Expression and kinetic analysis of carboxylesterase LmCesA1 from Locusta migratoria.

OBJECTIVE: To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria. RESULTS: We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the Km values of LmCesA1 for α-naphthyl acetate (α-NA) and ß-naphthyl acetate (ß-NA) were 0.08 ± 0.01 mM and 0.22 ± 0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for α-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7-10 and at 10-30 °C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected. CONCLUSIONS: These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.

DNA methyltransferase 3 participates in behavioral phase change in the migratory locust.

DNA methylation plays important roles in the behavioral plasticity of animals. The migratory locust, Locusta migratoria, displays striking density-dependent phenotypic plasticity that can reversely transit between solitarious and gregarious phases. However, the role and the mechanism through which DNA methylation is involved in locust phase change remain unknown. Here, we investigated the expression levels of three DNA methyltransferase genes and their roles in the regulation of locust phase changes. All three Dnmt genes, namely, Dnmt1, Dnmt2 and Dnmt3 showed high expression levels in the brains of gregarious locusts. By contrast, only Dnmt3 transcript rapidly responded to population density changes, decreasing during the isolation of gregarious locusts and steadily increasing upon the crowding of solitarious locusts. Dnmt3 knockdown significantly reduced the phase-related locomotor activity, rather than the attraction index, in gregarious and crowded solitarious locusts. Transcriptome analysis showed that Dnmt3 knockdown upregulated the genes related to metabolism and transporting activity and downregulated those associated with oxidative stress response. The expression level of the phase-core transcriptional factor, hormone receptor HR3, was significantly suppressed in the brain after Dnmt3 knockdown. Moreover, there was significant overlap in the differentially expressed genes between Dnmt3 RNAi and HR3 RNAi data sets, suggesting HR3 may act as key transcriptional factor mediating Dnmt3-controlled gene expression profiles in locust brains. These findings suggest that Dnmt3 transcription is involved in locust behavioral transition, implying the possible roles of DNA methylation in phase-related phenotypic plasticity in locusts.

Molecular identification and diapause-related functional characterization of a novel dual-specificity kinase gene, MPKL, in Locusta migratoria.

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.

Nuclear receptor hormone receptor 39 is required for locust moulting by regulating the chitinase and carboxypeptidase genes.

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.

Knockdown of cytochrome P450 CYP6 family genes increases susceptibility to carbamates and pyrethroids in the migratory locust, Locusta migratoria.

Insect cytochrome P450 monooxygenase (CYP) plays a key role in the detoxification of insecticides. In this study, four cDNA sequences of CYP6 genes were identified and characterized. Transcription levels of LmCYP6HC1 and LmCYP6HCL1 were high in first- and fourth-instar nymph stages, respectively. LmCYP6HN1 was primarily expressed in the egg to third-instar nymph stages, while LmCYP6HQ1 was predominantly expressed in the stages from fourth-instar nymph to the adult. The four CYP6 genes were predominantly distributed in the antenna, brain, fat body, integument, and hemolymph. Piperonyl butoxide exposure inhibited total CYP activity and synergized the toxicity of carbamates and pyrethroids. Knockdown of LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 increased nymph mortality following exposure to carbaryl, and silencing of LmCYP6HC1, LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 comprehensively raised nymph mortality following exposure to fluvalinate. Knockdown of LmCYP6HL1 or LmCYP6HN1 significantly increased nymph mortality following exposure to cypermethrin or fenvalerate, respectively. These results suggest that the CYP6 family plays a key role in determining the susceptibility of Locusta migratoria to both carbamates and pyrethroids.

Olive Leaf Extracts Toxicity to the Migratory Locust, Locusta migratoria: Histopathological Effects on the Alimentary Canal and Acetylcholinesterase and Glutathione S-Transferases Activity.

The migratory locust, Locusta migratoria (Linnaeus), is the most widespread locust species. Frequent applications of insecticides have inevitably resulted in environmental pollution and development of resistance in some natural populations of the locust. To find a new and safe alternative to conventional insecticides, experiments were conducted to assess the effect of olive leaf extracts on L. migratoria fifth instar larvae. The methanolic extracts were prepared from the leaves sampled during four phenological growth stages of olive tree which are as follows: Cluster formation (Cf), Swelling inflorescence buds (Sib), Full flowering (Ff), and Endocarp hardening (Eh). The most relevant result was noted with the extract prepared from the leaves collected at the Sib-stage. Results showed that treatment of newly emerged larvae resulted in a significant mortality with a dose-response relationship. The olive leaf extracts toxicity was also demonstrated by histopathological changes in the alimentary canal resulting in a considerable disorganization and serious damage of the midgut, ceca, and proventriculus structure. Epithelial cells alterations, less dense and degraded striated border, disintegrated regeneration crypts, vacuolarized cells, extrusion of cytoplasmic contents, and rupture of muscular layer were evident in the midgut and ceca of treated larvae. Data of biochemical analyzes showed that olive leaf extracts induced a significant decrease of the hemolymph metabolites (proteins, carbohydrates, and lipids). In a second series of experiments, we showed that the olive leaf extracts reduced the activity of acetylcholinesterase and induced the glutathione S-transferases with a dose-response relationship.

Contributions of dsRNases to differential RNAi efficiencies between the injection and oral delivery of dsRNA in Locusta migratoria.

BACKGROUND: The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive. RESULTS: We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut. CONCLUSION: The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management. © 2018 Society of Chemical Industry.

LmCht5-1 promotes pro-nymphal molting during locust embryonic development.

Chitinases, key enzymes involved in degradation of chitin, have been repeatedly shown to play an indispensable role during insect post-embryonic molting processes at stage transitions. However, how chitinases affect insect embryonic development remains to be analyzed. In this study, we investigated the role of chitinase 5-1 (LmCht5-1) during embryonic development of the hemimetabolous insect Locusta migratoria. LmCht5-1 transcript levels were high in pro-nymphs during late embryogenesis. The respective protein localized to both the pro-nymphal and, to a much lesser extent, the newly formed nymphal cuticle. After injection of double stranded RNA against LmCht5-1 into 8 days old embryos, LmCht5-1 transcripts were strongly reduced. Most of dsLmCht5-1-injected pro-nymphs failed to develop to first-instar nymphs and died at or before hatching. Histological analyzes showed that degradation of the pro-nymph cuticle was blocked in these animals. At the ultra-structural level, we found that LmCht5-1 was needed for the degradation of the lamellar procuticle, while the separation of the procuticle from the epicuticle and epidermis (apolysis) was independent of LmCht5-1 function. Taken together, our results indicate that LmCht5-1 and other yet unknown degrading enzymes act in parallel at distinct positions of the cuticle during molting of the pro-nymph to the first-instar nymph during locust embryogenesis.

Metabolic Activity of Cytochrome P450s Towards Four Pyrethroids in Midgut Tissue From Locusta migratoria (Orthoptera: Acrididae).

Cytochrome P450 monooxygenases (P450s) play important roles in metabolizing various insecticides and often contribute to the development of insecticide resistance in insects and other arthropod species. The objective of this study was to compare the metabolism of four commonly used pyrethroids including deltamethrin, fluvalinate, fenvalerate, and permethrin in the midgut tissue of Locusta migratoria Linnaeus (Orthoptera: Acrididae) by using synergism bioassay and ultra-performance liquid chromatography (UPLC)-mass spectrometer (MS) analyses. Our study showed that piperonyl butoxide (PBO, P450 enzyme inhibitor) can significantly synergize the toxicity of deltamethrin, fluvalinate, and fenvalerate with synergism ratios ranging from 1.30 to 1.70 folds. Preincubations of the midgut tissue with PBO followed by incubations with each of the four pyrethroids resulted in significantly higher amounts of unmetabolized deltamethrin and fluvalinate than those in the control (preincubation without PBO) as well as preincubations with other two detoxification enzyme inhibitors. These results indicate that P450s play important roles in metabolizing deltamethrin and fluvalinate in the midgut tissue. Our further study using deltamethrin as a representative pyrethroid and UPLC-MS techniques confirmed that the reduced amount of deltamethrin in the control (preincubation without PBO) was due to the metabolism of deltamethrin to yield hydroxydeltamethrin which is a major metabolite produced by P450-mediated aromatic hydroxylation of deltamethrim. These results provide new insights into differential metabolic activity of P450s towards different pyrethroids in the midgut tissue of L. migratoria.

Assessment of pasteurisation of edible insects using enzymatic tests (activity of alkaline phosphatase and lactoperoxidase) applied in dairy products.

Industrialising edible insects goes along with quality control and hazard analysis and critical control points. One of those steps is assessing heat treatment. For the present contribution, the potential of enzymatic heat assessment tests used in the dairy industry (alkaline phosphatase and lactoperoxidase) to detect heat treatment in several insect species ( Acheta domesticus, Gryllus assimilis, Gryllus bimaculatus, Locusta migratoria, Schistocerca gregaria, Chilecomadia moorei, Galleria mellonella, Bombyx mori, Pachnoda marginata, Tenebrio molitor, Zophobas atratus, Apis mellifera, and Hermetia illucens) was evaluated. Insect material was homogenised, diluted, and the enzymatic tests (Lactognost®, Peroxtesmo®) were carried with these liquids as if they were milk. All species but C. moorei, B. mori, P. marginata, and A. mellifera showed alkaline phosphatase activity in raw samples and none in heated (10 min at 100 ℃) ones, while only G. mellonella, T. molitor, and Z. atratus reacted accordingly with lactoperoxidase. In trial 2 focusing only on alkaline phosphatase activity, inactivation of the enzyme after 5, 10, and 15 min of heating occurred species specific within a range of 60-86 ℃, i.e. within ordinary pasteurisation schemes. Thus and for the time being, heat treatment in many edible insect species can be assessed using alkaline phosphatase activity test kits. In contrast to milk samples, positive results may display bluish or greenish colours, and the time until a reliable reading is possible is extended to 1-1.5 h (24 h in the case of Gryllidae).

Comparisons of microsomal cytochrome P450 content and enzymatic activity towards selected model substrates and insecticides in different tissues from the migratory locust (Locusta migratoria).

Cytochrome P450 monooxygenases (P450s) are important enzymes for biotransformations of various endogenous and xenobiotic substances in various organisms. In this study, we examined microsomal P450 protein content and enzymatic activity in four major detoxification tissues dissected from fifth-instar nymphs of the migratory locust (Locusta migratoria). The highest microsomal P450 protein content was found in the gastric caeca (a part of the midgut), followed by the midgut, Malpighian tubules and fat bodies. Microsomal P450s showed the highest aromatic hydroxylation, O-dealkylation and O-dearylation activities towards six of the seven model substrates examined in the fat bodies. Although the gastric caeca showed the highest P450 protein content, the enzymatic activities towards six of the seven model substrates were the lowest in this tissue. Further, the midgut, gastric caeca and fat bodies showed significant metabolic activities towards two pyrethroid insecticides (deltamethrin and fluvalinate), but no significant activities towards the other four insecticides (malathion, chlorpyrifos, carbaryl and methoprene). These results support our conclusions: 1) total P450 protein content alone cannot be reliably used to predict its enzymatic activity, and 2) insect P450 enzymatic activity is both tissue and substrate dependent.

Knockdown of NADPH-cytochrome P450 reductase increases the susceptibility to carbaryl in the migratory locust, Locusta migratoria.

BACKGROUND: NADPH-cytochrome P450 reductase (CPR) plays important roles in cytochrome P450-mediated metabolism of endogenous and exogenous compounds, and participates in cytochrome P450-related detoxification of insecticides. However, the CPR from Locusta migratoria has not been well characterized and its function is still undescribed. RESULTS: The full-length of CPR gene from Locusta migratoria (LmCPR) was cloned by RT-PCR based on transcriptome information. The membrane anchor region, and 3 conserved domains (FMN binding domain, connecting domain, FAD/NADPH binding domain) were analyzed by bioinformatics analysis. Phylogenetic analysis showed that LmCPR was grouped in the Orthoptera branch and was more closely related to the CPRs from hemimetabolous insects. The LmCPR gene was ubiquitously expressed at all developmental stages and was the most abundant in the fourth-instar nymphs and the lowest in the egg stage. Tissue-specific expression analysis showed that LmCPR was higher expressed in ovary, hindgut, and integument. The CPR activity was relatively higher in Malpighian tubules and integument. Silencing of LmCPR obviously reduced the enzymatic activity of LmCPR, and enhanced the susceptibility of Locusta migratoria to carbaryl. CONCLUSION: These results suggest that LmCPR contributes to the susceptibility of L. migratoria to carbaryl and could be considered as a novel target for pest control.

A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust.

Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.

Helicoidal Organization of Chitin in the Cuticle of the Migratory Locust Requires the Function of the Chitin Deacetylase2 Enzyme (LmCDA2).

In the three-dimensional extracellular matrix of the insect cuticle, horizontally aligned microfibrils composed of the polysaccharide chitin and associated proteins are stacked either parallel to each other or helicoidally. The underlying molecular mechanisms that implement differential chitin organization are largely unknown. To learn more about cuticle organization, we sought to study the role of chitin deacetylases (CDA) in this process. In the body cuticle of nymphs of the migratory locust Locusta migratoria, helicoidal chitin organization is changed to an organization with unidirectional microfibril orientation when LmCDA2 expression is knocked down by RNA interference. In addition, the LmCDA2-deficient cuticle is less compact suggesting that LmCDA2 is needed for chitin packaging. Animals with reduced LmCDA2 activity die at molting, underlining that correct chitin organization is essential for survival. Interestingly, we find that LmCDA2 localizes only to the initially produced chitin microfibrils that constitute the apical site of the chitin stack. Based on our data, we hypothesize that LmCDA2-mediated chitin deacetylation at the beginning of chitin production is a decisive reaction that triggers helicoidal arrangement of subsequently assembled chitin-protein microfibrils.

Identification and characterization of two CYP9A genes associated with pyrethroid detoxification in Locusta migratoria.

Cytochrome P450s (CYPs) constitute one of the largest gene super families and distribute widely in all living organisms. In this study, the full-length cDNA sequences of two LmCYP9A genes (LmCYP9AQ1 and LmCYP9A3) were cloned from Locusta migratoria. We analyzed the expression patterns of two LmCYP9A genes in various tissues and different developmental stages using real-time quantitative PCR. Then we evaluated the detoxification functions of the two LmCYP9A genes by testing mortalities with four kinds of pyrethroid treatment after RNA interference (RNAi), respectively. Combining with docking structure of two LmCYP9A genes, their detoxification properties were extensively analyzed. The full-length cDNAs of LmCYP9AQ1 and LmCYP9A3 putatively encoded 525 and 524 amino acid residues, respectively. Both LmCYP9A genes were expressed throughout the developmental stages. The expression of LmCYP9AQ1 in the brain was higher than that in other examined tissues, whereas the LmCYP9A3 was mainly expressed in the fat body. The mortalities of nymphs exposed to deltamethrin and permethrin increased from 27.7% to 77.7% and 27.7% to 58.3%, respectively, after dsLmCYP9A3 injection. While the mortalities of nymphs exposed to fluvalinate increased from 29.8% to 53.0% after LmCYP9AQ1 was silenced using RNA interference. Our results suggested that the two LmCYP9A genes may be involved in different pyrethroid insecticide detoxification in L. migratoria.

Identification and characteristic analysis of the catalase gene from Locusta migratoria.

Catalase (CAT) is a ubiquitous antioxidant enzyme in almost all living organisms exposed to atmosphere, which involved in decomposing harmful hydrogen peroxide, into oxygen and water. In this study, a full-length cDNA (1524bp) encoding the catalase gene (LmCAT) from Locusta migratoria was cloned (accession number KT716445). The open reading frame of the LmCAT gene encoded 507 amino acids and shared 57.8%-97.8% amino acid identities with other insect CATs. The coding region was interrupted by 9 introns, while its promoter region contained 15 putative binding sites for 5 kinds of transcriptional regulation factors. For the stage-specific expression profile, LmCAT was highly expressed in the fourth-instar nymphs. For the tissue-specific expression profile, the LmCAT transcripts were highest in the fat bodies, and relatively abundant in the gastric caecum, Malpighian tubules, ovary and integument. Moreover, the result showed that quercetin could significantly induce the expression level of LmCAT. The expression of LmCAT could be silenced by RNAi, but the moralities were not significantly different between control and RNAi groups. Our results would provide valuable information for further study on the ROS regulation mechanism in insect.

Alternative exon-encoding regions of Locusta migratoria muscle myosin modulate the pH dependence of ATPase activity.

Whereas the vertebrate muscle myosin heavy chains (MHCs) are encoded by a family of Mhc genes, most insects examined to date contain a single Mhc gene and produce all of the different MHC isoforms by alternative RNA splicing. Here, we found that the migratory locust, Locusta migratoria, has one Mhc gene, which contains 41 exons, including five alternative exclusive exons and one differently included penultimate exon, and potentially encodes 360 MHC isoforms. From the adult L. migratoria, we identified 14 MHC isoforms (including two identical isoforms): four from flight muscle (the thorax dorsal longitudinal muscle), three from jump muscle (the hind leg extensor tibiae muscle) and seven from the abdominal intersegmental muscle. We purified myosins from flight muscle and jump muscle and characterized their motor activities. At neutral pH, the flight and the jump muscle myosins displayed similar levels of in vitro actin-gliding activity, whereas the former had a slightly higher actin-activated ATPase activity than the latter. Interestingly, the pH dependences of the actin-activated ATPase activity of these two myosins are different. Because the dominant MHC isoforms in these two muscles are identical except for the two alternative exon-encoding regions, we propose that these two alternative regions modulate the pH dependence of L. migratoria muscle myosin.

LmCYP4G102: An oenocyte-specific cytochrome P450 gene required for cuticular waterproofing in the migratory locust, Locusta migratoria.

Cytochrome P450 superfamily proteins play important roles in detoxification of xenobiotics and during physiological and developmental processes. To contribute to our understanding of this large gene family in insects, we have investigated the function of the cytochrome P450 gene LmCYP4G102 in the migratory locust Locusta migratoria. Suppression of LmCYP4G102 expression by RNA interference (RNAi) does not interfere with moulting but causes rapid loss of body weight - probably due to massive loss of water, and death soon after moulting. Accordingly, maintaining these animals at 90% relative humidity prevented lethality. Consistently, RNAi against LmCYP4G102 provoked a decrease in the content of cuticular alkanes, which as an important fraction of cuticular hydrocarbons have been shown to confer desiccation resistance. In addition, the cuticle of LmCYP4G102-knockdown locusts was fragile and easier deformable than in control animals. Presumably, this phenotype is due to decreased amounts of cuticular water that is reported to modulate cuticle mechanics. Interestingly, LmCYP4G102 was not expressed in the epidermis that produces the cuticle but in the sub-epdiermal hepatocyte-like oenocytes. Together, our results suggest that the oenocyte-specific LmCYP4G102 plays a critical role in the synthesis of cuticular hydrocarbons, which are important for cuticle waterproofing and mechanical stability in L. migratoria.

Biochemical basis of synergism between pathogenic fungus Metarhizium anisopliae and insecticide chlorantraniliprole in Locusta migratoria (Meyen).

We challenged Locusta migratoria (Meyen) grasshoppers with simultaneous doses of both the insecticide chlorantraniliprole and the fungal pathogen, Metarhizium anisopliae. Our results showed synergistic and antagonistic effects on host mortality and enzyme activities. To elucidate the biochemical mechanisms that underlie detoxification and pathogen-immune responses in insects, we monitored the activities of 10 enzymes. After administration of insecticide and fungus, activities of glutathione-S-transferase (GST), general esterases (ESTs) and phenol oxidase (PO) decreased in the insect during the initial time period, whereas those of aryl acylamidase (AA) and chitinase (CHI) increased during the initial period and that of acetylcholinesterase (AChE) increased during a later time period. Activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreased at a later time period post treatment. Interestingly, treatment with chlorantraniliprole and M. anisopliae relieved the convulsions that normally accompany M. anisopliae infection. We speculate that locust mortality increased as a result of synergism via a mechanism related to Ca(2+) disruption in the host. Our study illuminates the biochemical mechanisms involved in insect immunity to xenobiotics and pathogens as well as the mechanisms by which these factors disrupt host homeostasis and induce death. We expect this knowledge to lead to more effective pest control.

Characteristics and expression patterns of histone-modifying enzyme systems in the migratory locust.

The density-dependent phase polyphenism in locusts offers an excellent model to investigate the epigenetic regulatory mechanisms underlying phenotypic plasticity. In this study, we identified histone-modifying enzymes mediating histone post-translational modifications, which serve as a major regulatory mechanism of epigenetic processes, on the basis of the whole genome sequence of the migratory locust, Locusta migratoria. We confirmed the existence of various functional histone modifications in the locusts. Compared with other sequenced insect genomes, the locust genome contains a richer repertoire of histone-modifying enzymes. Several locust histone-modifying enzymes display vertebrate-like characteristics, such as the presence of a Sirt3-like gene and multiple alternative splicing of GCN5 gene. Most histone-modifying enzymes are highly expressed in the eggs or in the testis tissues of male adults. Several histone deacetylases and H3K4-specific methyltransferases exhibit differential expression patterns in brain tissues between solitarious and gregarious locusts. These results reveal the main characteristics of histone-modifying enzymes and provide important cues for understanding the epigenetic mechanisms underlying phase polyphenism in locusts.