Resistant Field Strain Research Articles

Susceptibility of Field and Laboratory Bt-Susceptible and Resistant Strains of Helicoverpa zea (Boddie) to HearNPV.

During 2021 and 2022, eight field-collected and five laboratory Helicoverpa zea strains with varying susceptibility to different Bt proteins were evaluated for their responses against HearNPV using diet-overlay bioassays. The five laboratory strains included SS (susceptible to all Bt proteins), CRY-RR (resistant to Cry1 and Cry2), VIP-RR-70 (resistant to Vip3Aa), VIP-RR-15 (resistant to Vip3Aa), and TRE-RR (resistant to Cry1, Cry2, and Vip3Aa). Our findings showed that the susceptibility of TRE-RR, VIP-RR-70, and VIP-RR-15 strains to HearNPV was similar to that of the SS strain. However, the field and Cry-RR strains were more resistant to HearNPV compared to the SS strain. Because most feral H. zea strains in the southern U.S. have developed practical resistance to Cry Bt proteins but remain susceptible to Vip3Aa, the results suggest that the reduced susceptibility to HearNPV in H. zea may be associated with the resistance to Cry Bt proteins but not with the resistance to Vip3Aa. Correlation analysis confirmed that there was a significant positive relationship between Cry resistance and HearNPV resistance, but not between the Vip3Aa resistance and HearNPV resistance in H. zea. Our findings provide valuable insights into the relationship between susceptibility to HearNPV and resistance to Bt proteins in H. zea.

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella

BackgroundProtozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate.MethodsWe developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule.ResultsWe obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate.ConclusionsOur findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.Graphical abstract

Effects of copper and zinc oxide nanoparticles on German cockroach development, indoxacarb resistance, and bacterial load.

The German cockroach, Blattella germanica, is a ubiquitous and medically significant urban pest. The ongoing development of insecticide resistance in global populations of B. germanica has complicated control efforts and created a need for improved tools. We previously reported that disruption of the gut microbiota by oral administration of the antimicrobial doxycycline reduced resistance in an indoxacarb resistant field strain and also delayed nymphal development and reduced adult fecundity. However, the application of doxycycline for cockroach control in the field is impractical. Here, we sought to determine whether two metal nanoparticles with known antimicrobial properties, copper (Cu) and zinc oxide (ZnO), have similar effects to doxycycline on the physiology of B. germanica and could provide more practical alternatives for control. We found that dietary exposure to 0.1% Cu nanoparticles, but not ZnO, significantly delays the development of nymphs into adults. However, neither of the nanoparticles altered the fecundity of females, and ZnO surprisingly increased resistance to indoxacarb in a resistant field strain, in contrast to doxycycline. Semi-quantitative polymerase chain reaction (qPCR) further revealed that prolonged dietary exposure (14 days) to Cu or ZnO nanoparticles at the low concentration readily consumed by cockroaches (0.1%) does not reduce the load of the bacterial microbiota, suggesting alternative mechanisms behind their observed effects. Together, our results indicate that ingestion of Cu nanoparticles can impact German cockroach development through an undetermined mechanism that does not involve reducing the overall load of the bacterial microbiota. Therefore, Cu may have some applications in cockroach control as a result of this activity but antagonistic effects on insecticide resistance should be considered when evaluating the potential of nanoparticles for cockroach control. © 2023 Society of Chemical Industry.

Mechanism of Fusarium graminearum Resistance to Ergosterol Biosynthesis Inhibitors: G443S Substitution of the Drug Target FgCYP51A.

Fusarium head blight (FHB), caused by the Fusarium graminearum species complex, is a devastating fungal disease resulting in substantial yield and quality losses. Ergosterol biosynthesis inhibitors (EBIs) are the most popular chemicals for controlling FHB. Recently, the resistance of F. graminearum to EBIs has emerged in the field, and an amino acid substitution (G443S) of the sterol 14α-demethylase FgCYP51A was detected in the field resistant strains. To further illustrate the resistance mechanism of F. graminearum to EBIs, site-directed mutants conferring the G443S substitution of FgCYP51A were generated from the progenitor strain PH-1 via genetic transformation with site-directed mutagenesis. We found that the FgCYP51A-G443S substitution significantly decreased the sensitivity of F. graminearum to EBIs with EC50 values ranging from 0.1190 to 0.2302 μg mL-1 and EC90 values ranging from 1.3420 to 9.1119 μg mL-1 for tebuconazole. Furthermore, the FgCYP51A-G443S substitution decreased sexual reproduction and virulence, which will reduce the initial infection source of pathogen populations in the field, while the increase of sporulation capability may enhance the frequencies of the disease cycle, thereby contributing to epidemics of FHB disease. Surprisingly, the FgCYP51A-G443S substitution accelerated DON biosynthesis by upregulating TRI5 expression and enhancing the fluorescence intensity of TRI1-GFP, the marker protein of Fusarium toxisomes. Thus, we concluded that the FgCYP51A-G443S substitution regulates EBI-fungicide resistance and DON biosynthesis, increasing the risk of fungicide resistance development in the field, thereby threatening the control efficacy of EBIs against FHB.

Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella.

Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined.Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification.Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process.Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.

Antibacterial efficacy of in-house designed cell-penetrating peptide against multi-drug resistant strains of Salmonella Enteritidis and Salmonella Typhimurium.

The in vitro antibacterial efficacy of an in-house designed cell-penetrating peptide (CPP) variant of Cecropin A (1-7)-Melittin (CAMA) (CAMA-CPP) against the characterized multi-drug resistant (MDR) field strains of Salmonella Enteritidis and Salmonella Typhimurium were evaluated and compared with two identified CPPs namely, P7 and APP, keeping CAMA as control. Initially, the minimum inhibitory concentration (MIC) (μg ml-1 ) of in-house designed CAMA-CPP, APP and CAMA was determined to be 3.91, whereas that of P7 was 7.81; however, the minimum bactericidal concentration (MBC) of all the peptides were twice the MIC. CAMA-CPP and CAMA were found to be stable under different conditions (high-end temperatures, proteinase-K, cationic salts, pH and serum) when compared to the other CPPs. Moreover, CAMA-CPP exhibited negligible cytotoxicity in HEp-2 and RAW 264.7 cell lines as well as haemolysis in the sheep and human erythrocytes with no adverse effects against the commensal gut lactobacilli. In vitro time-kill assay revealed that the MBC levels of CAMA-CPP and APP could eliminate the intracellular MDR-Salmonella infections from mammalian cell lines; however, CAMA and P7 peptides were ineffective. CAMA-CPP appears to be a promising antimicrobial candidate and opens up further avenues for its in vivo clinical translation.

Novel Antiplasmodial Compounds Leveraged with Multistage Potency against the Parasite Plasmodium falciparum: In Vitro and In Vivo Evaluations and Pharmacokinetic Studies.

Hydroxyethylamine (HEA)-based novel compounds were synthesized and their activity against Plasmodium falciparum 3D7 was assessed, identifying a few hits without any apparent toxicity. Hits 5c and 5d also exhibited activity against resistant field strains, PfRKL-9 and PfC580Y. A single dose, 50 mg/Kg, of hits administered to the rodent parasite Plasmodium berghei ANKA exhibited up to 70% reduction in the parasite load. Compound 5d tested in combination with artesunate produced an additional antiparasitic effect with a prolonged survival period. Additionally, compound 5d showed 50% inhibition against hepatic P. berghei infection at 1.56 ± 0.56 μM concentration. This compound also considerably delayed the progression of transmission stages, ookinete and oocyst. Furthermore, the toxicity of 5d assessed in mice supported the normal liver and kidney functions. Altogether, HEA analogues (5a-m), particularly 5d, are nontoxic multistage antiplasmodial agents with therapeutic and transmission-blocking efficacy, along with favorable preliminary pharmacokinetic properties.

Oocysts Output of Broilers Experimentally Infected with Eimeria tenella And Treated with N- Butanol Leaf Extract of Khaya senegalensis

A study to determine the anticoccidial efficacy of Khaya senegalensis in relation to oocyst shedding by broilers experimentally infected with Eimeria tenella was conducted. The development of drug- resistant field strains of Eimeria species has prompted the exploitation of alternative methods for controlling coccidiosis and there is an increasing use of medicinal plants as alternatives to orthodox medicine. Fresh leaves of Khaya senegalensis (KS) were collected dried under shade and the extract prepared using the maceration method in 70% methanol. The dried crude extract was partitioned into petroleum ether, chloroform, n-butanol and aqueous portions, dried with phytochemical analysis conducted on them. One hundred and twenty birds reared under standard management practice were divided into six groups (A, B, C, D, E and F). All the groups except group F (uninfected untreated group) were infected at four weeks old with sporulated Eimeria tenella oocysts (1.0 x 105 sporulated oocysts / ml / bird) obtained locally from the intestinal scrapings of experimentally infected broilers. Groups A, B and C were given calculated three dose levels of 11mg/kg, 33mg/kg, 99mg/kg respectively of the prepared n-butanol methanol extract twice daily for 5 days, group D was given Amprolium while E and F were each given 0.2ml water. Faecal samples were collected daily for 4 weeks into clean well labelled polythene bags and analysed in the laboratory for oocyst count using the McMaster counting chamber and were expressed as oocysts count per gramme of faeces. The birds were observed for pathological lesions grossly and histopathologically and the survival rates were determined. Data collected were analysed using analysis of variance and chi square. Results from the Phytochemical studies showed the presence of phenolic compounds in Khaya senegalensis. Post-infection faecal examination revealed oocyst load of +++ in all the infected pens (A-E) on the 6th day. Comparison of the groups with time showed statistical significance (P˂0.05). High mean oocyst production (A; 156060 ± 67020, B; 261590 ± 144310, C; 211620 ± 114280, D; 276930 ± 233650 and E; 159230 ± 100970) among the infected groups one week post infection as well as irregular oocyst production were observed in the course of this study. The higher mean oocysts count obtained in the infected untreated group (1748849 ± 40869) than the extract treated groups in the first week post treatment indicated that the extract had some inhibitory effects on oocyst production. This however, was dose dependent. Among the extract treated group, the 99mg/kg had lower mean oocyst production 2 weeks post treatment (9720 ± 3180) and this was comparable to the group treated with normal dose of the conventional drug Amprolium (8600 ± 40). This was therefore seen as the effective dose. Grossly, the extract had a beneficial effect in alleviating the damages to the caecal epithelium of the infected treated groups compared to the shrunken caeca of the infected untreated groups. The survival percentage was higher in the treated groups compared to the infected un treated group (55%) though Amprolium was more efficacious in the in vivo study with the highest survival rate of 90%. The histopathological lesions observed in the infected birds in this study were consistent with those associated with E tenella infection in which the parasite induced very severe lesions including severe villous atrophy and fusion. The anticoccidial efficacy of Khaya senegalensis promises greater areas for research as it relates to drug development and it is recommended that Khaya senegalensis should be exploited further for its anticoccidial properties using other parts of the plant.
 Key words: Broiler chickens, Eimeria tenella, oocyst production, Khaya senegalensis, in vivo

Larvicidal potential of two silver nano-particles (Moringa oleifera and Ficus exasperata) against laboratory and field strains of Anopheles gambiae (Diptera: Culicidae) in Lagos, Nigeria

BackgroundThe interest in larvicides of plant origin is generally renewed in vector control because of their safety compared to synthetic larvicides. However, there are concerns about the relative safety dose of these phytochemicals on non-target organisms which led to the development of plant derived nanoparticles. In this study, we examined the bioefficacy of low doses of two green synthesized nanoparticles on immature stages of Anopheles mosquitoes in Nigeria. Aqueous plants (Moringa oleifera and Ficus exasperata) extracts were used in the biosynthesis. The prepared Ag-NPs were characterizations using Fourier-transform infrared spectroscopy (FT-IR), UV-Vis spectroscopy, and scanning electron microscopy (SEM). Third and early fourth instars of known susceptible laboratory strains of Anopheles gambiae s.s. (KISUMU strains) and pyrethroid resistant field strain of An. gambiae were exposed to serial dilutions of 0.25, 0.5, 0.75, 1.0 and 2.5 ppm of each phyto nanoparticles. Moribund and dead larvae were observed after 24 and 48 h post exposure, and the results were analysed with descriptive statistics.ResultsWith the laboratory mosquitoes, Moringa oleifera AgNP effected high mortalities of 88–100% (LC50 = 0.39 ppm; LC95 = 0.62 ppm) at 24 h post exposure except at the lowest concentration, while Ficus exasperate AgNP induced a 32–100% mortality (LC50 = 0.51 ppm; LC95 = 1.15 ppm) except at the lowest concentration. In the field populations, mortality in Moringa oleifera and Ficus exasperata was 23–93% (LC50 = 0.65 ppm; LC95 = 2.28 ppm) and 37–50% (LC50 = 1.51 ppm; LC95 = 391.64 ppm) respectively. There was no significant difference in mortality values between the laboratory and field strains (P < 0.05) at both 24 and 48 h post exposure times.ConclusionsOverall, the study demonstrates the bioefficacy and potential use of green synthesized nanoparticles, at very low concentrations for the control of Anopheles larvae even in areas where resistance to the current chemical insecticides have been reported.

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 β-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

Undefeated-Changing the phenamacril scaffold is not enough to beat resistant Fusarium.

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.

A Xenobiotic Detoxification Pathway through Transcriptional Regulation in Filamentous Fungi.

ABSTRACTFungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages.

Responses of phosphine susceptible and resistant strains of five stored-product insect species to chlorine dioxide

Adults of phosphine susceptible laboratory strains and phosphine resistant field strains of five stored-product insect species were exposed in vials with 0 or 10 g of wheat for different time periods to 0.54 g/m3 (200 ppm) of chlorine dioxide gas. After exposure, adult mortality was determined 5 d later at 28 °C and 65% r.h. The 5-d mortality was 100% in laboratory and field strains of the red flour beetle, Tribolium castaneum (Herbst); sawtoothed grain beetle, Oryzaephilus surinamensis (L.); lesser grain borer, Rhyzopertha dominica (F.); maize weevil, Sitophilus zeamais Motschulsky; and rice weevil, Sitophilus oryzae (L.) that were exposed in vials with 10 g of wheat to chlorine dioxide for 26, 16, 24–34, 18–24, and 15–18 h, respectively. Corresponding exposure durations for these species and strains in vials without wheat were 15, 3, 18–20, 7–15, and 5–7 h, respectively. Dosages of chlorine dioxide producing 99% mortality (LD99) of T. castaneum, O. surinamensis, R. dominica, S. zeamais, and S. oryzae strains in vials with wheat ranged from 14.79−22.57, 8.20–8.41, 15.79–21.60, 10.66–14.53, and 7.67–12.20 g-h/m3, respectively. In vials without wheat, corresponding LD99 values for T. castaneum, R. dominica, and S. zeamais strains were 6.51–8.66, 11.46–23.17, and 5.79–10.26 g-h/m3, respectively. LD99 values for O. surinamensis and S. oryzae could not be computed, because of 100% mortality after a 3–5 h exposure to chlorine dioxide. No adult progeny production of T. castaneum and O. surinamensis was observed after 8 weeks in control and chlorine dioxide-exposed samples. Adult progeny production of Sitophilus spp. was found only in the control samples. The dosage for 99% adult progeny reduction relative to control for R. dominica strains ranged from 10.07 to 18.11 g-h/m3. Chlorine dioxide gas is effective in killing phosphine susceptible and resistant strains of five stored-product insect species and suppressing adult progeny production of three out of the five species.

Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes.

BackgroundDengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance.Methodology/Principal FindingsGenome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb.Conclusion/significanceThe predominant over-expression of cytochrome P450s suggests that synergist-based (PBO) control tools could be utilised to improve control of this major dengue vector across Malaysia.

Involvement of Three Esterase Genes from Panonychus citri (McGregor) in Fenpropathrin Resistance.

The citrus red mite, Panonychus citri (McGregor), is a major citrus pest with a worldwide distribution and an extensive record of pesticide resistance. However, the underlying molecular mechanism associated with fenpropathrin resistance in this species have not yet been reported. In this study, synergist triphenyl phosphate (TPP) dramatically increased the toxicity of fenpropathrin, suggesting involvement of carboxylesterases (CarEs) in the metabolic detoxification of this insecticide. The subsequent spatiotemporal expression pattern analysis of PcE1, PcE7 and PcE9 showed that three CarEs genes were all over-expressed after insecticide exposure and higher transcripts levels were observed in different field resistant strains of P. citri. Heterologous expression combined with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay in Spodoptera frugiperda (Sf9) cells revealed that PcE1-, PcE7- or PcE9-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against fenpropathrin, demonstrating that PcEs probably detoxify fenpropathrin. Moreover, gene silencing through the method of leaf-mediated dsRNA feeding followed by insecticide bioassay increased the mortalities of fenpropathrin-treated mites by 31% (PcE1), 27% (PcE7) and 22% (PcE9), respectively, after individual PcE gene dsRNA treatment. In conclusion, this study provides evidence that PcE1, PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P. citri and enrich molecular understanding of CarEs during the resistance development of the mite.

Molecular analysis of cyenopyrafen resistance in the two-spotted spider mite Tetranychus urticae.

Cyenopyrafen is a recently developed acaricide with a new mode of action as a complex II inhibitor. However, it was recently shown that cross-resistance to cyenopyrafen can occur in resistant field strains of Tetranychus urticae, which might be linked to the previous use of classical METI acaricides. Here, we selected for cyenopyrafen resistance and studied the molecular mechanisms that underlie resistance. Selection for cyenopyrafen resistance confers cross-resistance to the complex II inhibitor cyflumetofen, but also to pyridaben, a frequently used complex I inhibitor. Cyenopyrafen resistance is highly synergised by piperonyl butoxide, and a 15-fold higher P450 activity was detected in the resistant strain. Target-site resistance was not detected. Genome-wide gene expression data, followed by a meta-analysis of previously obtained gene expression data, revealed the overexpression specifically of CYP392A11 and CYP392A12. Cyenopyrafen resistance is strongly linked to the overexpression of two P450s, which probably explains the observed cross-resistance. This information is highly valuable, as the novel complex II inhibitors cyenopyrafen and cyflumetofen are in the process of worldwide registration. The role of both CYP392A11 and CYP392A12 should be further supported by functional expression, but they are very promising candidates as molecular diagnostic markers for monitoring cyenopyrafen susceptibility in the field.

Field resistance to the Bacillus thuringiensis protein Cry1Ac expressed in Bollgard(®) hybrid cotton in pink bollworm, Pectinophora gossypiella (Saunders), populations in India.

Bollgard(®) cotton, expressing Cry1Ac insecticidal protein, was approved for commercial planting in India in 2002, and by 2009 constituted 87% of the Indian crop, reducing losses from lepidopteran pests, including pink bollworm (PBW), Pectinophora gossypiella. Inadequate control of PBW in fields of single-gene Bollgard cotton was reported in 2009; surveys revealed heavy infestations of PBW in Bollgard, restricted to Gujarat state, but not elsewhere in India. Bioassays of PBW strains from Bollgard bolls showed that, while susceptible PBW could not complete development to third and later instar at 10.0 µg Cry1Ac mL(-1) , 66.1% of larvae from Gujarat Bollgard strains could. A field-resistant strain, further selected in the laboratory, had susceptibility to Cry1Ac reduced by >2000-fold. Resistance to Cry1Ac did not confer cross-resistance to the Cry2Ab2 protein. In 2010, Bollgard fields in Gujarat continued to be infested with PBW, and many Bollgard fields in the adjoining states of Maharashtra and Madhya Pradesh showed high-level infestation by PBW. Inadequate planting of refuges for PBW is the likely explanation for the field resistance to Bt cotton observed in Gujarat. These findings underscore the higher vulnerability of single-gene Bt products relative to dual-gene products expressing Cry1Ac and Cry2Ab2, and the increased risk of resistance evolution with low refuge compliance.

Frequency distribution of non-specific esterase in susceptible, field and cypermethrin resistant strains of Culex pipiens mosquito

Frequency distribution of non-specific esterase in susceptible, field and cypermethrin resistant strains of Culex pipiens mosquito

Population Growth Potential of the Bed Bug, Cimex lectularius L.: A Life Table Analysis.

Experimental life tables were constructed and analyzed for three strains of the common bed bug: a pyrethroid-susceptible laboratory strain (HS), a highly resistant field strain (RR), and a field strain with a declining level of resistance (KR). Egg to adult survival in the RR strain was 94% compared with 79% and 69% in the HS and KR strains, respectively. The RR strain also developed significantly faster from egg to adult (∼35 days) than the other two strains (∼40 days). Analysis of a survivorship and fecundity life table for the RR strain produced the following results. The average life expectancy for a newly laid egg was ∼143 days, and that of a newly molted adult was ∼127 days. Females produced an average of 0.64 daughter eggs/day with the highest weekly production during the fifth week of adult life. Analysis of daily reproductive parity showed that females produced 1–3 and 4–6 eggs on 79 and 21% of the days, respectively, when egg laying occurred. The net reproductive rate (R0) of the RR strain was ∼35, which represents a 35-fold increase in the population per generation (∼92 days). The intrinsic rate of increase, r, was 0.054 indicating that the population multiplies 1.1 times/female/day (λ) and doubles in size every 13 days. The stable age distribution (cx) was dominated by nymphs (54%), followed by eggs (34%) and adults (12%). Reproductive values (vx) for the strain increased from egg to the adult stage.

Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log 10 cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 μs of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.