Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

Enzymatic characterization, molecular dynamics simulation, and application of a novel Bacillus licheniformis laccase.

A new laccase gene from newly isolated Bacillus licheniformis TCCC 111219 was actively expressed in Escherichia coli. This recombinant laccase (rLAC) exhibited a high stability towards a wide pH range and high temperatures. 170% of the initial activity was detected at pH 10.0 after 10-d incubation, and 60% of the initial activity was even kept after 2-h incubation at 70 °C. It indicated that only single type of extreme environment, such as strong alkaline environment (300 K, pH 12) or high temperature (370 K, pH 7), did not show obvious impact on the structural stability of rLAC during molecular dynamics simulation process. But the four loop regions of rLAC where the active site is situated were seriously destroyed when strong alkaline and high temperature environment existed simultaneously (370 K, pH 12) because of the damage of hydrogen bonds and salt bridges. Moreover, this thermo- and alkaline-stable enzyme could efficiently decolorize the structurally differing azo, triphenylmethane, and anthraquinone dyes with appropriate mediator at pH 3.0, 7.0, and 9.0 at 60 °C. These rare characteristics suggested its high potential in industrial applications to decolorize textile dyeing effluent.

Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac.

An extracellular laccase enzyme secreted from Sphingobacterium ksn-11 was purified to electrophoretic homogeneity, showing a molecular weight of 90 kDa. The purified enzyme was monomeric in nature confirmed by sodium dodecyl gel electrophoresis. The optimum temperature and pH were found to be 40 °C and 4.5 respectively. The enzyme showed highest substrate specificity for 2,2 azino-bis (ethylthiozoline-6-sulfonate) (ABTS), followed by syringaldazine. The Km value for ABTS was 2.12 mM with a Vmax value of 33.33 U/mg which was higher when compared with syringaldazine and guaiacol substrates. Sodium azide and EDTA inhibited the activity by 30%, whereas presence of Ca2+ and iron increased activity by 50%. The purified enzyme was immobilized in sodium alginate-silicon dioxide-polyvinyl alcohol beads and evaluated for diclofenac transformation studies. LC-MS analysis confirmed that immobilized laccase transformed diclofenac to 4-OH diclofenac after 4 h of incubation. 45 % of diclofenac was able to transform even at 3rd cycle of immobilized laccase use. Therefore, immobilized laccase can be used to transform or degrade several recalcitrant compounds from industrial effluents.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes.

Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 µM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.

Novel Fungicide 4-Chlorocinnamaldehyde Thiosemicarbazide (PMDD) Inhibits Laccase and Controls the Causal Agent of Take-All Disease in Wheat, Gaeumannomyces graminis var. tritici.

Our aim was to investigate the bioactivity and mode of action of a novel fungicide 4-chlorocinnamaldehyde thiosemicarbazide (PMDD). As a result of its efficacy against various plant pathogens, its protective fungicidal activity, and systemic transport after root treatment, PMDD could be a promising fungicide to control wheat root diseases. In a field assay, PMDD showed good control efficacy on wheat take-all disease. A biochemical study indicated that PMDD acts as a laccase inhibitor, a to date unique mode of fungicidal action. Moreover, a total of seven stable PMDD-resistant Gaeumannomyces graminis var. tritici (Ggt) mutants were generated and demonstrated no cross-resistance with any commercial fungicides used for take-all disease control, and the gene expression profile further confirmed that laccase could be the target of PMDD. Taken together, we conclude that PMDD is a laccase inhibitor and could be used on wheat to control take-all diseases. The current study could strongly benefit the registration and application of PMDD.

Synthesis and Structure-Activity Relationship Studies of Hydrazide-Hydrazones as Inhibitors of Laccase from Trametes versicolor.

A series of hydrazide-hydrazones 1-3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24-674 µM with the predicted and desirable competitive type of inhibition. The structure-activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.

Ecotoxicological test based on inhibition of fungal laccase activity: Application to agrochemicals and the monitoring of pesticide degradation processes.

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC50) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali.

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Poly(2-oxazoline)s with a 2,2'-Iminodiacetate End Group Inhibit and Stabilize Laccase.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.

Decolorization of Acid Green 25 by Surface Display of CotA laccase on Bacillus subtilis spores.

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN3, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

Enhanced decolorization of malachite green by a magnetic graphene oxide-CotA laccase composite.

In this study, a CotA laccase from Bacillus subtilis cjp3 was successfully immobilized onto magnetic graphene oxide (MGO) nanomaterials via covalent bonding with hydrochloride/N-hydroxysuccinimide (EDC/NHS). The morphology, structure, and properties of the MGO-laccase were then characterized by scanning-electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray-photoelectron spectroscopy (XPS), and a magnetic-property-measurement system (MPMS). The magnetic composite exhibited an extremely high binding capacity of ~145.04mg/g and maintained maximal relative enzyme activities at 25°C, pH7, and a reaction time of 2h. The pH, thermal, operational, and storage stabilities of MGO-laccase were significantly improved over those of free laccase. Moreover, MGO-laccase exhibited a higher tolerance than that of free laccase in the presence of organic solvents, inhibitors, metal ions, and salts. Furthermore, MGO-laccase showed good decolorization performance of malachite green (MG), with decolorization rates reaching 99% after 5h of reaction at 30°C and pH6. In addition, the maximum saturation magnetization of MGO-laccase was 27.7emu/g, allowing for rapid magnetic separation. Accordingly, magnetic separation allowed MGO-laccase to maintain 75% of its activity after ten consecutive decolorization cycles.

Biocatalytic characterization of free and immobilized laccase from Trametes versicolor in its activation zone.

This investigation may be of interest for researchers working on the determination of several biocatalytic properties of the laccase from Trametes versicolor. So, We will treated the effects of pH, temperature, several organic components and heavy metals by performing enzyme assays in the presence of a 2,6 dimethoxyphenol (DMP) as substrate on the laccase activity. The optimum activity and temperature are 4 and 40 °C, respectively. The maximum rate of the reaction is 124.53 U/mg and the Michaelis constant is in order of 1.23 mM. The effect of metal ions on the laccase activity with a final concentrations range varying from 1 to 5 mM show that the Cu2+ ions increase the activity for concentration inferiors to 4 mM and the other metal ions have a relative influence on the laccase activity. Four tri-block copolymers based on poly(ethylene oxide) and poly(propylene oxide) and two polyethylene glycols are used to study the synthetic polymers effects on the enzymatic activity. Also, we have demonstrated that the laccase keeps 95% of its initial activity at 60 °C in the PEGDA8000 and PEGDA6000 gel matrix. The maximum rate of the immobilized laccase is approximately around 21.03 and 47.22% smaller than the free one.

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization.

The gene encoding copper-dependent laccase from Bacillus subtilis strain R5 was cloned and expressed in Escherichia coli. Initially the recombinant protein was produced in insoluble form as inclusion bodies. Successful attempts were made to produce the recombinant protein in soluble and active form. The laccase activity of the recombinant protein was highly dependent on the presence of copper ions in the growth medium and microaerobic conditions during protein production. The purified enzyme exhibited highest activity at 55 °C and pH 7.0. The recombinant protein was highly thermostable, albeit from a mesophilic source, with a half-life of 150 min at 80 °C. Similar to temperature, the recombinant protein was stable in the presence of organic solvents and protein denaturants such as urea. Furthermore, the recombinant protein was successfully utilized for the degradation of various synthetic dyes reflecting its potential use in treatment of wastewater in textile industry. Abbreviations: ABTS,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CBB, Coomassie brilliant blue; SGZ, syringaldazine; DMP, 2,2-dimethoxy phenol.

Cell wall structure of secreted laccase-silenced strain in Lentinula edodes.

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble ß-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble ß-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of ß-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.

Expression and characterization of novel laccase gene from Pandoraea sp. ISTKB and its application.

In the present study, a non-blue laccase gene from previously reported lignin degrading bacterium, Pandoraea sp. ISTKB, was isolated, cloned and expressed in E. coli. Bioinformatics analysis of sequence discovered twin-arginine translocation signal sequence, copper binding motifs and presence of more random coil compare to helices and sheets in structure. The enzyme was found to be active on wide pH range and the pH optima was observed at pH 4 and 8 on substrate 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,6-Dimethoxyphenol respectively. This is a thermophilic enzyme with maximum activity around 50-70 °C. The enzyme was further characterized by spectroscopy, reaction kinetics and effect of metal ions and inhibitors were studied. Compared to laccase alone; the treatment of dyes with laccase plus mediator resulted in enhanced decolorization of crystal violet, methylene blue, azure B, carmine and Congo red but the effect of mediator was not observed on trypan blue. Laccase treatment triggered polymerization on vanillic acid (VA) and kraft lignin (KL). Laccase plus mediator treatment reversed the polymerization and resulted in transformation or degradation of VA and KL. This thermophilic and alkalophilic non-blue laccase from Pandoraea sp. ISTKB is promising with prospective biotechnological application.

Molecular Mechanisms Underlying Inhibitory Binding of Alkylimidazolium Ionic Liquids to Laccase.

Water-miscible alkylimidazolium ionic liquids (ILs) are "green" co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the TI Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C2~C6) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO4- showed higher biocompatibility than did Ac- or Cl-, probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase.

A novel laccase from white rot fungus Trametes orientalis: Purification, characterization, and application.

A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears as a monomeric protein with a molecular mass of 44.0kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80°C, respectively, and it retained more than 80% of its original activity after 2h incubation at 10°C to 50°C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn2+ was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN3, L-cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.

Systemic RNAi in the small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae), a serious pest of the European honey bee Apis mellifera.

BACKGROUND: Aethina tumida is a serious pest of the European honey bee (Apis mellifera) in North America and Australia. Here we investigate whether Laccase 2, the phenoloxidase gene essential for cuticle sclerotisation and pigmentation in many insects, and vacuolar-ATPase V-type subunit A, vital for the generation of proton gradients used to drive a range of transport processes, could be potential targets for RNAi-mediated control of A. tumida. RESULTS: Injection of V-ATPase subunit A (5 ng) and Laccase 2 (12.5 ng) dsRNAs resulted in 100% larval mortality, and qPCR confirmed significant decreases and enhanced suppression of transcript levels over time. Oral delivery of V-ATPase subunit A dsRNA in solutions resulted in 50% mortality; however, gene suppression could not be verified. We suggest that the inconsistent RNAi effect was a consequence of dsRNA degradation within the gut owing to the presence of extracellular nucleases. Target specificity was confirmed by a lack of effect on survival or gene expression in honey bees injected with A. tumida dsRNAs. CONCLUSION: This is the first study to show evidence for systemic RNAi in A. tumida in response to injected dsRNA, but further research is required to develop methods to induce RNAi effects via ingestion. © 2016 Crown copyright. Pest Management Science © 2016 Society of Chemical Industry.

RNA interference: a promising biopesticide strategy against the African Sweetpotato Weevil Cylas brunneus.

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.