In China, there is a unique edible insect species Clanis bilineata tsingtauica, soybean hawkmoth, which belongs to the Sphingidae family of Lepidoptera (Zhu and Wang, 1997). The larvae of soybean hawkmoth have high nutritional, medicinal, and economic value with abundant protein, vitamins, amino acids (especially essential amino acids (EAAs)), unsaturated fatty acids, microelements, chitin, etc. (Tian and Zhang, 2012). These nutrients can help people promote brain development, prevent the cell deterioration and maintain the endocrine balance (Qian et al., 2022). Currently, in the rural areas of China, especially in the north of Jiangsu Province, the artificial breeding of soybean hawkmoth has become a characteristic agricultural industry with an output of about 30,000 tons and 4.5 billion yuan every year (Guo et al., 2021). Under natural conditions, one generation of the soybean hawkmoth occurs each year in China. The old fifth-instar larvae burrow into the soil and keep still in early September, entering the diapause stage (Guo et al., 2021; Qian et al., 2023a).

Many insect species adaptively choose diapause, similar to hibernation or dormancy in mammals, to increase survival under adverse environmental conditions [e.g., low temperature, lack of food and water (Dittmer and Brucker, 2021)]. It is critical to provide adequate nutrition for diapause insects, and many species build up nutrient reserves in advance. Despite entering a stage of developmental arrest, diapause insects are able to maintain a stable body weight and nutritional status (Santana et al., 2017). However, the nutrient composition of some insects also varies with the intensity of diapause. Examples include lipids in diapause Arimania comaroffi, and glycerol, trehalose and glycogen in diapause Aphidius gifuensis (Bemani et al., 2012). During diapause, insects can self-regulate the composition and levels of nutrients, such as protein, fatty acids, EAAs and carbohydrates (Hahn and Denlinger, 2011). In addition to nutrient storage, insects also reduce their metabolism in order to meet energy requirements for diapause and to provide energy sources for post-diapause growth and development (Poelchau et al., 2013; Dittmer and Brucker, 2021). Despite the growing interest of entomologists in the regulatory mechanisms of insect diapause, the interaction between gut microbes and diapause insects, especially in aspects of nutrient and metabolic regulation, is still poorly understood (Engel and Moran, 2013; Douglas, 2015).

In numerous insect species, gut microbes are involved in nutrient metabolism, digestion, gut homeostasis and endocrine signaling to promote their development and growth, as noted by Zheng et al. (2017) and van Opstal and Bordenstein (2019). For instance, gut microbiota can provide the host insects with a steady supply of essential nutrients, including EAAs, carbohydrates and lipids, to sustain their metabolic processes (Fraune and Bosch, 2010). The gut microbiota assists in fixing atmospheric nitrogen and promoting amino acid metabolism, leading to a partial shift in the host’s amino acid composition pattern (Storelli et al., 2011). In addition, gut bacteria contribute significantly to lipid and protein digestion in Tenebrio molitor and Anticarsia gemmatalis (Genta et al., 2006; Visotto et al., 2009). They also exhibit enzymatic action on starch, lipids, pectin and esters in Helicoverpa armigera, Bombyx mori, Plutella xylostella and Antheraea assamensis (Anand et al., 2010; Gandotra et al., 2018). In Riptortus pedestris, the presence of Burkholderia in the gut results in higher levels of juvenile hormone (JH), leading to increased fecundity and storage proteins (Lee et al., 2019). Moreover, it has been demonstrated that Klebsiella oxytoca can boost the developmental stage of Drosophila suzukii by activating the glycolysis pathway (Gao et al., 2023). The gut microbiota in Drosophila melanogaster modulates the insulin signaling pathway, a key regulator of insect growth and nutrient homeostasis, to regulate nutrient allocation in the host (Shin et al., 2011).

In our previous study, 16S rRNA sequencing was conducted to analyze the gut microbes of diapause soybean hawkmoths. The results showed that Proteobacteria and Firmicutes were the most prominent taxa at the phylum level in the larval gut microbiota, with Enterococcus and Stenotrophomonas being the dominant genera. In addition, non-target metabolomics analysis on the intestinal contents showed that the most enriched pathways was mainly focused on amino acid metabolism (Qian et al., 2023b). Our findings suggest that the dominant gut bacteria may be involved in the nutritional physiology of insect hosts. However, the specific dominant strains of diapause soybean hawkmoths are still unidentified, not to mention their regulatory mechanisms for nutrient biosynthesis and metabolism in the host. This study identified the predominant strains within the larval gut via the metagenomic sequencing, and investigated the impact of diapause time on the diversity, species composition and gene functions of the gut microbiome. This study reports the first discovery of the dominant gut bacteria of soybean hawkmoths, as well as their influence on the nutrition of the hosts. Additionally, antimicrobial susceptibility tests were conducted for the most effective antibiotic to target the dominant bacterium in diapause larvae. Understanding how dominant gut bacteria regulate nutrient synthesis and metabolism in the soybean hawkmoth will help to explore and utilize the nutritional substances of edible insects, promote the development of the soybean hawkmoth industry in China’s rural areas, and further advance the implementation of the rural revitalization strategy.

As reported by Jing et al. (2020), the gut microbiota played a vital role in allocating and mobilizing nutrients in insects during diapause. This study employed metagenomic analysis to identify the dominant gut bacteria in the diapause larvae of soybean hawkmoths from Jiangsu Province. The dominant genera of gut bacteria found in diapause larvae were determined to be Enterococcus and Enterobacter. Our previous study on 16S rRNA sequencing analysis showed that Enterococcus and Stenotrophomonas were the predominant taxa at the genus level (Qian et al., 2023b). Thus, Enterococcus was certainly evidenced to be the predominant genus in the diapause larvae of soybean hawkmoths, while Enterobacter belongs to Proteobacteria, it was generally involved in the immunity and pathogenicity of insects. Xie et al. (2023) also reported that Enterococcus was the predominant genus found in soybean hawkmoths from Henan Province. Similar findings have been observed in other Lepidopteran species. For example, Kong et al. (2023) found that Enterococcus spp. were the predominant gut bacteria in Galleria mellonella larvae, with Enterobacter spp. being dominant in the pupae. However, Lv and Liu (2009) reported that Staphylococcus and Bacillus were the dominant bacterial genera in diapause soybean hawkmoths from Shandong Province by combining a combination of morphological identification and 16S DNA sequencing. Differences in results may be stem from variations in geographic populations and genetic differentiation, as well as advancements in next-generation sequencing (NGS) techniques. In recent years, metagenomic analysis has been widely used to study the gut microbiome of numerous species. The metagenomic analysis conducted in this study indicated that Enterococcus casseliflavus and Enterococcus pernyi, both belonging to the Enterococcus genus, were the dominant gut bacterial species detected in diapause larvae of soybean hawkmoth. It represents the first identification of such gut bacteria in soybean hawkmoths. This study advances our understanding of the interaction between gut microbes and insect hosts by identifying the dominant bacterium from genus to species levels.

The diapause stage is critical for insect development and presents challenges for both hosts and microbial partners (Dittmer and Brucker, 2021). This study examined how diapause time impacts composition, diversity and functionality of gut microbiomes present in soybean hawkmoths. The relative abundance of Enterococcus, including E. casseliflavus and E. pernyi, initially decreased on diapause D14 and slightly increased on D28. For the species diversity analysis, D14 was significantly separated from both D0 and D28, but D0 and D28 appear to overlap considerably. It suggested that the larvae may potentially survive harsh diapause conditions with the assistance of their dominant gut bacteria. Enterococcus is a predomiant gut commensal bacterium that plays a crucial role in the physiology and health of the major economic agricultural insect, B. mori (Zhang et al., 2022). Enterococcus faecium has been demonstrated to regulate the developmental genes in honey bees (Du et al., 2021). Kong et al. (2023) provided evidence that Enterococcus spp. significantly contributes to insect metamorphosis. Furthermore, Chao and ACE showed an increase with diapause time, indicating a greater abundance of gut microbiota in late diapause larvae.

Gut microbiota reportedly contributes significantly to insects’ nutritional metabolism, digestive function and endocrine signals (Fraune and Bosch, 2010; Dittmer and Brucker, 2021). Gut microbes are capable of consistently supplying insect hosts with some nutrients (e.g., EAAs, carbohydrates) which can boost metabolic production (Fraune and Bosch, 2010). During diapause, the axenic larvae of parasitic wasp Nasonia vitripennis consistently maintained lower levels of glucose and glycerol than conventional larvae (Dittmer and Brucker, 2021). In this study, the function of larval gut microbiota was primarily focused on metabolism, particularly in carbohydrate metabolism and amino acid metabolism. Chen B. et al. (2018) used shotgun metagenomic sequencing techniques to reveal that the gut microbiome of B. mori equally emphasizes the metabolism of both carbohydrates and amino acids. Additionally, the metabolic pathways of amino acids in the gut microbiota of soybean hawkmoths were up-regulated with extended diapause time, as indicated by the annotated data. An opposite trend was observed between the abundance of the dominant gut bacterium Enterococcus and the amino acid metabolic pathways with diapause time. Similarly, the relative abundance of Enterococcus in B. mori had a significant correlation with the concentration of amino acids (Li et al., 2022). Liang et al. (2022) reported that E. casseliflavus is capable of producing L-tryptophan in B. mori. It suggested that Enterococcus may upregulate amino acid metabolic pathways to maintain the nutritional balance in diapause soybean hawkmoths.

Based on the previous non-target metabolomics analysis on diapause soybean hawkmoths, it showed that the most enriched pathways were mainly those involved in amino acid metabolism. The differential metabolite D-glutamine was found to be involved in arginine biosynthesis pathways (Qian et al., 2023b). D-glutamine has been reported to assist in nutrient delivery (particularly amino acids) for the metabolism during insects’ immune response to adverse conditions, such as low temperatures or food restrictions (Dolezal et al., 2019). Moreover, the content of arginine in diapause larvae of soybean hawkmoths (Qian et al., 2023b) also exhibited an opposite alteration to the abundance of dominant gut bacterium E. casseliflavus. Therefore, it is speculated that soybean hawkmoths probably up-regulate arginine metabolism by reducing the abundance of E. casseliflavus, thus ensuring their nutritional balance during diapause.

E. casseliflavus, the dominant gut bacterium in diapause larvae of soybean hawkmoths, exhibits antibiotic resistance, complicating the investigation of its interaction with insect hosts. Tetracycline (TC), chloromycetin (CHL) and ampicillin (AMP) were screened as the most effective antibiotics for targeting it in this study. These results align with those found in other Lepidopteran insects. For example, Li (2022) applied TC and AMP to treat E. casseliflavus in Cnaphalocrocis medinalis, and TC was used to treat Tetranychus truncatus (Yang et al., 2020). Next, the mixed liquor of TC, CHL and AMP will be used to determine the nutritional function of E. casseliflavus affecting on C. bilineata tsingtauica. It will be helpful to the farming industry of C. bilineata tsingtauica via the manual intervention of intestinal microecology, and provide scientific basis for the resource exploitation and utilization of edible insects.

In summary, Enterococcus casseliflavus was identified as the dominant bacterium in diapause larvae of C. bilineata tsingtauica, with the gene function focusing on carbohydrate metabolism and amino acid metabolism. In addition, we demonstrated that diapause time reduced the relative abundance of E. casseliflavus, while concurrently enhancing the relative abundance of amino acid metabolic pathways. Building on our previous non-target metabolomics research, it is possible that diapause larvae of C. bilineata tsingtauica may up-regulate their arginine metabolism by reducing the levels of E. casseliflavus to maintain their nutritional balance. This not only extends our comprehension of dominant gut bacteria from the genus to the species level, but also offers an initial investigation of gene functionality in soybean hawkmoths. Furthermore, TC was screened as the most effective antibiotic target for E. casseliflavus in soybean hawkmoths. Nevertheless, further verification is required for the effect of TC in vivo experiments and the appropriate treatment concentrations. Next, we will systematically measure the gut microbiota of soybean hawkmoths from newly hatched larvae to adults, and investigate the interaction mechanism between E. casseliflavus and insect hosts.

The original contributions presented in the study are publicly available. This data can be found here: https://ncbi.nlm.nih.gov/bioproject/PRJNA1043846.

LQ: Conceptualization, Funding acquisition, Investigation, Writing – original draft. YW: Investigation, Methodology, Writing – original draft. PD: Investigation, Methodology, Writing – original draft. JZ: Investigation, Writing – original draft. YQ: Data curation, Investigation, Writing – original draft. ZL: Data curation, Investigation, Writing – original draft. HL: Project administration, Writing – review & editing. FC: Supervision, Writing – review & editing.