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1.
Molecules ; 29(8)2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38675573

ABSTRACT

The repellent capacity against Sitophilus zeamais and the in vitro inhibition on AChE of 11 essential oils, isolated from six plants of the northern region of Colombia, were assessed using a modified tunnel-type device and the Ellman colorimetric method, respectively. The results were as follows: (i) the degree of repellency (DR) of the EOs against S. zeamais was 20-68% (2 h) and 28-74% (4 h); (ii) the IC50 values on AChE were 5-36 µg/mL; likewise, the %inh. on AChE (1 µg/cm3 per EO) did not show any effect in 91% of the EO tested; (iii) six EOs (Bursera graveolens-bark, B. graveolens-leaves, B. simaruba-bark, Peperomia pellucida-leaves, Piper holtonii (1b*)-leaves, and P. reticulatum-leaves) exhibited a DR (53-74%) ≥ C+ (chlorpyrifos-61%), while all EOs were less active (8-60-fold) on AChE compared to chlorpyrifos (IC50 of 0.59 µg/mL). Based on the ANOVA/linear regression and multivariate analysis of data, some differences/similarities could be established, as well as identifying the most active EOs (five: B. simaruba-bark, Pep. Pellucida-leaves, P. holtonii (1b*)-leaves, B. graveolens-bark, and B. graveolens-leaves). Finally, these EOs were constituted by spathulenol (24%)/ß-selinene (18%)/caryophyllene oxide (10%)-B. simaruba; carotol (44%)/dillapiole (21%)-Pep. pellucida; dillapiole (81% confirmed by 1H-/13C-NMR)-P. holtonii; mint furanone derivative (14%)/mint furanone (14%)-B. graveolens-bark; limonene (17%)/carvone (10%)-B. graveolens-leaves.


Subject(s)
Cholinesterase Inhibitors , Insect Repellents , Oils, Volatile , Polycyclic Sesquiterpenes , Animals , Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/chemistry , Colombia , Insect Repellents/pharmacology , Insect Repellents/chemistry , Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Piper/chemistry , Plant Oils/pharmacology , Plant Oils/chemistry , Polycyclic Sesquiterpenes/chemistry , Polycyclic Sesquiterpenes/pharmacology , Weevils/enzymology , Weevils/drug effects , Sesquiterpenes, Eudesmane/chemistry , Sesquiterpenes, Eudesmane/pharmacology , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology
2.
Molecules ; 26(15)2021 Jul 27.
Article in English | MEDLINE | ID: mdl-34361693

ABSTRACT

Due to the rise of numerous legal restrictions as well as the increasing emergence of resistant populations, the number of available pesticides is decreasing significantly. One of the potential alternatives often described in the literature are essential oils (EOs). However, there is a lack of research addressing the potential emergence of resistance to this group of substances. In this paper, we investigated the multi-generational effects of sublethal concentrations of rosemary oil (Rosmarinus officinalis) on physiological and biochemical parameters of the cowpea weevil (Callosobruchus maculatus) such as egg laying, hatchability, oxygen consumption and acetylcholinesterase activity. Imago, which as larvae were exposed to EO at concentrations equivalent to LC25, showed significantly lower mortality. The results obtained indicate the potential development of resistance in insects exposed to EO in concentrations corresponding to LC25. In addition, in the case of the group treated with an EO concentration corresponding to LC3.12, a stimulation effect of the above-mentioned parameters was observed, which may indicate the occurrence of a hormesis effect. The obtained results may be an important reference for the development of future guidelines and EO-based insecticides.


Subject(s)
Drug Resistance/drug effects , Insecticides/pharmacology , Oils, Volatile/pharmacology , Weevils/drug effects , Acetylcholinesterase/metabolism , Animals , Female , Male , Oviposition/drug effects , Oxygen Consumption/drug effects , Signal Transduction/drug effects , Weevils/enzymology
3.
Arch Insect Biochem Physiol ; 106(2): e21760, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33231898

ABSTRACT

The Chinese white pine beetle Dendroctonus armandi (Tsai and Li) is a significant pest of the Qinling and Bashan Mountains pine forests of China. The Chinese white pine beetle can overcome the defences of Chinese white pine Pinus armandi (Franch) through pheromone-assisted aggregation that results in a mass attack of host trees. We isolated five full-length complementary DNAs encoding mevalonate pathway-related enzyme genes from the Chinese white pine beetle (D. armandi), which are acetoacetyl-CoA thiolase (AACT), geranylgeranyl diphosphate synthase (GGPPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MPDC), and phosphomevalonate kinase (PMK). Bioinformatic analyses were performed on the full-length deduced amino acid sequences. Differential expression of these five genes was observed between sexes, and within these significant differences among topically applied juvenile hormone III (JH III), fed on phloem of P. armandi, tissue distribution, and development stage. Mevalonate pathway genes expression were induced by JH III and feeding.


Subject(s)
Genes, Insect , Insect Proteins/genetics , Metabolic Networks and Pathways/genetics , Mevalonic Acid/metabolism , Transcriptome , Weevils/genetics , Amino Acid Sequence , Animals , Female , Insect Proteins/chemistry , Insect Proteins/metabolism , Larva/enzymology , Larva/growth & development , Male , Phylogeny , Pupa/enzymology , Pupa/growth & development , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Weevils/enzymology , Weevils/growth & development
4.
Curr Opin Insect Sci ; 43: 97-102, 2021 02.
Article in English | MEDLINE | ID: mdl-33359166

ABSTRACT

Bark beetles (family: Curculionidae; subfamily: Scolytinae) in the Dendroctonus and Ips genera are the most destructive forest pests in the Northern hemisphere. They use cytochromes P450 (P450s) to detoxify tree-produced terpenes to produce pheromones, in de novo pheromone production and to oxidize odorants on antennae. Many Dendroctonus spp. use trans-verbenol as an aggregation pheromone, and it is formed from host-tree produced α-pinene hydroxylated by CYP6DE1 during larval stages, stored as verbenyl ester of fatty acids, and then released when the female begins feeding on a new host tree. Ips spp. hydroxylate de novo produced myrcene to form ipsdienol. Subsequent steps form the appropriate enantiomeric composition of ipsdienol and convert ipsdienol to ipsenol. In this article we review recent progress in elucidating the functions of P450s in Ips and Dendroctonus species and in doing so provide insights into the role of these enzymes in host phytochemical detoxification and pheromone production.


Subject(s)
Cytochrome P-450 Enzyme System , Inactivation, Metabolic , Weevils/enzymology , Weevils/metabolism , Alcohols/metabolism , Animals , Bicyclic Monoterpenes/metabolism , Pheromones , Terpenes/metabolism , Tracheophyta/chemistry
5.
Arch Insect Biochem Physiol ; 105(2): e21730, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32737998

ABSTRACT

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.


Subject(s)
Digestive System/enzymology , Weevils/enzymology , Animals , Body Fluids/enzymology , Cellulases/chemistry , Cellulose/metabolism , Digestive System/growth & development , Head , Larva/enzymology , Larva/growth & development , Polygalacturonase/chemistry , Weevils/growth & development
6.
Int J Biol Macromol ; 147: 1029-1040, 2020 Mar 15.
Article in English | MEDLINE | ID: mdl-31751747

ABSTRACT

Red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the most destructive pests of cultivated palm trees. The application of synthetic insecticides is currently a main strategy for RPW control. In this study we estimated the distribution of acetylcholinesterase (AChE), as a detoxifying enzyme and the target site of inhibition by insecticides, using ASChI as substrate in different organs of the pest including whole gut, cuticle, fat body, head and haemolymph. The activity ranged from 314.9 to 3868 U in individual organs while the specific activity ranged from 99 to 340.8 U/mg proteins; the cuticle had the highest enzyme level. During larval development, the 11th instar larvae had the highest enzyme content with 5630 U in the cuticle, with a specific activity of 140 U/mg protein. The two major AChE isoenzymes were purified by chromatography on gel filtration and ion exchange columns. They had specific activities of 3504.3 and 2979 U/mg protein, molecular weights of 33 and 54 kDa and activation energies of 8.3 and 4.4 kcal/mol, respectively. Both isoenzymes had monomeric forms, optimum activity at pH 8.0 and 40 °C, were completely inhibited by Hg2+ and Cu2 and showed similar trends towards the inhibitors eserine, BW284C51 and iso-OMPA. The catalytic properties were compared with those previously recorded for different insect species. This work will pave the way for more studies for improving the understanding of insecticide resistance and developing the field application of synthetic insecticides for controlling R. ferrugineus to ensure successful application.


Subject(s)
Acetylcholinesterase/chemistry , Acetylcholinesterase/isolation & purification , Insect Proteins/chemistry , Insect Proteins/isolation & purification , Weevils/enzymology , Animals , Catalysis , Cations , Copper/chemistry , Hemolymph , Hydrogen-Ion Concentration , Hydrolysis , Insecticides , Isoenzymes/chemistry , Kinetics , Larva , Mercury/chemistry , Molecular Weight , Substrate Specificity , Temperature
7.
Sci Rep ; 9(1): 19580, 2019 12 20.
Article in English | MEDLINE | ID: mdl-31862955

ABSTRACT

In the last years, the production of ethanol fuel has started to change with the introduction of second-generation ethanol (2 G Ethanol) in the energy sector. However, in Brazil, the process of obtaining 2 G ethanol did not reach a basic standard to achieve relevant and economically viable results. Several studies have currently been addressed to solve these issues. A critical stage in the bioethanol production is the deployment of efficient and stable enzymes to catalyze the saccharification step into the process of biomass conversion. The present study comprises a screening for genes coding for plant biomass degradation enzymes, followed by cloning a selected gene, addressing its heterologous expression, and characterizing enzymatic activity towards cellulose derived substrates, with a view to second-generation ethanol production. A cDNA database of the Cotton Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae), an insect that feeds on cotton plant biomass, was used as a source of plant biomass degradation enzyme genes. A larva and adult midgut-specific ß-1,4-Endoglucanase-coding gene (AgraGH45-1) was cloned and expressed in the yeast Pichia pastoris. Its amino acid sequence, including the two catalytic domains, shares high identity with other Coleoptera Glycosyl Hydrolases from family 45 (GH45). AgraGH45-1 activity was detected in a Carboxymethylcellulose (CMC) and Hydroxyethylcellulose (HEC) degradation assay and the optimal conditions for enzymatic activity was pH 5.0 at 50 °C. When compared to commercial cellulase from Aspergillus niger, Agra GH45-1 was 1.3-fold more efficient to degrade HEC substrate. Together, these results show that AgraGH45-1 is a valid candidate to be engineered and be tested for 2 G ethanol production.


Subject(s)
Ethanol/metabolism , Glycoside Hydrolases/metabolism , Weevils/enzymology , Animals , Carboxymethylcellulose Sodium/metabolism , Cellulose/analogs & derivatives , Cellulose/metabolism , DNA, Complementary/metabolism , Weevils/metabolism
8.
Sci Rep ; 9(1): 14605, 2019 10 10.
Article in English | MEDLINE | ID: mdl-31601880

ABSTRACT

Infestation of phosphine (PH3) resistant insects threatens global grain reserves. PH3 fumigation controls rice weevil (Sitophilus oryzae) but not highly resistant insect pests. Here, we investigated naturally occurring strains of S. oryzae that were moderately resistant (MR), strongly resistant (SR), or susceptible (wild-type; WT) to PH3 using global proteome analysis and mitochondrial DNA sequencing. Both PH3 resistant (PH3-R) strains exhibited higher susceptibility to ethyl formate-mediated inhibition of cytochrome c oxidase than the WT strain, whereas the disinfectant PH3 concentration time of the SR strain was much longer than that of the MR strain. Unlike the MR strain, which showed altered expression levels of genes encoding metabolic enzymes involved in catabolic pathways that minimize metabolic burden, the SR strain showed changes in the mitochondrial respiratory chain. Our results suggest that the acquisition of strong PH3 resistance necessitates the avoidance of oxidative phosphorylation through the accumulation of a few non-synonymous mutations in mitochondrial genes encoding complex I subunits as well as nuclear genes encoding dihydrolipoamide dehydrogenase, concomitant with metabolic reprogramming, a recognized hallmark of cancer metabolism. Taken together, our data suggest that reprogrammed metabolism represents a survival strategy of SR insect pests for the compensation of minimized energy transduction under anoxic conditions. Therefore, understanding the resistance mechanism of PH3-R strains will support the development of new strategies to control insect pests.


Subject(s)
Electron Transport Complex IV/metabolism , Insecticide Resistance , Insecticides , Phosphines , Weevils/genetics , Animals , DNA, Mitochondrial/genetics , Electron Transport Complex IV/antagonists & inhibitors , Energy Metabolism , Formic Acid Esters , Mitochondria/metabolism , Mutation , Oxidation-Reduction , Polymorphism, Single Nucleotide , Proteome , Proteomics , Regression Analysis , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Weevils/enzymology
9.
Int J Mol Sci ; 20(16)2019 Aug 17.
Article in English | MEDLINE | ID: mdl-31426479

ABSTRACT

Bark beetles commonly produce de novo terpenoid pheromones using precursors synthesized through the mevalonate pathway. This process is regulated by Juvenile Hormone III (JH III). In this work, the expression levels of mevalonate pathway genes were quantified after phloem feeding-to induce the endogenous synthesis of JH III-and after the topical application of a JH III solution. The mevalonate pathway genes from D. rhizophagus were cloned, molecularly characterized, and their expression levels were quantified. Also, the terpenoid compounds produced in the gut were identified and quantified by Gas Chromatography Mass Spectrometry (GC-MS). The feeding treatment produced an evident upregulation, mainly in acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IPPI) genes, and males reached higher expression levels compared to females. In contrast, the JH III treatment did not present a clear pattern of upregulation in any sex or time. Notably, the genes responsible for the synthesis of frontalin and ipsdienol precursors (geranyl diphosphate synthase/farnesyl diphosphate synthase (GPPS/FPPS) and geranylgeranyl diphosphate synthase (GGPPS)) were not clearly upregulated, nor were these compounds further identified. Furthermore, trans-verbenol and myrtenol were the most abundant compounds in the gut, which are derived from an α-pinene transformation rather than de novo synthesis. Hence, the expression of mevalonate pathway genes in D. rhizophagus gut is not directed to the production of terpenoid pheromones, regardless of their frequent occurrence in the genus Dendroctonus.


Subject(s)
Eating , Gene Expression Regulation , Metabolic Networks and Pathways/genetics , Pheromones/biosynthesis , Weevils/genetics , Animals , Female , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/physiology , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , Male , Mevalonic Acid/metabolism , Terpenes/metabolism , Weevils/enzymology , Weevils/metabolism , Weevils/physiology
10.
BMC Genomics ; 20(1): 440, 2019 May 31.
Article in English | MEDLINE | ID: mdl-31151384

ABSTRACT

BACKGROUND: Cytochrome P450-dependent monooxygenases (P450s), constituting one of the largest and oldest gene superfamilies found in many organisms from bacteria to humans, play a vital role in the detoxification and inactivation of endogenous toxic compounds. The use of various insecticides has increased over the last two decades, and insects have developed resistance to most of these compounds through the detoxifying function of P450s. In this study, we focused on the red palm weevil (RPW), Rhynchophorus ferrugineus, the most devastating pest of palm trees worldwide, and demonstrated through functional analysis that upregulation of P450 gene expression has evolved as an adaptation to insecticide stress arising from exposure to the neonicotinoid-class systematic insecticide imidacloprid. RESULTS: Based on the RPW global transcriptome analysis, we identified 101 putative P450 genes, including 77 likely encoding protein coding genes with ubiquitous expression. A phylogenetic analysis revealed extensive functional and species-specific diversification of RPW P450s, indicating that multiple CYPs actively participated in the detoxification process. We identified highly conserved paralogs of insect P450s that likely play a role in the development of resistance to imidacloprid: Drosophila Cyp6g1 (CYP6345J1) and Bemisia tabaci CYP4C64 (CYP4LE1). We performed a toxicity bioassay and evaluated the induction of P450s, followed by the identification of overexpressed P450s, including CYP9Z82, CYP6fra5, CYP6NR1, CYP6345J1 and CYP4BD4, which confer cross-resistance to imidacloprid. In addition, under imidacloprid insecticide stress in a date palm field, we observed increased expression of various P450 genes, with CYP9Z82, CYP4BD4, CYP6NR1 and CYP6345J1 being the most upregulated detoxification genes in RPWs. Expression profiling and cluster analysis revealed P450 genes with multiple patterns of induction and differential expression. Furthermore, we used RNA interference to knock down the overexpressed P450s, after which a toxicity bioassay and quantitative expression analysis revealed likely candidates involved in metabolic resistance against imidacloprid in RPW. Ingestion of double-stranded RNA (dsRNA) successfully knocked down the expression of CYP9Z82, CYP6NR1 and CYP345J1 and demonstrated that silencing of CYP345J1 and CYP6NR1 significantly decreased the survival rate of adult RPWs treated with imidacloprid, indicating that overexpression of these two P450s may play an important role in developing tolerance to imidacloprid in a date palm field. CONCLUSION: Our study provides useful background information on imidacloprid-specific induction and overexpression of P450s, which may enable the development of diagnostic tools/markers for monitoring the spread of insecticide resistant RPWs. The observed trend of increasing tolerance to imidacloprid in the date palm field therefore indicated that strategies for resistance management are urgently needed.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Insecticides , Neonicotinoids , Nitro Compounds , Phoeniceae , Weevils/enzymology , Animals , Cytochrome P-450 Enzyme System/classification , Cytochrome P-450 Enzyme System/metabolism , Fat Body/enzymology , Gene Expression Profiling , Insecticide Resistance , Organ Specificity , RNA Interference , Survival Analysis , Weevils/genetics
11.
J Insect Sci ; 19(3)2019 May 01.
Article in English | MEDLINE | ID: mdl-31115475

ABSTRACT

Dendroctonus bark beetles (Scolytinae) are one of the most important disturbance agents of coniferous forests in North and Central America. These beetles spend their lives almost entirely under the tree bark, and their survival and reproductive success depend on their ability to overcome the toxic effect of the trees' oleoresin. The cytochromes P450 (CYPs) are associated with the detoxification process of xenobiotics, as well as other physiological processes. Different cytochromes (families 4, 6, and 9) in the Dendroctonus species have been expressed under several experimental conditions; nevertheless, the expression time-course of these genes is unknown. To explore the induction speed of CYPs, we evaluated the relative expression of the CYP6BW5, CYP6DG1, CYP6DJ2, CYP9Z18, and CYP9Z20 genes at the early hours of drilling and settling into a tree (1, 2, 4, 6, 8, 12, 18 h) both in females and males, solitary or paired, of the bark beetle Dendroctonus rhizophagus Thomas and Bright. Our findings show that the five genes were rapidly overexpressed in the early hours (1 to 6 h) in both sexes and in solitary and paired conditions, suggesting their participation in the detoxification process. Additionally, the CYPs expression shows up- and down-regulation patterns through these short times, suggesting their probable participation in other physiological processes as the biosynthesis of hormones, pheromones or compounds related to reproduction.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Weevils/enzymology , Animals , Cytochrome P-450 Enzyme System/genetics , Feeding Behavior , Female , Male , Trees , Weevils/genetics
12.
Insect Biochem Mol Biol ; 110: 80-89, 2019 07.
Article in English | MEDLINE | ID: mdl-31009678

ABSTRACT

RNA interference (RNAi) refers to the process of suppression of gene expression in eukaryotes, which has a great potential for the control of pest and diseases. Unfortunately, the efficacy of this technology is limited or at best variable in some insects. In the African sweet potato weevil (SPW) Cylas puncticollis, a devastating pest that affects the sweet potato production in Sub-Saharan Africa (SSA), the RNAi response was highly efficient when dsRNA was delivered by injection, but it showed a reduced response by oral feeding. In the present study, the role of nucleases in the reduced RNAi efficiency in C. puncticollis is investigated. Several putative dsRNases were first identified in the transcriptome of the SPW through homology search and were subsequently further characterized. Two of these dsRNases were specifically expressed in the gut tissue of the insect and we could demonstrate through RNAi experiments that these affected dsRNA stability in the gut. Furthermore, RNAi-of-RNAi studies, using snf7 as a reporter gene, demonstrated that silencing one of these nucleases, Cp-dsRNase-3, clearly increases RNAi efficacy. After silencing this nuclease, significantly higher mortality was observed in dssnf7-treated insects 14 days post-feeding as compared to control treatments, and the gene downregulation was confirmed at the transcript level via qPCR analysis. Taken together, our results demonstrate that the RNAi efficiency is certainly impaired by nuclease activity in the gut environment of the SPW Cylas puncticollis.


Subject(s)
Endonucleases/genetics , Insect Proteins/genetics , RNA Interference , Weevils/genetics , Amino Acid Sequence , Animals , Endonucleases/chemistry , Endonucleases/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Phylogeny , RNA, Double-Stranded/pharmacology , Sequence Alignment , Transcriptome , Weevils/enzymology , Weevils/metabolism
13.
Pest Manag Sci ; 75(4): 986-997, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30204286

ABSTRACT

BACKGROUND: Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of their host trees. Research on carboxylesterases (CarEs) has focused on their multiple functions in the metabolic detoxification of pesticides and plant allelochemicals, drug resistance, and juvenile hormone and pheromone degradation. RESULT: We identified eight new CarE genes in the Chinese white pine beetle (Dendroctonus armandi) and carried out bioinformatics analysis on the deduced full-length amino acid sequences. Differential transcript levels of CarE genes were observed between sexes; within these levels, significant differences were found among the different development stages, and between insects fed on the phloem of Pinus armandi and exposed to five stimuli [(-)-α-pinene, (-)-ß-pinene, (+)-3-carene, limonene and turpentine] at 8 and 24 h. CONCLUSION: Transcription levels of CarE genes suggest some relationship with the detoxification of terpenoids released by host trees. The functions of bark beetle esterase are mainly in hydrolyzing the host chemical defense and degrading odorant molecules during host selection and colonization. © 2018 Society of Chemical Industry.


Subject(s)
Carboxylic Ester Hydrolases/genetics , Insect Proteins/genetics , Monoterpenes/metabolism , Pinus/chemistry , Weevils/genetics , Amino Acid Sequence , Animals , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Female , Inactivation, Metabolic , Insect Proteins/chemistry , Insect Proteins/metabolism , Larva/enzymology , Larva/genetics , Larva/growth & development , Male , Phylogeny , Pupa/enzymology , Pupa/genetics , Pupa/growth & development , Sequence Alignment , Transcription, Genetic , Weevils/enzymology , Weevils/growth & development
14.
Pestic Biochem Physiol ; 149: 44-53, 2018 Jul.
Article in English | MEDLINE | ID: mdl-30033015

ABSTRACT

Plant essential oils with high bioactivity provide environmental friendly alternatives for synthetic pesticides. Melaleuca alternifolia essential oil and its main constituent terpinen-4-ol have high insecticidal activity to Sitophilus zeamais Motschulsky. Terpinen-4-ol may be metabolized by human and insect cytochrome P450s. However, little is known about how insects systemically respond to terpinen-4-ol. In this study, we used an RNA-seq approach to evaluate the global gene expression of S. zeamais after terpinen-4-ol fumigation. Duplicates of fumigated and control groups, for a total of four libraries, were collected for sequencing. A total of 36,117 unigenes with an average length of 1036 bp were generated in the de novo assembled transcriptome. Comparative analysis of S. zeamais libraries identified 592 differentially expressed genes (DEGs), of which 308 and 284 genes were up- and down-regulated in response to terpinen-4-ol fumigation, respectively. GO and KEGG functional analyses were performed for up and downregulated DEGs separately, showing these DEGs were enriched for terms related to catalytic activity, carbohydrate metabolism, and xenobiotics biodegradation and metabolism. DEGs encoding enzymes for detoxification were detected, including sixteen cytochrome P450s (P450s), eight glutathione S-transferase (GSTs), fourteen esterase (ESTs), ten UDP-glucuronosyltransferase (UGTs), and two ATP-binding cassette transporter (ABC transporter) genes. Real-time quantitative PCR confirmed that ten P450s, three GSTs and one EST were up-regulated dramatically after exposure to terpinen-4-ol at different concentrations and over a time course. The results provided a transcriptional overview of the changes in a stored-grain pest in response to terpinen-4-ol fumigation. The analysis revealed the expression levels of detoxification genes were altered, especially for P450s, and provided candidate genes for understanding systemic metabolic responses to terpinen-4-ol in insects.


Subject(s)
Enzymes/genetics , Fumigation , Gene Expression Profiling , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Fungal/drug effects , Terpenes/pharmacology , Weevils/enzymology , ATP-Binding Cassette Transporters/genetics , Animals , Down-Regulation , Inactivation, Metabolic/genetics , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , Up-Regulation , Weevils/genetics
15.
Int J Biol Macromol ; 112: 499-508, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29382578

ABSTRACT

Pectinases represent about one fifth of the enzyme worldwide market due their wide range of biotechnological applications. Current commercial pectinases are exclusively obtained from microbial sources, but here we report a pectin methylesterase (Sl-PME) and an endo-polygalacturonase (Sl-EPG) bioprospected from the sugarcane weevil, Sphenophorus levis, which revealed good potential for industrial applications. Sl-PME and Sl-EPG were overexpressed in Pichia pastoris, purified and enzymatically characterized. Sl-EPG presents optimal activity at pH 4-5 and 50 °C, showing that it can be used for juice extraction and clarification. On the other hand, Sl-PME presents optimal activity at pH 6-8 and 40 °C, and thus, suitable for both acidic and alkaline processing, such as coffee and tea fermentation. Sl-EPG shows Vmax = 3.23 mM/min, KM = 2.4 g/L and kcat = 418.6 s-1. While Sl-PME shows Vmax = 0.14 mM/min, KM = 4.1 g/L and kcat = 1.7 s-1. A PG inhibitor (PGIP2) weakly interfered in the Sl-EPG activity and Sl-PME was not affected by a usual PME inhibitor. Moreover, these enzymes manifested synergistic action towards methylesterified pectin. Here, we propose these enzymes as novel alternative tools for the current commercial pectinases.


Subject(s)
Carboxylic Ester Hydrolases/chemistry , Pectins/chemistry , Polygalacturonase/chemistry , Weevils/enzymology , Amino Acid Sequence/genetics , Animals , Biotechnology , Carboxylic Ester Hydrolases/genetics , Hydrogen-Ion Concentration , Pichia/genetics , Polygalacturonase/genetics , Saccharum/chemistry
16.
Sci Rep ; 7(1): 12483, 2017 10 02.
Article in English | MEDLINE | ID: mdl-28970561

ABSTRACT

Evolution of resistance among insects to action of pesticides has led to the discovery of several insecticides (neonicotinoids and organophosphates) with new targets in insect nervous system. Present study evaluates the mode of inhibition of acetylchlonesterase (AChE), biochemical efficacy, and molecular docking of 2,3-dimethylmaleic anhydride, against Periplaneta americana and Sitophilus oryzae. The knockdown activity of 2,3-dimethylmaleic anhydride was associated with in vivo inhibition of AChE. At KD99 dosage, the 2,3-dimethylmaleic anhydride showed more than 90% inhibition of AChE activity in test insects. A significant impairment in antioxidant system was observed, characterized by alteration in superoxide dismutase and catalase activities along with increase in reduced glutathione levels. Computational docking programs provided insights in to the possible interaction between 2,3-dimethylmaleic anhydride and AChE of P. americana. Our study reveals that 2,3-dimethylmaeic anhydride elicits toxicity in S. oryzae and P. americana primarily by AChE inhibition along with oxidative stress.


Subject(s)
Acetylcholinesterase/chemistry , Cholinesterase Inhibitors/pharmacology , Insect Proteins/antagonists & inhibitors , Insecticides/pharmacology , Maleic Anhydrides/pharmacology , Periplaneta/drug effects , Weevils/drug effects , Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Amino Acid Sequence , Animals , Catalase/antagonists & inhibitors , Catalase/metabolism , Catalytic Domain , Cholinesterase Inhibitors/chemistry , Dose-Response Relationship, Drug , Gene Expression , Glutathione/antagonists & inhibitors , Glutathione/metabolism , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/metabolism , Insecticides/chemistry , Kinetics , Maleic Anhydrides/chemistry , Molecular Docking Simulation , Nervous System/drug effects , Nervous System/enzymology , Oryza/parasitology , Oxidative Stress/drug effects , Periplaneta/enzymology , Periplaneta/genetics , Periplaneta/growth & development , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Sequence Alignment , Sequence Homology, Amino Acid , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/metabolism , Thermodynamics , Weevils/enzymology , Weevils/genetics , Weevils/growth & development
17.
Insect Mol Biol ; 26(4): 469-484, 2017 08.
Article in English | MEDLINE | ID: mdl-28556365

ABSTRACT

Plant cell wall degrading enzymes (PCWDEs) from insects were recently identified as a multigene family of proteins that consist primarily of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) and play essential roles in the degradation of the cellulose/hemicellulose/pectin network in the invaded host plant. Here we applied transcriptomic and degenerate PCR approaches to identify the PCWDEs from a destructive pest of palm trees, Rhynchophorus ferrugineus, followed by a gut-specific and stage-specific differential expression analysis. We identified a total of 27 transcripts encoding GH family members and three transcripts of the CE family with cellulase, hemicellulase and pectinase activities. We also identified two GH9 candidates, which have not previously been reported from Curculionidae. The gut-specific quantitative expression analysis identified key cellulases, hemicellulases and pectinases from R. ferrugineus. The expression analysis revealed a pectin methylesterase, RferCE8u02, and a cellulase, GH45c34485, which showed the highest gut enriched expression. Comparison of PCWDE expression patterns revealed that cellulases and pectinases are significantly upregulated in the adult stages, and we observed specific high expression of the hemicellulase RferGH16c4170. Overall, our study revealed the potential of PCWDEs from R. ferrugineus, which may be useful in biotechnological applications and may represent new tools in R. ferrugineus pest management strategies.


Subject(s)
Arecaceae , Cell Wall/metabolism , Herbivory , Weevils/enzymology , Animals , Esterases/metabolism , Female , Gastrointestinal Tract/metabolism , Gene Expression , Glycoside Hydrolases/metabolism , Male , Phylogeny , Real-Time Polymerase Chain Reaction , Weevils/genetics
18.
PLoS One ; 11(12): e0167748, 2016.
Article in English | MEDLINE | ID: mdl-27936192

ABSTRACT

BACKGROUND: The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. RESULTS: M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in mitochondrial respiratory chain. CONCLUSION: This is the first study to perform a comparative transcriptome analysis of S. zeamais in response to M. alternifolia essential oil fumigation. Our results provide new insights into the insecticidal mechanism of M. alternifolia essential oil fumigation against S. zeamais and eventually contribute to the management of this important agricultural pest.


Subject(s)
Fumigation/methods , Melaleuca/chemistry , Oils, Volatile/toxicity , Plant Oils/toxicity , Transcriptome/drug effects , Weevils/drug effects , Animals , Gene Expression Regulation/drug effects , Oils, Volatile/chemistry , Plant Oils/chemistry , Weevils/enzymology , Weevils/genetics
19.
Sci Rep ; 6: 38836, 2016 12 12.
Article in English | MEDLINE | ID: mdl-27941836

ABSTRACT

The African sweetpotato weevil Cylas brunneus is one of the most devastating pests affecting the production of sweetpotatoes, an important staple food in Sub-Saharan Africa. Current available control methods against this coleopteran pest are limited. In this study, we analyzed the potential of RNA interference as a novel crop protection strategy against this insect pest. First, the C. brunneus transcriptome was sequenced and RNAi functionality was confirmed by successfully silencing the laccase2 gene. Next, 24 potential target genes were chosen, based on their critical role in vital biological processes. A first screening via injection of gene-specific dsRNAs showed that the dsRNAs were highly toxic for C. brunneus. Injected doses of 200ng/mg body weight led to mortality rates of 90% or higher for 14 of the 24 tested genes after 14 days. The three best performing dsRNAs, targeting prosα2, rps13 and the homolog of Diabrotica virgifera snf7, were then used in further feeding trials to investigate RNAi by oral delivery. Different concentrations of dsRNAs mixed with artificial diet were tested and concentrations as low as 1 µg dsRNA/ mL diet led to significant mortality rates higher than 50%.These results proved that dsRNAs targeting essential genes show great potential to control C. brunneus.


Subject(s)
Pest Control, Biological/methods , RNA Interference , RNA, Double-Stranded/toxicity , Weevils/drug effects , Administration, Oral , Animals , Biological Control Agents , Insect Control/methods , Insect Proteins/antagonists & inhibitors , Insect Proteins/genetics , Insect Proteins/metabolism , Laccase/antagonists & inhibitors , Laccase/genetics , Larva , Lethal Dose 50 , Microinjections , Phenotype , RNA, Double-Stranded/administration & dosage , RNA, Double-Stranded/genetics , RNA, Double-Stranded/pharmacology , RNA, Small Interfering/genetics , Transcriptome , Weevils/enzymology , Weevils/genetics , Weevils/growth & development
20.
J Insect Sci ; 16(1)2016.
Article in English | MEDLINE | ID: mdl-27651423

ABSTRACT

The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) is a voracious pest of palm species. In recent decades its range has expanded greatly, particularly impacting the date palm industry in the Middle East. This has led to conjecture regarding the origins of invasive RPW populations. For example, in parts of the Middle East, RPW is commonly referred to as the "Pakistani weevil" in the belief that it originated there. We sought evidence to support or refute this belief. First reports of RPW in Pakistan were from the Punjab region in 1918, but it is unknown whether it is native or invasive there. We estimated genetic variation across five populations of RPW from two provinces of Pakistan, using sequences of the mitochondrial cytochrome oxidase subunit I gene. Four haplotypes were detected; two (H1 and H5) were abundant, accounting for 88% of specimens across the sampled populations, and were previously known from the Middle East. The remaining haplotypes (H51 and H52) were newly detected (in global terms) and there was no geographic overlap in their distribution within Pakistan. Levels of haplotype diversity were much lower than those previously recorded in accepted parts of the native range of RPW, suggesting that the weevil may be invasive in Pakistan. The affinity of Pakistani haplotypes to those reported from India (and the geographical proximity of the two countries), make the latter a likely "native" source. With regards the validity of the name "Pakistani weevil", we found little genetic evidence to justify it.


Subject(s)
DNA, Mitochondrial/genetics , Genetic Variation , Insect Proteins/genetics , Weevils/genetics , Animal Distribution , Animals , Introduced Species , Pakistan , Weevils/enzymology
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