of one scarab larvae, \u003cem\u003eProtaetia brevitarsis\u003c/em\u003e \u003ca href=\"#_ENREF_42\"\u003e\u003csup\u003e42\u003c/sup\u003e\u003c/a\u003e. We previously demonstrated that \u003cem\u003eH. oblita\u003c/em\u003e larvae gut isolates contributed to the relatively low susceptibility of young larvae to entomopathogens. \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e. However, as an important part of \u003cem\u003eH. oblita \u003c/em\u003edevelopmental stage, the microbiota of egg and egg cocoon has not been investigated before, and the role of the unique egg cocoon structure in pathogen infection remains poorly understood. To this end, we analyzed the microbiome of egg surface, egg cocoon and bulk soil in this work respectively. The average numbers of OTUs in egg surface (310.50 \u0026plusmn; 46.97) significantly decreased than egg cocoon (3261.00 \u0026plusmn; 310.52) and bulk soil (3544.71 \u0026plusmn; 124.14), indicating the microbial diversity of egg surface was less than soil. And the average Simpson indexes of egg surface, egg cocoon and bulk soil were 0.105 (\u0026plusmn; 0.024), 0.008 (\u0026plusmn; 0.002), and 0.008 (\u0026plusmn; 0.001) respectively, indicating the community unevenness of egg surface increased. The further microbial composition analysis demonstrated the community of egg surface was selective. At the genus level, the composition of \u003cem\u003eClostridium\u003c/em\u003e from\u003cem\u003e Firmicutes\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e\u003cem\u003e, Acinetobacter, Desulfovibrio, Enhydrobacter, Delftia, Sphingobium \u003c/em\u003efrom\u003cem\u003e Proteobacteria\u003c/em\u003e, \u003cem\u003eDysgonomonas\u003c/em\u003e\u003cem\u003e, Emticicia\u003c/em\u003e from \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eSebaldella \u003c/em\u003efrom\u003cem\u003e Fusobacteria\u003c/em\u003e, and \u003cem\u003eDeinococcus \u003c/em\u003efrom\u003cem\u003e Deinococcus-Thermus\u003c/em\u003e significantly increased in egg surface samples (Figure S4), some of which have been reported to exhibit antimicrobial activity. For example, the genus \u003cem\u003eClostridium\u003c/em\u003e mainly occurs in soil, and \u003cem\u003eC. butyricum\u003c/em\u003e of this genus has been shown to have antagonistic interaction against \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e spp., \u003cem\u003eSalmonella\u003c/em\u003e spp., \u003cem\u003eVibrio\u003c/em\u003e spp. and \u003cem\u003eHelicobacter pylori\u003c/em\u003e, as well as some fish pathogens \u003ca href=\"#_ENREF_43\"\u003e\u003csup\u003e43\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_44\"\u003e\u003csup\u003e44\u003c/sup\u003e\u003c/a\u003e. \u003cem\u003eAcinetobacter\u003c/em\u003e is an emerging nosocomial pathogen, with a growing prevalence worldwide \u003ca href=\"#_ENREF_45\"\u003e\u003csup\u003e45\u003c/sup\u003e\u003c/a\u003e. We previously reported that two \u003cem\u003eAcinetobacter\u003c/em\u003e strains isolated from the grub gut exhibited strong antibacterial activity against Bt strains \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e. Many species of genus \u003cem\u003ePseudomonas \u003c/em\u003ehave been proved synthesize a variety of compounds with antagonistic activity \u003ca href=\"#_ENREF_32\"\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_46\"\u003e\u003csup\u003e46\u003c/sup\u003e\u003c/a\u003e. Species of \u003cem\u003eSphingobium\u003c/em\u003e and \u003cem\u003eDelftia\u003c/em\u003e are also producing antimicrobial compounds, such as delftibactin A, which inhibit the growth of some common pathogenic microbes \u003ca href=\"#_ENREF_47\"\u003e\u003csup\u003e47\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_48\"\u003e\u003csup\u003e48\u003c/sup\u003e\u003c/a\u003e. These species may play an important role in the defense process of \u003cem\u003eH. oblita\u003c/em\u003e larvae against biocontrol agents. To further verify this hypothesis, we isolated 28 strains from egg and soil samples, and assessed the antimicrobial potential against Bt and Bb. All the four \u003cem\u003ePseudomonas\u003c/em\u003e isolates and seven \u003cem\u003eAlcaligenes\u003c/em\u003e isolates showed antimicrobial activity against both Bt and Bb.\u003c/p\u003e\n\u003cp\u003eScarab larvae gut isolates from \u003cem\u003eH. oblita\u003c/em\u003e, \u003cem\u003eH. parallela\u003c/em\u003e, and \u003cem\u003eA. corpulenta \u003c/em\u003ebelonged to four phlya, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e. Most of these gut isolates (81.25%, N=32) exhibited antibacterial activity against Bt strains. And \u003cem\u003eProteobacteria\u003c/em\u003e constituted the largest group (62.07%, N=29) \u003ca href=\"#_ENREF_18\"\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/a\u003e, which was also the dominant group among all samples in the current study (approximately 44.63%). \u003cem\u003eFirmicute \u003c/em\u003eand\u003cem\u003e Bacteroidetes\u003c/em\u003e were the most dominant phyla in egg surface, and \u003cem\u003eActinobacteria \u003c/em\u003ewas the dominant phylum in egg cocoon. Under natural conditions, the newly hatched larvae will eat the egg shells and feed on the soil around the eggs. During this process, the larvae may feed on these specific microorganisms, which could help them build a stable intestinal microbiota, and protect them from pathogen infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Key Research and Development Program of China (Grant: 2017YFD0201200), and the National Natural Science Foundation of China (No. 32070511).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.S., G.W. and J.Z. designed the study, C.L. and L.G. provided the insect and collected samples, Q.L. carried out the experimental work, K.W. did the data analysis, K.W. and C.S. wrote the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarrison, J. D. G. \u0026amp; Wingfield, M. J. A taxonomic review of white grubs and leaf chafers (Coleoptera: Scarabaeidae: Melolonthinae) recorded from forestry and agricultural crops in Sub-Saharan Africa. \u003cem\u003e Entomol. Res.\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 141-153 (2016).\u003c/li\u003e\n\u003cli\u003e\u0026aacute;lvarez, H. A., Carrillo-Ruiz, H. \u0026amp; Mor\u0026oacute;n, M. A. Record of scarabaeoidea larvae and adults associated with \u003cem\u003eAmaranthus hypochondriacus\u003c/em\u003e and living fences. \u003cem\u003eSouthwest. 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However, neither provides adequate control of scarab pests in the field. In this study, we used \u003cem\u003eHolotrichia oblita\u003c/em\u003e, an important scarab pest in China, as a model to explore the reason of difficult to control. Compared to other scarabs, \u003cem\u003eH. oblita\u003c/em\u003e could form a unique egg cocoon structure, in which the young larvae complete the initial development process. We analyzed the microbiome of eggs surface, egg cocoon and bulk soil respectively, and investigated the role of these microorganisms in pathogen infection. 16S rRNA gene analysis revealed low bacterial richness in egg surface with \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFusobacteria\u003c/em\u003e dominating in terms of OTUs number, and indicated that the microbial community structure varied among egg and soil samples. Some dominant species of the egg surface have been reported to exhibit antimicrobial activity, which may contribute to the low susceptibility of young larvae to entomopathogens. The further confrontation culture analysis verified this hypothesis. We found that 11 of 28 cultivable isolates possessed antimicrobial activity against both Bt and Bb, and all isolates from egg surface samples showed antagonistic ability. Four isolates belonging to \u003cem\u003ePseudomonas\u003c/em\u003e showed strong Bt- and weak Bb-antagonistic ability, and this genus significantly increased in egg surface samples. This study demonstrated that \u003cem\u003eH. oblita\u003c/em\u003e egg has a unique microbial community structure, containing microorganisms with antimicrobial activity against entomopathogens. These microorganisms protect the newly hatched larvae from pathogen infection.\u003c/p\u003e","manuscriptTitle":"Dominant Egg Surface Bacteria of Holotrichia Oblita (Coleoptera: Scarabaeidae) Inhibit The Multiplication of Bacillus Thuringiensis and Beauveria Bassiana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-18 20:53:46","doi":"10.21203/rs.3.rs-145611/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-03-04T11:31:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-19T14:30:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4aefad15-7824-4291-b3c6-2f949146c75f","date":"2021-01-14T11:10:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-13T05:39:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-12T16:04:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-12T11:45:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-12T11:03:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-01-12T07:08:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16393f71-0ab7-4cbf-b7cb-2ef4f79e15ad","owner":[],"postedDate":"January 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1902245,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-04-13T06:44:16+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-18 20:53:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-145611","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-145611","identity":"rs-145611","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}