Mosquito larvicidal activity of alkaloids from Zanthoxylum lemairei against the malaria vector Anopheles gambiae

Four alkaloids, 10-O-demethyl-17-O-methylisoarnottianamide 1, 6-acetonyl-N-methyl-dihydrodecarine 2, nitidine 3, and chelerythrine 4 were isolated from the plant Zanthoxylum lemairei (Rutaceae) and evaluated for mosquito larvicidal activity against the malaria vector Anopheles gambiae. The mortalities of the larvae were determined after 24 h. The results of the larvicidal tests demonstrated that compounds 1 and 2 were the most potent with mortality rates of 96.7% and 98.3% at a concentration of 250 mg/L, respectively. Compound 3 was less potent with a mortality of 28.3% at the same concentration. The percent mortality of 100% was observed at a concentration of 500 mg/L. The least potent of the four alkaloids was compound 4, which achieved 100% mortality at 1000 mg/L. These findings could be useful in the research for newer more selective, biodegradable and natural larvicidal compounds or can be used as lead compounds for the development of larvicides.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Mutated sodium channel genes and elevated monooxygenases are found in pyrethroid resistant populations of Sri Lankan malaria vectors

The present status of pyrethroid resistance in vectors of malaria; Anopheles culicifacies and Anopheles subpictus, was tested in two malarious Districts, Anuradhapura and Trincomalee, of Sri Lanka. Both species were resistant to permethrin and susceptible to cypermethrin and cyfluthrin. An. subpictus were resistant to deltamethrin. λ-Cyhalothrin and etofenprox resistance was shown only by Anuradhapura An. subpictus. Although there were no differences among the populations for esterase and glutathione S-transferase activities, increased monooxygenase levels were found among Trincomalee populations. The voltage-gated sodium channel gene, the target site gene of pyrethroids, was partially sequenced to screen for mutations previously associated with insecticide resistance. The classic leucine to phenylalanine substitution, TTA to TTT, was detected in An. subpictus. It appears that both kdr type and monooxygenase resistance underlie pyrethroid resistance in these two malaria vectors of Sri Lanka.     

Cytochrome P-based resistance mechanism and pyrethroid resistance in the field Anopheles albimanus resistance management trial

The relative rates of cytochrome P⁴⁵⁰ selection in southern Mexican Anopheles albimanus populations were investigated during a 3 years indoor residual house spraying intervention with a pyrethroid (PYR) or DDT, a mosaic of organophosphate (OP)-PYR, and the annual rotation of OP-PYR-carbamate (CARB). This insecticide resistance mechanism, initially evenly spread in the mosquito population, correlated with PYR resistance during the second treated year, when cytochrome P⁴⁵⁰ contents increased in most villages of the PYR, rotation and mosaic schemes. However, by the third year, mean cytochrome P⁴⁵⁰ contents declined to susceptible levels in mosquitoes of the rotation and one mosaic group but not in the PYR-treated villages. In DDT-treated villages, a continuous decrement of cytochrome P⁴⁵⁰ levels occurred since the first treatment year, and susceptible levels were observed at the end of the intervention. Most correlations of cytochrome P⁴⁵⁰ levels and PYR resistance were lost during the third year, indicating that another mechanism evolved in resistant mosquito populations.     

Absorption, translocation, and metabolism of propoxycarbazone-sodium in ALS-inhibitor resistant Bromus tectorum biotypes

Experiments were conducted to investigate the absorption, translocation, and metabolism of propoxycarbazone-sodium in acetolactate synthase-inhibitor resistant (AR and MR) and susceptible (AS and MS) Bromus tectorum biotypes. Absorption and translocation of ^l4C-propoxycarbazone-sodium were similar in all biotypes. One major and three minor metabolites were identified using reverse-phase high performance liquid chromatography. In all biotypes, 80% of the propoxycarbazone-sodium was metabolized by 72 h after treatment (HAT). However, propoxycarbazone-sodium was metabolized more rapidly in the MR biotype than in the other biotypes. The half-life of propoxycarbazone-sodium in the MR biotype was 8.9 h, which was 30, 36, and 40% shorter than in the AS, AR, and MS biotypes, respectively. When ¹⁴C-propoxycarbazone-sodium was applied with 1-aminobenzotriazole, a known cytochrome P450 inhibitor, metabolism decreased 20% 12 HAT in the MR biotype. These results indicate that resistance of the MR biotype to propoxycarbazone-sodium is due to a relatively rapid rate of propoxycarbazone-sodium metabolism compared to other B. tectorum biotypes and that cytochrome P450s may be involved in the metabolism. The fact that these populations evolved so quickly and with different resistance mechanisms is a concern as more ALS inhibitors are introduced into the production systems.     

The pyrethroid resistance status and mechanisms in Aedes aegypti from the Guerrero state,Mexico

Dengue is one of the most important vector-borne diseases worldwide and is a public health problem in Mexico. Most programs in dengue endemic countries rely on insecticides for Aedes control. In Mexico, pyrethroid insecticides (mainly permethrin and deltamethrin) have been extensively used over a decade as adulticides and represented a strong selection for insecticide resistance for dengue vectors in several parts of the country. We studied the type, frequency and distribution of insecticide resistance mechanisms in Aedes aegypti from six municipalities in the state of Guerrero selected on the basis of historically intense chemical control and a high risk for dengue transmission. Ae. aegypti eggs were collected from October 2009 to January 2010 using ovitraps. F₁ adults, emerged from these collections, were exposed to permethrin, deltamethrin and DDT in WHO diagnostic tests and showed high resistance levels to both pyrethroids and DDT. This was consistent with the presence of increased metabolic enzyme activities and target site insensitivity due to kdr mutations. Biochemical assays showed elevated esterase and glutathione S-transferase activities in the six municipalities. The V1016I kdr mutation on the IIS6 domain of the sodium channel gene was present in an overall frequency of 0.80. A second mutation, F1534C on the IIIS6 domain of the same gene was also detected, being the first report of this mutation in Guerrero. The multiple resistance mechanisms present in Ae. aegypti from Guerrero state represent a warning for the efficacy of the pyrethroid usage and consequently for the success of the dengue control program.     

Phenobarbital induction of permethrin detoxification and phenobarbital metabolism in susceptible and resistant strains of the beet armyworm Spodoptera exigua (Hübner)

The permethrin resistant strain (TR-strain) of the beet armyworm, Spodoptera exigua (Hübner), has 92.5-fold resistance to permethrin (at LD₅₀ level) compared to the permethrin susceptible strain (TS-strain). Bioassay involving permethrin mixed with piperonyl butoxide, an inhibitor of microsomal cytochrome P450s, significantly reduced the resistance ratio from 92.5- to 7.9-fold. However, S,S,S-tributylphosphorotrithioate and diethylmaleate which are inhibitors of esterases and glutathione S-transferase, respectively, did not affect the resistance level. These results indicate that the detoxification of permethrin in the TR-strain was primarily due to the cytochrome P450 monooxygenases. LD₅₀ for permethrin was increased to 4.5-fold by the pre-treatment of phenobarbital in the TS-strain. The effect of induction by phenobarbital was almost completely overcome by the piperonyl butoxide treatment. However, it was observed that phenobarbital treatment did not cause any change in the toxicity of permethrin to TR strain. Since this result deviated from the expectation that the metabolism of phenobarbital in the TR-strain should be greater than that in the TS-strain, it was deemed necessary to compare the metabolism of phenobarbital between the TS- and TR-strains. Comparison was made based on the concentration of phenobarbital in the hemolymph and whole body. The results showed no significant difference in phenobarbital treatment between the two strains used in this study suggesting the possibility that the induction system in TS-strain is different from the TR-strain.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

Identification of resistance-responsive proteins in larvae of Bactrocera dorsalis (Hendel), for pyrethroid toxicity by a proteomic approach

Insect resistance to the pyrethroid toxins has been examined previously using a number of traditional biochemical and molecular techniques. In this study, a proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and tandem mass spectrometry (MS/MS) were applied to examine changes in resistant stains larvae of Bactracera dorsalis Hendel induced by pyrethroid treatment over a 3 h, 6 h and 12 h time period, and a number of proteins changes were observed to change in the level of regulation. Out of total 15 proteins, 9 proteins were observed only after pyrethroid treatment; 6 proteins showed different expression. After MALDI-TOF analyses and peptide mapping method, the data was compared with those of the known proteins available in public databases. Sequence analyses revealed that resistance response correlates with up-regulation (glycerol-3-phosphate dehydrogenase) and down-regulation (ATP-ADP antiporter) of energy-related proteins. It indicated that increased metabolism and energy-indeed as a resistance response to pyrethroid toxins. The regulation of cytoskeleton proteins were possibly a B. dorsalis tissue repair response or in cell division. Up-regulation of protein synthesis would results in substantial bioenergetic enhancement, suggesting a trade-off insect resistance to pyrethroid. Down regulation of neural protein indicated that neural system was physically injured after pyrethroid stress. Some remaining proteins were not identifiable, suggesting these may be novel proteins. Oriental fruit fly proteomes of pesticide induced provide an integrative basis for consolidating our knowledge of insect resistance. The results pave the way for future investigation of the alteration of the insect resistance to chemical pesticides.     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

Cytochrome P450 mediated pyrethroid resistance in European populations of Meligethes aeneus (Coleoptera: Nitidulidae)

Pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae) is a major pest on several million hectares in European winter oilseed rape cultivation. Synthetic pyrethroids have been successfully used for many years to keep them under economic damage thresholds. Recently wide-spread resistance development to pyrethroids in pollen beetle populations was described in many European countries, including Germany, France, Poland, Denmark and others. Resistance monitoring is conducted by incubating beetles for 24 h in glass vials coated with different concentrations of lambda-cyhalothrin. Using such an assay format we were able to show cross-resistance to other pyrethroids, such as deltamethrin, cypermethrin, and to a somewhat lower extent bifenthrin, etofenprox and tau-fluvalinate. Here we also investigated in more detail in 27 different populations the biochemical mechanism of pyrethroid resistance. Synergism experiments revealed a high synergistic potential for piperonyl butoxide in vivo, whereas other compounds such as S,S,S-tributylphosphorotrithioate and diethylmaleimide failed to suppress pyrethroid resistance. Incubating microsomal fractions of pollen beetle with deltamethrin and subsequent LC–MS/MS analysis revealed 4-OH-deltamethrin as the major metabolite. Metabolite formation in vitro and pyrethroid resistance in vivo is correlated and inhibition trials with piperonyl butoxide, tebuconazole and aminobenzotriazole suggest the involvement of cytochrome P450′s. Furthermore we were able to show cross-resistance to tau-fluvalinate which is supported by the competitive inhibition of 4-OH-deltamethrin formation by increasing concentrations of tau-fluvalinate in microsomal hydroxylation assays. Although we provided clear experimental evidence for an oxidative mechanism of resistance in numerous populations, other mechanisms might be involved based on the data discussed.     

Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.     

Cytochrome P450 monooxygenase-mediated permethrin resistance confers limited and larval specific cross-resistance in the southern house mosquito, Culex pipiens quinquefasciatus

The cytochrome P450-dependent monooxygenases (P450s) are an important enzymatic system that metabolizes xenobiotics (e.g., pesticides), as well as endogenous compounds (e.g., hormones). P450-mediated metabolism can result in detoxification of insecticides such as pyrethroids, or can be involved in the bioactivation and detoxification of insecticides such as organophosphates. We isolated (from the JPAL strain) a permethrin resistant strain (ISOP450) of Culex pipiens quinquefasciatus, having 1300-fold permethrin resistance using standard backcrossing procedures. ISOP450 is highly related to the susceptible lab strain (SLAB) and the high resistance to permethrin is due solely to P450-mediated detoxification. This is the first time in mosquitoes that P450 monooxygenase involvement in pyrethroid resistance has been isolated and studied without the confounding effects of kdr. Resistance in ISOP450 is incompletely dominant (D = +0.3), autosomally linked, and monofactorally inherited. It is expressed in the larvae, but not in adults. Cross-resistance to pyrethroids lacking a 3-phenoxybenzyl moiety (tetramethrin, fenfluthrin, bioallethrin, and bifenthrin) ranged from 1.5- to 12-fold. ISOP450 had only limited (6.6- and 11-fold) cross-resistance to 3-phenoxybenzyl pyrethroids with an α-cyano group (cypermethrin and deltamethrin, respectively). Examination of cross-resistance patterns to organophosphate insecticides in ISOP450 showed an 8-fold resistance to fenitrothion, while low, but significant, levels of negative cross-resistance were found for malathion (RR = 0.84), temephos (RR = 0.73), and methyl-parathion (RR = 0.55). The importance and uniqueness of this P450 mechanism in insecticide resistance is discussed.     

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.)

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b₅ in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.     

Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.     

Potentiation between pyrethroid and organophosphate insecticides in resistant field populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan

The combined action of pyrethroids plus organophosphates was assessed on putatively resistant field populations of Helicoverpa armigera from Pakistan by using a leaf-dip bioassay. Ethion showed a good potentiation with bifenthrin, lambdacyhalothrin, cyfluthrin, betacyfluthrin, fenpropathrin, esfenvalerate, fluvalenate and tralomethrin. Profenofos was potentiating with bifenthrin but additive with lambdacyhalothrin. Methyl parathion also exhibited potentiation with bifenthrin. Contrarily, quinalphos produced an antagonism with bifenthrin. Chlorpyrifos potentiated lambdacyhalothrin in one population but had an additive effect in the other. A strong potentiation of pyrethroids by ethion in some populations indicates that esteratic detoxification is a key mechanism involved in imparting resistance to pyrethroids in Pakistani H. armigera.     

Spontaneous ultraweak photon emission from rice (Oryza sativa L.) and paddy weeds treated with a sulfonylurea herbicide

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emissions from organisms as a novel tool to investigate the physiological states of plants. In this study, we found ultraweak photon emissions from leaf segments of rice and several paddy weed species treated with a sulfonylurea herbicide. There is a definite difference in photon emissions among plant species, and rice (Oryza sativa), barnyardgrass (Echinochloa crus-galli) and Cyperus serotinus showed extremely strong enhancement of photon emissions. Photon emissions from these three species treated with sulfonylurea herbicide were suppressed when the leaf segments were treated with the cytochrome P450 monooxygenase (P450) inhibitors, piperonyl butoxide and malathion. These results suggest that P450 inhibitors affect the ultraweak photon emissions from plants.     

Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.