Chromosome-level genome assembly of the pygmy grasshopper Eucriotettix oculatus (Orthoptera: Tetrigoidea).

The pygmy grasshoppers, which belong to the superfamily Tetrigoidea, exhibit remarkable environmental adaptability. However, no study has yet reported a reference genome for this group. In this study, we assembled a high-quality chromosome-scale genome of Eucriotettix oculatus, which survive in the environment heavily polluted by heavy metals, achieved through Illumina and PacBio sequencing, alongside chromosome conformation capture techniques. The resulting genome spans 985.45 Mb across seven chromosomes (range: 71.55 to 266.65 Mb) and features an N50 length of 123.82 Mb. Chr5 is considered to be the single sex chromosome (X). This genome is composed of 46.42% repetitive elements and contains 14,906 predicted protein-coding genes, 91.63% of which are functionally annotated. Decoding the E. oculatus genome not only promotes future studies on environmental adaptation for the pygmy grasshopper, but also provides valuable resources for in-depth investigation on phylogeny, evolution, and behavior of Orthoptera.

Molecular strategies of the pygmy grasshopper Eucriotettix oculatus adapting to long-term heavy metal pollution.

To study the heavy metal accumulation and its impact on insect exterior and chromosome morphology, and reveal the molecular mechanism of insects adapting to long-term heavy metal compound pollution habitats, this study, in the Diaojiang river basin, which has been polluted by heavy metals(HMs) for nearly a thousand years, two Eucriotettix oculatus populations was collected from mining and non-mining areas. It was found that the contents of 7 heavy metals (As, Cd, Pb, Zn, Cu, Sn, Sb) in E. oculatus of the mining area were higher than that in the non-mining 1-11 times. The analysis of morphology shows that the external morphology, the hind wing type and the chromosomal morphology of E. oculatus are significant differences between the two populations. Based on the heavy metal accumulation，morphological change, and stable population density, it is inferred that the mining area population has been affected by heavy metals and has adapted to the environment of heavy metals pollution. Then, by analyzing the transcriptome of the two populations, it was found that the digestion, immunity, excretion, endocrine, nerve, circulation, reproductive and other systems and lysosomes, endoplasmic reticulum and other cell structure-related gene expression were suppressed. This shows that the functions of the above-mentioned related systems of E. oculatus are inhibited by heavy metal stress. However, it has also been found that through the significant up-regulation of genes related to the above system, such as ATP2B, pepsin A, ubiquitin, AQP1, ACOX, ATPeV0A, SEC61A, CANX, ALDH7A1, DLD, aceE, Hsp40, and catalase, etc., and the down-regulation of MAPK signalling pathway genes, can enhanced nutrient absorption, improve energy metabolism, repair damaged cells and degrade abnormal proteins, maintain the stability of cells and systems, and resist heavy metal damage so that E. oculatus can adapt to the environment of heavy metal pollution for a long time.

Chromosomal-Level Reference Genome for the Chinese Endemic Pygmy Grasshopper, Zhengitettix transpicula, Sheds Light on Tetrigidae Evolution and Advancing Conservation Efforts.

The pygmy grasshopper, Zhengitettix transpicula, is a Chinese endemic species with an exceedingly limited distribution and fragile population structure, rendering it vulnerable to extinction. We present a high-continuity, chromosome-scale reference genome assembly to elucidate this species' distinctive biology and inform conservation. Employing an integrated sequencing approach, we achieved a 970.40 Mb assembly with 96.32% coverage across seven pseudo-chromosomes and impressive continuity (N50 > 220 Mb). Genome annotation achieves identification with 99.2% BUSCO completeness, supporting quality. Comparative analyses with 14 genomes from Orthoptera-facilitated phylogenomics and revealed 549 significantly expanded gene families in Z. transpicula associated with metabolism, stress response, and development. However, genomic analysis exposed remarkably low heterozygosity (0.02%), implying a severe genetic bottleneck from small, fragmented populations, characteristic of species vulnerable to extinction from environmental disruptions. Elucidating the genetic basis of population dynamics and specialization provides an imperative guideline for habitat conservation and restoration of this rare organism. Moreover, divergent evolution analysis of the CYP305m2 gene regulating locust aggregation highlighted potential structural and hence functional variations between Acrididae and Tetrigidae. Our chromosomal genomic characterization of Z. transpicula advances Orthopteran resources, establishing a framework for evolutionary developmental explorations and applied conservation genomics, reversing the trajectory of this unique grasshopper lineage towards oblivion.

Reassessment of the Phylogenetics of Two Pygmy Grasshopper Generic Groups Tetrix and Systolederus through Mitochondrial Phylogenomics Using Four New Mitochondrial Genome Assemblies.

Mitochondrial genomes offer pragmatic genetic markers to reconstruct evolutionary relationships and inform taxonomic classifications. Here, we present complete mitochondrial sequences for four Chinese pygmy grasshoppers (Tetrigidae), aiming to reevaluate phylogenetic patterns and morphological taxonomy. Our 17,643 bp, 16,274 bp, 15,086 bp, and 15,398 bp mitogenomes of Exothotettix guangxiensis, Formosatettix longwangshanensis, Euparatettix sinufemoralis and Systolederus zhengi, respectively, exhibit archetypal Tetrigidae architecture. We constructed phylogenies using 13 protein-coding loci from 39 Tetrigidae mitogenomes, revealing several genus-level clusters with statistically solid support, conflicts regarding Ex. guangxiensis, F. longwangshanensis merging into Tetrix, and two subclades of Systolederus. The dated divergence analysis indicates over 150 Mya of Tetrigidae ancestry, tracing the Systolederus generic group splits up to ~75 million years ago. Moreover, the Tetrix generic group radiated over 14 Mya across vast distributions, consistent with rapid adaptive dispersals. Our mitochondrial reconstructions suggest that Synstolederus is taxonomically overextended for a single genus, while the distinctiveness of Ex. guangxiensis and F. longwangshanensis from Tetrix appears questionable, and the Tetrix generic group comprises a potential tRNA-Ile coding region. Our integrative mitogenomic approaches will help resolve issues stemming from morphological taxonomy that is reliant on traits that are prone to convergence. This investigation enhances comprehension of Tetrigidae phylogeny and accentuates molecular systematics.

On the specific status of Scelimenaspicupennis and a new record of S.discalis from China with mitochondrial genome characterization (Orthoptera, Tetrigidae).

The genus Scelimena Serville (Orthoptera: Tetrigidae) from China is reviewed. One species, Scelimenaspicupennis Zheng & Ou, 2003 (China: Yunnan) is redescribed, and a new record of Scelimenadiscalis (Hancock, 1915) from China is given. An annotated identification key for Chinese species of the genus Scelimena is provided. Mitochondrial genes of S.spicupennis and S.discalis were sequenced and annotated. The sizes of the two sequenced mitogenomes are 17,552 bp (S.discalis), and 16,069 bp (S.spicupennis), respectively. All of the PCGs started with the typical ATN (ATT, ATC or ATG) or TTG codon and most ended with complete TAA or TAG codon, with the exception of the ND5 gene, which terminated with an incomplete T. The mitochondrial genomes for these two recorded species are provided, and the constructed phylogenetic tree supports their morphological taxonomic classification. The topology of the phylogenetic tree showed that three species of Scelimena were clustered into one branch and formed a monophyletic and a holophyletic group.

Review of the genus Formosatettix Tinkham (Orthoptera: Tetrigidae) with description of new taxa, new synonymy and an updated key to species.

The genus Formosatettix Tinkham is reviewed. Seven new species from China, Formosatettix cliva Deng, sp. nov., Formosatettix guangyuanensis Deng, sp. nov., Formosatettix shuimogouensis Deng, sp. nov., Formosatettix strictivertex Deng, sp. nov., Formosatettix tangjiaheensis Deng, sp. nov., Formosatettix yueqingensis Deng, sp. nov., Formosatettix zheminzhengi Deng, sp. nov. are described and illustrated. One new name is proposed: Formosatettix latifemurus Deng, nom. nov. One new combination is established: Formosatettix nyalamensis (Zheng & Lin, 2015), comb. nov. The following new synonyms are established: Bolivaritettix circocephalus Zheng, 1992 = Formosatettix torulosinota Zheng & Mao, 2002, syn. nov., Criotettix bispinosus (Dalman, 1818) = Formosatettix hainanensis Zheng, 2012, syn. nov., Epitettix guangxiensis (Zheng & Jiang, 1994) = Formosatettix guangxiensis Zheng & Jiang, 1998, syn. nov., Formosatettix longwangshanensis Zheng, 1998 = Formosatettix tianmushanensis Zheng & Li, 2001, syn. nov., Formosatettix serrifemora Deng, 2019 = Formosatettix wulongensis Zha & Ding, 2020, syn. nov., Formosatettix huapingensis Zheng & Jiang, 1997 = Formosatettix nanlingensis Zheng & Cao, 2011, syn. nov. = Formosatettix undulatifemura, Zheng, 2012, syn. nov. = Formosatettix guposhanensis Deng, 2019, syn. nov. In addition, Formosatettix leigongshanensis Zha & Ding, 2020 is briefly commented.

Pygmy grasshoppers of the genus Xistra Bolvar, 1887 (Orthoptera: Tetrigidae: Metrodorinae).

The genus Xistra Bolvar is reviewed. Two new species from China, Xistra hainanensis Deng, sp. nov. and Xistra nigriabdominis Deng, sp. nov. are described and illustrated. Three new combinations are established: Xistra emeiensis (Zheng, 1998), comb. nov., Xistra guangxiensis (Zheng, 1998), comb. nov. and Xistra lochengensis (Zheng, 2005), comb. nov. Four new synonyms are established: Bannatettix ruiliensis Zheng, 1993 = Xistra klinnema Zheng Zeng, 2011, syn. nov., Systolederus emeiensis Zheng, 1998 = Xistra yaanensis Zheng, 2009, syn. nov., Systolederus guangxiensis Zheng, 1998 = Xistra longzhouensis Zheng Jiang, 1998, syn. nov., Mazarredia lochengensis Zheng, 2005 = Xistra oculata Li, Deng Zheng, 2014, syn. nov. In addition, an updated key to all species of the genus Xistra is given.

Two new species of the genus Zhengitettix (Orthoptera: Tetrigidae: Scelimeninae) from China.

New species, Zhengitettix convexa Deng , sp. nov. from Yunnan and Zhengitettix fengyangshana Deng, sp. nov. from Zhejiang, are described and illustrated. Z. convexa can be easily distinguished from other species of the genus by the disc of pronotum slightly convex between shoulders and interspersed with sparse carinae on the protuberance, slightly concave behind shoulders. Z. fengyangshana is similar to Z. transpicula Zheng Jiang, 2002, but differs in narrow frontal ridge, with interhumeral carinae, shape of median carina of pronotum and posterior angles of pronotal lateral lobes. An updated key to all species of the genus Zhengitettix is given.

New genus and new species of leaf-mimic pygmy grasshoppers from China (Orthoptera: Tetrigidae: Cladonotinae).

Many species of pygmy grasshoppers (Tetrigidae) exhibit leaf-like appearance, leaf-mimic pygmy grasshoppers mainly characterized by having high pronotal crest (= elevated median carina). A new genus of leaf-mimic Cladonotinae, Deltonotusoides gen. nov., and a new species, Deltonotusoides strictivertex sp. nov. are described from Motuo, Xizang, China.

The complete chloroplast genome of Saussurella borneensis (Orthoptera: Tetrigoidea) from China and its phylogenetic analysis.

The mitochondrial genome (mitogenome) of Saussurella borneensis (Orthoptera: Tetrigoidea) was determined and analyzed. The complete mitochondrial genome is 16,006 bp in length, consisting of 37 genes, including 13 protein-coding (PCGs), 22 tRNA, 2 rRNA genes as well as an A + T-rich region. Ten PCGs initiated with a typical ATN codon (one with ATC, two with ATA, two with ATT, and five with ATG) and 13 terminated with complete stop codons. The overall nucleotide composition was 42.97% for A, 17.61% for C, 11.62% for G, and 27.8% for T. Phylogenetic analysis of S. borneensis fully resolved it in a basal branch sister to Tripetaloceroides tonkinensis. This data increase the bioinformatics of the Tetrigidae, and improves our understanding of the phylogenetic status of S. borneensis in the Tetrigoidea.

Effect of heavy metals pollution on the composition and diversity of the intestinal microbial community of a pygmy grasshopper (Eucriotettix oculatus).

Heavy metal pollution in the environment is mostly manifested as a multi-elemental compound pollution. The effect of the long-term exposure to heavy metal pollution on the gut microbes of insects has remained unknown. For the current work, the population of Eucriotettix oculatus living in mining areas around the Diaojiang River with a history of hundreds of years of pollution, was selected along with the similar species living in non-mining areas to conduct a comparative study of their gut microbes. The microbial communities were analyzed using 16S rRNA amplicon sequencing. The results showed Proteobacteria to be dominant among gut microbes of E. oculatus, but the abundance of Proteobacteria was significantly increased when the insects were exposed to the environment with heavy metal pollution. The symbiotic bacteria belonging to genus Wolbachia were found to be dominant among the insect population from the non-mining area group, while the pathogenic bacteria belonging to Aeromonas were dominant among the insect population of the mining area group. The diversity analysis showed that the gut microbial community diversity of E. oculatus was reduced in the heavy metal pollution habitat. The analysis of the differences in the gut microbial population and metabolic pathways of the two groups showed that the heavy metal pollution caused the increase in pathogenic bacteria among the gut microbes of E. oculatus, which might have a negative impact on the health of the host. At the same time, probiotics and the beneficial metabolism pathways were also found to increase and enhance, helping the host to resist the damage caused by heavy metal stress. This might be one of the strategies used by E. oculatus to adapt to heavy metal pollution.

Description of a new species of the genus Pseudepitettix Zheng (Orthoptera: Tetrigoidea: Cladonotinae) from China.

Pseudepitettix convexa Deng, sp. nov. is described with detailed illustrations of external morphology. Comparison between new species and other species of the genus Pseudepitettix Zheng, 1995 is compiled into a table for identification. An updated key to species of the genus is given.

Characterization of the complete mitochondrial genome of Tripetaloceroides tonkinensis (Orthoptera: Tetrigoidea) from China and its phylogenetic analysis.

The mitochondrial genome (mitogenome) of the Tripetaloceroides tonkinensis (Orthoptera: Tetrigoidea) was sequenced and annotated. The complete mitogenome has a length of 16,696 bp and consists of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a A + T-rich region. Thirteen PCGs started with typical ATN codon and ended with complete stop codons (five with TAG, eight with TAA). The overall nucleotide composition was 42.7% of A, 10.34% of G, 25.87% of T, and 21.08% of C. The phylogenetic trees in the current study confirmed that T. tonkinensis was clustered with other Tetrigoidea species, and the study would improve our understanding for the mitogenomes of Tetrigoidea.

New genus and new species of the subfamily Metrodorinae from China (Orthoptera: Tetrigidae).

The genus Concavetettix Deng, gen. nov. (type species: Concavetettix yunnanensis Deng, sp. nov.) is described from Daweishan, Pingbian County, Yunnan Province, China. The genus Macromotettix Günther, 1939 is reviewed. Three new species of the genus, M. microptera Deng, sp. nov., M. zhengi Deng, sp. nov. and M. brachyptera Deng, sp. nov. are described with detailed illustrations of external morphology. One new name is proposed: Macromotettix napoensis Deng, nom. nov.. Additionally, an updated key to species of the genus Macromotettix is given.

First record of mitochondrial genome of Teredorus nigropennis (Orthoptera: Tetrigidae) and phylogenetic analysis.

The nearly complete mitochondrial genome (mitogenome) of Teredorus nigropennis was determined and analyzed. This mitogenome was 14,652 bp in size and encoded 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes. The most common start codon is ATN, the most common termination codon is TAA and two genes have incomplete termination codon T (TA). The overall nucleotide composition was 45.2% of A, 10.2% of G, 28.6% of T, and 16.1% of C. The data will increase the basic information of Tetrigidae phylogenetic research and can help to better understand the phylogenetic status of T. nigropennis in Tetrigiodea.

Sequence and phylogenetic analysis of the mitochondrial genome for the groundhopper Mazarredia convexa (Orthoptera: Tetrigidae).

Using next generation sequencing (NGS), we determined the mitochondrial genome of Mazarredia convexa. The assembled mitochondrial genome of M. convexa was found to be 15,089 bp, consisting of 37 genes (13 protein-coding genes, 22 tRNA genes, and two rRNA genes). The region that we failed to sequence was between rrnS and trnI, and generally contained a putative AT-rich region. Its gene composition and order were similar to all reported tetrigid species. The overall nucleotide composition was 43.4% of A, 30.6% of T, 9.4% of G, and 16.6% of C. The data can help to better understand the phylogenetic status of M. convexa in Tetrigidae.

Molecular data provide new insights into the phylogeny of Cladonotinae (Orthoptera: Tetrigoidea) from China with the description of a new genus and species.

Considerable effort has been devoted to elucidating the phylogenetic relationships of tetrigides. However, there is still no commonly accepted phylogenetic hypothesis. Therefore, the phylogenetic relationships among some subfamilies remain unclear; e.g., Cladonotinae is a controversial group, in which the phylogenetic relationships between genera and the boundaries of some of the included genera are unclear, causing some of the taxa to be difficult to identify. Therefore, an in-depth phylogenetic analysis of Cladonotinae is urgently needed. In this study, a robust phylogenetic framework for the tetrigides was reconstructed based on the combined mitochondrial cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and nuclear 18S ribosomal RNA (18S rRNA) gene sequences of 25 species belonging to 16 genera of Tetrigoidea from China, which included 13 species from 8 genera of Cladonotinae. Phylogenetic inferences were performed using the combined dataset and Bayesian inference (BI) and Maximum Parsimony (MP) methods, and the phylogenetic tree of Cladonotinae was reconstructed. All inferences based on the results of the present study supported the Cladonotinae subfamily as a polyphyletic group; within the Cladonotinae subfamily, Tetradinodula, and Tuberfemurus were closely related to Tetriginae, while Austrohancockia and Gibbotettix showed a close relationship to the Scelimenidae subfamily. Additionally, a new genus and new species of the Cladonotinae subfamily are described and illustrated: Hainantettix Deng, gen. nov. and Hainantettix strictivertex Deng, sp. nov.

Taxonomic revision of the subfamily Cladonotinae (Orthoptera: Tetrigidae) from China with description of three new species.

The Chinese genera and species of the subfamily Cladonotinae are reviewed. A checklist is given for all genera and species. Three new species are described from China: Pseudepitettix hainanensis sp. nov. (Hainan prov.), Pseudepitettix strictivertex sp. nov. (Hainan prov.) and Tuberfemurus convexa sp. nov. (Zhejiang prov.). New synonymies are proposed: Deltonotus hainanensis Zheng Liang, 1985 = Deltonotus wuzhishanensis Lin Zheng, 2014, syn. nov., Gibbotettix emeiensis Zheng, 1992 = Gibbotettix serrifemura Deng, Zheng Wei, 2016, syn. nov., Gibbotettix emeiensis Zheng, 1992 = Gibbotettix longivalva Zha Li, 2018, syn. nov., Pseudepitettix nigritibis Zheng Jiang, 2000 = Tuberfemurus liboensis Deng, Zheng Wei, 2009, syn. nov., Yunnantettix bannaensis Zheng, 1995 = Yunnantettix variabilis Zha Wen, 2016, syn. nov., Yunnantettix thaicus Storozhenko Pushkar, 2015 = Yunnantettix nanensis Zha Wen, 2015, syn. nov. Four new combinations are established: Epitettix guangxiensis (Zheng Jiang, 1994), comb. nov., Epitettix yunnanensis (Zheng, 1995), comb. nov., Gibbotettix undatimarginis (Deng Zheng, 2012), comb. nov., Pseudepitettix torulosinota (Zheng Lin, 2016), comb. nov.

New species and new synonyms of Macromotettixoides (Orthoptera: Tetrigidae) with an updated key.

The genus Macromotettixoides Zheng, Wei Jiang, 2005 is reviewed. Four new species of the genus, M. daiyunshanensis Deng, sp. nov., M. curvicarina Deng, sp. nov., M. convexa Deng, sp. nov. and M. shengtangshanensis Deng, sp. nov. are described with detailed illustrations of external morphology. Two new synonyms are established: M. jinggangshanensis, syn. nov. is synonymized with M. jiuwanshanensis Zheng, Wei Jiang, 2005; M. parvula Zha Wen, 2017, syn. nov. is synonymized with M. undulatifemura Deng, Zheng Yang, 2012. Additionally, an updated key to species of the genus is given.

Characterization of the complete mitochondrial genome of Ergatettix serrifemora (Orthoptera: Tetrigidae) from China and its phylogenetic analysis.

The mitochondrial genome (mitogenome) of the Ergatettix serrifemora (Orthoptera: Tetrigidae: Tetriginae) was sequenced and annotated. The assembled mitochondrial genome was 14,947 bp, containing 45.8% of A, 15.7% of C, 9.6% of G and 28.9% of T, respectively, which is the classical structure for insect mitogenome. The region that we failed to sequence was between rrnS and trnI, and generally contained a putative AT-rich region. Twelve PCGs started with typical ATN codon and eleven ended with complete stop codons (three with TAG, eight with TAA). The phylogenetic trees in the current study confirmed that E. serrifemora was clustered with other Tetriginae species, and this study would improve our understanding for the mitogenomes of Tetrigoidea.