Isolation and characterization of FMRFamide-like peptides in the venoms of solitary sphecid wasps.

Purification of small peptide components in the venoms of the solitary sphecid wasps, Sphex argentatus argentatus and Isodontia harmandi, led to the isolation of several major peptides. Analysis of MS/MS spectra by MALDI-TOF/TOF revealed the sequence of a new peptide Sa112 (EDVDHVFLRF-NH2), which is structurally very similar to leucomyosupressin (pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-like peptides from cockroach and locust, respectively. Indeed, this new peptide, like SchistoFLRFamide, inhibited the frequency and amplitude of spontaneous contractions of the locust oviduct in a dose-dependent manner. A non-amidated peptide Sa12b (EDVDHVFLRF) was also isolated, but this peptide had no effect on spontaneous locust oviduct contraction. This is the first example of a FMRF-like peptide to be found in solitary wasp venom. Additionally, a truncated form of the myosuppressins, which has previously been synthesized and tested for biological activity, DVDHVFLRF-NH2 (Sh5b), was found for the first time as a natural product. Four other novel peptides were isolated and characterized as Sa81 (EDDLEDFNPTVS), Sa10 (EDDLEDFNPTIA), Sh41 (DDLSDFNPKV), and Sh42 (EDDLSDFNPKV). They are structurally related to each other, having a high content of acidic amino acids, but no structural similarity to any known peptides. Ion channel associated activities of Sh41 and Sh42 were tested, but did not show any activity for Na+, K+, Ca2+ channels.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

4-Vinylanisole is an aggregation pheromone in locusts.

Locust plagues threaten agricultural and environmental safety throughout the world1,2. Aggregation pheromones have a crucial role in the transition of locusts from a solitary form to the devastating gregarious form and the formation of large-scale swarms3,4. However, none of the candidate compounds reported5-7 meet all the criteria for a locust aggregation pheromone. Here, using behavioural assays, electrophysiological recording, olfactory receptor characterization and field experiments, we demonstrate that 4-vinylanisole (4VA) (also known as 4-methoxystyrene) is an aggregation pheromone of the migratory locust (Locusta migratoria). Both gregarious and solitary locusts are strongly attracted to 4VA, regardless of age and sex. Although it is emitted specifically by gregarious locusts, 4VA production can be triggered by aggregation of four to five solitary locusts. It elicits responses specifically from basiconic sensilla on locust antennae. We also identified OR35 as a specific olfactory receptor of 4VA. Knockout of OR35 using CRISPR-Cas9 markedly reduced the electrophysiological responses of the antennae and impaired 4VA behavioural attractiveness. Finally, field trapping experiments verified the attractiveness of 4VA to experimental and wild populations. These findings identify a locust aggregation pheromone and provide insights for the development of novel control strategies for locusts.

Cloning and expression of OdTx12, a ß excitatory toxin from Odontobuthus doriae, in Escherichia coli and evaluation of its bioactivity in Locusta migratoria.

The venom of Odontobuthus doriae contains several peptide toxins that interfere with the sodium channel function of cell membranes, some of which specifically act on the insect's sodium channel without affecting mammalian cells. In this study sodium channel toxins of Odontobuthus doriae were aligned to other closely related toxins by BLAST and ClustalW servers. Among these toxins, NaTx12 (OdTx12) showed more than 90% similarity to the most known beta excitatory toxin, AaHIT1; furthermore, our modeling studies confirmed high tertiary structure similarity of these proteins. OdTx12 was cloned and expressed in E.coli, using pET26-b and pET28-a expression vectors. Tris-tricine SDS-PAGE and Western blot analysis showed OdTx12 expression by pET28-a, only. After purification, bioactivity of the purified protein was analyzed by injection and oral administration to Locusta migratoria larvae, and toxicity to mammals was tested on mice. Injection of OdTx12 resulted in the killing of larvae with LD50 of 0.4 and 0.2 after 48 and 72 h respectively, but oral administration of OdTx12 had no significant effect on Locusta migratoria, nor did the injection to mice show any signs of toxicity. These results showed that OdTx12, as a novel ß excitatory toxin can be considered as a candidate for insect control purposes.

Neonicotinoid and sulfoximine pesticides differentially impair insect escape behavior and motion detection.

Insect nervous systems offer unique advantages for studying interactions between sensory systems and behavior, given their complexity with high tractability. By examining the neural coding of salient environmental stimuli and resulting behavioral output in the context of environmental stressors, we gain an understanding of the effects of these stressors on brain and behavior and provide insight into normal function. The implication of neonicotinoid (neonic) pesticides in contributing to declines of nontarget species, such as bees, has motivated the development of new compounds that can potentially mitigate putative resistance in target species and declines of nontarget species. We used a neuroethologic approach, including behavioral assays and multineuronal recording techniques, to investigate effects of imidacloprid (IMD) and the novel insecticide sulfoxaflor (SFX) on visual motion-detection circuits and related escape behavior in the tractable locust system. Despite similar LD50 values, IMD and SFX evoked different behavioral and physiological effects. IMD significantly attenuated collision avoidance behaviors and impaired responses of neural populations, including decreases in spontaneous firing and neural habituation. In contrast, SFX displayed no effect at a comparable sublethal dose. These results show that neonics affect population responses and habituation of a visual motion detection system. We propose that differences in the sublethal effects of SFX reflect a different mode of action than that of IMD. More broadly, we suggest that neuroethologic assays for comparative neurotoxicology are valuable tools for fully addressing current issues regarding the proximal effects of environmental toxicity in nontarget species.

Transcriptome analysis of antennal cytochrome P450s and their transcriptional responses to plant and locust volatiles in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) constitute a large superfamily of heme-thiolate proteins that are involved in the biosynthesis or degradation of endogenous compounds and detoxification of exogenous chemicals. It has been reported that P450s could serve as odorant-degrading enzymes (ODEs) to inactivate odorants to avoid saturating the antennae. However, there is little information about P450s in the antennae of Locusta migratoria. In the current work, we conducted an antenna transcriptome analysis and identified 92 P450s, including 68 full-length and 24 partial sequences. Phylogenetic analysis showed that 68 full-length P450s were grouped into four clans: CYP2, CYP3, CYP4, and mitochondria clans. Tissue, stage, and sex-dependent expressions of these 68 P450s were investigated. The results showed that 4 P450s were antenna-specific, whereas others were antenna-rich but also expressed in other tissues, implying their various potential roles in the antennae. In addition, the responses of seven selected P450s to five gramineous plant volatiles and four locust volatiles were determined. CYP6MU1 could be induced by almost all compounds tested, suggesting its important roles in odorant processing. Different P450s exhibited diverse responses to odorants, indicating that specific regulation of P450 expression by odorants might modulate the sensitivity of the olfactory responses to various chemicals.

An odorant-binding protein mediates sexually dimorphic behaviors via binding male-specific 2-heptanone in migratory locust.

Migratory locusts (Locusta migratoria) frequently aggregate into huge swarms that cause serious economic losses for the agricultural sector. Differential behaviors of male and female insects may contribute to such population explosions. However, the key olfactory mechanisms underlying different behaviors associated with sex-related pheromones are unclear. Here, we report that male-specific odor, 2-heptanone plays different roles in relation to the behavior of migratory locust males and females, and that this sexual dimorphism involves a soluble odorant-binding protein (OBP) in the peripheral olfactory processes. This odor strongly binds to LmigOBP4, a novel OBP, present in antennal trichoid sensilla, and elicits opposite locomotor tendencies between the sexes: attracting females and repelling males. Furthermore, an adult male group mimicked a high dosage of 2-heptanone by promoting their attractiveness to single females. Additionally, RNAi suppression of Lmigobp4 expression reduced the physiological responses to 2-heptanone to levels that were indistinguishable between the sexes. This suppression reversed the adult behavioral responses to 2-heptanone, i.e., females were repelled and males were attracted. We conclude that LmigOBP4 is associated with olfactory recognition of male-specific 2-heptanone, which plays dual roles that differ between adult male and female migratory locusts.

Metabolism of selected model substrates and insecticides by recombinant CYP6FD encoded by its gene predominately expressed in the brain of Locusta migratoria.

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.

Neural conduction, visual motion detection, and insect flight behaviour are disrupted by low doses of imidacloprid and its metabolites.

While neonicotinoid insecticides impair visually guided behaviours, the effects of their metabolites are unknown and measurements of environmental concentrations of neonicotinoids, typically lower than those required to elicit toxic effects, tend to exclude metabolites. Here we examined the contributions of imidacloprid and two of its metabolites, imidacloprid-olefin and 5-hydroxy-imidacloprid, on neural conduction velocity, visual motion detection and flight in the locust (Locusta migratoria) using a combination of electrophysiological and behavioural assays. We show reduced visual motion detection and impaired flight behaviour following treatment of metabolite concentrations equal to sublethal doses of the parent compound. Additionally, we show for the first time that imidacloprid and its metabolites result in a decrease in conduction velocity along an unmyelinated axon. We suggest that secondary effects of the insecticide on the biophysical properties of the axon may result in decreased neural conduction. As these metabolites display neurotoxicity similar to the parent compound they should be considered when quantifying environmental concentrations.

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.

Structural glycoprotein LmAbd-9 is required for the formation of the endocuticle during locust molting.

Insect cuticle is a composite made of chitin filaments embedded in a proteinaceous matrix, consisting mainly of structural cuticular proteins. In the present study, an endocuticle structural glycoprotein gene, LmAbd-9, was characterized based on the Locusta migratoria transcriptome. LmAbd-9 encodes a glycoprotein with a chitin binding domain 4, belonging to RR-1 subclass of the CPR family, which has two potential O-linked glycosylation sites (S115 and T137) at which glycosylation modification may occur. LmAbd-9 was highly expressed in the integument and showed periodic expression during molting. The expression levels of LmAbd-9 were significantly down-regulated after injection with 20-hydroxyecdysone (20E) for 6, 12, and 24 h, whereas it was upregulated after double-stranded RNA-mediated RNA interference of the 20E receptor gene LmEcR and LmFTZ-F1beta at day 2 of fifth instar nymphs for 48 h. After injection of dsLmAbd-9 on day 2 of fifth instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than in the control. Thus, LmAbd-9 that negatively regulated by the 20E signaling pathway was involved in the formation of the endocuticle in L. migratoria.

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis.

Insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are widely used as environmentally friendly insecticides. As the only known Cry protein with insecticidal activity against Locusta migratoria manilensis, a locust subspecies that causes extensive destruction of crops, the Cry7Ca1 protein from Bt strain BTH-13 identified in our previous study is of particular interest to locust prevention and control. However, the three-dimensional structure of Cry7Ca1 toxin (the active form of the Cry7Ca1 protein) and the mechanisms of the Cry7Ca1 insecticidal specificity remain largely elusive. Here, we report a 2.3 Å crystal structure of the Cry7Ca1 toxin and carry out a systematic comparison of all available Cry toxins structures. A cluster of six loops in Cry toxin domain II, named Apex here, are the most variable structural elements and were documented to contribute in insecticidal specificity. The Cry7Ca1 toxin Apex loops are different from those of other Cry toxins in length, conformation, and sequence. Electrostatic potential analysis further revealed that Cry7Ca1 is the only structure-available Cry toxin that does not have a high contrast of surface electrostatic potentials in the Apex. We further suggest that the L1/L2 loops in the center of the Cry7Ca1 Apex may be worthy of attention in future efforts to unravel the Cry7Ca1 insecticidal specificity as they exhibit unique features not found in the corresponding regions of other Cry toxins. Our work highlights the uniqueness of the Apex in the Cry7Ca1 toxin and may assist exploration of the insecticidal mechanism of the Cry7Ca1 against Locusta migratoria manilensis.

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.

High-level expression and purification of active scorpion long-chain neurotoxin BjαIT from Pichia pastoris.

As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pastoris transformants showing high levels of secretory recombinant (r)BjαIT. Secreted rBjαIT was expressed at levels as high as 340 mg/L following methanol induction in a fed-batch reactor, with ∼21 mg of pure rBjαIT obtained from 200-mL fed-batch culture supernatant by Ni2+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Injection of purified rBjαIT induced neurotoxicity symptoms in locust (Locusta migratoria) larvae, and the half-lethal dose of rBjαIT for locusts at 24-h post-injection ranged from 11 to 14 µg/g body weight. These results demonstrated that large amounts of active rBjαIT were efficiently prepared from P. pastoris, suggesting this system as efficacious for determining rBjαIT insecticidal activity against other agricultural insect pests.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli.

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl ß-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.

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.

Two distinctive ß subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one ß subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third ß subunits (Locß3) was identified in this study, which reveals at least three ß subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with Kd values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locß1, Locß2 and Locß3 respectively. Specific immunodepletion of Locß1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locß3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locß2 had no significant effect on the specific [3H]imidacloprid binding. Taken together, these data indicated that Locß1 and Locß3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlß1 was in two binding sites for imidacloprid. The involvement of two ß subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in ß subunits in L. migratoria.

A sublethal dose of a neonicotinoid insecticide disrupts visual processing and collision avoidance behaviour in Locusta migratoria.

Neonicotinoids are known to affect insect navigation and vision, however the mechanisms of these effects are not fully understood. A visual motion sensitive neuron in the locust, the Descending Contralateral Movement Detector (DCMD), integrates visual information and is involved in eliciting escape behaviours. The DCMD receives coded input from the compound eyes and monosynaptically excites motorneurons involved in flight and jumping. We show that imidacloprid (IMD) impairs neural responses to visual stimuli at sublethal concentrations, and these effects are sustained two and twenty-four hours after treatment. Most significantly, IMD disrupted bursting, a coding property important for motion detection. Specifically, IMD reduced the DCMD peak firing rate within bursts at ecologically relevant doses of 10 ng/g (ng IMD per g locust body weight). Effects on DCMD firing translate to deficits in collision avoidance behaviours: exposure to 10 ng/g IMD attenuates escape manoeuvers while 100 ng/g IMD prevents the ability to fly and walk. We show that, at ecologically-relevant doses, IMD causes significant and lasting impairment of an important pathway involved with visual sensory coding and escape behaviours. These results show, for the first time, that a neonicotinoid pesticide directly impairs an important, taxonomically conserved, motion-sensitive visual network.

Transcriptional Analysis of The Adaptive Digestive System of The Migratory Locust in Response to Plant Defensive Protease Inhibitors.

Herbivorous insects evolved adaptive mechanisms to compensate for the presence of plant defensive protease inhibitors (PI) in their food. The underlying regulatory mechanisms of these compensatory responses remain largely elusive. In the current study, we investigated the initiation of this adaptive response in the migratory locust, Locusta migratoria, via microarray analysis of gut tissues. Four hours after dietary uptake of PIs, 114 and 150 transcripts were respectively found up- or downregulated. The results suggest a quick trade-off between compensating for potential loss of digestive activity on the one hand, and stress tolerance, defense, and structural integrity of the gut on the other hand. We additionally addressed the role of a group of related upregulated hexamerin-like proteins in the PI-induced response. Simultaneous knockdown of corresponding transcripts by means of RNA interference resulted in a reduced capacity of the locust nymphs to cope with the effects of PI. Moreover, since insect hexamerins have been shown to bind Juvenile Hormone (JH), we also investigated the effect of JH on the proteolytic digestion in L. migratoria. Our results indicate that JH has a stimulatory effect on the expression of three homologous chymotrypsin genes, while knocking down the JH receptor (methoprene tolerant) led to opposite effects.

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.