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.

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.

Biomechanical strategies to reach a compromise between stiffness and flexibility in hind femora of desert locusts.

Insect cuticle can reach a wide range of material properties, which is thought to be the result of adaptations to applied mechanical stresses. Biomechanical mechanisms behind these property variations remain largely unknown. To fill this gap, here we performed a comprehensive study by simultaneous investigation of the microstructure, sclerotization and the elasticity modulus of the specialized cuticle of the femora of desert locusts. We hypothesized that, considering their different roles in jumping, the femora of fore-, mid- and hind legs should be equipped with cuticles that have different mechanical properties. Surprisingly, our results showed that the hind femur, which typically bears higher stresses, has a lower elasticity modulus than the fore and mid femora in the longitudinal direction. This is likely due to the lower sclerotization and different microstructure of the hind femur cuticle. This allows for some deformability in the femur wall and is likely to reduce the risk of mechanical failure. In contrast to both other femora, the hind femur is also equipped with a set of sclerotized ridges that are likely to provide it with the required stiffness to withstand the mechanical loads during walking and jumping. This paper is one of only a few comprehensive studies on insect cuticle, which advances the current understanding of the relationship between the structure, material property and function in this complex biological composite. STATEMENT OF SIGNIFICANCE: Insect cuticle is a biological composite with strong anisotropy and wide ranges of material properties. Using an example of the femoral cuticle of desert locusts, we measured the elasticity modulus, microstructure and sclerotization level of the cuticle. Our results show that, although the hind femur withstands most of the stress during locomotion, it has a lower elasticity modulus than the fore and mid femora. This is likely to be a functional adaption to jumping, in order to allow small deformations of the femur wall and reduce the risk of material failure. Our results deepen the current understanding of the structure-material-function relationship in the complex insect cuticle.

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.

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.

Elucidating the complex organization of neural micro-domains in the locust Schistocerca gregaria using dMRI.

To understand brain function it is necessary to characterize both the underlying structural connectivity between neurons and the physiological integrity of these connections. Previous research exploring insect brain connectivity has typically used electron microscopy techniques, but this methodology cannot be applied to living animals and so cannot be used to understand dynamic physiological processes. The relatively large brain of the desert locust, Schistercera gregaria (Forksȧl) is ideal for exploring a novel methodology; micro diffusion magnetic resonance imaging (micro-dMRI) for the characterization of neuronal connectivity in an insect brain. The diffusion-weighted imaging (DWI) data were acquired on a preclinical system using a customised multi-shell diffusion MRI scheme optimized to image the locust brain. Endogenous imaging contrasts from the averaged DWIs and Diffusion Kurtosis Imaging (DKI) scheme were applied to classify various anatomical features and diffusion patterns in neuropils, respectively. The application of micro-dMRI modelling to the locust brain provides a novel means of identifying anatomical regions and inferring connectivity of large tracts in an insect brain. Furthermore, quantitative imaging indices derived from the kurtosis model that include fractional anisotropy (FA), mean diffusivity (MD) and kurtosis anisotropy (KA) can be extracted. These metrics could, in future, be used to quantify longitudinal structural changes in the nervous system of the locust brain that occur due to environmental stressors or ageing.

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.

Anatomical and ultrastructural analysis of the posterior optic tubercle in the locust Schistocerca gregaria.

Locusts, like other insects, partly rely on a sun compass mechanism for spatial orientation during seasonal migrations. To serve as a useful guiding cue throughout the day, however, the sun's apparent movement has to be accounted for. In locusts, a neural pathway from the accessory medulla, the circadian pacemaker, via the posterior optic tubercle, to the protocerebral bridge, part of the internal sky compass, has been proposed to mediate the required time compensation. Toward a better understanding of neural connectivities within the posterior optic tubercle, we investigated this neuropil using light and electron microscopy. Based on vesicle content, four types of synaptic profile were distinguished within the posterior optic tubercle. Immunogold labeling showed that pigment-dispersing hormone immunoreactive neurons from the accessory medulla, containing large dense-core vesicles, have presynaptic terminals in the posterior optic tubercle. Ultrastructural examination of two Neurobiotin-injected tangential neurons of the protocerebral bridge revealed that these neurons are postsynaptic in the posterior optic tubercle. Our data, therefore, support a role of the posterior optic tubercles in mediating circadian input to the insect sky compass.

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.

Feedback inhibition and its control in an insect olfactory circuit.

Inhibitory neurons play critical roles in regulating and shaping olfactory responses in vertebrates and invertebrates. In insects, these roles are performed by relatively few neurons, which can be interrogated efficiently, revealing fundamental principles of olfactory coding. Here, with electrophysiological recordings from the locust and a large-scale biophysical model, we analyzed the properties and functions of GGN, a unique giant GABAergic neuron that plays a central role in structuring olfactory codes in the locust mushroom body. Our simulations suggest that depolarizing GGN at its input branch can globally inhibit KCs several hundred microns away. Our in vivorecordings show that GGN responds to odors with complex temporal patterns of depolarization and hyperpolarization that can vary with odors and across animals, leading our model to predict the existence of a yet-undiscovered olfactory pathway. Our analysis reveals basic new features of GGN and the olfactory network surrounding it.

Cuticle sclerotization determines the difference between the elastic moduli of locust tibiae.

A striking characteristic of insect cuticle is the wide range of its material property values, with respect to stiffness, strength and toughness. The elastic modulus of cuticle, for instance, ranges over seven orders of magnitude in different structures and different species. Previous studies suggested that this characteristic is influenced by the microstructure and sclerotization of cuticle. However, the relative role of the two factors in determining the material properties of cuticle is unknown. Here we used a combination of scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and nanoindentation, to investigate the effect of microstructure and sclerotization on the elastic modulus of tibiae of desert locusts. Our results showed that tibial cuticle is an anisotropic material with the highest elastic modulus along the tibial axis. This is likely because majority of the fibers in the cuticle are oriented along this axis. We also found that the hind tibia has a significantly higher elastic modulus, compared with the fore and mid tibiae. This is likely due to the higher sclerotization level of the hind tibia cuticle, and seems to be an adaptation to the locust locomotion by jumping, in which axial loads in the hind tibiae may reach several times the insect body weight. Our results suggest that while sclerotization determines the difference between the elastic moduli of the tibiae, anisotropic properties of each tibia is controlled by the specific fiber orientation. Our study provides one of only a few comprehensive investigations on insect cuticle, and helps to better understand the structure-material-function relationship in this complex biological composite. STATEMENT OF SIGNIFICANCE: Insect cuticle is a biological composite with strong anisotropy and wide ranges of material properties. Using an example of the tibial cuticle of desert locusts, we examined the role of two influential factors on the elastic modulus of cuticle: microstructure and sclerotization. Our results suggested the strong influence of sclerotization on the variation of the elastic modulus among fore, mid and hind tibiae, and that of the microstructure on the anisotropy of each tibia. Our results deepens the current understanding of the structure-material-function relationship in complex insect cuticle.

Accessory glands of male reproductive system in Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera: Acrididae): A light and electron microscopic study.

The accessory glands of male reproductive system in insects play a significant role in the reproduction process by protecting sperm in spermatheca, preventing female to accept other males after mating and stimulating oviposition. The number, structure, and arrangement of the tubules of accessory glands can change from species to species. In this study, the accessory glands belonging the male reproductive system in Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) were examined with stereomicroscope, light microscope, scanning (SEM), and transmission (TEM) electron microscopes at Gazi University, Faculty of Science in 2017-2019. P. parallelus parallelus is a widespread species that is located at the extending areas from Italy to the Northern Europe and also in Turkey. The accessory glands of P. parallelus parallelus' male reproductive system are composed of about 10 tubules. The tubules can be classified into two groups according to the thickness of their muscle tissues. Both groups have single layered epithelial cells with mitochondria, well-developed endoplasmic reticulum, spherical nucleus with electron dense chromatin, secretory vesicles and multivesicular bodies in their cytoplasm. In addition, apocrine type secretion is seen in epithelial cells.

High-Strength Nanostructured Films Based on Well-Preserved α-Chitin Nanofibrils Disintegrated from Insect Cuticles.

The α-chitin nanofibril is an alternative to nanocellulose as a building-block for strong films and other nanomaterials. The hypothesis of high film strength for films based on mildly treated insect cuticles was tested. Fibrils from the cuticle of Ruspolia differens (a long-horned bush cricket grasshopper locally known as senene) are disintegrated by a mild process, subsequently characterized by transmission electron microscopy, NMR, Fourier transform infrared spectroscopy, and XRD, and used to prepare strong and transparent films. A mild process (with 20% NaOH treatment for 2 weeks and at room temperature) was used to largely remove the strongly bound protein associated with chitin. The purpose was to reduce chitin degradation. The native structure of chitin was indeed well preserved and close to the native state, as is supported by data for degree of acetylation, molar mass, crystallinity, and crystallite dimensions. The diameter of the smallest chitin fibrils was as small as 3-7 nm (average 6 nm) with lengths larger than or around 1 µm. A stable and well-dispersed colloidal chitin fibril suspension in water was achieved. A nanostructured chitin film prepared by filtration showed high optical transmittance (∼90%) and very high tensile strength (220 MPa). The high tensile strength was attributed to the well-preserved chitin structure, high intrinsic fibril strength, and high colloidal stability of the fibril suspension. Strong, transparent insect chitin films offer interesting alternatives to nanocellulose films because of different resource origins, surface chemistries, and potential antimicrobial properties.

Comparative analysis of the multivariate genetic architecture of morphological traits in three species of Gomphocerine grasshoppers.

Evolutionary change is the change in trait values across generations, and usually occurs in multidimensional trait space rather than along isolated traits. Genetic covariation influences the magnitude and direction of evolutionary change and can be statistically summarized by the additive genetic (co)variance matrix, G. While G can affect the response to selection, it is exposed to evolutionary change by selection and genetic drift, but the magnitude and speed of these changes are poorly understood. We use comparative G matrix analyses to assess evolution of the shape and orientation of G over longer timescales in three species of Gomphocerine grasshoppers. We estimate 10 × 10 G matrices for five morphological traits expressed in both sexes. We find low-to-moderate heritabilities (average 0.36), mostly large cross-sex correlations (average 0.54) and moderate between-trait correlations (average 0.34). G matrices differ significantly among species with wing length contributing most to these differences. Wing length is the trait that is most divergent among species, suggesting it has been under selection during species divergence. The more distantly related species, Pseudochorthippus parallelus, was the most different in the shape of G. Projection of contemporary genetic variation into the divergence space D illustrates that the major axis of genetic variation in Gomphocerippus rufus is aligned with divergence from Chorthippus biguttulus, while the major axis of genetic variation in neither of the species is aligned with the divergence between Pseudochorthippus parallelus and the other two species. Our results demonstrate significant differences in G matrices with a phylogenetic signal in the differentiation.

Neuroarchitecture of the central complex of the desert locust: Tangential neurons.

The central complex (CX) comprises a group of midline neuropils in the insect brain, consisting of the protocerebral bridge (PB), the upper (CBU) and lower division (CBL) of the central body and a pair of globular noduli. It receives prominent input from the visual system and plays a major role in spatial orientation of the animals. Vertical slices and horizontal layers of the CX are formed by columnar, tangential, and pontine neurons. While pontine and columnar neurons have been analyzed in detail, especially in the fruit fly and desert locust, understanding of the organization of tangential cells is still rudimentary. As a basis for future functional studies, we have studied the morphologies of tangential neurons of the CX of the desert locust Schistocerca gregaria. Intracellular dye injections revealed 43 different types of tangential neuron, 8 of the PB, 5 of the CBL, 24 of the CBU, 2 of the noduli, and 4 innervating multiple substructures. Cell bodies of these neurons were located in 11 different clusters in the cell body rind. Judging from the presence of fine versus beaded terminals, the vast majority of these neurons provide input into the CX, especially from the lateral complex (LX), the superior protocerebrum, the posterior slope, and other surrounding brain areas, but not directly from the mushroom bodies. Connections are largely subunit- and partly layer-specific. No direct connections were found between the CBU and the CBL. Instead, both subdivisions are connected in parallel with the PB and distinct layers of the noduli.

Transepithelial transport of P-glycoprotein substrate by the Malpighian tubules of the desert locust.

Extrusion of xenobiotics is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in xenobiotic extrusion. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. To determine the quantity of the P-glycoprotein substrate extruded we developed a simple and cheap method as an alternative to liquid chromatography-mass spectrometry, radiolabelled alkaloids or confocal microscopy. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins contribute to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves.

Evolutionarily conserved anatomical and physiological properties of olfactory pathway through fourth-order neurons in a species of grasshopper (Hieroglyphus banian).

Olfactory systems of different species show variations in structure and physiology despite some conserved features. We characterized the olfactory circuit of the grasshopper Hieroglyphus banian of family Acrididae (subfamily: Hemiacridinae) and compared it to a well-studied species of locust, Schistocerca americana (subfamily: Cyrtacanthacridinae), also belonging to family Acrididae. We used in vivo electrophysiological, immunohistochemical, and anatomical (bulk tract tracing) methods to elucidate the olfactory pathway from the second-order neurons in antennal lobe to the fourth-order neurons in ß-lobe of H. banian. We observe conserved anatomical and physiological characteristics through the fourth-order neurons in the olfactory circuit of H. banian and S. americana, though they are evolutionarily divergent (~ 57 million years ago). However, we found one major difference between the two species-there are four antennal lobe tracts in H. banian, while only one is reported in S. americana. Besides, we have discovered a new class of bilateral neurons which respond weakly to olfactory stimuli, even though they innervate densely downstream of Kenyon cells.

Revealing hidden density-dependent phenotypic plasticity in sedentary grasshoppers in the genus Schistocerca Stål (Orthoptera: Acrididae: Cyrtacanthacridinae).

Comparative quantification of reaction norms across closely related species in a clade is rare, but such a study can reveal valuable insights into understanding how reaction norms evolve along phylogeny. The grasshopper genus Schistocerca Stål (Orthoptera: Acrididae: Cyrtacanthacridinae) is an ideal group to study the evolution of density-dependent phenotypic plasticity because it includes both swarming locusts and non-swarming sedentary grasshoppers, which show varying degrees of plastic reaction norms in many traits. The swarming locusts exhibit locust phase polyphenism in which cryptically colored and solitary individuals can transform into conspicuously colored and highly gregarious individuals in response to increases in population density. The sedentary grasshoppers do not swarm in nature, and thus it has been assumed that they have little or no expression of plastic reaction norms in many traits, except for color, which has been shown to be a phylogenetically conserved trait. In this study, we have quantified density-dependent reaction norms in behavior, color, body size, and morphometric ratio in the nymphs of four sedentary species within Schistocerca by conducting explicit rearing experiments to induce potential phenotypic changes in response to isolation and crowding. In contrast to our previous assumption, we find that all four species show a certain level of density-dependent plastic reaction norms, which implies that these sedentary species have hidden reaction norms that can only be induced experimentally, some components of which must be phylogenetically conserved. Furthermore, we demonstrate that rearing density differentially affects the expression of reaction norms in different species, suggesting that different reaction norms must have followed independent evolutionary trajectories.