Type catalogue of Oedipodinae (Orthoptera: Acrididae) present in Naturalis Biodiversity Center Leiden (Netherlands).

A type catalogue of Oedipodinae in the collection of Naturalis Biodiversity Center is presented altogether 82 type specimens including 13 primary types and 5 junior synonyms: holotypes (4 species), neotype (1 species), lectotypes (2 species, 1 subspecies), and syntypes (5 species). Furthermore 50 additional secondary type specimens were recorded. Here, we present the full type material catalogue including a locality map of all species and pictures of the 15 primary type species.

Amblytropidia paranaensis n. sp., a new species of grasshopper (Orthoptera: Acrididae: Gomphocerinae) from Paran State, Brazil, including new records, a distribution map and a key to the Brazilians species.

A new species of Amblytropidia Stl, 1873 (Orthoptera, Acrididae, Gomphocerinae), named Amblytropidia paranaensis n. sp., is delineated from the Trs Barras do Paran municipality, Paran State, Brazil. Detailed morphological delineations, accompanied by illustrations highlighting key diagnostic traits of both male and female external genitalia, are provided. Amblytropidia paranaensis n. sp. is distinguished from its congeners by distinctive body coloration, particularly evident in the head and thorax regions, as well as larger-sized anchorae compared to anterior projections, and uniquely trilobated and sclerotized lophi. Moreover, this study offers insights into the species habitat preferences, documented occurrences, a distribution map, and an updated taxonomy key specific to Brazilian Amblytropidia species.

Pseudoscopas carbonelli n. sp. (Orthoptera: Acrididae: Melanoplinae) from southern Brazil, including chromosome complement.

New species of Pseudoscopas (Orthoptera, Acrididae, Melanoplinae). Pseudoscopas carbonelli n. sp. from São Francisco de Paula, State of Rio Grande do Sul, Brazil is described, and a key to the species of Pseudoscopas Hebard, 1931 is added. Morphological descriptions are provided together with illustrations emphasizing the most significant diagnostic features of external morphology and male genitalia. Pseudoscopas carbonelli n. sp. differs from the other species known by epiproct with six protuberances in the median region, and extremities of the apical region of the epiproct sclerotized, as well as the lophi with sclerotization. Chromosome analyses were performed using standard staining procedures, showing diploid number of 2n = 23, X0♂/24, XX♀, and the karyotype made up of exclusively acrocentric chromosomes, including a medium-size megameric chromosome. Information is given about type specimens, material examined and geographic distribution.

A revision and first record of the genus Oxyina Hollis, 1975 (Acrididae: Oxyinae) from India, with description of a new species.

The genus Oxyina make up a total of three known species and a new species. In the present study an extensive survey was carried out from August, 2018 to September, 2020. The collected specimens comprehend all the three species viz., Oxyina bidentata, Oxyina sinobidentata, Oxyina javana and a new species: Oxyina kashmira n. sp. is illustrated. Oxyina javana is rediscovered, as this species has never been reported since its original description. On account of this, genus Oxyina now includes four species. Moreover the genus Oxyina is being reported first time from India. Principally based on genitalic characters, a brief diagnosis and a key to the species is provided.

Taxonomic review of the genus Assamacris (Orthoptera: Acrididae: Catantopinae) with a new species and a newly discovered female.

Taxonomic review of the genus Assamacris Uvarov, 1942 is given. The genus Traulitonkinacris You Bi, 1983 is synonymized with Assamacris. Traulitonkinacris bifurcatus You Bi, 1983 is transferred to Assamacris as a new combination. Assamacris splendida sp. nov. is described as new species. A newly discovered female of A. curticerca (Huang, 1981) is introduced. A key to the species is updated.

On the exact position of the tribe Incolacridini in the modern classification of grasshoppers (Orthoptera: Acrididae).

Incolacridinae is considered as a separate subfamily, not as a tribe of subfamily Catantopinae. It consists of four genera: Incolacris C. Willemse, 1932, stat. resurr., Stolzia C. Willemse, 1930, Bettotania C. Willemse, 1933, and Asymmetritania gen. nov. (type species: Stolzia vietnamensis Storozhenko, 2020). New combinations are proposed: Incolacris jianfengensis (Zheng et Ma, 1989), comb. nov., Asymmetritania asymmetrica (Ingrisch, 1989), comb. nov., and A. vietnamensis (Storozhenko, 2020), comb. nov. The diagnosis of subfamily and genera, a key to genera and an annotated list of the species of Incolacridinae are also given.

Molecular phylogeny of the grasshopper family Pyrgomorphidae (Caelifera, Orthoptera) reveals rampant paraphyly and convergence of traditionally used taxonomic characters.

The grasshopper family Pyrgomorphidae is one of the most colorful orthopteran lineages, and includes biologically fascinating and culturally important species. Recent attempts to reconstruct the phylogeny of this family have resulted in a large degree of conflicts between a morphology-based study and a molecular-based study, mainly due to convergent morphological traits that affected phylogenetic reconstruction. In this study, a molecular phylogeny of Pyrgomorphidae based on 32 ingroup species and mitochondrial genome data is proposed, which is used to test the monophyly of the taxonomic groupings used in the current classification scheme. Using the ancestral character state reconstruction analyses and character mapping, we demonstrate that some of the morphological characters, including the male genitalia, which were considered to be taxonomically important, have evolved convergently across the phylogeny. We discuss the discrepancies between our phylogeny and the previous studies and propose an approach to establish a natural classification scheme for Pyrgomorphidae.

Songs and morphology in grasshoppers of the Stenobothrus eurasius group (Orthoptera: Acrdidae: Gomphocerinae) from Russia and adjacent countries: clarifying of taxonomic status.

On the basis of the song and morphological analyses, we revised the status of the subspecies Stenobothrus eurasius eurasius Zubowsky, 1898, and S. eurasius hyalosuperficies Vorontsovskii, 1927. The status of the subspecies S. eurasius hyalosuperficies Vor. has been changed to the species level. The most striking difference between S. eurasius and S. hyalosuperficies lies in the song parameters. The calling songs differ not only in temporal parameters but are also produced by different mechanisms. S. eurasius generates calling songs by common leg stridulation, whereas S. hyalosuperficies produces sound by wing clapping. The courtship songs of both species are complex (contain several elements) and very different in temporal structure. The morphological differences between the two species are not as striking as the differences in bioacoustics: we found the only differences in the hind wing venation. At the same time, we suggest these differences to be important since they might be due to different mechanisms of sound production. We revised the ranges of the two species on the territory of Ukraine, Russia and Kazakhstan. We also reviewed the type localities of S. eurasius Zub. and designated lectotype and paralectotype of this species.

Demographic consequences of dispersal-related trait shift in two recently diverged taxa of montane grasshoppers.

Although the pervasiveness of intraspecific wing-size polymorphism and transitions to flightlessness have long captivated biologists, the demographic outcomes of shifts in dispersal ability are not yet well understood and have been seldom studied at early stages of diversification. Here, we use genomic data to infer the consequences of dispersal-related trait variation in the taxonomically controversial short-winged (Chorthippus corsicus corsicus) and long-winged (Chorthippus corsicus pascuorum) Corsican grasshoppers. Our analyses revealed lack of contemporary hybridization between sympatric long- and short-winged forms and phylogenomic reconstructions supported their taxonomic distinctiveness, rejecting the hypothesis of intraspecific wing polymorphism. Statistical evaluation of alternative models of speciation strongly supported a scenario of Pleistocene divergence (<1.5 Ma) with ancestral gene flow. According to neutral expectations from differences in dispersal capacity, historical effective migration rates from the long- to the short-winged taxon were threefold higher than in the opposite direction. Although populations of the two taxa present a marked genetic structure and have experienced parallel demographic histories, our coalescent-based analyses suggest that reduced dispersal has fueled diversification in the short-winged C. c. corsicus. Collectively, our study illustrates how dispersal reduction can speed up geographical diversification and increase the opportunity for allopatric speciation in topographically complex landscapes.

Selection for functional performance in the evolution of cuticle hardening mechanisms in insects.

Calcified tissues have repeatedly evolved in many animal lineages and show a tremendous diversity of forms and functions. The cuticle of many insects is enriched with elements other than Calcium, a strategy of hardening that is taxonomically widespread but apparently poorly variable among clades. Here, we investigate the evolutionary potential of the enrichment with metals in insect cuticle at different biological levels. We combined experimental evidence of Zinc content variation in the mandibles of a target species (Chorthippus cazurroi [Bolívar]) with phylogenetic comparative analyses among grasshopper species. We found that mandibular Zinc content was repeatable among related individuals and was associated with an indicator of fitness, so there was potential for adaptive variation. Among species, Zinc enrichment evolved as a consequence of environmental and dietary influences on the physical function of the jaw (cutting and chewing), suggesting a role of natural selection in environmental fit. However, there were also important within and transgenerational environmental sources of similarity among individuals. These environmental influences, along with the tight relationship with biomechanics, may limit the potential for diversification of this hardening mechanism. This work provides novel insights into the diversification of biological structures and the link between evolutionary capacity and intra- and interspecific variation.

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world's poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Phylogenomic analysis sheds light on the evolutionary pathways towards acoustic communication in Orthoptera.

Acoustic communication is enabled by the evolution of specialised hearing and sound producing organs. In this study, we performed a large-scale macroevolutionary study to understand how both hearing and sound production evolved and affected diversification in the insect order Orthoptera, which includes many familiar singing insects, such as crickets, katydids, and grasshoppers. Using phylogenomic data, we firmly establish phylogenetic relationships among the major lineages and divergence time estimates within Orthoptera, as well as the lineage-specific and dynamic patterns of evolution for hearing and sound producing organs. In the suborder Ensifera, we infer that forewing-based stridulation and tibial tympanal ears co-evolved, but in the suborder Caelifera, abdominal tympanal ears first evolved in a non-sexual context, and later co-opted for sexual signalling when sound producing organs evolved. However, we find little evidence that the evolution of hearing and sound producing organs increased diversification rates in those lineages with known acoustic communication.

Phylogeny and species delimitation of the genus Longgenacris and Fruhstorferiola viridifemorata species group (Orthoptera: Acrididae: Melanoplinae) based on molecular evidence.

Phylogenetic positions of the genus Longgenacris and one of its members, i.e. L. rufiantennus are controversial. The species boundaries within both of L. rufiantennus+Fruhstorferiola tonkinensis and F. viridifemorata species groups are unclear. In this study, we explored the phylogenetic positions of the genus Longgenacris and the species L. rufiantennus and the relationships among F. viridifemorata group based on the 658-base fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI) barcode and the complete sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the nuclear ribosomal DNA. The phylogenies were reconstructed in maximum likelihood framework using IQ-TREE. K2P distances were used to assess the overlap range between intraspecific variation and interspecific divergence. Phylogenetic species concept and NJ tree, K2P distance, the statistical parsimony network as well as the generalized mixed Yule coalescent model (GMYC) were employed to delimitate the species boundaries in L. rufiantennus+F. tonkinensis and F. viridifemorata species groups. The results demonstrated that the genus Longgenacris should be placed in the subfamily Melanoplinae but not Catantopinae, and L. rufiantennus should be a member of the genus Fruhstorferiola but not Longgenacris. Species boundary delimitation confirmed the presence of oversplitting in L. rufiantennus+F. tonkinensis and F. viridifemorata species groups and suggested that each group should be treated as a single species.

Genomic footprints of an old affair: Single nucleotide polymorphism data reveal historical hybridization and the subsequent evolution of reproductive barriers in two recently diverged grasshoppers with partly overlapping distributions.

Secondary contact in close relatives can result in hybridization and the admixture of previously isolated gene pools. However, after an initial period of hybridization, reproductive isolation can evolve through different processes and lead to the interruption of gene flow and the completion of the speciation process. Omocestus minutissimus and O. uhagonii are two closely related grasshoppers with partially overlapping distributions in the Central System mountains of the Iberian Peninsula. To analyse spatial patterns of historical and/or contemporary hybridization between these two taxa and understand how species boundaries are maintained in the region of secondary contact, we sampled sympatric and allopatric populations of the two species and obtained genome-wide single nucleotide polymorphism data using a restriction site-associated DNA sequencing approach. We used Bayesian clustering analyses to test the hypothesis of contemporary hybridization in sympatric populations and employed a suite of phylogenomic approaches and a coalescent-based simulation framework to evaluate alternative hypothetical scenarios of interspecific gene flow. Our analyses rejected the hypothesis of contemporary hybridization but revealed past introgression in the area where the distributions of the two species overlap. Overall, these results point to a scenario of historical gene flow after secondary contact followed by the evolution of reproductive isolation that currently prevents hybridization among sympatric populations.

Comparative analysis of mitogenomes among six species of grasshoppers (Orthoptera: Acridoidea: Catantopidae) and their phylogenetic implications in wing-type evolution.

The degree of wing development has a close relationship with insects' movement ability and range, and it should also be closely related to mitochondrial-related genes. The complete mitochondrial genomes of six species of Catantopidae were sequenced, annotated and analyzed. Then, combined with 37 mitogenomes of grasshoppers, the ratio of nonsynonymous substitution to synonymous substitution (Ka/Ks) of the combined sequences of protein coding genes (PCGs) was calculated by DnaSP5, and the phylogenetic relationships were reconstructed by maximum likelihood (ML) and Bayesian (BI) methods based on PCGs+rRNAs. The results showed that the sizes of the six complete mitogenomes are Stenocatantops mistshenkoi Willemse F., 1968, 15,573 bp; Traulia lofaoshana Tinkham, 1940, 15,645 bp; Sinopodisma rostellocerca You, 1980, 15,622 bp; Anapodisma miramae Dovnar-Zapolskij, 1932, 15,189 bp; Qinlingacris elaeodes Yin & Chou, 1979, 15,221 bp; and Eozubovskya planicaudata Zhang & Jin, 1985, 15,830 bp; their structures are the same as those of Acridoidea. The AT bias of the wing-degenerated group (lobiform and apterous) is higher than that of the longipennate group, and more nonsynonymous substitutions accumulated in the wing-degenerated group than in the longipennate group (P = 0.000), which indicates that the wing-degenerated group has undergone weaker evolutionary selection than the longipinnate group. The phylogenetic tree shows that the wing-degenerated group in the Catantopidae are multiorigin and present parallel evolution.

An alpine grasshopper radiation older than the mountains, on Ka Tiritiri o te Moana (Southern Alps) of Aotearoa (New Zealand).

In New Zealand, 13 flightless species of endemic grasshopper are associated with alpine habitats and freeze tolerance. We examined the phylogenetic relationships of the New Zealand species and a subset of Australian alpine grasshoppers using DNA sequences from the entire mitochondrial genome, nuclear 45S rRNA and Histone H3 and H4 loci. Within our sampling, the New Zealand alpine taxa are monophyletic and sister to a pair of alpine Tasmanian grasshoppers. We used six Orthopteran fossils to calibrate a molecular clock analysis to infer that the most recent common ancestor of New Zealand and Tasmanian grasshoppers existed about 20 million years ago, before alpine habitat was available in New Zealand. We inferred a radiation of New Zealand grasshoppers ~13-15 Mya, suggesting alpine species diversification occurred in New Zealand well before the Southern Alps were formed by the mountain building events of the Kaikoura Orogeny 2-5 Mya. This would suggest that either the ancestors of today's New Zealand grasshoppers were not dependent on living in the alpine zone, or they diversified outside of New Zealand.

Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera).

Acrididae family is characterized by diverse phylogenetic uncertainties, with different paraphyletic subfamilies. This study characterized the mitogenome of the grasshopper Rhammatocerus brasiliensis and determined its phylogenetic position in the family Acrididae. Sequencing was performed on an Illumina platform. The Short Oligonucleotide Analysis Package (SOAP) was used for genome assembly and the MITOS Web Server for annotation. Phylogenetic analysis was performed using mtDNA nucleic acid and protein sequences of R. brasiliensis and more 63 species belonging to 12 subfamilies of Acrididae. Phylogenetic trees were reconstructed using Bayesian inference with a relaxed molecular clock to estimate the speciation divergence time between taxa. The mitochondrial genome of R. brasiliensis has 15,571 bp of length, is rich in AT (72%), and contains 37 genes, including 13 protein-encoding genes, 22 genes encoding transfer RNA and two genes encoding ribosomal RNA. In addition, we also have annotated intergenic spacers and gene overlaps. The phylogenetic trees based on nucleic acid and amino acid sequences showed similar topologies. Phylogenetic analysis revealed that R. brasiliensis is grouped as an early offset of the Acrididae family. Phylogenetic analyses also corroborated the presence of several paraphyletic subfamilies in the family Acrididae including Gomphocerinae. The positioning of R. brasiliensis in the mtDNA phylogenetic tree further supports paraphyly of this subfamily. Moreover, the basal position of R. brasiliensis suggests that Gomphocerinae probably originated in South America.

Molecular Identification of Oxyspirura Petrowi Intermediate Hosts by Nested PCR Using Internal Transcribed Spacer 1 (ITS1).

Recently, the heteroxenous eyeworm, Oxyspirura petrowi, has gained attention due to its prevalence in the declining game bird, Northern bobwhite (Colinus virginianus), but the intermediate hosts of many nematodes remain unknown. However, identifying the intermediate host of O. petrowi with traditional techniques would be difficult and time-consuming, especially considering there are more than 80 potential orthopteran hosts just in Texas. To screen a large number of samples quickly and effectively, primers for nested PCR (nPCR) were developed using the internal transcribed spacer 1 (ITS1) region. Then the nPCR was used to identify which of the 35 species collected from the Order Orthoptera were potential intermediate hosts of O. petrowi. With this technique, 18 potential intermediate hosts were identified. Later, we collected live specimens of species that tested positive to confirm the presence of larvae, but larvae were not found in the live specimens, nor in the extra tissue of the species that had tested positive for O. petrowi DNA. Despite this, this study demonstrated that nPCR is more sensitive than traditional techniques and can be a valuable tool in determining the intermediate hosts of parasites.

The gut bacterial communities across six grasshopper species from a coastal tallgrass prairie.

Insect microbiomes play an important role in the health and fitness of insect hosts by contributing to nutrient absorption, immune health, and overall ecological fitness. As such, research interests in insect microbiomes have focused on agriculturally and industrially important organisms such as honey bees and termites. Orthopterans, on the other hand, have not been well explored for their resident microbial communities. Grasshoppers are an integral part of grassland ecosystems and provide important ecosystem services. Conversely, grasshoppers can be an agricultural pest requiring management with broad spectrum pesticides. However, little is known about the microbiomes of grasshoppers and their potential contribution to grasshopper biology. Here we examine the gut microbiome of six species of grasshoppers (n = 60) from a coastal tallgrass prairie ecosystem to gain a better understanding of the microbial communities present across the orthopteran order in this ecosystem. We found that there are bacterial phyla common to all six grasshopper species: Actinobacteria, Proteobacteria, Firmicutes, and to a lesser degree, Tenericutes. Although the grasshopper species shared a high relative abundance of these groups, there were notable shifts in dominant phyla depending on the grasshopper species. Moreover, measures of alpha diversity revealed a more diverse microbiome in males than females. Our observations support the hypothesis that there is a "core" group of bacterial families in these grasshopper species and factors such as trophic behaviors and the evolution of the host may contribute to the shifts in prevalence among these core microbial groups.

A second intracellular copper/zinc superoxide dismutase and a manganese superoxide dismutase in Oxya chinensis: Molecular and biochemical characteristics and roles in chlorpyrifos stress.

A second intracellular copper/zinc superoxide dismutase (icCuZnSOD2) and manganese SOD (MnSOD) were cloned and characterized in Oxya chinensis. The open reading frame (ORF) of OcicCuZnSOD2 and OcMnSOD are 462 and 672 bp encoding 153 and 223 amino acids, respectively. OcicCuZnSOD2 contains two signature sequences, one potential N-glycosylation site, and seven copper/zinc binding sites. OcMnSOD includes a mitochondria targeting sequence of 7 amino acids at N-terminal, one signature sequence, two N-glycosylation sites, and four manganese binding sites. The secondary structure and homology model of OcicCuZnSOD2 include nine ß sheets, two Greek-key motifs, and one electrostatic loop. OcMnSOD contains nine α-helices and three ß-sheets. Phylogenetic analysis shows that OcMnSOD is evolutionarily conserved while OcicCuZnSOD2 may be gene duplication and is paralogous to OcicCuZnSOD1. OcMnSOD expressed widely in all tissues and developmental stages. OcicCuZnSOD2 showed testis-specific expression and expressed highest in the 5th-instar nymph and the adult. The optimum temperatures and pH values of the recombinant OcicCuZnSOD2 and OcMnSOD were 40 °C and 8.0. They were stable at 25-55 °C and at pH 5.0-12.0 and pH 6.0-12.0, respectively. The activity and mRNA expression of each OcSOD were assayed after chlorpyrifos treatments. Total SOD and CuZnSOD activities first increased then declined under chlorpyrifos stress. Chlorpyrifos induced the mRNA expression and activity of OcMnSOD as a dose-dependent manner and inhibited OcicCuZnSOD2 transcription. The role of each OcSOD gene in chlorpyrifos stress was investigated using RNAi and disc diffusion assay with Escherichia coli overexpressing OcSOD proteins. Silencing of OcMnSOD significantly increased ROS content in chlorpyrifos-exposed grasshoppers. Disc diffusion assay showed that the plates with E. coli overexpressing OcMnSOD had the smaller inhibition zones around the chlorpyrifos-soaked filter discs. These results implied that OcMnSOD played a significant role in defense chlorpyrifos-induced oxidative stress.