Bioaccessibility of trace elements and Fe and Al endogenic nanoparticles in farmed insects: Pursuing quality sustainable food.

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.

Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

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.

Insights into the molecular mechanism of yellow cuticle coloration by a chitin-binding carotenoprotein in gregarious locusts.

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 ß-carotene-binding protein, BBP. HPLC and Raman spectroscopy confirmed that recombinant BBP avidly binds ß-carotene, inducing the unusual vibronic structure of its absorbance spectrum, just like native BBP extracted from the locust cuticles. Bound to recombinant BBP, ß-carotene exhibits pronounced circular dichroism and allows BBP to withstand heating (T0.5 = 68 °C), detergents and pH variations. Using bacteria producing distinct xanthophylls we demonstrate that, while ß-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.

A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), 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 m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.

The effects of dietary amino acid balance on post-embryonic development in a lubber grasshopper.

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.

Isolation and characterization of intestinal bacteria associated with cellulose degradation in grasshoppers (Orthoptera).

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.

Low Glycolysis Is Neuroprotective during Anoxic Spreading Depolarization (SD) and Reoxygenation in Locusts.

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 CO2 output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO2 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.

PLIN2-induced ectopic lipid accumulation promotes muscle ageing in gregarious locusts.

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 ß-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.

The chemical ecology of locust cannibalism.

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

A chemical defense deters cannibalism in migratory locusts.

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.

Identification and analysis of odorant receptors expressed in the two main olfactory organs, antennae and palps, of Schistocerca americana.

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.

Protein storage and reproduction increase in grasshoppers on a diet matched to the amino acids of egg yolk protein.

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.

Neuroendocrinal and molecular basis of flight performance in locusts.

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.

Variable stoichiometric and macronutrient responses to lizard predation in Ozark glade grasshopper communities.

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.

Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.

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.

A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts.

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.

Piwi/piRNAs control food intake by promoting neuropeptide F expression in locusts.

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.

Locust density shapes energy metabolism and oxidative stress resulting in divergence of flight traits.

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.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

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 EC50 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.

Four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 in Oxya chinensis.

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 CuCl2 and OcicCuZnSOD1b was significantly up-regulated by 14.67 mM CuCl2. OcicCuZnSOD1a was significantly inhibited by 19.13 mM ZnSO4 while OcicCuZnSOD1d were significantly induced by 22.61 mM ZnSO4. Disc diffusion assay showed that the four isoforms of OcicCuZnSOD1 made the killing zones smaller surrounding the CdCl2-soaked filter discs. However, the reduction ratios of OcicCuZnSOD1a were the highest. These results implied that the four transcripts played roles in defense against CdCl2-induced oxidative stress while OcicCuZnSOD1a had stronger antioxidant capacity.