Background: DNA barcoding has been developed as a useful tool for species discrimination. Several sequence-based species delimitation methods, such as Barcode Index Number (BIN), REfined Single Linkage (RESL), Automatic Barcode Gap Discovery (ABGD), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP), were used. Our aim was to estimate Chinese katydid biodiversity using standard DNA barcode cytochrome c oxidase subunit I (COI-5P) sequences. Results: Detection of a barcoding gap by similarity-based analyses and clustering-base analyses indicated that 131 identified morphological species (morphospecies) were assigned to 196 BINs and were divided into four categories: (i) MATCH (83/131 = 64.89%), morphospecies were a perfect match between morphospecies and BINs (including 61 concordant BINs and 22 singleton BINs); (ii) MERGE (14/131 = 10.69%), morphospecies shared its unique BIN with other species; (iii) SPLIT (33/131 = 25.19%, when 22 singleton species were excluded, it rose to 33/109 = 30.28%), morphospecies were placed in more than one BIN; (iv) MIXTURE (4/131 = 5.34%), morphospecies showed a more complex partition involving both a merge and a split. Neighbor-joining (NJ) analyses showed that nearly all BINs and most morphospecies formed monophyletic cluster with little variation. The molecular operational taxonomic units (MOTUs) were defined considering only the more inclusive clades found by at least four of seven species delimitation methods. Our results robustly supported 61 of 109 (55.96%) morphospecies represented by more than one specimen, 159 of 213 (74.65%) concordant BINs, and 3 of 8 (37.5%) discordant BINs. Conclusions: Molecular species delimitation analyses generated a larger number of MOTUs compared with morphospecies. If these MOTU splits are proven to be true, Chinese katydids probably contain a seemingly large proportion of cryptic/undescribed taxa. Future amplification of additional molecular markers, particularly from the nuclear DNA, may be especially useful for specimens that were identified here as problematic taxa.

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.

Hagloidea Handlirsch, 1906 was an ancient group of Ensifera, that was much more diverse in the past extending at least into the Triassic, apparently diminishing in diversity through the Cretaceous, and now only represented by a few extant species. In this paper, we report the complete mitochondrial genome (mitogenome) of Tarragoilus diuturnus Gorochov, 2001, representing the first mitogenome of the superfamily Hagloidea. The size of the entire mitogenome of T. diuturnus is 16144 bp, containing 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and one control region. The order and orientation of the gene arrangement pattern is identical to that of D. yakuba and most ensiferans species. A phylogenomic analysis was carried out based on the concatenated dataset of 13 PCGs and 2 rRNA genes from mitogenome sequences of 15 ensiferan species, comprising four superfamilies Grylloidea, Tettigonioidae, Rhaphidophoroidea and Hagloidea. Both maximum likelihood and Bayesian inference analyses strongly support Hagloidea T. diuturnus and Rhaphidophoroidea Troglophilus neglectus as forming a monophyletic group, sister to the Tettigonioidea. The relationships among four superfamilies of Ensifera were (Grylloidea, (Tettigonioidea, (Hagloidea, Rhaphidophoroidea))).

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.