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.

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.

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.

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.

First mitogenomic characterization of Macromotettixoides (Orthoptera, Tetrigidae), with the descriptions of two new species.

Classification of species is commonly based on morphological, molecular, and distribution depending on the taxa. Macromotettixoides Zheng, Wei & Jiang, 2005 and Formosatettix Tinkham, 1937 are both wingless types of Tetrigidae with extremely similar morphological characteristics, and in the current taxonomic system they are placed in two different subfamilies, Metrodorinae and Tetriginae, respectively. It is difficult to clearly identify the species of these two genera by morphological characteristics, and molecular data is often needed to assist identification. Here, the complete mitogenomes of two new species were sequenced and assembled, with that of Macromotettixoidesorthomargina. Molecular data of species of Formosatettix were used to test the monophyly of Macromotettixoides and to re-assess the generic characters, and also to test whether Macromotettixoides belongs to the Asian Metrodorinae or Tetriginae. Furthermore, mitochondrial characteristics were analyzed and the phylogeny of the Tetrigidae reconstructed based on mitochondrial protein-coding genes (PCGs). The results indicated that the two new species were clustered with Macromotettixoides rather than Formosatettix, and the anterior margin of the fastigium and pronotum of the two new species usually had the humeral angle different from that of Formosatettix. Therefore, after integrating morphological and molecular data, the two new species were placed in the genus Macromotettixoides, M.maoershanensissp. nov. and M.brachycornasp. nov. Finally, a phylogenetic reconstruction supported Macromotettixoides being assigned to Tetriginae rather than Metrodorinae, in contrast to the previous classification of this genus.

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.

Twenty-three new synonyms of the Eastern common groundhopper, Tetrixjaponica (Bolívar, 1887) (Orthoptera, Tetrigidae).

The Eastern common groundhopper, Tetrixjaponica, is a pygmy grasshopper species widely distributed in the Eastern Palearctic region, and shows a high degree of phenotypic variation. The classification of Tetrixjaponica is difficult and frequently involved errors. Among the many species of Tetrigidae that have been described in China within the last decades, many synonyms of Tetrixjaponica were found. The type specimens of many species deposited in the Chinese museums have been re-examined and as a result, Tetrixjaponica is systematically revised. Based on the results of this review, 23 new synonyms of Tetrixjaponica are proposed: Coptotettixcircinihumerus Zheng & Deng, 2004, syn. nov.; Coptotettixemeiensis Zheng, Lin & Zhang, 2012, syn. nov.; Euparatettixrongshuiensis Zheng, 2005, syn. nov.; Euparatettixzayuensis Zheng, Zeng & Ou, 2011, syn. nov.; Macromotettixnigritubercle Zheng & Jiang, 2006, syn. nov.; Macromotettixyaoshanensis Zheng & Jiang, 2000, syn. nov.; Tetrixalbistriatus Yao & Zheng, 2006, syn. nov.; Tetrixalbomaculatus Zheng & Jiang, 2006, syn. nov.; Tetrixalbomarginis Zheng & Nie, 2005, syn. nov.; Tetrixcenwanglaoshana Zheng, Jiang & Liu, 2005, syn. nov.; Tetrixcliva Zheng & Deng, 2004, syn. nov.; Tetrixduolunensis Zheng, 1996, syn. nov.; Tetrixgrossovalva Zheng, 1994, syn. nov.; Tetrixjiuwanshanensis Zheng, 2005, syn. nov.; Tetrixlatipalpa Cao & Zheng, 2011, syn. nov.; Tetrixliuwanshanensis Deng, Zheng & Wei, 2007, syn. nov.; Tetrixqinlingensis Zheng, Huo & Zhang, 2000, syn. nov.; Tetrixrectimargina Zheng & Jiang, 2004, syn. nov.; Tetrixruyuanensis Liang, 1998, syn. nov.; Tetrixxianensis Zheng, 1996, syn. nov.; Tetrixxinchengensis Deng, Zheng & Wei, 2007, syn. nov.; Tetrixyunlongensis Zheng & Mao, 2002, syn. nov.; Tetrixzhoushanensis Gao, Liu & Yin, 2022, syn. nov. It is expected that there will be the discoveries of more synonyms of this and other Tetriginae species from the Eastern Palearctic.

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.

Review of the Chinese species of the genus Scelimena Serville, 1838 (Tetrigidae: Scelimeninae: Scelimenini).

A brief preliminary revision of the genus Scelimena Serville, 1838 (Tetrigidae: Scelimeninae: Scelimenini) from PR China is presented. Scelimena pyrroma Lao, Kasalo, Gao, Deng et Skejo sp. nov. is described from Hainan, Ding'an, based on a female holotype and a male paratype, and an additional photograph of a female in the natural habitat from Wuzhi National Nature Reserve (Wuzhi-shan Mountain). Specimens of this new species were hitherto reported from Hainan as S. dentiumeris (Hancock, 1907), a species endemic to Borneo. The new species is easily distinguished from its congeners by the presence of prominent yellow humeral tubercles and by the lateral pronotal spines directed forwards. The new species from Hainan and S. chinensis (Hancock, 1915) from Vietnam are assigned to Scelimena producta species group based on the similarity with S. producta (Serville, 1838), which is characterized by the humeral angles with tubercles, wide vertex, and toothed ventral margins of hind femora, while S. boettcheri Günther, 1938 from Palawan is assigned to Scelimena discalis species group, based on the tuberculated median carina. Scelimena dammermanni Günther, 1938, stat. nov., hitherto regarded as a subspecies of S. producta, is elevated to a species level because it lacks ventral spines on the hind legs, present in all S. producta individuals. New synonymy is established: Scelimena melli Günther, 1938, =Scelimena brevispina Cao et Zheng, 2011, syn. nov.; =Scelimena wulingshana Zheng, 1993, syn. nov.; =Eufalconoides guizhouensis Zheng et Shi, 2006, syn. nov. Scelimena melli, S. guangxiensis Zheng et Jiang, 1994 from southern China and S. kempi (Hancock, 1915) from NE India are assigned to S. bellula species group on the basis of similarity to S. bellula Storozhenko et Dawwrueng, 2015. Further research should determine whether S. spicupennis Zheng & Ou, 2003 represents a valid species or a synonym of another Scelimena species, as well as if S. nitidogranulosa Günther, 1938 and S. guangxiensis Zheng & Jiang, 1994 represent separate species or whether they are conspecific. Specimens of S. nitidogranulosa from Hainan reported by Liang & Zheng in 1998 are in fact Platygavialidium sinicum Günther, 1939. Scelimena songkrana Zha et Wen, 2017 is recorded from China for the first time. A key to Scelimena species found in PR China is also provided.

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.

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.

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.

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.

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.

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.

Mitochondrial genomes of eight Scelimeninae species (Orthoptera) and their phylogenetic implications within Tetrigoidea.

Scelimeninae is a key member of the pygmy grasshopper community, and an important ecological indicator. No mitochondrial genomes of Scelimeninae have been reported to date, and the monophyly of Scelimeninae and its phylogenetic relationship within Tetrigidae is still unclear. We sequenced and analyzed eight nearly complete mitochondrial genomes representing eight genera of Scelimeninae. These mitogenomes ranged in size from 13,112 to 16,380 bp and the order of tRNA genes between COII and ATP8 was reversed compared with the ancestral order of insects. The protein-coding genes (PCGs) of tetrigid species mainly with the typical ATN codons and most terminated with complete (TAA or TAG) stop codons. Analyses of pairwise genetic distances showed that ATP8 was the least conserved gene within Tetrigidae, while COI was the most conserved. The longest intergenic spacer (IGS) region in the mitogenomes was always found between tRNASer(UCN) and ND1. Additionally, tandem repeat units were identified in the longest IGS of three mitogenomes. Maximum likelihood (ML) and Bayesian Inference (BI) analyses based on the two datasets supported the monophyly of Tetriginae. Scelimeninae was classified as a non-monophyletic subfamily.

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.