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The gut microbial community structure of the oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) affects the the virulence of the entomopathogenic fungus Metarhizium rileyi

BMC Microbiology volume 25, Article number: 232 (2025) Cite this article

Abstract

Mythimna separata, the oriental armyworm, is a lepidopteran pest that threatens cereal crops. In the current study, two strains (XSBN200920 and JHML200710) of entomopathogenic fungus Metarhizium rileyi were tested for virulence against oriental armyworms. When treated with spore suspensions of both strains at a concentration of 1.0 × 108 spores/mL, the 3rd instar larvae's survival rate was considerably different (P < 0.01). The median lethal time of the insects exposed to XSBN200920 was about 3 d longer than that of JHML200710. The results of 16S ribosomal RNA sequencing showed that Chao1 richness in the JHML200710 treatment group was significantly decreased compared with the CK ( 0.02% Tween 80). The dominant gut bacteria species at the phylum level were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota in the three groups. The CK group had a much higher associated abundance of cyanobacteria than the other two fungal treatment groups. Sixteen genera revealed significant variations in the gut bacteria of the insects at the genus level. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional gene and enzyme analysis showed that when compared with the CK group, the XSBN200920 treatment group showed a significant reduction in six aspects, including betalain biosynthesis, spliceosome, and neuroactive ligand-receptor interaction. These findings suggested that healthy and fungus-infected insects' intestinal microbial community structure differed significantly. And the virulence of M. rileyi is closely linked to its ability to alter the structure of the intestinal microbiome of insects. The results offer a starting point for examining the relationship between the gut microbial diversity of oriental armyworms and variations in the virulence of pathogenic fungi.

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Introduction

The oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae), is a major migratory pest of cereal crops in East Asia, South Asia, and Australia [1, 2]. The insect primarily harms pasture, rice, wheat, and corn [3]. Its traits include polyphagy, movement, and rapid reproduction [4]. However, due to the long-term and large-scale use of chemical insecticides, it has developed a high resistance to many traditional insecticides [5]. As a result, it is imperative to explore novel control approaches to mitigate and regulate [6].

Metarhizium rileyi, also known as Nomuraea rileyi [7], is a worldwide filamentous fungus that plays an indispensable role in the field control of Lepidoptera pests [8], such as Spodoptera frugiperda (J. E. Smith) [9, 10]. The M. rileyi conidium penetrates the insect epidermis and quickly colonizes the host blood cavity, forming an appressorium and germ [11]. In this complex process, multiple factors jointly affect its virulence [12, 13].

Numerous types of microorganisms with intricate community structures occupy the guts of insects. These microbes are involved in various biological functions, including host nutrition metabolism, energy balancing, immunological defense, intraspecies communication, copulation, and reproduction [14, 15]. Studies have shown that the insect gut microbiome plays a crucial role in the endocrine system and influences a variety of physiological processes, such as the supply of nutrients, the efficiency of the transmission of disease, the degradation of toxic compounds, the protection of the host against pesticides, parasites, and pathogens, and the promotion of host growth and development [16,17,18,19,20]. Studies suggest that habitat, food sources, and the age of host insects can profoundly influence gut microbiota, helping digest, detoxify, and defend against natural enemies [21].

The significant heterogeneity in the gut microbiota of lepidopterans may be influenced by various factors, which can operate alone or synergistically, including environmental conditions, dietary intake, developmental stage, and intestinal physiology [21]. Host microorganisms can act on the surface of the insect skin, in the gut, in specific insect microbial host structures, and within cells, including a range of facultative and/or obligate exosymbionts and endosymbionts [22]. Some insects acquire microorganisms that may specialize in producing antifungal compounds to fight infection by (entomopathogenic) fungi [23, 24]. Numerous studies indicate that diverse insects exhibit alterations in their gut microbial communities following infection by entomopathogenic fungus [25, 26]. For example, in the early stages of Helicoverpa armigera (Hübner, 1808) being infected with M. rileyi, gut bacteria are induced and transferred into the hemolymph, eliminating them due to increased antimicrobial activity [27]. Different bacterial groups can promote the infection of entomopathogenic fungi in the host [28, 29].

Moreover, after B. bassiana kills its host, its metabolite oosporin limits the growth of the bacteria [30]. Several insect bacteria undergo modifications due to pathogenic processes caused by entomopathogenic fungi, which depend on environmental variables, including temperature [31]. Despite recent studies on the gut microbial communities in oriental armyworms [32], the structure and diversity of the communities after infection with entomopathogenic fungi are still unknown. As previous studies have not focused on gut microbial communities in oriental armyworms infected by M. rileyi, we attempted to explore this.

The virulence differences between M. rileyi strains XSBN200920 and JHML200710 against oriental armyworms were determined in this investigation. The gut microbial community structure of healthy and fungus-infected oriental armyworm was examined to understand better the intrinsic relationship between fungal virulence and the gut microbes of M. separata infected by pathogenic fungi. Moreover, the relationship between the gut microbial community diversity of the insects and the virulence difference in the M. rileyi strains was further revealed. The results will offer an initial assessment of the gut microbial community of oriental armyworms infected with entomopathogenic fungi and an understanding of the process behind the link between the virulence of pathogenic fungi and the gut bacteria of insects.

Materials and methods

Fungi and media

The M. rileyi strains XSBN200920 and JHML200710 that were used in this experiment were collected from infected S. frugiperda larvae on maize plants in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China, in 2020 [33]. These two strains of fungi were preserved in our laboratory. The fungi were inoculated and cultured on Sabouraud maltose agar medium plates (SMAY: 1% peptone, 1% yeast extract, 4% maltose, and 1.5% agarose) in a constant temperature incubator at 25 °C with a photoperiod of 12L:12D in a culture chamber. M. rileyi, grown on SMAY plates for 14 days, was employed for producing conidial suspensions.

Collection and feeding of the insects

The oriental armyworms were established and maintained from insects initially collected from the rice variety Dianheyou 615 cultivated in dry fields in Lancang County, Pu'er City, Yunnan Province, China (Fig. 1A). Oriental armyworms were reared indoors in artificial climate chambers (27 ± 0.5℃, photoperiod 16 h L: 8 h D, RH 70% ± 5%) with fresh corn leaves for 3 generations. Fresh corn leaves are used to feed the insects during their larval stage. 1–3 instar larvae crown fresh corn leaves every 3 days. For 3–5 instars, replace fresh corn leaves once a day. For adult rearing, replace fresh maize leaves every other day while keeping the adults in a cage (50 × 50 × 50 cm).

Fig. 1
figure 1

virulence of M. rileyi XSBN200920 and JHML200710 in oriental armyworm. A Oriental armyworm from Dianheyou 615, Lancang County, Pu 'er City, Yunnan Province, China. B Phenotypes of cadavers after infection with M. rileyi XSBN200920 and JHML200710 at 3 and 6 days of M. separata third instar larvae, respectively. C 108 spore/mL conidia suspension of M. rileyi XSBN200920 and JHML200710 Survival of M. separata third instar larvae. D 108 spore/mL conidia suspension of M. rileyi XSBN200920 and JHML200710 Half-lethal time (n = 3) for M. separata third instar larvae. Tukey's honestly significant difference [HSD]: P < 0.01; error bars: standard deviation

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Bioassay

The 3rd instar larvae of over five generations of laboratory-cultured oriental armyworms were employed to determine their virulence. The two strains' conidia were combined to create a suspension of 108 spores/mL, with 0.02% Tween 80 solution as the control (CK) [33]. The virulence of healthy 3rd instar larvae of the same size was determined by the dip method, according to Pang et al. [33]. The above experiment was repeated three times, with three treatments (the two strains separately and as one spore suspension) for each experiment and no less than 35 samples for each treatment. After the treatment, they were fed in an artificial climate box with a temperature of 25℃, relative humidity of 75%, and photocycle of 16:8. The daily count of deceased insects was documented, and they were preserved in a petri dish for moisture analysis to ascertain potential infection by the test strain.

DNA extraction and PCR amplification

In the treatment group, a 1.0 × 108 spores/mL spore suspension of the M.rileyi strains XSBN200920 or JHML200710 was inoculated into the 3rd instar larvae of oriental armyworm using the dipping method. During the five days post-inoculation, the intestines of the infected worms were carefully dissected, while the intestines of the 3rd instar larvae were examined in the control group. Isolation of genomic DNA from insect intestinal microbes facilitated by Majorbio Co. Ltd., Shanghai [29]. The PCR reaction system and reaction procedure were referred to by Peng et al. [29]. From every group, three samples were amplified three times. Following manufacturer directions, the PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and then quantified using Quantus™ Fluorometer (Promega, USA).

Illumina MiSeq sequencing

Purified amplicons were combined in equimolar concentrations and subjected to paired-end sequencing on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA) using standard methods established by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Data processing

Using an in-house Perl script to process raw FASTQ files, fastp version 0.19.6, FLASH version 1.2.7, and UPARSE 7.1. The most prevalent sequence for each OTU was designated as the representative sequence.

Enzyme activity assay of insect gut

Two strains of fungi were inoculated with 3-instar Oriental Armyworm larvae by spray method (108/mL). Control group (CK) was treated with 0.02% Tween-80. During the feeding with feed. After 5 days of treatment, the larvae were placed in a sterile petri dish and starved for 24 h. The larvae were dissected, and the intestines were removed from the tail of the worms with sterile tweezers and put into a centrifuge tube with 1 mL of sterile PBS buffer. A mixed sample of the gut from each of the six insects was treated as a single. Each strain was treated at least three times. Each trial was set up for 3 replicates. Lipase extraction kit (Beijing Boxengong Technology Co., LTD., China), protease extraction kit (Shanghai Fanwei Biotechnology Co., LTD., China) and amylase extraction kit (Shanghai Fanwei Biotechnology Co., LTD., China) were determined according to the kit method. The unit of enzyme activity is U·g−1 protein.

Data analysis

An unpaired t-test was used to compare data that were homogeneous in variance between the two groups and according to a normal distribution. Tukey's post hoc test was performed after univariate and bivariate analysis of variance (ANOVA) comparisons across several groups. If P is less than 0.05, then there is a significant difference. The gut microbiota was investigated through bioinformatics using the Majorbio Cloud platform (https://cloud.majorbio.com). The co-occurrence networks were constructed based on the Spearman’s correlation matrices using the Majorbio Cloud platform. Only OTUs with top 200 were included in the network construction. We then loaded the correlation table into Gephi software to calculate the nodes and links. Gephi software was also used to determine network topological features, and visualization of co-occurrence networks was presented accordingly. The analyses employed OTU data, rarefaction curves, alpha diversity indices, and observed OTUs. Including comparing the relative abundance of the intestinal microbes between the groups, α multiplicity-Chao richness [34, 35], and principal coordinate analysis (PCoA) [36], which were analyzed by Peng et al. [29]. Species with significant differences in sample classification were identified using linear discriminant analysis (LDA ≥ 2, P < 0.05). Tax4Fun was used to obtain three levels of metabolic pathway information and pathway abundance.

Results

Differences in the virulence between the M. rileyi strains XSBN200920 and JHML200710 against the 3rd instar larvae of oriental armyworm

The virulence test results showed that the survival rate of the 3rd instar larvae that were treated with JHML200710 was significantly lower than that for the larvae that were treated with XSBN200920 (Fig. 1B) (P < 0.001). The median lethal time (LT50) of the 3rd instar larvae that were treated with JHML200710 was also significantly lower than that of the larvae that were treated with XSBN200920 (Fig. 1D) (t = 5.839, df = 4, P = 0.0043). According to the moisture culture data, white mycelia formed on the body surface of the insects treated with XSBN200920 and JHML200710 approximately 72 h after death. It was discovered that the insect body surface was infected with M. rileyi at 144 h because it was covered in green spores (Fig. 1C). The CK group did not produce green spores after being dipped in sterile 0.02% Tween80.

Sequencing data Statistics and clustering

The sequence numbers were 9.66 ± 4.51, 8.92 ± 1.93, and 8.37 ± 2.68 in the CK, JHML200710, and XSBN200920 groups (Table 1), respectively. Which were retained after quality control, and all sequences were longer than 200 (Table 1). Table. 1 displays that all effective sequences were clustered into OTUs (CK: 166.67 ± 9.07, JHML200710: 137.67 ± 16.17, and XSBN200920: 159.00 ± 8.54). The rarefaction curves of these sequences are usually flat, suggesting that the sequencing process has a sufficient sample size (Fig. 2).

Table 1 The OTUs clustered by effective sequences
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Fig. 2
figure 2

OTU rarefaction curve derived from 16S rDNA sequencing

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Analysis of the abundance and diversity of the intestinal bacteria in oriental armyworm

The intestines of the insects treated with CK, XSBN2009, and JHML200710 revealed 32, 25, and 20 distinct microbial species, as shown in the Veen diagram (Fig. 3A). Comparatively to the CK group, the gut of the insects treated with XSBN2009 counted 168 microorganisms at the Operational Taxonomic Unit level (Fig. 3A). There were 159 microorganisms in the gut of the JHML200710-treated insects compared to the control group. There were 158 microorganisms in the gut of insects treated with the XSBN200920 and JHML200710 strains. The total number of gut microbial species shared among the three groups was 144. The Chao index (Fig. 3B) and ace index (Figure S1A) richness in the JHML200710 treatment group were significantly decreased compared to that in the CK group, respectively. The Chao index and ace index of the XSBN200920 treatment group decreased, but there was no significant difference. There were no significant differences in the Shannon Index among all groups (Figure S1B).The Simpson index (Figure S1C) richness in the XSBN200920 treatment group was significantly decreased compared to that in the CK group. The Simpson index in the JHML200710 treatment group was no significant difference compared to that in the CK group.

Fig. 3
figure 3

Effects of M. rileyi infection on intestinal microbial diversity of oriental armyworm larvae. A Veen map of OTU numbers of gut microbes in healthy and infected insects infected with M. rileyi XSBN200920 and JHML200710. B Intestinal microalpha diversity-Chao index of insects. Tukey's honestly significant difference [HSD]: P < 0.05 (*). Error bars: standard deviation (n = 3). C Beta diversity-PCoA analysis of insect gut microbes. D The relative abundance of gut microbes at the Phylum level in infected and healthy insects infected with M. rileyi XSBN200920 and JHML200710 (n = 3). E Significant differences in cyanobacteria between intestinal microbes of infected and healthy insects by M. rileyi XSBN200920 and JHML200710 (Kruskal–Wallis rank sum test (n = 3): P < 0.01 (**)), error bars: standard deviation

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The beta diversity index-PCoA results (Fig. 3C) indicated no substantial overlap between the CK group and the JHML200710 treatment group, as determined by the weighted UniFrac distance matrix, signifying a differentiated gut microbial community structure for each cohort (P > 0.05). At the phylum level, the dominant intestinal bacteria of the three groups were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota, and Proteobacteria was the dominant phylum in the CK, XSBN200920, and JHML200710 groups (Fig. 3D). Compared to the Bacteroidota species in the CK group, the proportion of species in the JHML200710 and JHML200710 treatment groups gradually increased. The Cyanobacteria exhibited a significant difference between the CK group and the two fungus-treated groups (P = 0.0379); its mean proportion diminished with the expansion of M. rileyi virulence (Fig. 3E).

Abundance and diversity analysis of the intestinal bacteria in oriental armyworm at the genus level

The analysis of the Top 20 species at the genus level indicated (Fig. 4A) minimal variations in the constituents of the gut microbial community between the CK group and the treatment groups. At the genus level, the dominant intestinal bacteria species of the three treatment groups were Enterococcus, Enterobacter, Acinetobacter, Glutamicibacter, Brachybacterium, Allorhizobium-Neorhizobium, Pararhizobium-Rhizobium, Ochrobactrum, Leucobacter, Corynebacterium, Brevibacterium, Pseudomonas, Dietzia, unclassified_o_Micrococcales, unclassified_f_Micrococcaceae, Salana, Paracoccus, Devosia, Sphingobacterium, Brevundimonas, and Sphingomonas, among others. The dominant CK, XSBN200920, and JHML200710 genera were Enterococcus, Enterobacter, and Enterobacter, respectively.

Fig. 4
figure 4

Effects of M. rileyi on intestinal microorganisms of 3.rd instar larvae of oriental armyworm. A The relative abundance of the Top20 gut microbes in healthy and infected insects infected with M. rileyi XSBN200920 and JHML200710 at the generic level (n = 3). B Genera with significant differences between infected and healthy insects infected by M. rileyi XSBN200920 and JHML200710. C LDA revealed the most featured intestinal bacteria in oriental armyworms. Kruskal–Wallis rank sum test: P < 0.01 (**)

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Among the intestinal bacteria of the three groups, 16 genera showed significant differences, including Corticicoccus, unclassified Alcaligenaceae, Bosea, Ottowia, Parapusillimonas, unclassified_f_Bacillaceae, Nocardiopsis, Shinella, unclassified_f_Microbacteriaceae, Aureimonas, Camelimonas, Cellulosimicrobium, methylobacterium-methylorubrum, Nocardioides, and Sphingomonas (Fig. 4B). Sphingomonas was in the top 20 genera (Fig. 4B). Notably, the mean proportion of unclassified_f_Bacillaceae, Nocardiopsis, Shinella, unclassified_f_Microbacteriaceae, Camelimonas, Cellulosimicrobium, methylobacterium-methylorubrum, Nocardioides, and Sphingomonas decreased with the increase of Metarhizium rileyi virulence (Fig. 4B). However, the mean fraction of Corticicoccus increased with the virulence of Metarhizium rileyi (Fig. 4B).

LDA revealed the most featured intestinal bacteria in oriental armyworm

We performed LDA on bacterial abundance profiles to identify the featured bacteria associated with M.rileyi infecting. As shown in Fig. 4C, 1 featured phylum (Cyanobacteria), 1 featured class (Cyanobacteria), 4 featured order (Propionibacteriales, Sphingomonadales, and Streptosporangiales), 6 featured family (Beijerinckiaceae, Nocardioidaceae, Nocardiopsaceae, Promicromonosporaceae, and Sphingomonadaceae), and 10 featured genera (Camelimonas, Cellulosimicrobium, Corticicoccus, Methylobacterium-Methylorubrum, Nocardioides, Nocardiopsist, Ottowia, Parapusillimonas, Shinella, Sphingomonas, unclassified_f__Alcaligenaceae, and unclassified_f__Microbacteriaceae) were identified in the control oriental armyworm. Aureimonas and Parapusillimonas genera were featured in the XSBN200920 strain treatment group. At the JHML200710 strain treatment group, Corticicoccus and Ottowia were the featured genera.

Effects of M. rileyi infection on intestinal microbial co-occurrence networks of oriental armyworm larvae

The network was assigned to six modules with CK, XSBN200920, and JHML200710 groups. M. rileyi infection decreased the nodes and links levels compared to CK group (Figure S1D). The modularity increased from 0.696 in CK group to 0.789 and 0.711 in XSBN200920 and JHML200710 groups, respectively (Table S3). The more proportion in CK, XSBN200920, and JHML200710 groups was M3 (30.5%), M1 (22.11%), and M0 (30.5%), respectively. Moreover, the ratios of positive correlations to negative correlations were decreased from 0.68 in CK group to 0.55 and 0.32 in XSBN200920 and JHML200710 groups, respectively. Notably, the ratios of positive correlations to negative correlations decrease with increasing the virulence of M. rileyi.

Prediction of the gut microbial functions in oriental armyworm

To gain a more thorough understanding of the role played by the gut microbiota of oriental armyworms, we used PICRUSt2 to sequence 16S ribosomal RNA. The top six predicted functions for each group were identified by comparing the results with the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database to examine further the relationships between the populations and functions (Fig. 5A and Table S1). These results showed that the CK and treatment groups XSBN200920 and JHML200710 exhibited significant biosynthesis of 12-, 14- and 16-membered macrolides and betalain biosynthesis. The JHML200710 treatment group demonstrated a substantial decrease in the abundance of functional genes associated with neuroactive ligand-receptor interaction compared to the control group. Functional genes related to leishmaniasis, the NF-kappa B signaling system, and the spliceosome were substantially more abundant in the CK than in the JHML200710 treatment group. When compared with the CK group, the XSBN200920 treatment group showed a significant reduction in six aspects, including the biosynthesis of 12-, 14- and 16-membered macrolides, betalain biosynthesis, spliceosome, NF-kappa B signaling pathway, neuroactive ligand-receptor interaction, and leishmaniasis.

Fig. 5
figure 5

KEGG function analysis of intestinal microbes in healthy and Metarhizium rileyi infected insects. A KEGG analysis predicts the possible life activities of insect gut microbes. B KEGG analysis of enzymes that indicate the function of insect gut microbes. Kruskal–Wallis rank sum test: P < 0.05

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Compared with the CK group, the KEGG analysis results (Fig. 5B and Table S2) showed that 32 enzymes in the insect intestines treated with JHML200710 were substantially different. In contrast, there was a significant difference in 32 enzymes in the XSBN200920 therapy group. The JHML200710 treatment group had 20 substantially different enzymes from the XSBN200920 treatment group. These included pseudaminic acid synthase, tetrahydrosarcinapterin synthase, L-arabinose l-dehydrogenase, mannosylfructose-phosphate synthase, prostaglandin-endoperoxide synthase, glycerate 2-kinase, 4-phosphopantoate-beta-alanine ligase, arylmalonate decarboxylase, all-trans-nonaprenyl diphosphate synthase (geranylgeranyl-diphosphate specific), all-trans-nonaprenyl diphosphate synthase (geranyl-diphosphate specific), and opine dehydrogenase. Notably, the relative abundance of 32 enzymes was elevated with the increase of M. rileyi virulence (Fig. 5B and Table S2).

The lipase, amylase and total protease activity in the insect gut infected by XSBN200920 were significantly lower than those of JHML200710

In order to further verify the relationship between the level of intestinal enzyme activity and the virulence of fungi. Lipase, amylase and total protease activity in insect intestine were measured. The results showed (Fig. 6) that the intestinal lipase (Fig. 6A), amylase (Fig. 6B) and total protease enzyme activity (Fig. 6C) of insects infected by XSBN200920 were significantly lower than those of JHML200710.

Fig. 6
figure 6

Comparison of lipase, amylase and total protease activity in intestinal tract of insects infected by two fungal strains. A-C was the activity of lipase, amylase and total protease in the gut of insects infected with M. rileyi XSBN200920 and JHML200710 (the 5th day), respectively. Tukey's honestly significant difference [HSD]: P < 0.01; error bars: standard deviation

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Discussion

In this study, the M. rileyi JHML200710 and XSBN200920 strains (1 × 108 connidia/mL) achieved 50% mortality against the oriental armyworm on days 5 and 8, respectively (Figs. 1C and D). In comparison, the mortality rate of Beauveria bassiana and Isaria fumosorosea (6 × 108 spores/mL) reached 50% on days 4 and 5–6, respectively [37]. This indicates that the strain M. rileyi JHML200710 possesses significant promise for the biological control of oriental armyworms.

When used against oriental armyworms, XSBN200920 had a median fatal time of approximately 8 days. According to earlier research, armyworms treated with XSBN200920 spore suspension at the same dose had a median lethal period of about 4.5 days [33]. This suggests that M. rileyi has a different virulence against different insects of the Lepidoptera. These results may be because the two insects have various defense mechanisms against the same pathogenic fungi [38]. For example, Metarhizium anisopliae (Sorokin) infection in oriental armyworm caused changes in the activity of the detoxification enzymes glutathione S-transferase, acetylcholinesterase, and esterase [39].

Regarding the virulence differences and the gut microbial diversity, the Chao index (Chao1 richness estimator) of the insects treated with M. rileyi JHML200710 was significantly lower than that of the CK group (Fig. 3B). The treatment group that received XSBN200920 did not experience any significant variations. Thus, not every entomopathogenic fungal species induces alterations in the microbial composition of insect guts. Only when the virulence of the entomopathogenic fungi reaches a specific threshold is it likely to impact the composition of the microbial community in the insect gut.

The Cyanobacteria showed significant differences between the CK group and XSBN200920 and JHML200710 treatment groups (Fig. 3E), although they were not the dominant class. This suggests that Cyanobacteria are crucial for defense and health in the gut of insect larvae. Previous studies have also shown that Cyanobacteria are essential in the gut of mosquito larvae [40]. Different feeding habits also affect the relative abundance of Cyanobacteria in the insect gut [41]. Infection with Metarhizium Robertsii resulted in a decrease in the diversity of insect intestines, including cyanobacteria. The alterations in community structure were insignificant, except for the proliferation of Serratia [42]. A significant decline in cyanobacterial abundance is associated with compromised feeding by the insects. This study involved feeding fresh maize leaves to several insect groups, with the leaves providing essential nutrients for the survival of cyanobacteria. The proliferation of cyanobacteria in the infected insects was reduced significantly due to impaired food consumption, resulting in insufficient nutritional availability for the intestinal cyanobacteria. This investigation revealed variations in the genera Camelimonas, Cellulosimicrobium, Methylorubrum, Nocardioides, Sphingomonas, and Nocardiopsis. The abundance of the bacteria Shigella was significantly reduced in the three groups (CK, XSBN200920, and JHML200710). The intestinal symbiote Sphingomonas plays a central role in pest resistance to insecticides, as they can mediate the resistance of cotton aphids to imidacloprid through hydroxylation and nitro reduction [43]. This shows that after infection by pathogenic fungi, these species of bacteria, particularly Sphingomonas bacteria, are threatened, leading to significant modifications in the phenotypes between the pathogenic fungi with varied virulence. This result confirms that Sphingomonas bacteria plays an important role in the host defense system to some extent. One of its limitations in the current study is its inability to investigate whether Sphingomonas bacteria are directly responsible for the variations in M. rileyi's virulence.

According to the KEGG enrichment analysis and functional gene predictions, notable discrepancies were seen in the NF-kappa B signaling pathway, neuroactive ligand-receptor interaction, and leishmaniasis (Fig. 5A). The alteration or depletion of metabolites directly influences insect growth, development, and longevity [44]. Furthermore, the enzymes, such as aldose reductase, were elevated in the treatment groups of the CK and JHML200710 strains. This demonstrated that infection with entomopathogenic fungus substantially influenced the metabolic rate of the oriental armyworm. Various entomopathogenic fungi such as B. bassiana [45] and M. anisopliae [46], M. Robertsii [47], and M. rileyi [48] are used to control lepidoptera pests. In our study, the relationship between insect gut enzyme activity levels and fungal virulence was validated. M. rileyi XSBN200920 was significantly more virulent to insects than JHML200710 (Fig. 1). Lipase, amylase and total protease in the gut of insects infected with M. rileyi XSBN200920 were significantly lower than those of JHML200710 (Fig. 6). The above two experimental results are consistent with each other. This result also confirmed the prediction results of intestinal microbial function to a certain extent (Fig. 5). This study clarifies why M. rileyi exterminates insects and accounts for variations in virulence among distinct strains from the host gut microbial diversity standpoint. This prompted the pursuit of an alternative management strategy to regulate this pest.

Conclusions

In conclusion, this investigation is the first to describe the composition and alterations in the gut microbial community following M. rileyi infection in oriental armyworms. The gut microbiota of healthy and infected oriental armyworms was preliminary summarised by comparing and contrasting the fungus-treated groups (XSBN200920 and JHML200710 strains) with the uninfected group (CK). These findings provide a foundation for further research on the relationship between virulence differences in pathogenic fungi and the gut microbial diversity of oriental armyworms. It also offers new insights for understanding the virulence differences of entomopathogenic fungi.

Data availability

Gut bacteria raw data have been deposited in the NCBI Sequence Read Archive (https://submit.ncbi.nlm.nih.gov/subs/sra/) under the accession number of PRJNA1130424.

Abbreviations

CK:

0.02% Tween 80 solution treatment

ANOVA:

Analysis of variance

PCoA:

Principal coordinate analysis

OUT:

Operational taxonomic unit

PCA:

Principal component analysis

LDA:

Linear discriminant analysis

KEGG:

Kyoto Encyclopedia of Genes and Genomes

AMF:

Arbuscular mycoeehizal fungi

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Funding

This work and the article processing charges (APC) were supported by the Major Science and Technology Project of Yunnan and Kunming (202202AE090036), Major Science and Technology Project of Yunnan Province (202402AE090026) and Science and Technology Department of Yunnan Province Basic Research Project (202301AT070487, 202401AT070244).

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  1. Yuejin Peng and Xv Zhang contributed equally to this work.

Authors and Affiliations

  1. Yunnan State Key Laboratory of Conservation and Utilization of Biological Resources, College of Plant Protection, Yunnan Agricultural University, Kunming, 650201, China

    Yuejin Peng, Xv Zhang, Guang Wang, Zhengfei Li, Xinling Lai, Baoyun Yang, Bin Chen & Guangzu Du

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Contributions

J.P.: investigation, formal analysis, validation; X.Z.: formal analysis, visualization; G. W.: Writing—original draft and editing; Z.L.: investigation; X.L.: investigation; B.Y.: formal analysis and investigation; B.C.: conceptualization, funding acquisition, project administration, supervision; G.D.: conceptualization, funding acquisition, project administration, supervision, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Bin Chen or Guangzu Du.

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This study was conducted at the College of Plant Protection, Yunnan Agricultural University. All relevant institutional, national and international guidelines and regulations were followed. The plant material Dianheyou615 involved in this study is allowed to be used in any ordinary experiments. All the methods were carried out in accordance with relevant guidelines and regulations.

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Peng, Y., Zhang, X., Wang, G. et al. The gut microbial community structure of the oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) affects the the virulence of the entomopathogenic fungus Metarhizium rileyi. BMC Microbiol 25, 232 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03875-y

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