Impact of nitrogen fertilizer sustainability on corn crop yield: the role of beneficial microbial inoculation interactions.

BACKGROUND: Considering the challenges posed by nitrogen (N) pollution and its impact on food security and sustainability, it is crucial to develop management techniques that optimize N fertilization in croplands. Our research intended to explore the potential benefits of co-inoculation with Azospirillum brasilense and Bacillus subtilis combined with N application rates on corn plants. The study focused on evaluating corn photosynthesis-related parameters, oxidative stress assay, and physiological nutrient use parameters. Focus was placed on the eventual improved capacity of plants to recover N from applied fertilizers (AFR) and enhance N use efficiency (NUE) during photosynthesis. The two-year field trial involved four seed inoculation treatments (control, A. brasilense, B. subtilis, and A. brasilense + B. subtilis) and five N application rates (0 to 240 kg N ha-1, applied as side-dress). RESULTS: Our results suggested that the combined effects of microbial consortia and adequate N-application rates played a crucial role in N-recovery; enhanced NUE; increased N accumulation, leaf chlorophyll index (LCI), and shoot and root growth; consequently improving corn grain yield. The integration of inoculation and adequate N rates upregulated CO2 uptake and assimilation, transpiration, and water use efficiency, while downregulated oxidative stress. CONCLUSIONS: The results indicated that the optimum N application rate could be reduced from 240 to 175 kg N ha-1 while increasing corn yield by 5.2%. Furthermore, our findings suggest that replacing 240 by 175 kg N ha-1 of N fertilizer (-65 kg N ha-1) with microbial consortia would reduce CO2 emission by 682.5 kg CO2 -e ha-1. Excessive N application, mainly with the presence of beneficial bacteria, can disrupt N-balance in the plant, alter soil and bacteria levels, and ultimately affect plant growth and yield. Hence, highlighting the importance of adequate N management to maximize the benefits of inoculation in agriculture and to counteract N loss from agricultural systems intensification.

DIMBOA-induced gene expression, activity profiles of detoxification enzymes, multi-resistance mechanisms, and increased resistance to indoxacarb in tobacco cutworm, Spodoptera litura (Fabricius).

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is one of the most destructive insect pests owned strong resistance to different insecticides. Indoxacarb as a novel oxadiazine insecticide becomes the main pesticide against S. litura. DIMBOA [2,4-dihydroxy-7-methoxy-2 H-1,4-benz-oxazin-3(4 H)-one] is involved in important chemical defense processes in corn plants. However, the insects' adaptation mechanism to insecticides when exposed to defensive allelochemicals in their host plants remains unclear. Here, we assessed multi-resistance, and resistance mechanisms based on S. litura life history traits. After 18 generations of selection, indoxacarb resistance was increased by 61.95-fold (Ind-Sel) and 86.06-fold (Dim-Sel) as compared to the Lab-Sus. Also, DIMBOA-pretreated larvae developed high resistance to beta-cypermethrin, chlorpyrifos, phoxim, chlorantraniliprole, and emamectin benzoate. Meanwhile, indoxacarb (LC50) was applied to detect its impact on thirty-eight detoxification-related genes expression. The transcripts of SlituCOE073, SlituCOE009, SlituCOE074, and SlituCOE111 as well as SlGSTs5, SlGSTu1, and SlGSTe13 were considerably raised in the Ind-Sel strain. Among the twenty-three P450s, CYP6AE68, CYP321B1, CYP6B50, CYP9A39, CYP4L10, and CYP4S9v1 transcripts denoted significantly higher levels in the Ind-Sel strain, suggesting that CarEs, GSTs and P450s genes may be engaged in indoxacarb resistance. These outcomes further highlighted the importance of detoxification enzymes for S. litura gene expression and their role in responses to insecticides and pest management approaches.

Insecticidal and Synergistic Potential of Three Monoterpenoids against the Yellow Fever Mosquito, Aedes aegypti (Diptera: Culicidae), and the House Fly, Musca domestica (Diptera: Muscidae).

As resistance to the limited number of insecticides available for medical and veterinary pests becomes more widespread, there is an urgent need for new insecticides and synergists on the market. To address this need, we conducted a study to assess the toxicity of three monoterpenoids-carvone, menthone, and fenchone-in comparison to permethrin and methomyl against adults of two common pests: the yellow fever mosquito (Aedes aegypti) and the house fly (Musca domestica). We also examined the potential for these monoterpenoids to enhance the effectiveness of permethrin and methomyl when used together. Finally, we evaluated the ability of each monoterpenoid to inhibit acetylcholinesterase, comparing them to methomyl. While all three monoterpenoids performed relatively poorly as topical insecticides (LD50 > 4000 ng/mg on M. domestica; >6000 ng/mg on Ae. aegypti), they synergized both permethrin and methomyl as well as or better than piperonyl butoxide (PBO). Carvone and menthone yielded synergistic co-toxicity factors (23 and 29, respectively), which were each higher than PBO at 24 h. Currently, the mechanism of action is unknown. During preliminary testing, symptoms of acetylcholinesterase inhibition were identified, prompting further testing. Acetylcholinesterase inhibition did not appear to explain the toxic or synergistic effects of the three monoterpenoids, with IC50 values greater than 1 mM for all, compared to the 2.5 and 1.7 µM for methomyl on Aedes aegypti and Musca domestica, respectively. This study provides valuable monoterpenoid toxicity and synergism data on two pestiferous insects and highlights the potential for these chemistries in future pest control formulations.

Dual-Wavelength Fluorescence Polarization Immunoassay for Simultaneous Detection of Sulfonamides and Antibacterial Synergists in Milk.

Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC50 values for 23 SAs below 100 µg L-1 and 5 ASGs below 50 µg L-1. The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.

Resistance Mechanisms of Sitobion miscanthi (Hemiptera: Aphididae) to Malathion Revealed by Synergist Assay.

A resistant strain (MRS) of Sitobion miscanthi was cultured by continuous selection with malathion for over 40 generations. The MRS exhibited 32.7-fold resistance to malathion compared to the susceptible strain (MSS) and 13.5-fold, 2.9-fold and 4.8-fold cross-resistance for omethoate, methomyl and beta-cypermethrin, respectively. However, no cross-resistance was found to imidacloprid in this resistant strain. The realized heritability for malathion resistance was 0.02. Inhibitors of esterase activity, both triphenyl phosphate (TPP) and S,S,S,-tributyl phosphorotrithioate (DEF) as synergists, exhibited significant synergism to malathion in the MRS strain, with 11.77-fold and 5.12-fold synergistic ratios, respectively, while piperonyl butoxide (PBO) and diethyl maleate (DEM) showed no significant synergism in the MRS strain. The biochemical assay indicated that carboxylesterase activity was higher in MRS than in MSS. These results suggest that the increase in esterase activity might play an important role in S. miscanthi resistance to malathion. Imidacloprid could be used as an alternative for malathion in the management of wheat aphid resistance.

Insights into the Use of Eco-Friendly Synergists in Resistance Management of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the most notorious insect pest of potato globally. Injudicious use of insecticides for management of this pest has resulted in resistance to all major groups of insecticides along with many human, animal health, and environmental concerns. Additionally, the input cost of insecticide development/discovery is markedly increasing because each year thousands of chemicals are produced and tested for their insecticidal properties, requiring billions of dollars. For the management of resistance in insect pests, synergists can play a pivotal role by reducing the application dose of most insecticides. These eco-friendly synergists can be classified into two types: plant-based synergists and RNAi-based synergists. The use of plant-based and RNAi-based synergists in resistance management of insect pests can give promising results with lesser environmental side effects. This review summarizes the resistance status of CPB and discusses the potential advantage of plant-based and RNAi-based synergists for CPB resistance management. It will motivate researchers to further investigate the techniques of using plant- and RNAi-based synergists in combination with insecticides.

Synergistic property of piperonyl butoxide, diethyl maleate, triphenyl phosphate and verapamil hydrochloride with deltamethrin and ivermectin against Rhipicephalus microplus ticks.

The present study was taken up to evaluate the synergistic properties of piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP) and verapamil (VER) with deltamethrin (DLM) and ivermectin (IVM) against DLM and IVM resistant tick populations collected from Madhya Pradesh and Punjab states of India. The collected field tick populations were resistant to DLM (Resistance Factor [RF] in the range of 21.71-32.98) and IVM (RF in the range of 1.89-4.98). A strong synergism between DLM and, IVM with PBO and IVM with VER was noticed. The synergistic efficacy of PBO and VER with IVM in reducing the lethal concentration 50 (LC50) value (1.69-5.72 times for PBO and 3.00-10.62 times for VER) of IVM in resistant ticks suggest that a combination of these synergists with IVM can significantly enhance the effectiveness of IVM against IVM-resistant Rhipicephlaus microplus populations gradually establishing in the different parts of the country. The synergistic efficiency of PBO with DLM in reducing the LC50 value was 2.65 and 18.01 times, respectively, against DLM- resistant two R. microplus populations (KTN and LDH). The study revealed the gradual establishment of DLM and IVM resistant populations in the surveyed states suggesting the need to adopt required resistance management strategies. The use of synergists with DLM and IVM has emerged as an effective approach for controlling the acaricide-resistant ticks.

Patterns of Kdr-L995F Allele Emergence Alongside Detoxifying Enzymes Associated with Deltamethrin Resistance in Anopheles gambiae s.l. from North Cameroon.

Understanding how multiple insecticide resistance mechanisms occur in malaria vectors is essential for efficient vector control. This study aimed at assessing the evolution of metabolic mechanisms and Kdr L995F/S resistance alleles in Anopheles gambiae s.l. from North Cameroon, following long-lasting insecticidal nets (LLINs) distribution in 2011. Female An. gambiae s.l. emerging from larvae collected in Ouro-Housso/Kanadi, Be-Centre, and Bala in 2011 and 2015, were tested for susceptibility to deltamethrin + piperonyl butoxide (PBO) or SSS-tributyl-phosphoro-thrithioate (DEF) synergists, using the World Health Organization's standard protocol. The Kdr L995F/S alleles were genotyped using Hot Ligation Oligonucleotide Assay. Tested mosquitoes identified using PCR-RFLP were composed of An. arabiensis (68.5%), An. coluzzii (25.5%) and An. gambiae (6%) species. From 2011 to 2015, metabolic resistance increased in Ouro-Housso/Kanadi (up to 89.5% mortality to deltametnrin+synergists in 2015 versus <65% in 2011; p < 0.02), while it decreased in Be-Centre and Bala (>95% mortality in 2011 versus 42-94% in 2015; p < 0.001). Conversely, the Kdr L995F allelic frequencies slightly decreased in Ouro-Housso/Kanadi (from 50% to 46%, p > 0.9), while significantly increasing in Be-Centre and Bala (from 0-13% to 18-36%, p < 0.02). These data revealed two evolutionary trends of deltamethrin resistance mechanisms; non-pyrethroid vector control tools should supplement LLINs in North Cameroon.

Perspective: It is time to consider new ways to attack unpesticidable (undruggable) target sites by designing peptide pesticides.

Evolved resistance and regulatory deregistration have severely limited farmers' pesticide options. Many potential new pesticide target sites have been elucidated using targeted gene suppression and mutational tools, but few small molecules could be found that inhibit the target enzymes; the targets were considered 'undruggable'. Some organisms from all biological kingdoms use toxic peptides to ward off or kill enemies, and the agrochemical industry has used a few peptide analogs (glufosinate and bialophos) for field application. Conversely, pharmaceutical scientists have been using three-dimensional target protein structure to discover and synthesize short peptides that bind tightly to the surfaces of, and inhibit previously undruggable targets. New computational tools to quickly elucidate 3-D protein structure from amino acid sequence have just emerged. They replace crystallizing target proteins and performing X-ray crystallography to elucidate 3-D structure. These new tools allow prediction of peptides that will bind to the target proteins. They have further modified such peptides to enhance penetration, translocation and temperature stability. There is reason to assume that the same pioneering techniques can be used to develop peptide pesticides as well as pesticide synergists that act against undruggable targets and have excellent environmental and toxicological profiles. © 2022 Society of Chemical Industry.

Genetics and mechanism of resistance to chlorantraniliprole in Musca domestica L. (Diptera: Muscidae).

The house fly, Musca domestica L. is an important mechanical vector of different pathogens of medical and veterinary importance. It is an organism well-known for its ability to develop insecticide resistance. In the current study, we investigated the genetic basis and mechanism of chlorantraniliprole resistance in a field strain of house fly by selecting it artificially in the laboratory with a commercial formulation of chlorantraniliprole (CTPR-SEL). After seven generations of consecutive selection with chlorantraniliprole, CTPR-SEL strain developed a 644-fold resistance compared with the Susceptible strain and a 3-fold resistance compared with the field strain. Reciprocal crossing between the CTPR-SEL and Susceptible homozygous strains revealed an autosomal and incomplete dominant mode of resistance to chlorantraniliprole. A direct test using a monogenic inheritance model based on chi-square analysis revealed that the resistance was governed by more than one gene. Bioassays with synergists indicated that esterases might be involved in the resistance of house fly to chlorantraniliprole. These findings may be helpful to the development of an improved strategy for chlorantraniliprole resistance management in house fly.

Potential metabolic resistance mechanisms to ivermectin in Anopheles gambiae: a synergist bioassay study.

BACKGROUND: Despite remarkable success obtained with current malaria vector control strategies in the last 15 years, additional innovative measures will be needed to achieve the ambitious goals for malaria control set for 2030 by the World Health Organization (WHO). New tools will need to address insecticide resistance and residual transmission as key challenges. Endectocides such as ivermectin are drugs that kill mosquitoes which feed on treated subjects. Mass administration of ivermectin can effectively target outdoor and early biting vectors, complementing the still effective conventional tools. Although this approach has garnered attention, development of ivermectin resistance is a potential pitfall. Herein, we evaluate the potential role of xenobiotic pumps and cytochrome P450 enzymes in protecting mosquitoes against ivermectin by active efflux and metabolic detoxification, respectively. METHODS: We determined the lethal concentration 50 for ivermectin in colonized Anopheles gambiae; then we used chemical inhibitors and inducers of xenobiotic pumps and cytochrome P450 enzymes in combination with ivermectin to probe the mechanism of ivermectin detoxification. RESULTS: Dual inhibition of xenobiotic pumps and cytochromes was found to have a synergistic effect with ivermectin, greatly increasing mosquito mortality. Inhibition of xenobiotic pumps alone had no effect on ivermectin-induced mortality. Induction of xenobiotic pumps and cytochromes may confer partial protection from ivermectin. CONCLUSION: There is a clear pathway for development of ivermectin resistance in malaria vectors. Detoxification mechanisms mediated by cytochrome P450 enzymes are more important than xenobiotic pumps in protecting mosquitoes against ivermectin.

Inheritance, stability, cross-resistance, and life history parameters of a clothianidin-selected strain of house fly, Musca domestica Linnaeus.

The house fly, Musca domestica L., is a cosmopolitan insect pest of public and animal health importance that serves as a mechanical vector of pathogens. Aimed at prospective resistance management to reduce environmental pollution, we characterized the inheritance pattern, realized heritability, fitness cost, cross resistance, stability and mechanism of clothianidin resistance in M. domestica that were collected from the poultry farm. By continuous selection with clothianidin for 11 generations, the clothianidin selected M. domestica strain (Clotha-SEL) developed a 3827-fold resistance compared to a susceptible strain. However, resistance to clothianidin was proved to be unstable when selection with clothianidin was removed for five generations (G7 to G12). Inheritance pattern analysis at G8 of Clotha-SEL (RR = 897) revealed that resistance to clothianidin was polygenic, autosomal and incompletely dominant. Realized heritability (h2) for resistance value was 0.38 (at G11) in the tested strain. Synergist bioassays showed that microsomal oxidases and esterases might not contribute significantly in resistance evolution. Fitness costs of clothianidin resistance were present, for example, reduction in growth potential of the Clotha-SEL strain in comparison to the untreated counterpart strain (UNSEL) was observed. No cross resistance to bifenthrin and fipronil and a very low cross-resistance to spinosad were observed. These insecticides could be alternated with clothianidin as an insecticide resistance management tool to sustain its efficacy for a longer time period. These results shall be utilized to devise a proactive resistance management strategy for use of clothianidin against M. domestica that will be helpful to alleviate the allied threats to environmental and human health.

20-Hydroxyecdysone (20E) signaling as a promising target for the chemical control of malaria vectors.

With the rapid development and spread of resistance to insecticides among anopheline malaria vectors, the efficacy of current World Health Organization (WHO)-approved insecticides targeting these vectors is under threat. This has led to the development of novel interventions, including improved and enhanced insecticide formulations with new targets or synergists or with added sterilants and/or antimalarials, among others. To date, several studies in mosquitoes have revealed that the 20-hydroxyecdysone (20E) signaling pathway regulates both vector abundance and competence, two parameters that influence malaria transmission. Therefore, insecticides which target 20E signaling (e.g. methoxyfenozide and halofenozide) may be an asset for malaria vector control. While such insecticides are already commercially available for lepidopteran and coleopteran pests, they still need to be approved by the WHO for malaria vector control programs. Until recently, chemicals targeting 20E signaling were considered to be insect growth regulators, and their effect was mostly studied against immature mosquito stages. However, in the last few years, promising results have been obtained by applying methoxyfenozide or halofenozide (two compounds that boost 20E signaling) to Anopheles populations at different phases of their life-cycle. In addition, preliminary studies suggest that methoxyfenozide resistance is unstable, causing the insects substantial fitness costs, thereby potentially circumventing one of the biggest challenges faced by current vector control efforts. In this review, we first describe the 20E signaling pathway in mosquitoes and then summarize the mechanisms whereby 20E signaling regulates the physiological processes associated with vector competence and vector abundance. Finally, we discuss the potential of using chemicals targeting 20E signaling to control malaria vectors.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification.

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Bacteria-mediated modification of insecticide toxicity in the yellow fever mosquito, Aedes aegypti.

The incidence of mosquito-borne disease poses a significant threat to human and animal health throughout the world, with effective chemical control interventions limited by widespread insecticide resistance. Recent evidence suggests that gut bacteria of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may also play a significant role in facilitating insecticide resistance. This study investigated the extent to which bacteria contribute to the general esterase and cytochrome P450 monooxygenase (P450)-mediated detoxification of the insecticides propoxur and naled, as well as the insecticidal activity of these chemistries to the yellow fever mosquito, Aedes aegypti. Experiments conducted using insecticide synergists that reduce general esterase and P450 activity demonstrate a role for both groups of enzymes in the metabolic detoxification of propoxur and naled. Furthermore, reduction of bacteria in mosquito larvae using broad-spectrum antibiotics was found to decrease the metabolic detoxification of propoxur and naled, suggesting that the bacteria themselves may be contributing to the in vivo metabolic detoxification of these insecticides. This was supported by in vitro assays using culturable gut bacteria isolated from mosquito larvae which demonstrated that the bacteria were capable of reducing insecticide toxicity. More work is needed, however, to fully elucidate the contribution of bacteria in Ae. aegypti larvae to the metabolic detoxification of insecticides.

Integrated Management of Chive Gnats (Bradysia odoriphaga Yang & Zhang) in Chives Using Entomopathogenic Nematodes and Low-Toxicity Insecticides.

Bradysia odoriphaga is a major pest that causes damage to chive production, and which has developed resistance to highly toxic chemical insecticides. Entomopathogenic nematodes (EPN) show a high potential for B. odoriphaga control. This study aimed to develop an effective management method against B. odoriphaga larvae, using EPN with low-toxicity insecticides. Fourteen selected insecticides had no significant effects on the survival and infectivity of Steinernema feltiae SN and Heterorhabditis indica LN2. Synergistic interactions were observed for imidacloprid and osthole with S. feltiae SN against B. odoriphaga larvae. Steinernema feltiae SN was more effective than H. indica LN2 against B. odoriphaga at 15 and 20 °C, and the addition of imidacloprid at 1/10 recommended concentration (RC) significantly increased the efficacy of S. feltiae SN. The year-round occurrence of the B. odoriphaga larvae in chive fields treated by EPN and imidacloprid at 1/10 RC was studied. Results showed that the application of EPN with imidacloprid at 1/10 RC successfully suppressed larval populations of B. odoriphaga in chive fields, thus significantly increasing the yield of chive. The practical method of applying EPN-imidacloprid combinations provided a cost-effective and environmental safety strategy for controlling B. odoriphaga larvae in chive production, which can reduce the usage of toxic chemical insecticides.

Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

BACKGROUND: Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor. METHODS: Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations. RESULTS: Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids. CONCLUSIONS: In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.

Resistance to insecticides and synergistic and antagonistic effects of essential oils on dimefluthrin toxicity in a field population of Culex quinquefasciatus Say.

In this work, we firstly tested five spatial repellent pyrethroids, meperfluthrin, dimefluthrin, heptafluthrin, metofluthrin and transfluthrin, to determine the susceptibility of pyrethroids to field strains of Culex quinquefasciatus using adult topical bioassay. The results showed that though field strains exhibited the highest resistance to dimefluthrin among the selected five pyrethroids, it still can be considered low resistance in the scale of Cui et al. (2006; 2007). Then, the aim of this study was to optimise the synergistic efficacy of essential oils combined with dimefluthrin and explore the major contribution composition of eucalyptus oil, basil oil and cinnamon oil as natural synergist of dimefluthrin against the field populations of C. quinquefasciatus. GC-MS analysis showed 1,8-cineole, eugenol and trans-cinnamaldehyde were the main chemical components of eucalyptus oil, basil oil and cinnamon oil, respectively. The results of bioactivity showed that eucalyptus oil and 1,8-cineole have highly fumigant knock-down activity to the adults, showing KT50 (the median knockdown time) of 5.76 and 4.27 min at the concentration of 24.2 µL/L; basil oil and eugenol, cinnamon oil and trans-cinnamaldehyde have highly fumigant toxicity to the adults, showing LD50 of 1.00 and 0.79, 1.26 and 1.03 µL/L, respectively. Three effective main essential oil components were selected to prepare binary mixtures, which combined with dimefluthrin against the field population of Culex quinquefasciatus. 1,8-cineole+eugenol (9:1, w/w), 1,8-cineole+trans-cinnamaldehyde (1:1, w/w) and trans-cinnamaldehyde+eugenol (9:1, w/w) combined with dimefluthrin (10:1, w/w) were the most synergistic interaction, showed SR (synergistic ratio) values of 1.2471, 1.5709 and 1.1969; KT50 of 11.68, 9.51 and 10.67 min respectively, by quadrate box method. In addition, to validate the stable synergistic interaction of 1,8-cineole+trans-cinnamaldehyde (1:1, w/w) combined with dimefluthrin (10:1, w/w), the SR values were about 1.3, and KT50 values were 38.72-50.26 min by simulated house method. Overall, our results pointed out the promising potential of these essential oils to increase the efficacy of dimefluthrin. It might be expected that these essential oils could be developed to a useful botanical synergist of dimefluthrin for the control of the field populations of C. quinquefasciatus.

Broad-spectrum monoclonal antibody and a sensitive multi-residue indirect competitive enzyme-linked immunosorbent assay for the antibacterial synergists in samples of animal origin.

To monitor the abuse of antibacterial synergists, a hapten, trimethoprim carboxylic derivative (TMPCOOH), was designed by using molecular modelling technology. A broad-spectrum monoclonal antibody (mAb) TMP/2G1 was prepared, for which the IC50 values of trimethoprim, diaveridine, aditoprim, baquiloprim, ormetoprim, and brodimoprim were 0.232, 0.527, 1.479, 4.354, 0.965, and 0.119 µg L-1, respectively. Based on the broad spectrum mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to determine the residues of antibacterial synergists. The limit of detection regarding the developed ic-ELISA for antibacterial synergists ranged from 0.025 to 1.126 µg L-1 in milk, honey and edible animal tissues. The recoveries ranged from 81.4% to 107.7%, with a coefficient of variation less than 20%. A good correlation (R2 = 0.994) between the ic-ELISA and HPLC-MS/MS showed the reliability of the developed ic-ELISA.

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra.

The possible involvement of the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra in the biodegradation of the insecticide chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP), in pure cultures and in plant surfaces (tomato fruits) was investigated. Higher biodegradation rates were observed as the concentration of chlorpyrifos and the inoculum of the microorganisms were increased, while the yeasts proved to be more active at 25 and 15 °C. The presence of glucose in the mineral nutrient medium, as an extra source of carbon, delayed the biodegradation by Rhodotorula glutinis, while Rhodotorula rubra proved to be more active. The detection and quantification of the parent compound and TCP was successfully achieved using a LC/MS/MS chromatographic system. The in vitro enzymatic assays applied suggested that esterases may be involved in the biodegradation of chlorpyrifos, a fact that was further enhanced after the addition of the synergists triphenyl phosphate, diethyl maleate and piperonyl butoxide in the biodegradation trials. The decrease of chlorpyrifos residues on tomato fruits confirmed the corresponding on pure cultures, resulting in the suggestion that the yeasts R. glutinis and R. rubra can possibly be used successfully for the removal or detoxification of chlorpyrifos residues on tomatoes.