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This study investigated the in vitro bioaccessibility of aluminum, copper, iron, manganese, lead, selenium, and zinc in three important species of farmed insects: the yellow mealworm (Tenebrio molitor), the house cricket (Acheta domesticus) and the migratory locust (Locusta migratoria). Results show that all three insect species constitute excellent sources of essential elements (Fe, Cu and Zn) for the human diet, contributing to the recommended dietary allowance, i.e., 10%, 50%, and 92%, respectively. A higher accumulation of Se (≥1.4 mg Se/kg) was observed with increasing exposure concentration in A. domesticus, showing the possibility of using insects as a supplements for this element. The presence of Al and Fe nanoparticles was confirmed in all three species using single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The results also indicate that Fe bioaccessibility declines with increasing Fe-nanoparticle concentration. These findings contribute to increase the nutritional and toxicological insights of farmed insects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

RNA N 6 -methyladenosine (m 6 A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m 6 A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m 6 A functions in migratory locusts clearly demonstrated the crucial roles of the m 6 A pathway in effectively modulating aggregation behaviors., (© 2023. Science China Press.)

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects., (© 2024. The Author(s).)

Carotenoids are hydrophobic pigments binding to diverse carotenoproteins, many of which remain unexplored. Focusing on yellow gregarious locusts accumulating cuticular carotenoids, here we use engineered Escherichia coli cells to reconstitute a functional water-soluble β-carotene-binding protein, BBP. HPLC and Raman spectroscopy confirmed that recombinant BBP avidly binds β-carotene, inducing the unusual vibronic structure of its absorbance spectrum, just like native BBP extracted from the locust cuticles. Bound to recombinant BBP, β-carotene exhibits pronounced circular dichroism and allows BBP to withstand heating (T 0.5  = 68 °C), detergents and pH variations. Using bacteria producing distinct xanthophylls we demonstrate that, while β-carotene is the preferred carotenoid, BBP can also extract from membranes ketocarotenoids and, very poorly, hydroxycarotenoids. We show that BBP-carotenoid complex reversibly binds to chitin, but not to chitosan, implying the role for chitin acetyl groups in cuticular BBP deposition. Reconstructing such locust coloration mechanism in vitro paves the way for structural studies and BBP applications., (© 2024. The Author(s).)

Effects of dietary protein quality on insect development (not just growth) are unclear. Dietary amino acid blends matching yolk proteins support reproduction and juvenile development in Drosophila melanogaster. We matched amino acids to vitellogenin and tested development of juvenile male lubber grasshoppers, which do not produce vitellogenin. Last instars were fed classic dry diets with amino acids substituted for proteins. Matching amino acids to vitellogenin allowed molting to adulthood, while an unmatched isonitrogenous diet did not. Health on dry diets was poor, so we developed wet diets with agar, horse feed, and amino acids. Juveniles fed these diets matched to vitellogenin developed comparably to juveniles fed lettuce. However, wet diets with amino acids dissimilar to vitellogenin (low-quality) slowed development but maintained size at adulthood. We observed no compensatory feeding on low-quality diets. Theory suggests accumulation of proteins permits development. To detect a threshold, we started last juvenile instars on high-quality diets, then abruptly switched them to low-qualities diets. When switched to the poor-quality diet at 6d, grasshoppers molted at a similar age (∼17d) to grasshoppers continuously on the high-quality diet. Total hemolymph proteins levels were unaffected by the timing of diet switches. Last, methionine is essential but can be noxious at high levels. Diets with low-quality protein except for methionine slowed growth early but did not alter the time or size at molt. Overall, the feeding threshold is solely due to essential amino acids, and low-quality protein diets slowed development but did not affect adult size., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Migratory locusts enter a reversible hypometabolic coma to survive environmental anoxia, wherein the cessation of CNS activity is driven by spreading depolarization (SD). While glycolysis is recognized as a crucial anaerobic energy source contributing to animal anoxia tolerance, its influence on the anoxic SD trajectory and recovery outcomes remains poorly understood. We investigated the effects of varying glycolytic capacity on adult female locust anoxic SD parameters, using glucose or the glycolytic inhibitors 2-deoxy-d-glucose (2DG) or monosodium iodoacetate (MIA). Surprisingly, 2DG treatment shared similarities with glucose yet had opposite effects compared with MIA. Specifically, although SD onset was not affected, both glucose and 2DG expedited the recovery of CNS electrical activity during reoxygenation, whereas MIA delayed it. Additionally, glucose and MIA, but not 2DG, increased tissue damage and neural cell death following anoxia-reoxygenation. Notably, glucose-induced injuries were associated with heightened CO 2 output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO 2 output and gradually increasing it throughout the recovery period. Given the discrepancies between effects of 2DG and MIA, the current results require cautious interpretations. Nonetheless, our findings present evidence that glycolysis is not a critical metabolic component in either anoxic SD onset or recovery and that heightened glycolysis during reoxygenation may exacerbate CNS injuries. Furthermore, we suggest that locust anoxic recovery is not solely dependent on energy availability, and the regulation of metabolic flux during early reoxygenation may constitute a strategy to mitigate damage., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Wang et al.)

Insect gut bacteria play an essential role in the nutritional metabolism, growth, and development of insects. Grasshoppers (Orthoptera) are cellulose-rich plant-feeding pests. Although the biological potential of grasshopper gut microorganisms to assist cellulose decomposition is well established, microbial resources for efficient degradation of cellulose biomass are still scarce and need to be developed. In this study, we used selective media to isolate cellulose-degrading bacteria from the intestines of Atractomorpha sinensis, Trilophidia annulata, Sphingonotus mongolicus, and Calliptamus abbreviatus. Phylogenetic analysis based on the maximum likelihood method using 16S rDNA sequencing sequences to identify bacteria revealed the isolation of 11 strains belonging to 3 genera, including Klebsiella, Aeromonas, and Bacillus. The degradability of the isolates to cellulose was then determined by the DNS colorimetric method, and the results showed that Bacillus had the highest degradation rate. The elucidation of microbial cellulose degradation capacity in grasshoppers not only contributes to the understanding of multiple plant-insect-microbe interactions, but also provides a valuable microbial resource for solving the biomass conversion of cellulose species problem., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

Ageing plasticity represents the flexibility of the ageing process in response to non-genetic factors, occurring commonly in animals. However, the regulatory mechanisms underlying ageing plasticity are largely unclear. The density-dependent polyphenism of locusts, Locusta migratoria, displays dramatic lifespan divergence between solitary and gregarious phases, providing a useful system for studying ageing plasticity. Here, we found that gregarious locusts displayed faster locomotor deficits and increased muscle degeneration on ageing than solitary locusts. Comparative transcriptome analysis in flight muscles revealed significant differences in transcriptional patterns on ageing between two phases. RNA interference screening showed that the knockdown of the upregulated PLIN2 gene significantly relieved the ageing-related flight impairments in gregarious locusts. Mechanistically, the gradual upregulation of PLIN2 could induce the accumulation of ectopic lipid droplets and triacylglycerols in flight muscles during the ageing process. Further experiments suggested that ectopic lipid accumulation led to an ageing-related β-oxidation decline through limiting fatty acid transport and content. These findings reveal the key roles of lipid metabolism in the differences of muscle ageing between solitary and gregarious locusts and provide a potential mechanism underlying environment-induced muscle ageing plasticity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.

An anticannibalistic signaling pathway offers a new understanding of locust swarm formation.

Locusts depend upon their sense of smell and provide useful models for understanding olfaction. Extending this understanding requires knowledge of the molecular and structural organization of the olfactory system. Odor sensing begins with olfactory receptor neurons (ORNs), which express odorant receptors (ORs). In insects, ORNs are housed, in varying numbers, in olfactory sensilla. Because the organization of ORs within sensilla affects their function, it is essential to identify the ORs they contain. Here, using RNA sequencing, we identified 179 putative ORs in the transcriptomes of the two main olfactory organs, antenna and palp, of the locust Schistocerca americana. Quantitative expression analysis showed most putative ORs (140) are expressed in antennae while only 31 are in the palps. Further, our analysis identified one OR detected only in the palps and seven ORs that are expressed differentially by sex. An in situ analysis of OR expression suggested ORs are organized in non-random combinations within antennal sensilla. A phylogenetic comparison of OR predicted protein sequences revealed homologous relationships among two other Acrididae species. Our results provide a foundation for understanding the organization of the first stage of the olfactory system in S. americana, a well-studied model for olfactory processing., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The diets of animals are essential to support development, and protein is key. Accumulation of stored nutrients can support developmental events such as molting and initiation of reproduction. Agricultural studies have addressed how dietary protein quality affects growth, but few studies have addressed the effects of dietary protein quality on developmental transitions. Studies on how dietary quality may affect protein storage and development are possible in arthropods, which store proteins in the hemolymph. We hypothesized that diets with a composition of amino acids that matches the precursor of egg yolk protein (vitellogenin, Vg) will be high quality and support both egg production and accumulation of storage proteins. Grasshoppers were fed one of two isonitrogenous solutions of amino acids daily: Vg-balanced (matched to Vg) or Unbalanced (same total moles of amino acids, but not matched to egg yolk). We measured reproduction and storage protein levels in serial hemolymph samples from individuals. The Vg-balanced group had greater reproduction and greater cumulative levels of storage proteins than did the Unbalanced group. This occurred even though amino acids fed to the Vg-balanced group were not a better match to storage protein than were the amino acids fed to the Unbalanced group. Further, oviposition timing was best explained by a combination of diet, age at the maximum level of storage protein hexamerin-270 and accumulation of hexamerin-90. Our study tightens the link between storage proteins and commitment to reproduction, and shows that dietary protein quality is vital for protein storage and reproduction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The General Stress Paradigm (GSP) predicts that prey body compositions should shift under chronic predation as prey increase body carbon and decrease body nitrogen content through dietary changes, heightened metabolism, reduced dietary efficiency, and the breakdown of nitrogen rich tissues to make labile carbohydrates available. In our study, we explored how the elemental and macronutrient content along with the morphology of three abundant Ozark glade grasshopper species differed between glades with and without predatory collared lizard (Crotaphytus collaris) populations. Our results indicated that lichen grasshoppers (Trimerotropis saxatilis) increased body C:N ratios in response to predators. Scudder's short-wing grasshoppers (Melanoplus scudderi) increased both body %C and %protein content, while the handsome grasshoppers (Syrbula admirabilis) did not significantly respond to the presence of collared lizards. None of the three grasshopper species showed morphological responses to predation. We also found that elemental and macronutrient content of grasshoppers was not always significantly correlated and was not associated with the same environmental factors, indicating a need to incorporate both perspectives in future research and utilize more accurate macromolecular assays. Overall, we found support for some aspects of the GSP in field-active animals and add to the growing body of evidence that predator-induced changes in prey body composition are more complex than predicted by the original GSP., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Insect flight is a complex physiological process that involves sensory and neuroendocrinal control, efficient energy metabolism, rhythmic muscle contraction, and coordinated wing movement. As a classical study model for insect flight, locusts have attracted much attention from physiologists, behaviorists, and neuroendocrinologists over the past decades. In earlier research, scientists made extensive efforts to explore the hormone regulation of metabolism related to locust flight; however, this work was hindered by the absence of molecular and genetic tools. Recently, the rapid development of molecular and genetic tools as well as multi-omics has greatly advanced our understanding of the metabolic, molecular, and neuroendocrinal basis of long-term flight in locusts. Novel neural and molecular factors modulating locust flight and their regulatory mechanisms have been explored. Moreover, the molecular mechanisms underlying phase-dependent differences in locust flight have also been revealed. Here, we provide a systematic review of locust flight physiology, with emphasis on recent advances in the neuroendocrinal, genetic, and molecular basis. Future research directions and potential challenges are also addressed., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

Animal feeding, which directly affects growth and metabolism, is an important physiological process. However, the contribution of PIWI proteins and PIWI-interacting RNAs (piRNAs) to the regulatory mechanism of animal feeding is unknown. Here, we report a novel function of Piwi and piRNAs in regulating food intake in locusts. Our study shows that the locust can serve as a representative species for determining PIWI function in insects. Knockdown of Piwi1 expression suppresses anabolic processes and reduces food consumption and body weight. The reduction in food intake by knockdown of Piwi1 expression results from decreased expression of neuropeptide NPF1 in a piRNA-dependent manner. Mechanistically, intronic piRNAs might enhance RNA splicing of NPF1 by preventing hairpin formation at the branch point sites. These results suggest a novel nuclear PIWI/piRNA-mediated mechanism that controls food intake in the locust nervous system., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Flight ability is essential for the enormous diversity and evolutionary success of insects. The migratory locusts exhibit flight capacity plasticity in gregarious and solitary individuals closely linked with different density experiences. However, the differential mechanisms underlying flight traits of locusts are largely unexplored. Here, we investigated the variation of flight capacity by using behavioral, physiological, and multiomics approaches. Behavioral assays showed that solitary locusts possess high initial flight speeds and short-term flight, whereas gregarious locusts can fly for a longer distance at a relatively lower speed. Metabolome-transcriptome analysis revealed that solitary locusts have more active flight muscle energy metabolism than gregarious locusts, whereas gregarious locusts show less evidence of reactive oxygen species production during flight. The repression of metabolic activity by RNA interference markedly reduced the initial flight speed of solitary locusts. Elevating the oxidative stress by paraquat injection remarkably inhibited the long-distance flight of gregarious locusts. In respective crowding and isolation treatments, energy metabolic profiles and flight traits of solitary and gregarious locusts were reversed, indicating that the differentiation of flight capacity depended on density and can be reshaped rapidly. The density-dependent flight traits of locusts were attributed to the plasticity of energy metabolism and degree of oxidative stress production but not energy storage. The findings provided insights into the mechanism underlying the trade-off between velocity and sustainability in animal locomotion and movement., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The majority of long noncoding RNAs (lncRNAs) contain transposable elements (TEs). PAHAL , a nuclear-retained lncRNA that is inserted by a Gypsy retrotransposon, has been shown to be a vital regulator of phenylalanine hydroxylase ( PAH ) gene expression that controls dopamine biosynthesis and behavioural aggregation in the migratory locust. However, the role of the Gypsy retrotransposon in the transcriptional regulation of PAHAL remains unknown. Here, we identified a Gypsy retrotransposon (named Gypsy element) as an inverted long terminal repeat located in the 3' end of PAHAL , representing a feature shared by many other lncRNAs in the locust genome. The embedded Gypsy element contains a RNA nuclear localization signal motif, which promotes the stable accumulation of PAHAL in the nucleus. The Gypsy element also provides high-affinity SRSF2 binding sites for PAHAL that induce the recruitment of SRSF2, resulting in the PAHAL -mediated transcriptional activation of PAH . Thus, our data demonstrate that TEs provide discrete functional domains for lncRNA organization and highlight the contribution of TEs to the regulatory significance of lncRNAs.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria . We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC 50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

In this study, we obtained four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 (icCuZnSOD1) in Oxya chinensis. OcicCuZnSOD1a has all common characteristics of CuZnSOD family and is a canonical CuZnSOD. OcicCuZnSOD1b is missing a Zn binding site. OcicCuZnSOD1c lacks Zn ion and is a Cu-only SOD. OcicCuZnSOD1d is missing a CuZnSOD conserved sequence and lacks the E-loop, a conserved disulfide bond, and an active site arginine. OcicCuZnSOD1a was the most heat-resistant and OcicCuZnSOD1c was the most unstable at high temperatures above 55 °C. They were stable at a wide pH range, especially in alkaline conditions. The four variants expressed at the throughout developmental stages and had various tissue expression patterns. OcicCuZnSOD1a and OcicCuZnSOD1d were significantly induced by 8.79 mM CuCl 2 and OcicCuZnSOD1b was significantly up-regulated by 14.67 mM CuCl 2 . OcicCuZnSOD1a was significantly inhibited by 19.13 mM ZnSO 4 while OcicCuZnSOD1d were significantly induced by 22.61 mM ZnSO 4 . Disc diffusion assay showed that the four isoforms of OcicCuZnSOD1 made the killing zones smaller surrounding the CdCl 2 -soaked filter discs. However, the reduction ratios of OcicCuZnSOD1a were the highest. These results implied that the four transcripts played roles in defense against CdCl 2 -induced oxidative stress while OcicCuZnSOD1a had stronger antioxidant capacity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gomphocerus sibiricus L., the dominant insect species in the alpine and subalpine grassland, overwinters with diapause at egg stage. In this study, cold tolerance and related cryoprotectants of G. sibiricus eggs were investigated. In particular, the supercooling point (SCP), water content, carbohydrates (trehalose, glucose, fructose, glycogen), polyols (glycerol, inositol, sorbitol), fat, and amino acids contents were evaluated at different developmental stages of G. sibiricus eggs collected under natural conditions. The SCPs of eggs were very low (-32.83 to -22.61°C) at mid-diapause. Water content gradually increased during development. The fructose, glycerol, and sorbitol contents were significantly higher in diapausing eggs than in early embryogenesis stage and post-diapause development stage. Glycogen content was high throughout the whole developmental period. The trehalose, glucose, and inositol contents were low during diapause compared to that in early embryogenesis stage and post-diapause development stage. There were no significant differences in the fat content of eggs among all development stages. The total amino acid contents in eggs in the early embryogenesis and at the start of diapause were higher than that in post-diapause eggs. The contents of Glu, Asp, Leu, Pro and Arg during diapause were significantly higher than those during post-diapause development. Results indicate that G. sibiricus eggs have a high supercooling capacity. Successful overwintering can be attributed to the accumulation of glycerol, fructose, sorbitol, and amino acids (Glu, Asp, Leu, Pro and Arg). These findings provide insight into the mechanisms underlying the adaptation of G. sibiricus to cold conditions., (© 2021 The Authors. Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC.)

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust ( Schistocerca gregaria ) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC 50 = 0.80 μM at -75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC 50 (half-maximal inhibitory concentration) = 0.13 μM at -75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC 50 = 0.44 μM at -75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC 50 = 1.71 nM at -75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Olfaction plays an important role in the behavioural ecology of the desert locust Schistocerca gregaria (Orthoptera:Acrididae). Different locust life stages and sexes use olfactory cues for different behaviours such as grouping, mating, oviposition, feeding, maturation and gregarization, which can be exploited for management of the desert locust. However, the full spectrum of the chemistry of volatiles released by the desert locust remains unknown. Here, we compared the volatile emissions of different life stages of a natural wild population reared in the laboratory for one generation with those of a population that has been reared in the laboratory for seven generations. Coupled gas chromatography-mass spectrometric analysis was used to identify captured volatiles. Analysis of similarities (ANOSIM) and non-metric multidimensional scaling of the volatile organic compounds (VOCs) showed significant chemo-diversity between different life stages, and sexes for the adults of the two populations. Additionally, chemical analysis showed that nymphs and adults of the field population released ~4- to 40-fold more volatiles, and they were compositionally richer than their laboratory counterparts. These results demonstrate the composition and variation in odors of field and laboratory populations of the desert locust, and that the discriminating odors warrant further investigation to determine their roles in the bioecology and management of this locust species., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The central complex is a group of highly interconnected neuropils in the insect brain. It is involved in the control of spatial orientation, based on external compass cues and various internal needs. The functional and neurochemical organization of the central complex has been studied in detail in the desert locust Schistocerca gregaria. In addition to classical neurotransmitters, immunocytochemistry has provided evidence for a major contribution of neuropeptides to neural signaling within the central complex. To complement these data, we have identified all orcokinin-immunoreactive neurons in the locust central complex and associated brain areas. About 50 bilateral pairs of neurons innervating all substructures of the central complex exhibit orcokinin immunoreactivity. Among these were about 20 columnar neurons, 33 bilateral pairs of tangential neurons of the central body, and seven pairs of tangential neurons of the protocerebral bridge. In silico transcript analysis suggests the presence of eight different orcokinin-A type peptides in the desert locust. Double label experiments showed that all orcokinin-immunostained tangential neurons of the lateral accessory lobe cluster were also immunoreactive for GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase. Two types of tangential neurons of the upper division of the central body were, furthermore, also labeled with an antiserum against Dip-allatostatin I. No colocalization was found with serotonin immunostaining. The data provide additional insights into the neurochemical organization of the locust central complex and suggest that orcokinin-peptides of the orcokinin-A gene act as neuroactive substances at all stages of signal processing in this brain area., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure-property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r 2  > 0.5, with the descriptors log(P*MW -0.5 ) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW -0.5 ) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

One hundred years ago in 1921, Sir Boris Uvarov recognized that two locust species are one species but appearing in two different phases, a solitarious and a gregarious phase. As locust swarms are still a big problem affecting millions of people, basic research has tried to understand the causes for the transition between phases. This phenomenon of phase polymorphism, now called polyphenism, is a very complex multifactorial process and this short review will draw attention to this important aspect of insect research.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH 2 ). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II β-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI β-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH 2 , and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II β turn exhibited by cycloAKH from the type VI β turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations., (Published by Elsevier Ltd.)

Apoptosis plays a major role in development, tissue renewal and the progression of degenerative diseases. Studies on various types of mammalian cells reported a pro-apoptotic function of acetylcholinesterase (AChE), particularly in the formation of the apoptosome and the degradation of nuclear DNA. While three AChE splice variants are present in mammals, invertebrates typically express two ache genes that code for a synaptically located protein and a protein with non-synaptic functions respectively. In order to investigate a potential contribution of AChE to apoptosis in insects, we selected the migratory locust Locusta migratoria. We established primary neuronal cultures of locust brains and characterized apoptosis progression in vitro. Dying neurons displayed typical characteristics of apoptosis, including caspase-activation, nuclear condensation and DNA fragmentation visualized by TUNEL staining. Addition of the AChE inhibitors neostigmine and territrem B reduced apoptotic cell death under normal culture conditions. Moreover, both inhibitors completely suppressed hypoxia-induced neuronal cell death. Exposure of live animals to severe hypoxia moderately increased the expression of ace-1 in locust brains in vivo. Our results indicate a previously unreported role of AChE in insect apoptosis that parallels the pro-apoptotic role in mammalian cells. This similarity adds to the list of apoptotic mechanisms shared by mammals and insects, supporting the hypothesized existence of an ancient, complex apoptosis regulatory network present in common ancestors of vertebrates and insects.

Malpighian tubules, analogous to vertebrate nephrons, play a key role in insect osmoregulation and detoxification. Tubules can become infected with a protozoan, Malpighamoeba, which damages their epithelial cells, potentially compromising their function. Here we used a modified Ramsay assay to quantify the impact of Malpighamoeba infection on fluid secretion and P-glycoprotein-dependent detoxification by desert locust Malpighian tubules. Infected tubules have a greater surface area and a higher fluid secretion rate than uninfected tubules. Infection also impairs P-glycoprotein-dependent detoxification by reducing the net rhodamine extrusion per surface area. However, due to the increased surface area and fluid secretion rate, infected tubules have similar total net extrusion per tubule to uninfected tubules. Increased fluid secretion rate of infected tubules likely exposes locusts to greater water stress and increased energy costs. Coupled with reduced efficiency of P-glycoprotein detoxification per surface area, Malpighamoeba infection is likely to reduce insect survival in natural environments.

Acrididae are diverse in size, body shape, behavior, ecology and life history; widely distributed; easy to collect; and important to agriculture. They represent promising model candidates for functional genomics, but their extremely large genomes have hindered this research; establishing a reference transcriptome for a species is the primary means of obtaining genetic information. Here, two Acrididae species, Gomphocerus licenti and Mongolotettix japonicus, were selected for full-length (FL) PacBio transcriptome sequencing. For G. licenti and M. japonicus, respectively, 590,112 and 566,165 circular consensus sequences (CCS) were generated, which identified 458,131 and 428,979 full-length nonchimeric (FLNC) reads. After isoform-level clustering, next-generation sequencing (NGS) short sequences were used for error correction, and remove redundant sequences with CD-HIT, 17,970 and 16,766 unigenes were generated for G. licenti and M. japonicus. In addition, we obtained 17,495 and 16,373 coding sequences, 1,082 and 813 transcription factors, 11,840 and 10,814 simple sequence repeats, and 905 and 706 long noncoding RNAs by analyzing the transcriptomes of G. licenti and M. japonicus, respectively, and 15,803 and 14,846 unigenes were annotated in eight functional databases. This is the first study to sequence FL transcriptomes of G. licenti and M. japonicus, providing valuable genetic resources for further functional genomics research.

Adipokinetic hormones (AKHs), the neurohormones synthesized in the insect corpora cardiaca are known to mobilize lipids and carbohydrates for energy-consuming activities including reproduction. However, both inhibitory and stimulatory effects of AKHs on insect reproduction have been reported, and the underlying mechanisms remain elusive. Using the migratory locust, Locusta migratoria, as a model system, we report here that AKHs are expressed in response to rhythmic diel change, and AKH III expression increases markedly at photophase. Diurnal injection of AKH III but not AKH I or AKH II in adult females stimulates vitellogenesis and egg development. In contrast, AKH treatment at scotophase represses female reproduction. RNA interference-mediated knockdown of AKH receptor (AKHR) results in significantly reduced vitellogenin (Vg) expression in the fat body at photophase along with reduced Vg deposition in the ovary. AKHR knockdown also leads to decreased expression of Brummer, triacylglycerol lipase and trehalose transporter, accompanied by suppressed mobilization of triacylglycerol and trehalose. We propose that in addition to stimulating Vg expression at photophase, AKH/AKHR signalling is likely to regulate ovarian uptake of Vg via triacylglycerol mobilization and trehalose homeostasis. This study provides new insights into the understanding of AKH/AKHR signalling in the regulation of insect reproduction., (© 2020 The Royal Entomological Society.)

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain., Competing Interests: No authors have competing interests.

Olfaction plays a critical role in mediating environmental chemical signals to affect behavior. Locusts are voracious feeders that can cause substantial agricultural and environmental damage. Here, we have identified 3473 proteins in the antennal proteome of the migratory locust, Locusta migratoria. Comparative analysis of sex variation in the antennal proteomes of male and female locusts using iTRAQ proteomics identified 260 differentially abundant proteins (DAPs). In relation to olfaction, 29 chemosensory proteins (CSPs), 9 odorant binding proteins (OBPs), 31 odorant receptors (ORs), and 8 ionotropic receptors (IRs) were identified, in the male and female antennal proteomes. Two new previously undescribed CSP proteins were identified in antennae and the expression of their respective gene coding sequences verified. In addition, sex-biased proteins were found significantly enriched in melanogenesis and riboflavin metabolism in males and in ribosome and metabolic pathways in females. The sex- and tissue-specific gene expression profiles of 29 CSPs, 31 ORs and 8 IRs were investigated by quantitative real-time PCR (qRT-PCR), revealing distinct pattern of expression or these genes in different locust tissues including the antennae, legs, wing, and palps. Our data identify sex-biased antennal proteome profiles and indicate expression of putative olfaction genes in tissues beyond the antennae. BIOLOGICAL SIGNIFICANCE: Little is known concerning sexual dimorphism in olfaction related processes at the level of the antennal proteome of insects. Using iTRAQ-based proteomic analyses the protein repertoire of the antennae of male and female locusts were determined. These data show sex-specific differences, identifying a subset of proteins, including two novel CSPs, that likely help define male versus female olfactory responses that contributes to our understanding of chemoreception and downstream behavioral consequences. The characterization of antennal and related olfactory proteins identifies potential biomarkers that can contribute to the development of control strategies against locusts and orthologous insect pests., Competing Interests: Declaration of competing interest The authors declare they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There is still a scarcity of data on the transfer of naturally occurring radionuclides to wildlife in various ecosystems. In the present study, concentration ratios (CRwo-media) of 238U and 226Ra were obtained for grasshoppers, frogs and newts in terrestrial and freshwater ecosystems. Soil, water and animal samples were collected for 2 years in the vicinity of the closed uranium mine at Ningyo-toge, Japan. Three sites with different 238U and 226Ra levels were of interest: (i) pond and its shore (PO); (ii) low-level stream and its shore near overburden dump (OD); and (iii) uranium mill tailings pond and its shore (MP). The activity concentrations in both soil and water were PO ≈ OD < MP for 238U, and PO < OD < MP for 226Ra. Regarding the wildlife, 238U was able to be determined for all samples, but the detection of 226Ra was observed only for part of the samples. The means and standard deviations of CRwo-soil or CRwo-water were then calculated and may indicate the insignificant dependence of CRwo-media on environmental conditions characterized by the tested sites. The present data on CRwo-media were compared to the corresponding data or surrogate data from the IAEA's database, showing both agreement and discrepancy. Our data contribute to enhancing the available data for those radionuclides and animals. In particular, the transfer to amphibians, one of the main links in common food webs, is reported here for the first time., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Heat processing is commonly used to prepare edible insects for consumption. This study aimed at determining the effect of boiling and subsequent oven roasting on Ruspolia differens' nutrient composition, colour and odor compounds. Boiling leads to: a significant increase in protein and decrease in fat content on a dry matter basis; a minimal influence on its amino and fatty acids profile; a significant reduction in its ash content due to leaching of phosphorus, potassium and sodium; a significant increase in iron, zinc, copper, manganese and calcium content; and a fivefold reduction in the amount of vitamin B12. Roasting leads to a relative increase in the amount of calcium and trace mineral elements but doesn't affect other nutrients. Roasting results into a more uniform colour intensity when green and brown polymorphs are roasted together. Lipid oxidation is responsible for the colour and aroma of heat processed R. differens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium (NaV) channels, encoded by the gene para, play a critical role in the rapid processing and propagation of visual information related to collision avoidance behaviors. We investigated their localization by immunostaining the optic lobes and central brain of the grasshopper Schistocerca americana and the vinegar fly Drosophila melanogaster with an antibody that recognizes the channel peptide domain responsible for fast inactivation gating. NaV channels were detected at high density at all stages of development. In the optic lobe, they revealed stereotypically repeating fascicles consistent with the regular structure of the eye. In the central brain, major axonal tracts were strongly labeled, particularly in the grasshopper olfactory system. We used the NaV channel sequence of Drosophila to identify an ortholog in the transcriptome of Schistocerca. The grasshopper, vinegar fly, and human NaV channels exhibit a high degree of conservation at gating and ion selectivity domains. Comparison with three species evolutionarily close to Schistocerca identified splice variants of Para and their relation to those of Drosophila. The anatomical distribution of NaV channels molecularly analogous to those of humans in grasshoppers and vinegar flies provides a substrate for rapid signal propagation and visual processing in the context of visually-guided collision avoidance.

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to β-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to β-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Insects are considered edible food resources with sufficient nutrients, but their nutrient composition and safety evaluation have not been fully investigated yet. In this study, we investigated the nutrient composition and the acute and sub-chronic toxicity of locust powder in male rats. In the acute oral toxicological experiment, rats were administered locust powder at a dose of 10 or 20 g/kg/dose, followed by monitoring general signs of toxicity for 14 days. In the sub-chronic toxicological experiments, rats were fed with a diet containing 1% and 3% locust powder for 28 and 90 days. General signs of toxicity, body weight, plasma and blood components, weight and fat accumulation in tissues, and fecal fat excretion were investigated. The locust powder was rich in proteins, essential amino acids, minerals, and polyunsaturated fatty acids. In the acute toxicological experiment, no general signs of acute toxicity were observed at a dose of 20 g/kg. In the sub-chronic toxicological experiments, parameters related to red blood cell were lowered by the 3% locust powder for 28 days, but not for 90 days. Liver lipid accumulation and fecal fat excretion were increased by the 3% locust powder for 90 days, but the liver lipids contents were considered to be within a nontoxic level. Cecum contents and cecum short-chain fatty acids were lowered by the locust powder, which can be caused by its fiber and fiber-like components. In conclusion, acute and sub-chronic intake of locust powder had little effect on general, biochemical, and hematological signs of toxicity in rats. PRACTICAL APPLICATION: Edible insects are increasingly viewed as new sustainable protein sources for human foods and livestock feeds worldwide because of their high nutritional balance, high food conversion rate, and environmental merits. Here, we have clarified that a locust powder contains high levels of protein, polyunsaturated functional fatty acids, and minerals (iron, zinc, and magnesium), and intake of locust powder (3% in diet) had little effects on general, biochemical, and hematological signs of toxicity in male rats. Locust as an edible insect, in powder form, can contribute to human dietary needs., (© 2020 Institute of Food Technologists®.)

In insects, pheromones are detected by olfactory sensory neurons (OSNs) of the antennae that co-express pheromone receptors (PRs) and the "sensory neuron membrane protein 1" (SNMP1). Beyond its relevance for pheromone detection via the antenna, little is known about a potential expression and functional role of SNMP1 in cells of other chemosensory appendages. Here, we report that in the desert locust Schistocerca gregaria, SNMP1 is also expressed in the labial and maxillary palps of the mouthparts. In the palps, the SNMP1-positive cells were situated next to the so-called terminal sensilla that are considered as chemosensory. Moreover, the SNMP1-positive cells of the palps expressed the "odorant receptor co-receptor" (Orco), a marker for OSNs endowed with odorant receptors (ORs), suggesting that these cells are olfactory. With respect to an olfactory function of the SNMP1-positive cells, further analyses examining a possible expression of ORs (notably putative PRs) in the labial and maxillary palps revealed that several members of a particular OR subfamily from S. gregaria, the b-OR group, are co-expressed with SNMP1 in cells of the palps. Interestingly, b-OR types co-expressed with SNMP1 in antennal OSNs were also co-expressed with SNMP1 in cells of the palps, indicating a specific pairing in the expression of SNMP1 and given ORs in both antennae and palps. The co-expression of SNMP1 and certain b-ORs that are regarded as candidate PRs opens up the possibility that chemosensory cells on the palps of the desert locust may contribute to pheromone detection.

Chemosensory proteins (CSPs) are thought to play roles in the insect olfactory system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. The band-winged grasshopper, Oedaleus asiaticus Bei-Bienko, is one of the most important grasshopper pests in northern China, but there is little information about its olfactory system. In order to investigate the olfactory functions of CSPs in this pest, three CSP genes (OasiCSP4, OasiCSP11 and OasiCSP12) were expressed in Escherichia coli, and the binding affinities of the three recombinant CSP proteins were measured for 16 volatiles from the host plant (Stipa krylovii), fecal material and body of live adult O. asiaticus using fluorescence competitive binding assays. To further verify their olfactory functions, RNA interference (RNAi) and electrophysiological recording were conducted. The three recombinant proteins displayed different degrees of binding to various volatiles in ligand-binding assays, with OasiCSP12 having higher binding affinities for more volatiles than OasiCSP4 and OasiCSP11. OasiCSP12 exhibited strong binding affinities (Ki < 20 μΜ) for five host plant volatiles and one volatile from the live body of adult O. asiaticus. The transcript levels of the three OasiCSP genes were significantly lower after silencing the individual genes by RNAi, which in turn reduced the EAG responses in adults of both sexes to most tested compounds. Our study indicates that these three OasiCSPs are involved in the detection of volatile semiochemicals, and may play important roles in finding host plants and in aggregation in O. asiaticus.

The pathways of insect melatonin (MEL) biosynthesis apparently follow the same routes as those identified in vertebrates but information on MEL synthesis variations related with serotonin (5-HT), 5-hydroxy-indole acetic acid (5HIAA), and N-acetylserotonin (NAS) levels, as well as 5-HT N-acetyltransferase (NAT) activity throughout the day, is very limited in the insect nervous system. In the present study, the levels of MEL, metabolites (5-HT, NAS, and 5-HIAA) and enzyme NAT were determined in the optic lobes and the midbrain of the grasshopper Oedipoda caerulescens, in conditions of light and darkness. In both tissues, a different pattern of MEL synthesis was observed over the light/dark cycle. Variations in the levels of 5-HT, NAS and NAT activity related to the synthesis of cerebral MEL follow a pattern very similar to that observed in the pineal of mammals, with a peak of synthesis in the first half of the scotophase. Also, we observed differences in the metabolism of 5-HT between the optic lobes and the midbrain light/dark-dependent., (© 2019 Wiley Periodicals, Inc.)

Vitellogenin receptor (VgR) mediates the intake of vitellin via oocytes, thus exerting an important role in vitellogenesis. In this study, reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends techniques were adopted to clone the CiVgR gene, namely the VgR gene of Calliptamus italicus, i.e., Orthopteran. The full length of CiVgR was 5,589 bp, and the open reading frame was estimated to be 5,265 bp, which encoded 1,754 amino acids (aa). Sequence alignment analysis showed that CiVgR belonged to the superfamily of low-density lipoprotein receptor genes, which contained several conserved domains, including ligand-binding domains, epidermal growth factor precursor homology domains, transmembrane domains, and cytoplasmic domains. However, no O-linked sugar domain was identified. Phylogenetic analysis showed that CiVgR had the closest genetic relationship to Blattarias. RT-PCR showed that CiVgR was only specifically expressed in the ovarian tissue of females. quantitative real time polymerase chain reaction showed that the transcription of CiVgR already appeared in the fourth-instar nymph of C. italicus, which gradually increased after adult emergence, peaked at the previtellogenesis stage, and then started to decrease. The expression pattern of CiVgR was closely associated with vitellogenesis. The findings of this study further our understanding of the molecular mechanisms involved in the reproduction of C. italicus, and provide new ideas to control this insect., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

The excellent rapid jumping and kicking of locusts are largely attributed to the power amplification mechanisms due to the semi-lunar processes (SLP) at their distal metathoracic femurs, especially dorsal-core (i.e., portion II) and ventral-core parts (i.e., portion III). The physiological range of strain rates at the two portions of locust SLP is quite broad in the periods of energy storage and release (approximately three orders). However, it still remains elusive how the mechanical properties of the two SLP portions change with the strain rate. We identified the elastic moduli and material compositions of SLP portions II and III by using nanoindentation and confocal laser scanning microscope. Apparent and creep-corrected reduced elastic moduli were calculated to represent the total energy absorption and storage, respectively. The results revealed that both portions II and III exhibit strain rate-sensitive elastic moduli, regardless of water content. The efficiency of elastic energy storage is only 51-70% in the case of low strain rate. This work can deepen our understanding in the energy storage and release mechanisms in locust locomotion and further provide guidelines for biomimetic design of power amplification apparatus in jumping robots., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Juvenile hormones (JH) are key endocrine regulators produced by the corpora allata (CA) of insects. Together with ecdysteroids, as well as nutritional cues, JH coordinates different aspects of insect postembryonic development and reproduction. The function of the recently characterized JH receptor, Methoprene-tolerant (Met), appears to be conserved in different processes regulated by JH. However, its functional interactions with other hormonal signalling pathways seem highly dependent on the feeding habits and on the developmental and reproductive strategies employed by the insect species investigated. Here we report on the effects of RNA interference (RNAi) mediated SgMet knockdown during the first gonadotrophic cycle in female desert locusts (Schistocerca gregaria). This voracious, phytophagous pest species can form migrating swarms that devastate field crops and harvests in several of the world's poorest countries. A better knowledge of the JH signalling pathway may contribute to the development of novel, more target-specific insecticides to combat this very harmful swarming pest. Using RNAi, we show that the JH receptor Met is essential for ovarian maturation, vitellogenesis and associated ecdysteroid biosynthesis in adult female S. gregaria. Interestingly, knockdown of SgMet also resulted in a significant decrease of insulin-related peptide (SgIRP) and increase of neuroparsin (SgNP) 3 and 4 transcript levels in the fat body, illustrating the existence of an intricate regulatory interplay between different hormonal factors. In addition, SgMet knockdown in females resulted in delayed display of copulation behaviour with virgin males, when compared with dsGFP injected control animals. Moreover, we observed an incapacity of adult dsSgMet injected female locusts to oviposit during the time of the experimental setup. As such, SgMet is an essential gene playing crucial roles in the endocrine communication necessary for successful reproduction of the desert locust.

The yellow protein of the takeout family (YPT) controls the development of yellow body color in the desert locust. This study focused on two aspects related to YPT in the locust. We first examined the expression pattern of YPT during nymphal stages because yellowing was not obvious during the early instars. YPT expression levels were extremely low in the second and third instars compared with the last two nymphal instars. Warm rearing temperature and juvenile hormone (JH) injection, which stimulated YPT expression in the late instars, had little effect in the second instar, suggesting that YPT expression during the early instars was suppressed and could not be stimulated by either of these factors. We also investigated delayed yellowing in fasting male adults, under the hypothesis that fasting decreased the JH titers and delayed the onset of YPT expression. Yellowing was delayed in fasting adults compared with well-fed adults and YPT expression was stimulated by JH injections at Day 15. However, we failed to obtain evidence that fasting significantly influenced the expression levels of YPT and the JH early-inducible gene Krüppel homolog 1 at Days 15 and 20 post-adult emergence. Results suggest that a YPT-independent mechanism possibly induces delayed yellowing in fasting males., (© 2019 Wiley Periodicals, Inc.)

Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity., Competing Interests: NO authors have competing interests.

Odorant-binding proteins (OBPs) are essential molecular elements of the insect chemosensory system, which is composed of the antennae and the mouthpart palps (maxillary and labial). In this study, we have analysed the expression and the sensilla specificity of 14 OBP subtypes in the palps of the desert locust Schistocerca gregaria. The locust palps comprise only a low number of sensilla basiconica but a high number of sensilla chaetica. Employing a variety of approaches, we found that only a subset of the antennal OBP repertoire was expressed in both palp types. These OBPs were previously shown to be expressed either in sensilla basiconica or sensilla chaetica of the antennae. Comparing the expression pattern in the two chemosensory organs revealed similarities and differences; most remarkably, two OBP subtypes, OBP6 and OBP8, were found in both sensilla types on palps, whereas on the antennae they were solely expressed in one sensillum type. Together, the data indicate a differential, but partly overlapping, expression of OBPs in the two sensilla types of the palps. The differences in the expression pattern of OBP subtypes between antennae and palps might be indicative for distinct functions of the OBPs in the two chemosensory organs., (© 2018 The Authors Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) genes are responsible for the regulation of insect insulin-like pathway (ILP), cells growth, metabolism initiation, gene transcription and observing immune response. Signal transduction in insect cell is also associated with PTPs and PTKs. The grasshopper (Oedaleus asiaticus) 'Bey-Bienko' were treated with dsRNA of protein tyrosine non-receptor type 4 (PTPN4) and protein tyrosine kinase 5 (PTK5) along with control (water). Applying dsPTK5 treatments in 5th instar of Oedaleus asiaticus, significant reduction was recorded in body dry mass, growth rate and overall performance except survival rate. Whereas with PTPN4, no such significant impact on all of these growth parameters was recorded. Expression of genes in ILP 5th instar of Oedaleus asiaticus by the application of dsPTPN4 and dsPTK5 revealed that PTK, INSR (insulin receptor), IRS (insulin receptor substrate), PI3K (phosphoinositide 3-kinase), PDK (3-phosphoinositide-dependent protein kinase), Akt (protein kinase B) and FOXO (forkhead transcription factor) significantly expressed with downregulation except PTPN4, which remained non-significant. On the other hand, the phosphorylation level of ILP four proteins in O. asiaticus with the treatment of dsPTPN4 and dsPTK5 significantly affected P-IRS and P-FOXO, while P-INSR and P-AKT remained stable at the probability level of 5%. This indicated that the stress response in the O. asiaticus insulin-like signalling pathway (ILP) reduced. Regarding association of protective enzymatic activities, ROS (relative oxygen species), CAT (catalase) and PO (phenol oxidase) increased significantly with exposure to dsPTK5 as compared to dsPTPN4 and control, while exposure of 5th instar of O. asiaticus to dsPTPN4 treatment slightly raised CAT and PO activities with but significant contribution. No such significant effect on MFO and POD was seen using dsPTPN4 and dsPTK5. This showed that in the ILP of O. asiaticus, PTK5 was detrimental to growth, body mass and overall performance, which ultimately benefited insect detoxification with high-energy cost.

Many aggregating animals use aposematic signals to advertise their toxicity to predators. However, the coordination between aposematic signals and toxins is poorly understood. Here, we reveal that phenylacetonitrile (PAN) acts as an olfactory aposematic signal and precursor of hypertoxic hydrogen cyanide (HCN) to protect gregarious locusts from predation. We found that PAN biosynthesis from phenylalanine is catalyzed by CYP305M2 , a novel gene encoding a cytochrome P450 enzyme in gregarious locusts. The RNA interference (RNAi) knockdown of CYP305M2 increases the vulnerability of gregarious locusts to bird predation. By contrast, the elevation of PAN levels through supplementation with synthetic PAN increases the resistance of solitary locusts to predation. When locusts are attacked by birds, PAN is converted to HCN, which causes food poisoning in birds. Our results indicate that locusts develop a defense mechanism wherein an aposematic compound is converted to hypertoxic cyanide in resistance to predation by natural enemies.

Juvenile hormone (JH) is known to promote cell polyploidization for insect vitellogenesis and egg production, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that the expression of cyclin-dependent kinase 6 (Cdk6) and adenovirus E2 factor-1 (E2f1), the core mediators in cell cycle progression is regulated by JH and its receptor Methoprene-tolerant (Met). JH acts through its receptor complex comprised of Met and Taiman to directly activate the transcription of Cdk6 and E2f1. Depletion of Cdk6 or E2f1 results in significantly decreased ploidy, precocious mitotic entry and increased cell numbers in the fat body, accompanied by substantial reduction of Vitellogenin gene expression, blocked ovarian growth and arrested oocyte maturation. These findings indicate a crucial role of Cdk6 and E2f1 in JH-regulated polyploidization and vitellogenesis as well as a novel regulatory machinery for endocycling in insects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)