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Myosin light chain kinase (MLCK) is a dedicated kinase of myosin regulatory light chain (RLC), playing an essential role in the regulation of muscle contraction and cell motility. Much of the knowledge about MLCK comes from the study of vertebrate MLCK, and little is known about insect MLCK. Here, we identified the single MLCK gene in the locust Locusta migratoria, which spans over 1400 kb, includes 62 exons and accounts for at least five transcripts. We found that the five distinct transcripts of the locust MLCK gene are expressed in a tissue‐specific manner, including three muscle‐specific isoforms and two generic isoforms. To characterise the kinase activity of locust MLCK, we recombinantly expressed LmMLCK‐G, the smallest locust MLCK isoform, in insect Sf9 cells. We demonstrated that LmMLCK‐G is a Ca2+/calmodulin‐dependent kinase that specifically phosphorylates serine 50 of locust muscle myosin RLC (LmRLC). Additionally, we found that almost all LmRLC molecules in the flight muscle and the hindleg muscles of adult locusts are phosphorylated. [ABSTRACT FROM AUTHOR]

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N‐terminal SH3‐like domain, one lip of the nucleotide‐binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9‐baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1‐HMM displayed the highest level of actin‐activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1‐HMM, we produced a number of chimeras of FL1‐HMM and AB4‐HMM. Substitution with the relay of AB4‐HMM (encoded by exon‐14c) substantially decreased the ATPase activity of FL1‐HMM, and conversely, the relay of FL1‐HMM (encoded by exon‐14a) enhanced the ATPase activity of AB4‐HMM. Mutagenesis showed that the exon‐14a‐encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1‐HMM. Those results indicate that the alternative relay encoded by exon‐14a/c play a key role in regulating the ATPase activity of FL1‐HMM and AB4‐HMM. [ABSTRACT FROM AUTHOR]

BACKGROUND: The fungal genera Metarhizium contain many important multiple species that are used as biocontrol agents and as model organisms for exploring insect–fungal interactions. Metarhizium spp. exhibit different traits of pathogenicity, suggesting that the pathogenesis can be quite distinctive. However, the underlying differences in their pathogenesis remain poorly understood. RESULTS: Pathogenicity analysis showed that Metarhizium anisopliae (strain CQMa421) displayed higher virulence against oriental migratory locusts, Locusta migratoria manilensis (Meyen), than the acridid‐specific specie Metarhizium acridum (strain CQMa102). Relative to M. acridum, M. anisopliae possessed a higher conidial hydrophobicity, increased ability to penetrate the host, accelerated growth under hypoxia and enhanced ability for the utilization of different carbon sources. Different distributions of carbohydrate epitopes at cell wall surface of M. anisopliae might also contribute to successful evasion of host immune defenses. Comparative genomics showed that M. anisopliae has 98 more virulence‐related secreted proteins (133) than M. acridum (35), which can be functionally classified as hydrolases, virulence effectors, cell wall degradation and stress tolerance‐related proteins, and helpful to the cuticle penetration and host internal environment adaption. In addition, differences in genomic clusters specifically related to secondary metabolites, including the clusters of Indole‐NRPS hybrid, T1PKS‐NRPS like hybrid, Betalactone, Fungal‐Ripp and NRPS‐Terpene hybrid, may lead to differences in core virulence‐related secondary metabolite genes in M. acridum (18) and M. anisopliae (36). CONCLUSION: The comparative study provided new insights into the different infection strategies between M. anisopliae and M. acridum, and further facilitate the identification of virulence‐related genes for the improvement of mycoinsecticides. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The oriental migratory locust is a major crop pest across eastern and south‐eastern Asia. Metarhizium anisopliae is an effective biopesticide agent used for locust control, but its performance is temperature dependent, and thus can be more variable than chemical pesticide performance. To predict biopesticide performance for the control of the oriental migratory locust, we adapted a previous temperature‐dependent model and validated it using field trial data. To increase the applicability of this model, we explored the use of readily available temperature variables, as well as our own satellite‐derived canopy temperature variable, to run the model. RESULTS: Compared to collected in situ temperature data, our canopy temperature variable most accurately represented the ambient temperature experienced by the locust. When the biopesticide performance model was run using this canopy temperature and compared to field trials results, the model predictions were more accurate than when the model was run with the other temperature variables. The accuracy of the biopesticide performance model was impacted by vegetation cover, but across the areas most associated with locust oviposition, growth and migration, the model predictions were satisfactorily accurate to guide biopesticide operational use. CONCLUSION: We validated the model in six provinces in China, representing the three agro‐ecological zones largely representative of the oriental migratory locust problem areas in China, Thailand, Cambodia and Vietnam. Whilst further validation work is needed, this model could be used in these countries to assess, at a fine spatial scale, the appropriateness of M. anisopliae for controlling the oriental migratory locust. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The Sahara Desert is one of the major ecological barriers of the Palearctic‐Africa bird migration system. We analysed the behaviour of GPS‐tagged booted eagles (Aquila pennata) breeding in Spain and wintering in Tropical Africa, focusing on the Sahara crossing. We searched for anomalous track segments recorded during the desert crossing, finding that non‐directional and/or slow movements occur in 35% of the migratory journeys and last up to 31 days (average: 5.9). The analyses show that this behaviour is not triggered by adverse weather conditions, and is shown especially by females during spring. The length of the stopping periods affects the arrival date to the breeding grounds only in males, which means that females buffer the potential delay with an earlier migration start, suggesting that this behaviour is followed deliberately. These wandering movements occur in hyper‐arid areas without vegetation cover. A similar behaviour has not been previously recorded in any other trans‐Saharan raptor and is probably related to the ability of booted eagles to take advantage of food resources unexploited by other predators, such as migratory songbirds stopping in the desert during the day, and migratory locusts. If confirmed by further research involving higher resolution tracking and field observations, our findings may show a new way of combining foraging and migration, without a well‐defined directed trajectory, nor stopping in a restricted spot, but instead wandering over wide areas. [ABSTRACT FROM AUTHOR]

BACKGROUND: Locusts and grasshoppers are pests of many agricultural crops, and their frequent outbreaks worldwide threaten food security. Microbial control agents are currently used to suppress the early (nymphal) stages of pests, but they are often less effective against adults, which are primarily responsible for locust plagues. The fungal pathogen Aspergillus oryzae XJ‐1 has high pathogenicity in locust nymphs. To assess its potential for controlling locust adults, we evaluated the virulence of A. oryzae XJ‐1 (i.e., locust Aspergillus, LAsp) in locust adults using laboratory, field‐cage experiments, and a field trial. RESULTS: The lethal concentration of LAsp in adult Locusta migratoria was 3.58 ± 0.09 × 105 conidia mL−1 15 days after inoculation in the laboratory. A field‐cage experiment showed that the mortalities of adult L. migratoria were 92.0 ± 4.6% and 90.1 ± 3.2% 15 days after inoculation with 3 × 105 and 3 × 103 conidia m−2 of LAsp, respectively. A large‐scale field trial of 666.6 ha was conducted, in which a LAsp water suspension was applied at a concentration of 2 × 108 conidia mL−1 in 15 L ha−1 by aerial spraying via drones. The densities of mixed populations of L. migratoria and Epacromius spp. were significantly reduced by 85.4 ± 7.9%–94.9 ± 5.1%. In addition, the infection rates of surviving locusts collected from the treated plots were 79.6% and 78.3% on the 17th and 31st day after treatment, respectively. CONCLUSION: These results indicate that A. oryzae XJ‐1 is highly virulent in adult locusts and that it has high potential for the control of locusts. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The Clustered Regularly Interspaced Short Palindromic Repeats‐associated (CRISPR‐Cas) system has been used as a powerful diagnostic tool because of its trans‐cleavage ability to accurately recognize and cleave specific nucleic acid targets. However, its application in insect pest detection has rarely been reported. The aim of the current study was to develop a new molecular identification method for insect based on CRISPR‐Cas13a system. In this study, we have developed a highly specific and sensitive detection system that combines polymerase chain reaction, CRISPR‐Cas13a system and lateral flow dipstick (PCR‐Cas13a‐LFD) to rapidly detect the presence of Locusta migratoria manilensis (Orthoptera: Acrididae) DNA and discriminate it from other species, by using material from different stages of development. A pair of specific primers and crRNA were designed based on the Gene DEAD‐box DDX10 mRNA sequence. For visual readout, a FAM‐RNA‐biotin reporter was used for detection. The CRISPR‐Cas13a‐based detection method utilizes the visual identification of L. migratoria manilensis at different developmental stages or with the use of incomplete samples. The test results could be distinguished within 2 h. The sensitivity of the PCR‐Cas13a‐LFD detection system reached 0.1 ng/μL, a 10‐fold increase compared with that of the PCR with electrophoresis. The LFD coupled with CRISPR‐Cas13a (Cas13a‐LFD) is promising for the portable, onsite and fast identification of insect pests. This study provides a reference for the development of a new method for insect identification. [ABSTRACT FROM AUTHOR]

Hypoxia challenges aerobic organisms in numerous environments, and hypoxic conditions may become more severe under future climate‐change scenarios. The impact of hypoxia on the development of terrestrial insect embryos is not well understood. Here, to address this gap, embryonic life‐history traits of migratory locust Locusta migratoria from low‐altitude and high‐altitude regions were compared under 2 oxygen levels: normoxia (i.e., 21 kPa oxygen partial pressure and mild hypoxia (i.e., 10 kPa oxygen partial pressure). Our results demonstrated that, whether reared under normoxia or mild hypoxia, L. migratoria from high‐altitude populations had longer developmental times, reduced weight, and lower mean relative growth rate as compared with those from low‐altitude populations. When transferred from normoxia to mild hypoxia, nearly all the tested life‐history traits presented significant negative changes in the low‐altitude populations, but not in the high‐altitude populations. The factor 'strain' alone explained 18.26%–54.59% of the total variation for traits, suggesting that the phenotypic differences between L. migratoria populations from the 2 altitudes could be driven by genetic variation. Significant genetic correlations were found between life‐history traits, and most of these showed differentiation between the 2 altitudinal gradients. G‐matrix comparisons showed significant structural differences between L. migratoria from the 2 regions, as well as several negative covariances (i.e., trade‐offs) between traits in the low‐altitude populations. Overall, our study provides clear evidence that evolutionary divergence of embryonic traits between L. migratoria populations from different altitudes has occurred. [ABSTRACT FROM AUTHOR]

Paranosema (Antonospora) locustae (Canning) is a microsporidium with an extensive host range including >100 reported insect hosts from the order Orthoptera. The susceptibility of two species of locusts (Orthoptera: Acrididae) – the migratory locust, Locusta migratoria subsp. migratorioides (Fairmaire & Reiche), and the desert locust, Schistocerca gregaria Forsskål – to P. locustae was compared under laboratory conditions at a decreased temperature of 27 °C to enhance susceptibility to infection. Locusta migratoria was found highly vulnerable as infection percentages exceeded 70% at 104, 105, and 106 spores per insect, and mortality increased with increasing dosage. Conversely, P. locustae spores were not found in S. gregaria throughout the experiment. Only at 107 spores per insect, as many as 20% of S. gregaria were infected. This suggests that the desert locust is resistant to P. locustae infection, as opposed to the migratory locust. The laboratory models of these parasite–host systems may be useful for elucidating mechanisms of insect immunity to microsporidia. [ABSTRACT FROM AUTHOR]

Lower plant resistance to herbivores following domestication has been suggested as the main cause for higher feeding damage in crops than in wild progenitors. While herbivore compensatory feeding has also been proposed as a possible mechanism for raised damage in crops with low nutritional quality, predictions regarding the effects of plant domestication on nutritional quality for herbivores remain unclear. In particular, data on primary metabolites, even major macronutrients, measured in the organs consumed by herbivores, are scarce. In this study, we used a collection of 10 accessions of wild ancestors and 10 accessions of modern progenies of Triticum turgidum to examine whether feeding damage and selectivity by nymphs of Locusta migratoria primarily depended on five leaf traits related to structural resistance or nutrient profiles. Our results unexpectedly showed that locusts favored wild ancestors over domesticated accessions and that leaf toughness and nitrogen and soluble protein contents increased with the domestication process. Furthermore, the quantitative relationship between soluble protein and digestible carbohydrates was found to poorly meet the specific requirements of the herbivore, in all wheat accessions, both wild and modern. The increase in leaf structural resistance to herbivores in domesticated tetraploid wheat accessions suggested that resource allocation trade‐offs between growth and herbivory resistance may have been disrupted by domestication in the vegetative organs of this species. Since domestication did not result in a loss of nutritional quality in the leaves of the tetraploid wheat, our results rather provides evidence for a role of the content of plants in nonnutritive nitrogenous secondary compounds, possibly deterrent or toxic, at least for grasshopper herbivores. [ABSTRACT FROM AUTHOR]

Clathrin heavy chain (Chc) is a constituent of clathrin‐coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but appears to have physiological roles also at the organismal level. Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells. However, the physiological functions of Chc in insects remain poorly understood. Here, we identified a Chc ortholog from a Locusta migratoria transcriptome database. RT‐qPCR revealed that LmChc was constitutively expressed in fifth‐instar nymphs. In this developmental stage, LmChc showed the highest expression in the ovary followed by hemolymph, testis, hindgut, midgut, and foregut. In isolated hemocytes, we detected the Chc protein in patches at the plasma membrane. To examine the role of LmChc in L. migratoria during development, RNA interference was performed by injecting dsRNA into the early fifth‐instar nymphs. Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult. In addition, silencing of LmChc resulted in abnormal development of the ovaries, the size of which was significantly smaller than that in controls. Taken together, our results suggest that LmChc is a vital gene in L. migratoria that plays an important role in growth, development, and reproduction. LmChc may be used as an efficient RNAi target gene for developing dsRNA‐based biological insecticides to manage insect pests. [ABSTRACT FROM AUTHOR]

The ovipositor comprises the external genitalia of female insects, which plays an important role in the mating and ovipositing process of insects. However, it remains rudimentary of regional gene expression and physiological function in the ovipositor during structural development. Here, we analysed the basic structure and characteristics of the ovipositor in the migratory locust Locusta migratoria. RNA‐seq analysis revealed the specialization of chitin metabolism, lipids synthesis and transport, tanning and cuticular protein genes in the ovipositor. Among them, two cuticle protein genes, LmCP8 and LmACP79, were identified, which are specifically expressed in the ovipositor. Functional analysis based on RNA interference showed that deficiency of LmCP8 affected the structural development of the ovipositor resulting in the retention of a large number of remaining unproduced oocysts in the ovary of the locusts. Our results provide a fundamental resource to investigate the structural development and physiological function of the ovipositor in L. migratoria. [ABSTRACT FROM AUTHOR]

BACKGROUND: The selective aphicide flonicamid is known to cause symptoms in aphids that are like those of chordotonal organ TRPV channel modulator insecticides such as pymetrozine, pyrifluquinazon and afidopyropen. Flonicamid is classified by the Insecticide Resistance Action Committee as a chordotonal organ modulator with an undefined target site. However, although it has been shown not to act on TRPV channels, flonicamid's action on chordotonal organs has not been documented in the literature. RESULTS: Flonicamid causes locusts to extend their hindlegs, indicating an action on the femoral chordotonal organ. In fruit flies, it abolishes negative gravitaxis behavior by disrupting transduction and mechanical amplification in antennal chordotonal neurons. Although flonicamid itself only weakly affects locust chordotonal organs, its major animal metabolite 4‐trifluoromethylnicotinamide (TFNA‐AM) potently stimulates both locust and fly chordotonal organs. Like pymetrozine, TFNA‐AM rapidly increases Ca2+ in antennal chordotonal neurons in wild‐type flies, but not iav1 mutants, yet the effect is nonadditive with the TRPV channel agonist. CONCLUSIONS: Flonicamid is a pro‐insecticide form of TFNA‐AM, a potent chordotonal organ modulator. The functional effects of TFNA‐AM on chordotonal organs of locusts and flies are indistinguishable from those of the TRPV agonists pymetrozine, pyrifluquinazon and afidopyropen. Because our previous results indicate that TFNA‐AM does not act directly on TRPV channels, we conclude that it acts upstream in a pathway that leads to TRPV channel activation. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are important enzymes that boost the hydrolysis of recalcitrant polysaccharides, such as chitin. They are found extensively in different insect species and are classified as auxiliary activities family 15 (AA15) LPMOs (LPMO15). Some of them were identified from the insect midgut and proven to act on chitin. However, knowledge about their physiological roles during insect growth and development remains limited. Here, we found that midgut‐specific LPMO15s are widely distributed in different insect orders, such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori. Using L. migratoria as a model insect, the function of midgut‐specific LmLPMO15‐3 during development was investigated. Double‐stranded RNA‐mediated downregulation of LmLPMO15‐3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes. Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix (PM), which suggested that LmLPMO15‐3 is essential for the deconstruction of the PM during molting. This study provides direct evidence of the physiological importance of a midgut‐specific LPMO15 during insect development. As L. migratoria is one of the most destructive agricultural pests, LmLPMO15‐3 is a potential target for pest management. [ABSTRACT FROM AUTHOR]

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT‐qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third‐instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl‐injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor‐like structures. Our results indicated that LmLgl may act as a tumor‐suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA‐based biological pesticides for managing insect pests. [ABSTRACT FROM AUTHOR]

BACKGROUND: CreA has been proved to be a core gene in asexual conidiation in Metarhizium acridum, which regulates the shift of normal conidiation and microcycle conidiation. At present, research on CreA in fungi has focused on carbon source metabolism. There is a lack of research on the effect of CreA in virulence of pathogenic fungi. RESULTS: The virulence of the MaCreA disrupted strain (ΔMaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of ΔMaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of ΔMaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. α‐1,3‐Glucan and β‐1,3‐glucan are more exposed on the surface of ΔMaCreA conidia than on the wild type. Lmspätzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of ΔMaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by ΔMaCreA in vitro. CONCLUSION: The main reasons for the loss of virulence of ΔMaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of ΔMaCreA. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The troponin (Tn) complex, the sensor for Ca2+ to regulate contraction of striated muscle, is composed of three subunits, that is, TnT, TnI and TnC. Different isoforms of TnI and TnC are expressed in the thorax dorsal longitudinal muscle (flight muscle) and the hind leg extensor tibiae muscle (jump muscle) of the migratory locust, Locusta migratoria. The major Tn complexes in the flight muscle and the jump muscle are Tn‐171 (TnT1/TnI7/TnC1) and Tn‐153 (TnT1/TnI5/TnC3), respectively. Here, we prepared a number of recombinant Tn complexes and the reconstituted thin filaments, and investigated their regulation on thin filament. Although both Tn‐171 and Tn‐153 regulate thin filament in a Ca2+‐dependent manner, the extent of Ca2+ activation mediated by Tn‐171 was significantly lower than that by Tn‐153. We demonstrated that TnC1 and TnC3, rather than TnI5 and TnI7, are responsible for the different levels of the thin filament activation. Mutagenesis of TnC1 and TnC3 shows that the low level of TnC1‐mediated thin filament activation can be attributed to the noncanonical residue Leu60 in the EF‐hand‐II of TnC1. We therefore propose that, in addition to Ca2+, other regulatory mechanism(s) is required for the full activation of locust flight muscle. [ABSTRACT FROM AUTHOR]

Ecdysone‐induced protein 93F (E93) plays important roles during the metamorphosis process in insects. In this study, a cDNA of the LmE93 gene was identified from the transcriptome of Locusta migratoria, which consists of the 3378‐nucleotide open‐reading frame (ORF) and encodes 1125 amino acids with helix–turn–helix (HTH) motifs. Reverse transcription quantitative polymerase chain reaction analysis revealed that LmE93 was highest expressed in ovary. The LmE93 expression level was markedly low from the 3rd to 4th instar nymphs, and greatly increased in 1‐day‐old 5th instar nymphs with a peak on middle nymphal days, then declined in the late nymphal days. Moreover, injected dsLmE93 into 4th and 5th instar nymphs greatly reduced LmE93 transcripts, respectively, and prevented the process of metamorphosis, causing supernumerary nymphal stages. Hematoxylin–eosin staining of the integument showed that the apolysis occurred in advance in 4th instar nymphs, and old cuticle degradation was decreased in dsLmE93‐injected locusts of 5th instar nymphs. Smaller and no fully developed wings with reduced columns between the anterior and posterior regions were found in N6 and N7 supernumerary nymphs. In addition, the development of the ovary in dsLmE93‐injected locusts was severely blocked, the yolk was almost not formed and there was no development of ovarioles. The results indicated that LmE93 play key roles in the metamorphosis, cuticle, wing and ovarian development of locusts. [ABSTRACT FROM AUTHOR]

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue‐dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double‐stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria. [ABSTRACT FROM AUTHOR]

Insects prevent uncontrolled penetration of water and xenobiotics by producing an impermeable cuticle. The major component of the cuticle is chitin that adopts a crystalline structure thereby contributing to cuticle stability. Our understanding of the contribution of chitin to the cuticle barrier function is limited. Here, we studied the role of the DOMON domain protein Knickkopf (LmKnk) that is involved in chitin organization and cuticle permeability in the migratory locust Locusta migratoria. We show that LmKnk localizes to the chitin layer in the newly produced cuticle. Injection of double‐stranded RNA targeting LmKnk (dsLmKnk) in locust nymphs caused failure of moulting to the next stage. Histological experiments revealed that apolysis, i.e., the detachment of the old cuticle from the body surface, was normal; however, the newly synthesized cuticle was thinner than the cuticle of the control insects. Indeed, chitin content dropped after suppression of LmKnk expression. As seen by transmission electron microscopy, crystalline chitin organization was lost in dsLmKnk‐treated insects. In addition, the structure of pore canals, which are lipid transporting routes in the cuticle, was abnormal. Consistently, their content was reduced and, probably by consequence, lipid deposition on the cuticle was decreased after injection of dsLmKnk. Suppression of LmKnk transcript levels rendered L. migratoria more susceptible to each of four selected insecticides including malathion, chlorpyrifos, carbaryl and deltamethrin. Overall, our data show that LmKnk is needed for correct chitin amounts and organization, and their changes ultimately affect cuticular permeability in L. migratoria. [ABSTRACT FROM AUTHOR]

Background: Vacuolar (H+)‐ATPase (V‐ATPase) is a multi‐subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V‐ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V‐ATPase‐c functions in insects. Results: We identified and characterized the V‐ATPase‐c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV‐ATPase‐c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV‐ATPase‐c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV‐ATPase‐c. In addition, silencing of LmV‐ATPase‐c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double‐stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV‐ATPase‐c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. Conclusion: Our results indicate that LmV‐ATPase‐c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV‐ATPase‐c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

With the wide application of genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model system, we developed a non‐invasive method to accurately identify genome‐edited mutants by using DNA from insect exuviae. We compared the quantity and quality of genomic DNA from exuviae in five instar hoppers and found that the 1st instar exuviae had the highest DNA yield and content, while the 3rd instar exuviae had the best quality. Consensus genotypes were identified from genomic DNA of hoppers at different developmental stages in the same individuals. Moreover, we demonstrated that the amplification products from DNA extracted from locust exuviae are the consensus sequences with those from the hemolymph and foreleg pre‐tarsus. Therefore, non‐invasive samples provide the same genotyping results as minimally invasive and invasive samples of the same individuals. Furthermore, this identification method that uses genomic DNA from exuviae can be used for early screening of positive genome‐edited individuals in each generation for adult crossing. In our study, the non‐invasive identification method was not only simpler and provided results earlier than existing methods, but also had a better reproducibility and accuracy. This non‐invasive identification approach using genomic DNA from exuviae can be adapted to meet the growing demand for genetic analysis and will find wide application in insect genome editing research. [ABSTRACT FROM AUTHOR]

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th‐instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20‐hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down‐regulated, while that of LmChsI was substantially up‐regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation. [ABSTRACT FROM AUTHOR]

The vacuolar‐type H+‐ATPase (V‐ATPase) is an ATP‐dependent proton pump, which regulates various cellular processes. To date, most functional studies on V‐ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV‐ATPase a were identified in Locusta migratoria. RT‐qPCR analysis revealed that LmV‐ATPase a1 and LmV‐ATPase a2 are differentially expressed in various tissues and developmental stages. Injection of dsRNA for the common region of LmV‐ATPase a1 and LmV‐ATPase a2 into third‐instar nymphs resulted in a significant suppression of LmV‐ATPase a. The injected nymphs ceased feeding, lost body weight and finally died at a mortality of 98.6%. Furthermore, aberrations of midgut epithelial cells, the accumulation of electron‐lucent vesicles in the cytoplasm, and a partially damaged brush border were observed in dsLmV‐ATPase a‐injected nymphs using transmission electron microscopy. Especially, the mRNA level of wingles, and notch genes were dramatically down‐regulated in the dsLmV‐ATPase a‐injected nymphs. Taken together, our results suggest that LmV‐ATPase a is required for survival and midgut development of L. migratoria. Hence, this gene could be a good target for RNAi‐based control against locusts. [ABSTRACT FROM AUTHOR]

Locusta migratoria is a potential alternative food and animal feed additive in South Korea and has already been registered in the European Union. We investigated the optimal conditions to improve hatchability of L. migratoria eggs under varying hatching conditions for improved artificial rearing. The parameters such as temperature, photoperiod, illuminance, or relative humidity were altered, while the other factors were kept constant. Among the four different temperature treatments (25, 27, 30, and 33°C), the 33°C temperature showed the highest total hatching rate of 85.6% at days 8–12 after oviposition. For photoperiod, hatchability was the highest (81.2%) at 9 L/15D, followed by 80.0% at 12 L/12D, and 72.6% at 15 L/9D. Under illuminance conditions of 600, 1,800, and 3,500 lux, 3,500 lux displayed the highest total hatching rate of 76.1% at days 7–11 after oviposition. Among the relative humidity (RH) levels of 50%, 65%, and 80%, 65% RH provided the highest hatching rate of 86.4% at days 8–13 post‐oviposition. In short, the most effective conditions for hatchability were 33°C, 9 L/15D, 65% RH, and 3,500 lux. Under these conditions, the hatching rate increased significantly to 98.8%. Application of these conditions will contribute to improve hatchability, which will be necessary for industrialization of L. migratoria. [ABSTRACT FROM AUTHOR]

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th‐instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue‐specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental processes remains uninvestigated. In this study, we discovered functional binding sites for miR‐184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR‐184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR‐184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down‐regulation of miR‐184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR‐184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control. [ABSTRACT FROM AUTHOR]

Trichomes function in plant defenses against biotic and abiotic stresses; examination of glabrous lines, which lack trichomes, has revealed key aspects of trichome development and function. Tests of allelism in 51 glabrous rice (Oryza sativa) accessions collected worldwide identified OsSPL10 and OsWOX3B as regulators of trichome development in rice. Here, we report that OsSPL10 acts as a transcriptional regulator controlling trichome development. Haplotype and transient expression analyses revealed that variation in the approximately 700‐bp OsSPL10 promoter region is the primary cause of the glabrous phenotype in the indica cultivar WD‐17993. Disruption of OsSPL10 by genome editing decreased leaf trichome density and length in the NIL‐HL6 background. Plants with genotype OsSPL10WD‐17993/HL6 generated by crossing WD‐17993 with NIL‐HL6 also had fewer trichomes in the glumes. HAIRY LEAF6 (HL6) encodes another transcription factor that regulates trichome initiation and elongation, and OsSPL10 directly binds to the HL6 promoter to regulate its expression. Moreover, the transcript levels of auxin‐related genes, such as OsYUCCA5 and OsPIN‐FORMED1b, were altered in OsSPL10 overexpression and RNAi transgenic lines. Feeding tests using locusts (Locusta migratoria) demonstrated that non‐glandular trichomes affect feeding by this herbivore. Our findings provide a molecular framework for trichome development and an ecological perspective on trichome functions. [ABSTRACT FROM AUTHOR]

Summary: The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin BjαIT, the cuticle‐degrading protease Pr1A, and a double‐stranded RNA (dsRNA) that targets host gnbp3, individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor, Locusta migratoria, Plutella xylostella and Galleria mellonella, whereas the binary transgenic strain expressing both pr1A and BjαIT had increased virulence to T. molitor and L. migratoria, with no change in virulence against P. xylostella and G. mellonella. Importantly, all ternary transgenic strains simultaneously expressing pr1A, BjαIT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect‐specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests. [ABSTRACT FROM AUTHOR]

Wings are an important flight organ of insects and their morphogenesis depends on a series of cell‐to‐cell and cell‐to‐extracellular matrix interactions. Integrin as a transmembrane protein receptor mediates cell‐to‐cell adhesion, cell‐to‐extracellular matrix interactions and signal transduction. In the present study, we characterized an integrin gene that encodes integrinβ‐PS protein in Locusta migratoria. LmIntegrinβ‐PS is highly expressed in the wing pads and the middle stages of 5th instar nymphs. Immunohistochemical analysis revealed that the LmIntegrinβ‐PS protein was localized at the cell base of the two layers of wings. After suppression of LmIntegrinβ‐PS by RNA interference, the wing pads or wings were unable to form normally, with a blister wing appearance during nymph to nymph transition and nymph to adult transition. We further found that the dorsal and ventral epidermis of the wings after dsLmIntegrinβ‐PS injection were improperly connected and formed huge cavities revealed by hematoxylin and eosin staining. Furthermore, the morphology and structure of the wing cuticle was significantly disturbed which affected the stable arrangement and attachments of the wing epidermis. Moreover, the expression of related cell adhesion genes was significantly decreased in LmIntegrinβ‐PS‐suppressed L. migratoria, suggesting that LmIntegrinβ‐PS is required for the morphogenesis and development of wings during molting by stabilizing cell adhesion and maintaining the cytoskeleton of these cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: Diapause plays a vital role in the survival and population reproduction of the migratory locust in temperate regions. Although some studies have suggested that photoperiod and temperature are two important environmental factors involved in the induction and termination of embryonic diapause in locusts, the independent or synergetic roles of these two factors are still unclear. RESULTS: We designed crossover experiments to investigate the effects of photoperiod, temperature and treatment time on the induction and termination of embryonic diapause in locusts by optical coherence tomography (OCT), which can track the entire embryonic developmental process via noninvasive 3D real‐time imaging. Diapause induction experiments showed that a short photoperiod (in adults) and a low egg treatment temperature are the most important prerequisites for inducing egg diapause. The diapause rate reached 90.7% when the adults were reared under a short photoperiod (8 L:16 D) and the eggs were treated at 22 °C. However, the adult rearing temperature had no significant effect on diapause induction. Analysis of variance also confirmed that there was a significant interactive effect between parental photoperiod and egg treatment temperature. Moreover, diapause termination experiments showed that 13 °C (near the developmental threshold temperature of 15 °C) for 20 days is the most effective condition for terminating diapause. CONCLUSION: Our study provides accurate photoperiod and temperature thresholds for inducing and terminating diapause in the migratory locust, suggesting potential applications in the prediction and control of locust plagues. © 2021 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Even though Paranosema locustae is widely used in China as a biological agent for controlling grasshoppers, the mortality rate is initially quite low. This study sought to determine whether the simultaneous use of P. locustae and Beauveria bassiana would be a more effective control strategy. Additionally, changes in the intestinal microbial communities of migratory locusts infected with the two pathogens were analyzed to investigate the roles of gut microbes in pathogen–host interactions. The mortality rate of locusts inoculated with B. bassiana and P. locustae simultaneously was not significantly higher than expected, but the mortality rates of locusts inoculated with B. bassiana 3, 6, and 9 days after inoculation with P. locustae were significantly higher than if their effects were additive, indicating synergism. A MiSeq analysis found that Weissella was the most common bacterium, representing 41.48% and 51.62% of the total bacteria in the mid‐ and hindguts, respectively, and the bacterial declines were greatest during dual infections with B. bassiana and P. locustae. The appropriately timed combined application of P. locustae and B. bassiana was more effective against locusts than either treatment alone. Moreover, the combined inoculation of the two pathogens changed the gut microflora of locusts, indicating the potential relevancy of their synergistic effects on locust control. [ABSTRACT FROM AUTHOR]

The objective of this study is to identify potential insect species comparing with commonly used protein sources based on efficiency of the in vitro digestibility on dry matter (DMd), organic matter (OMd) and crude protein (CPd) in broiler chickens, black‐meat chickens (Native breed) and quails. Each of gastric mucosa, pancreas and duodenal mucosa were obtained from proventriculus, pancreas and duodenum, respectively. Crude digestive enzyme extracts (CTE) were extracted from these organs to perform in vitro digestibility. Eighteen insect samples and six commonly used protein sources were served as substrates which were evaluated on DMd, OMd and CPd in triplicate for each substrate. The CTE from gastric mucosa was used to simulate proventriculus, whereas small intestine was simulation by adding the CTE from pancreas and duodenum. The large variation of chemical composition between insect meals was presented. For commonly used protein sources, animal proteins were higher on digestibility than plant proteins (p <.001). Quails represented a great potential to digest insect meals comparing other animals. Based on CPd results, there were potential insect species for broiler chickens (Achroia grisella: AG, Tenebrio molitor: TM and Musca domestica), black‐meat chickens (Patanga succincta, TM and AG) and quails (Hermetia illucens, Acheta domesticus and Locusta migratoria; p <.001). The evidences from this study suggest that these insect species contain a great potential to use as alternative protein sources promoting an animal production with sustainability. However, the in vivo experimentation must be performed to confirm in further study. [ABSTRACT FROM AUTHOR]

Lipids of the insect cuticle have important roles in resistance against the arid environment and invasion of foreign substances. Fatty acid synthase (FAS) is an important enzyme of the insect lipid synthesis pathway. In the present study, we identified three FAS genes from transcriptome data of the migratory locust, Locusta migratoria, based on bioinformatics analyses. Among them, two FAS genes (LmFAS1 and LmFAS3) are highly expressed in the integument of fifth instar nymphs. Suppression of LmFAS1 and LmFAS3 by RNA interference caused lethality during ecdysis or shortly after moulting. The weight of the locusts and the content of lipid droplets were reduced compared with those of the control. The results of gas chromatography–mass spectrometry analysis showed that knockdown of LmFAS3 led to a decrease of both cuticular hydrocarbons and inner hydrocarbons (CHCs and IHCs) contents, especially the content of methyl branched hydrocarbons. By contrast, knockdown of LmFAS1 only resulted in a decrease in the IHC content, but not that of CHCs. By consequence, in LmFAS1‐ and LmFAS3‐suppressed locusts, hydrocarbon deficiency reduced desiccation resistance and enhanced cuticle permeability and sensitivity to insecticides. These results indicate that LmFAS1 and LmFAS3 are essential for hydrocarbon production and cuticle permeability, which play influential roles in waterproofing the insect cuticle. [ABSTRACT FROM AUTHOR]

The article discusses deserts locust swarms have devastated agricultural fields in east Africa, parts of the Middle East, as well as India and Pakistan and continue to spread. Topics include the young, flightless locusts congregate to gigantic marching bands of hoppers; and the locusts flew for the Red Sea and to Ethiopia.

BACKGROUND Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. RESULTS: We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT‐qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference‐mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. CONCLUSION: Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND Knickkopf (Knk) proteins play crucial roles in the formation of insect cuticle. Recent studies in the holometabolous insect red flour beetle (Tribolium castaneum) have shown that three Knk genes encoding TcKnk, TcKnk2 and TcKnk3 play different but essential roles at different developmental stages and in different tissues. However, the functions of Knk genes had not been fully explored in hemimetabolous insects such as the migratory locust Locusta migratoria. RESULTS: We identified three transcripts of LmKnk‐like genes LmKnk2 and LmKnk3 with the full‐length cDNA sequences, which were named as LmKnk2, LmKnk3‐FL and LmKnk3‐5′. These three transcripts were highly expressed before molting and mainly expressed in the integument. Among these genes, silencing only LmKnk3‐5′ by RNA interference (RNAi) caused molting defects and high mortality of the locusts. Injection of dsLmKnk3‐5′ dramatically decreased chitin content, but did not affect cuticle laminar ultra‐structures in the integument. After the knockdown of LmKnk3‐5′ transcript, lipid deposition on the cuticle surface was impeded, and locusts exhibited increased susceptibility to each of four insecticides in three different classes. However, no visible phenotypic changes were observed after LmKnk2 or LmKnk3‐FL was silenced by RNAi. CONCLUSION: We demonstrate that LmKnk3‐5′ is essential for cuticle formation in L. migratoria. This contrasts the findings that the cognate protein in T. castaneum TcKnk3‐5′ is dispensable for cuticle formation and survival. Hence, we provide some evidence that the function of Knk‐type proteins may be species‐specific. We therefore think that LmKnk3‐5′ may be a good target for the application of RNAi‐based technologies for species‐specific insect pest management. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND PPO (prophenoloxidase) cascade plays an important role in resisting invasion of entomogenous fungus. The 20‐hydroxyecdysone (20E) exerts potent effect on the innate immunity in many insects. However, whether 20E controls the PPO cascade system against fungi and the regulatory mechanism in insects remains unclear. RESULTS : In this study, both the proteome and transcriptome of Locusta migratoria were determined followed by the induction of 20E. Pattern recognition receptor GNBP‐2 (Gram‐negative binding proteins) has been identified that responded to 20E at both messenger RNA (mRNA) and protein levels. The PPO gene expression in fat body and PO (phenoloxidase) activity in plasma was found significantly induced after 20E injection and during the high‐20E developmental stage. However, when 20E signal was blocked by RNA interference (RNAi) of ecdysone receptor, the expression level of PPO and PO activity failed to be increased by 20E. Thus, 20E could not significantly induce the expression of PPO gene and PO activity after RNAi of GNBP‐2. Furthermore, 20E treatment notably enhanced the resistance of L. migratoria against Metarhizium anisopliae. Followed by of GNBP‐2 silencing, the mortality of nymphs was significantly increased under the stress of Metarhizium anisopliae, and 20E injection could not increase the resistance. CONCLUSION : The 20E regulates the PPO system to resist fungal invasion via regulating GNBP‐2 in worldwide pest L. migratoria. Our results provide insight into the mechanism of how 20E enhances the antimicrobial immunity, and will be beneficial for modification of entomogenous fungi targeting on hormones and the immune system. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Continuous jumping behavior, a kind of endurance locomotion, plays important roles in insect ecological adaption and survival. However, the methods used for the efficient evaluation of insect jumping behavior remain largely lacking. Here, we developed a locomotion detection system named JumpDetector with automatic trajectory tracking and data analysis to evaluate the jumping of insects. This automated system exhibits more accurate, efficient, and adjustable performance than manual methods. By using this automatic system, we characterized a gradually declining pattern of continuous jumping behavior in 4th‐instar nymphs of the migratory locust. We found that locusts in their gregarious phase outperformed locusts in their solitary phase in the endurance jumping locomotion. Therefore, the JumpDetector could be widely used in jumping behavior and endurance locomotion measurement. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1–serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label‐free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double‐stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests. [ABSTRACT FROM AUTHOR]

The endemic locust species on the Tibetan plateau, Locusta migratoria tibetensis Chen can grow and breed successfully under the plateau temperature environment. This study explored the molecular response process of Tibetan locusts to high and low temperatures by comparing its transcriptome under different temperature conditions. A total of 66,256 unigenes were obtained through RNA‐Seq, among which 30,051 were successfully annotated. In detail, there were 778 differentially expressed genes (DEGs) at high temperatures and 1,787 DEGs at low temperatures. Under high temperatures, the metabolic pathways of saturated and unsaturated fatty acids and amino acids were up‐regulated, producing a significant amount of fatty acids and amino acids. However, under low temperatures, the chitinase activity was enhanced, causing chitin to decompose into small sugar molecules. Meanwhile, various sugar metabolism pathways were up‐regulated when a significant amount of small sugar molecules were produced. Then, large quantities of alpha‐linolenic acid and arachidonic acid were accumulated to maintain the normal physiological functions of cells. The analysis of annotated genes revealed that heat shock proteins (Hsp) were associated with the response to temperature stress, and mainly involved in the synthesis and transportation of proteins, preventing protein damage and the decomposition of damaged proteins. These results provide a basis for the further understanding of the adaptation mechanism of Tibetan locusts to high and low temperatures, laying a foundation for further understanding the adaptability of Tibetan locusts on Tibetan plateaus. [ABSTRACT FROM AUTHOR]

Migratory insect pests pose a substantial challenge to global food security. These issues are particularly acute when pest incursions occur considerably beyond the expected range, through natural migration or human‐aided transport, because the lack of species‐specific control strategies and a potential absence of species‐specific natural enemies in the newly‐invaded area may lead to rapid establishment of a new pest.One such threat is posed by the Oriental migratory locust Locusta migratoria manilensis in China, which, historically, has been restricted to eastern China from the Bohai Gulf southwards, and now threatens to expand its range into the agriculturally important region of northeast China.We analyzed data from a recent outbreak of migratory locusts in Heilongjiang Province (extreme northeast China), > 700 km north of its current known range, and identified the source region, timing of arrival and probable migratory routes of this incursion.We further show that warming temperatures in this region will likely allow subsequent invasions to establish permanent populations in northeast China, and thus authorities in this important crop‐producing region of East Asia should be vigilant to the threat posed by this species. [ABSTRACT FROM AUTHOR]

BACKGROUND: Metarhizium acridum, is a specific acridid pathogen developed for use against the migratory locust (Locusta migratoria manilensis). Adenylate‐forming reductases (AFRs) include enzymes that are involved in natural product biosynthesis. Here, we genetically characterize the functions of a class IV AFR in M. acridum (MaAfrIV) on fungal development and virulence. RESULTS: Gene expression analyses indicated MaAfrIV was induced on locust wings early during the infection process. Surprisingly, loss of MaAfrIV increased virulence (25.20% decrease in the median lethal time) against the locust in topical bioassays but was no different than the wild type when the cuticle was bypassed by direct infection of conidia into the insect hemocoel. Virulence markers including protease (Pr1) expression and appressorial turgor pressure were higher in the mutant than the parent strain. No difference was seen in the expression of host immune genes (Toll pathway) or in polyphenol oxidase (PPO) activity in locusts infected by the ΔMaAfrIV or wild type strains. However, the ΔMaAfrIV strain was unable to successfully sporulate on dead cadavers. CONCLUSION: Disruption of MaAfrIV increased fungal virulence by promoting insect cuticle invasion without altering host immune response or fungal immune evasion. Although loss of MaAfrIV conferred an apparent benefit to the fungus in terms of enhanced virulence, a significant trade‐off was seen in the inability of the fungus to sporulate on the cadaver. As conidiation on the cadaver is essential for subsequent propagation in the environment, loss of MaAfrIV can reduce the engineering strains survivability in the field and improve the safety. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background: Entomogenous fungi are important factors in biological control, but innate immunity of insects restricts the efficiency of fungus infection. 20‐hydroxyecdysone (20E) is involved in regulating the immune response of insects. Our previous studies have revealed that 20E enhances the expression of antibacterial peptides in the worldwide pest Locusta migratoria. However, the mechanism by which 20E controls innate immunity against entomogenous fungi is still unknown. Results: In the present study, based on the transcriptome of L. migratoria fat bodies challenged by 20E, immulectin‐1 (LmIML‐1) was screened and identified to be involved in modulating antifungal immunity. Spatio‐temporal expression analysis showed LmIML‐1 was highly expressed in the fifth instar nymph stage, and mainly distributed in the fat bodies and hemolymph. Both exogenous and endogenous 20E could increase the transcription of LmIML‐1. In contrast, transcription of LmIML‐1 did not increase when the 20E signal was blocked by RNAi of LmEcR (ecdysone receptor). The expressed recombinant protein rLmIML‐1 possessed agglutination activity and promoted the encapsulation. RNA interference of LmIML‐1 reduced the encapsulation of hemocytes, decreased the antifungal activity of plasma against Metarhizium anisopliae and accelerated the death of nymphs under the stress of entomogenous fungus. Meanwhile, 20E did not increase the antifungal activity with silence of LmIML‐1 in L. migratoria. Conclusion: 20E enhances antifungal immunity by activating immulectin‐1 in L. migratoria. Our findings indicate a potential mechanism of 20E systematically regulating innate immune response to resist pathogens and provide a well‐defined molecular target for improving biological control. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Diapause is an important overwintering strategy enabling Locusta migratoria to survive under stressed conditions. We identified a novel dual‐specificity kinase gene that is differentially expressed between long and short day‐treated L. migratoria. To determine its function on photoperiodic diapause induction, we cloned the specific gene. Interestingly, phylogenetic analysis shows that this dual‐specificity kinase is of the mycetozoa protein kinase‐like (MPKL) type and may have been transferred horizontally from Mycetozoa to L. migratoria. RNA interference results confirm that MPKL promotes photoperiodic diapause induction of L. migratoria. Furthermore, MPKL significantly inhibits Akt and FOXO (i.e. forkhead box protein O) phosphorylation levels in ovaries, and also enhances reactive oxygen species, superoxide dismutase and catalase activities, whereas peroxidase activity is decreased under both photoperiodic regimes. The findings of the present study offer insight into the molecular mechanism responsible for dual‐specificity kinase‐induced diapause in insects. [ABSTRACT FROM AUTHOR]

BACKGROUND: The aryl hydrocarbon receptor (AhR) belongs to the bHLH‐PAS (basic Helix–Loop–Helix – Period/ARNT/Single minded) family of transcription factors. AhR is a ligand‐activated transcription factor, which participates in the sensing and transmitting stimuli of endogenous and exogenous chemicals, and subsequently activates the transcription of genes related to various physiological and detoxification functions. RESULT: In this study, a single full‐length LmAhR sequence was cloned and characterized. RNA interference (RNAi) and insecticide bioassays showed that LmAhR plays a vital role in chlorpyrifos susceptibility. To better identify aryl hydrocarbon receptor from locusta migratoria (LmAhR)‐regulated genes involved in chlorpyrifos susceptibility, a comparative transcriptome analysis was performed using double‐stranded (ds)GFP‐ and dsLmAhR‐injected Locusta migratoria. Differential gene expression analysis identified 145 down‐regulated and 67 up‐regulated genes (P ≤ 0.05 and fold change ≥2) in dsLmAhR‐knockdown insects. We selected 27 down‐regulated genes and verified their expression levels using reverse transcription quantitative PCR. Finally, one glutathione S‐transferase (GST) gene (LmGSTd7) was selected as a candidate detoxification gene and was further validated via RNAi and chlorpyrifos bioassays. CONCLUSION: Our data suggest that AhR is associated with chlorpyrifos susceptibility via the regulation of LmGSTd7 expression in L. migratoria. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Thermoregulation is a central aspect of animal physiology. Mobile ectotherms have the potential to influence their temperature through their location and orientation. Behavioural thermoregulation has been extensively studied in insects, particularly in the migratory locust Locusta migratoria. However, most field studies are confined to daytime observations typically using invasive thermocouples with obvious potential to disrupt natural behaviour.We demonstrate that miniature radiotransmitters represent an alternative and less invasive method to study insect thermoregulation. We discuss how this method can be used to study the thermal behaviour of free‐ranging animals for extended periods. Specifically, we show that there is a close correlation between temperature recordings from implanted thermocouples in locusts L. migratoria and externally mounted radiotransmitters on the same animals.Our experiments match earlier observations of locust thermoregulatory behaviour confirming that the locusts with transmitters exhibit 'normal' thermoregulatory responses to feeding and to infections (behavioural fever).Finally, we demonstrate the practicality of a radiotransmitter‐based system by recording natural thermoregulatory behaviour of locusts in a semi‐field setting. Our field study showed locusts actively chose warm microclimates during the day and cold microclimates at night. We conclude that the use of radiotelemetry in studies of behavioural thermoregulation in wild insects could provide unique continuous recordings of body temperature over several days. Such data will provide researchers with a more complete understanding of how insects use behavioural thermoregulation in nature. [ABSTRACT FROM AUTHOR]

The nuclear receptor‐mediated 20‐hydroxyecdysone (20E) signalling pathway plays crucial roles in insects by initiating and regulating moulting and metamorphosis. In the present study, we identified and characterized a cDNA encoding a putative nuclear receptor protein (Locusta migratoria hormone receptor 39, LmHR39) based on L. migratoria transcriptomics data. Reverse‐transcription quantitative PCR (RT‐qPCR) revealed that LmHR39 shows low‐level expression in the early days of fifth‐instar nymphs, and peak expression occurs on day 5, which is followed by a decrease before ecdysis. LmHR39 transcription could be induced by 20E in vivo and was significantly suppressed by knocking down the expression of the L. migratoria ecdysone receptor gene and early‐late gene LmHR3. After RNA interference of LmHR39 with double‐stranded RNA (dsRNA), 85% of the insects showed abnormal morphology, with curly wings after moulting and delayed eclosion time. Haematoxylin and eosin staining indicated that apolysis of the integument and wing pad cuticle in the dsLmHR39‐treated insects was delayed compared to that in the dsRNA for green fluorescent protein‐injected control. Furthermore, RNA‐sequencing and RT‐qPCR analysis showed the expression level of carboxypeptidase genes (Carboxypeptidase A (CPA) and Carboxypeptidase M (CPM)) and chitin degrading genes (LmChitinase5 (LmCHT5) and LmChitinase10 (LmCHT10)) dramatically declined in the dsLmHR39‐treated insects, implying that the LmHR39‐mediated 20E signalling pathway is involved in the regulation of carboxypeptidase genes (CPA and CPM) and chitinase genes (LmCHT5 and LmCHT10), and participated in apolysis of the integument and wing pads during locust moulting. [ABSTRACT FROM AUTHOR]

The arthropod communities are influenced by both local conditions and features of the surrounding landscape. Landscape complexity and stand factors may both influence arthropod communities in poplar forests, but the multiscale effects of these factors on poplar defoliators and natural enemies are still poorly understood. We collected poplar arthropods at 30 sampling sites within five forest landscapes in Xinjiang, China, and assessed whether landscape complexity and stand factors influence species abundance and diversity of poplar arthropods. Landscape complexity was quantified by several independent metrics of landscape composition, configuration, and connectivity at three spatial scales. We also determined the most powerful explanatory variables and the scale effect of each arthropod. Results found that landscape complexity and stand factors had different effects on different poplar arthropod communities. Landscape complexity promoted natural enemies at different spatial scales, but it inhibited the population of poplar defoliators at the scale of 200 m. Specifically, the abundance and diversity of all defoliators decreased with increasing proportion of nonhost plants. Landscape diversity only had a negative effect on defoliator abundance. The shape complexity of habitat patches increased the abundance of carabid beetles but reduced the abundance of green leafhoppers and migratory locusts. The abundance and diversity of predators increased with increasing structural connectivity of forest landscape. Additionally, both the abundance and diversity of all defoliators were positively correlated with the average height of herbaceous plants. Diversity of all defoliators increased with increasing size of host trees. The distance from sampling site to the nearest village positively influenced the abundance and diversity of all predators. Arthropod abundance and diversity in poplar forests were driven by stand factors and landscape complexity. Therefore, maintaining complex shape and structural connectivity of habitat patches and keeping poplar stands away from the village are crucial for management of forest landscape to enhance natural enemies. And in order to reduce the abundance of defoliators in poplar forest, the diversity of surrounding habitat types should be promoted within 200 m radii. [ABSTRACT FROM AUTHOR]