Comparative study of the gut microbial community structure of Spodoptera frugiperda and Spodoptera literal (Lepidoptera)

Introduction

The fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797), is a destructive invasive pest originating from tropical and subtropical America (Todde & Poole, 1980). In 2016, S. frugiperda invaded Africa from America and spread to more than 100 countries, including India, Bangladesh, Sri Lanka, Thailand, and Myanmar (CABI, 2019). In January 2019, S. frugiperda was first reported in Jiangcheng, Pu’er City, Yunnan Province in China (Jing et al., 2020). Since then, S. frugiperda has spread rapidly to 26 provinces (Wang, Chen & Lu, 2019). S. frugiperda larvae attack more than 350 host plant species belonging to 76 plant families, including maize, rice, sugarcane, sorghum, peanut, buckwheat, lettuce, and cotton, but the greatest damage is observed in maize (Montezano et al., 2018). In China, the larvae of S. frugiperda are causing 20–30% crop loss in maize, leading to extensive economic losses and threatening food security (Mao, 2019).

S. litura (Fabricius, 1775) is native to China and closely related to S. frugiperda. After S. frugiperda invaded China, it usually co-occurs with S. litura in maize fields (Zhao, Luo & Sun, 2019). Because they share the host species, the two pests’ frequency of occurrence, and morphological characteristics are very similar. Therefore, it is challenging to formulate different control strategies for the native and the invasive species. In an earlier study, we found that cytochrome P450 (CYP) genes are more expanded in S. frugiperda than in S. litura and six other Lepidopteran species, and the expanded CYP genes from S. frugiperda showed very short time divergence (Gui et al., 2020). Moreover, compared to S. litura, GSTs in certain branches showed obvious expansion, identifying several amino acid mutations, some of which were predicted to affect protein function (Huang et al., 2019; Zhu et al., 2020). These characteristics help S. frugiperda deal with a wider variety of exogenous toxic and odorous substances, which may be one of the reasons for its rapid invasion, giving it a competitive advantage over native species.

The gut microbiome is a complex ecosystem dominated by bacteria, forming a close symbiotic relationship with the host (Clemente et al., 2012). Insect gut bacterial community structure differs greatly between insect species and even among individuals. Gut bacteria can help insects digest food, improve protection against pathogens and pesticides, regulate mating and reproductive functions, degrade toxic substances in host plants, and affect intraspecific and interspecific substance exchange (Zhou et al., 2020; Chen, Lu & Shao, 2017; Zilber-Rosenberg & Rosenberg, 2008). For instance, the bessbug’s (Odontotaenius disjunctus; a wood-feeding insect) gut structure and microbial composition contribute to the decomposition of lignocellulose, which helps O. disjunctus digest food (Ceja-Navarro et al., 2019). In the gut of S. frugiperda larvae, a variety of bacteria such as Klebsiella, Staphylococcus, Bacillus, and Acinetobacter can produce cellulase, xylanase, pectinase, and metabolize phenol, improving its adaptability to the host plants (Chen et al., 2022). Furthermore, a causal link exists between gut microbial composition and insect resistance to insecticides. Gut bacteria may prevent or restore damage to insect immune systems caused by insecticides rather than bacterial degradation, constituting the mechanism of acquired resistance (Shao et al., 2014).

Studies on the competition between invasive species and closely related native species have focused on the differences in the adaptability of insects to the environment, while little is known about the differences in and roles of gut symbiotic microbes (Huang et al., 2019; Zhu et al., 2020). Food is an important factor in the interspecific competition (Farjana, Tuno & Higa, 2012), and food is also one of the most important factors affecting the gut bacterial community structure (Yun et al., 2014). Therefore, we wanted to explore differences in the gut bacterial structure between S. frugiperda and S. litura under the influence of the same host plant, and the potential effects these differences may have on their competitive relationship.

Here, we used 16S rRNA sequence profiling to characterize the diversity of gut microbiota associated with S. frugiperda and S. litura that fed on maize leaves, reporting gut bacterial species. Then, we preliminarily explored the commonalities and differences between the gut bacteria communities of these two insect species. Functional bacterial species especially play an essential role in the host fitness (Armitage et al., 2022). Our findings provide a basis for future studies to develop novel pest management strategies, especially by formulating specific gut microecological regulation strategies.

Materials & Methods

Results

Sequence splicing assembly and OTU clustering analysis

The Illumina MiSeq/HiSeq sequencing platform was used to sequence the V3-V4 region of the 16S rDNA of maize-fed S. frugiperda and S. litura. The average number of optimized sequences obtained for S. frugiperda (65,031) was lower than that for S. litura (66,532), with an average of 79,678 and 79,619 pairs of reads obtained for S. frugiperda and S. litura samples, representing 97.8% and 97.9% of the valid sequences. The sequencing accuracy of the samples was satisfactory and met the analysis requirements. The number of OTUs of S. frugiperda samples was higher than that of the S. litura samples (Figs. 1A–1B).

Species difference analysis

The three phyla with the highest abundances in S. frugiperda and S. litura samples were Firmicutes (50.7 and 55.6%, respectively), Proteobacteria (22.4 and 16.9%) and Bacteroidetes (16.2 and 12.6%) (Figs. 2A–2B). The ten genera with the highest abundances were Enterococcus, ZOR0006, Enterobacter, Erysipelatoclostridium, Bacteroides, Escherichia, Prevotella.9, Lactobacillus, Ruminoccaceae.UCG.014, and Sphingomonas. Phylogenetic analysis was conducted based on the top 10 OTUs in all samples at the genus level (Fig. S1). In S. frugiperda, Enterococcus (27.4%), ZOR0006 (9.4%), Escherichia (3.5%), Bacteroides (2.2%), and Lactobacillus (1.5%) were the five species with the highest abundances. In S. litura, Enterococcus (34.5%), Erysipelatoclostridium (8.8%), ZOR0006 (1.9%), Enterobacter (1.8%), and Bacteroides (1.7%) had the highest abundances (Figs. 2C–2D).

Analysis of S. frugiperdaandS. litura larval gut microbiome biomarkers

To further analyze the differences in gut bacteria between samples and to find key microbiome elements, the abundance profiles of OTUs obtained after species annotation were analyzed for linear discriminant analysis effect values (LEfSe) to find species that differed significantly in abundance between pest species (i.e., biomarkers). The number of biomarkers at different taxonomic levels that differed significantly between S. frugiperda and S. litura was 29 and 3 on genus and phylum level, respectively (Fig. 3A). There were more biomarkers in S. frugiperda. The biomarkers for S. litura included Erysipelatoclostridium, Firmicutes, and Enterobacter. The biomarkers for S. frugiperda were dominated by the phyla Actinobacteria, Flavobacteria, Proteobacteria, Bacteroidetes and Firmicutes, including Micrococcaceae, Leucobacter, Bifidobacterium, Flavobacteriaceae, Pseudochrobactrum, Ochrobactrum, Muribaculaceae, Escherichia, Brucellaceae, Bacteroidales, ZOR0006, Lactobacillus, Peptostreptococcaceae, and Clostridiales (Fig. 3A). Because of different taxonomic levels of biomarkers, the evolutionary relationships of these biomarkers were shown in Fig. 3B and Table S1.

Functional predictions and differences in the gut microbiome of S. frugiperdaandS. litura

We compared the COG function terms and KEGG pathways between S. frugiperda and S. litura and visualized the results using STAMP software. The two pest species had 15 significantly different KEGG function terms (Figs. 4A–4B). The dominant five functional categories were transcription, cell growth and death, excretory system, cardiovascular diseases, and circulatory system. COG pathway analysis showed 13 significantly different metabolic pathways between the two species. Further, PCA illustrated functional differences between the two groups (Fig. 4C).

Discussion

S. litura is the native species most closely related to S. frugiperda. The two species may compete in the same environment due to their similar ecological niche (Montezano et al., 2018; Tang et al., 2022). In species competition, one species may crowd out or even replace other species, and if invasive species crowd out native species, native species diversity is affected. In this study, the diversity and richness of the gut bacterial communities of S. frugiperda and S. litura were studied, functions of the gut bacterial community were predicted, and differences in the structure and function of the gut bacterial community between the two species were found. This is significant for analyzing differences in environmental adaptation between invasive species and their native relatives and, ultimately, managing invasive species.

We found that the gut bacterial communities of S. frugiperda and S. litura were dominated by Firmicutes, Proteobacteria, and Bacteroidetes at the phylum levels (Figs. 2A–2B). This aligns with findings on other Lepidoptera, Hymenoptera, and Coleoptera e.g., Bombyx mori, Lymantria dispar, Helicoverpa armigera, Apis, and Holotrichia oblita and may be the result of long-term insect evolution (Sheng et al., 2012; Mason et al., 2016; Kumar et al., 2019; Frago, Dicke & Godfray, 2012; Martinson, Moy & Moran, 2012). Herbivorous insects need carbohydrate hydrolases to help them digest and degrade host plants (Ceja-Navarro et al., 2019), and the enzymes that degrade cellulose, hemicellulose, and pectin are mainly encoded by the bacterial genes of Firmicutes and Proteobacteria (Dantur et al., 2015). They play a vital role in maintaining their normal physiological functions.

At the genus level, Enterococcus had the highest abundance of gut bacteria in both insect species (Figs. 2C–2D). Enterococcus is abundant in many insect guts and plays an important role in detoxifying pesticides and providing a natural defense system (Shao et al., 2014; Vilanova et al., 2016). Enterococcus contributed 82% to gut bacterial abundance in silkworms, effectively preventing the colonization of pathogenic bacteria and maintaining the balance of the gut microbiome (Zhang et al., 2022). Enterococcus isolated from the gut of Brithys crini can effectively degrade plant terpenes and reduce their toxic effect on insects (Vilanova et al., 2016). Enterococcus from Plutella xylostella can significantly enhance this moth’s resistance to chlorpyrifos insecticides (Hadi et al., 2021). In the gut of S. litura, Enterococcus is metabolically active and can consume plant cellulose quickly, releasing glucose as an energy source for both the host insect and itself (Devi et al., 2022). The abundance of Enterococcus was significantly higher in the gut of S. litura than in that of S. frugiperda. Enterococcus can form a biofilm on the gut epidermis of S. litura larvae, acting as a shield against foreign potentially harmful microorganisms (Shao et al., 2014).

There were significantly fewer biomarkers in S. litura than in S. frugiperda, and Enterobacteria and Erysipelatoclostridium were common gut strains of S. litura. Biomarkers for S. frugiperda included Micrococcaceae, Leucobacter, and Bifidobacterium belong to the Actinobacteria (Fig. 3A), which are prevalent in the guts of various animals and insects and can help the host degrade toxic substances produced by oxygen-mediated respiration (Bottacini et al., 2012). Flavobacteriaceae, a common symbiote of insects, can help insects synthesize essential amino acids and complete phylogeny (Short et al., 2017). Muribaculaceae, Brucellaceae, Ochrobactrum, Pseudochrobactrum, and Escherichia belong to the Proteobacteria. Ochrobactrum can degrade pesticides and help Termitidae degrade lignin, cellulose, and hemicellulose (Fathollahi et al., 2021). Brucellaceae may be related to the metabolism of heavy metals in insects (Wu et al., 2020). Bacteroidales of Bacteroidetes can help insects produce specific enzymes for nitrogen metabolism under a nitrogen-deficient diet (Desai & Brune, 2012). Peptostreptococcaceae, Clostridiales, ZOR0006, and Lactobacillus belong to the Firmicutes. During the rainy season, ZOR0006 increased in relative abundance in S. frugiperda. This may be related to S. frugiperda’s temperature regulation (Higuita Palacio et al., 2021). Lactobacillus is one of the most common probiotics in humans and one of the main symbiotic bacteria of insects. It assists in the physiological development of Drosophila larvae (Table S1) (Storelli et al., 2011).

Through functional analysis using the KEGG, it was found that S. frugiperda had the higher abudance of genes that participated in most metabolic pathways than that of S. litura. The gut bacteria of S. frugiperda were enriched in genes with nutrient metabolism function. These genes mainly exercise metabolic functions regarding amino acid, vitamin, and lipid metabolism (Figs. 4A–4B). Amino acids and other nutrients have low concentrations in plants and often cannot fully meet insects’ growth and development requirements. However, insect gut bacteria can synthesize some of these nutrients. This phenomenon has been confirmed in studies of Plutella xylostella (Indiragandhi et al., 2007). COG enrichment analysis showed similar results, with significant differences between S. frugiperda and S. litura in 13 COG functional classifications. The most significant differences occurred in post-translational modification, cytoskeleton, lipid transport and metabolism, and RNA processing. The relatively high abundance of modifications indicated that the gut bacteria of S. frugiperda have strong anabolism and transportation functions (Fig. 4A). This phenomenon may be due to the long evolution and adaptation of gut bacteria in insects, as well as the development of strategies to cope with environmental stress. Based on β diversity analysis, the survival requirements of the two species led to a differentiation of gut bacterial communities and functions (Fig. 4C). S. frugiperda had significantly more functional genes than S. litura, which may give S. frugiperda a greater potential for adaptation to host plants and a competitive advantage.

Conclusions

In conclusion, our systematic comparison between S. frugiperda and S. litura gut microbiomes showed that distinctive S. frugiperda and S. litura gut bacterial strains possess different functions, possibly related to their inter-species diversity. This study laid the foundation for further research on the function of gut bacteria in S. frugiperda and S. litura and adaptation mechanisms to host plants. The comparison between invasive and native species is conducive to differential pest control, and S. frugiperda may have a competitive advantage over S. litura regarding the functional characteristics of the gut microbiome.

Supplemental Information