Insect microbial symbioses play a critical role in insect lifecycle, and insect gut microbiome could be influenced by many factors. Studies have shown that host diet and taxonomy have a strong influence on insect gut microbial community. In this study, we performed sequencing of V3-V4 region of 16S rRNA gene to compare the composition and diversity of 12 Ensifera from 6 provinces of China. Moreover, the influences of feeding habits and taxonomic status of insects on their gut bacterial community were evaluated, which might provide reference for further application research. The results showed that Proteobacteria (45.66%), Firmicutes (34.25%) and Cyanobacteria (7.7%) were the predominant bacterial phyla in Ensifera. Moreover, the gut bacterial community composition of samples with different feeding habits was significantly different, which was irrespective of their taxa. The highest diversity of gut bacteria was found in the omnivorous Ensifera. Furthermore, common and unique bacteria with biomarkers were found based on the dietary characteristics of the samples. However, the bacterial community structure of the Ensifera samples was significantly different from that of Caelifera. Therefore, we concluded that feeding habits and taxonomic status jointly affect the gut bacterial community composition of the samples from Orthoptera. However, the influence of feeding habit dominates when taxonomy category below the suborder level. In addition, the dominant, common and unique bacterial community structure could be used to predict the contrastic feeding habits of insects belonging to Ensifera.

Feeding habits were the primary factor affecting the gut bacterial communities in Ensifera. However, the interaction mechanism between the gut microbiota and feeding characteristics is not precisely understood. Here, the gut microbiota of Ensifera with diverse feeding habits was analyzed by shotgun metagenomic sequencing to further clarify the composition and function of the gut microbiota and its relationship with feeding characteristics. Our results indicate that under the influence of feeding habits, the gut microbial communities of Ensifera showed specific characteristics. Firstly, the gut microbial communities of the Ensifera with different feeding habits differed significantly, among which the gut microbial diversity of the herbivorous Mecopoda niponensis was the highest. Secondly, the functional genes related to feeding habits were in high abundance. Thirdly, the specific function of the gut microbial species in the omnivorous Gryllotalpa orientalis showed that the more diverse the feeding behavior of Ensifera, the worse the functional specificity related to the feeding characteristics of its gut microbiota. However, feeding habits were not the only factors affecting the gut microbiota of Ensifera. Some microorganisms’ genes, whose functions were unrelated to feeding characteristics but were relevant to energy acquisition and nutrient absorption, were detected in high abundance. Our results were the first to report on the composition and function of the gut microbiota of Ensifera based on shotgun metagenomic sequencing and to explore the potential mechanism of the gut microbiota’s association with diverse feeding habits.

The insect gut harbors a diverse array of functional microorganisms that warrant further exploration and utilization. However, there is currently a paucity of research reports on the discovery of protease-producing microorganisms with industrial application value in the gut. Here, we employed microbial culturing to screen and identify the protease-producing microorganisms in the gut extract of Gryllotalpa orientalis. Based on morphological, physiological, and biochemical characterization, 16S rRNA sequencing, as well as ANI and dDDH values of whole genome, the protease-producing strains isolated from the insect gut were identified as Priestia aryahattai DBM-1 and DX-4, P. megaterium DX-3, and Serratia surfactantfaciens DBM-5. According to whole-genome analysis, strain DBM-5, which exhibited the highest enzyme activity, possesses abundant membrane transport genes and carbohydrate metabolism enzymes. In contrast, strains DX-3 and DX-4 not only have the ability to hydrolyze proteins but also demonstrate the capability to hydrolyze plant materials. Furthermore, strains that are closely related tend to have similar metabolic product gene clusters in their genomes. The screening and identification of protease resources are essential for the subsequent development and utilization of gut functional microorganisms and genetic resources in insects.

An insect’s gut microbiome is an essential “organ” in their life cycle, playing a crucial role by aiding food digestion and nutrient absorption. This study employed both culture-independent and culture-dependent methods to explore the protease resources present in the gut of the omnivorous insect Gryllotalpa orientalis. The findings revealed that the gut extract of G. orientalis contained a diverse array of proteases, including cysteine proteases, pepsin, serine proteases, and trypsin, as well as some unidentified proteases. Furthermore, the protease gene htpX, derived from gut bacterium Priestia megaterium DX-3, has been cloned and recombinantly expressed. The recombinant DX-3-htpX protease exhibited a 61.9-fold increase in fermentation level compared to the DX-3 protease. This protease was characterized as a neutral, heat-resistant metalloprotease with an M48 peptidase domain, and it was observed that the binding of Ca²⁺ to the recombinant protease resulted in the formation of the largest active pocket. This study provides technical support for further development and utilization of functional protein resources in insect gut.

This paper describes two new species of the genus Xizicus Gorochov, 1993 (Tettigoniidae: Meconematini) from China (Guangxi and Sichuan), i.e. Xizicus (Xizicus) rectangulatus sp. nov., Xizicus (Haploxizicus) carinicaudatus sp. nov., reports Xizicus (Xizicus) daedalus Gorochov, 2011 from China for the first time. In addition, the first description of the female of Acosmetides dilobosa Chang, Wang & Shi, 2021 is presented.