Harnessing the potential of microalgae-based systems for mitigating pesticide pollution and its impact on their metabolism.

Due to increased pesticide usage in agriculture, a significant concentration of pesticides is reported in the environment that can directly impact humans, aquatic flora, and fauna. Utilizing microalgae-based systems for pesticide removal is becoming more popular because of their environmentally friendly nature, ability to degrade pesticide molecules into simpler, nontoxic molecules, and cost-effectiveness of the technology. Thus, this review focused on the efficiency, mechanisms, and factors governing pesticide removal using microalgae-based systems and their effect on microalgal metabolism. A wide range of pesticides, like atrazine, cypermethrin, malathion, trichlorfon, thiacloprid, etc., can be effectively removed by different microalgal strains. Some species of Chlorella, Chlamydomonas, Scenedesmus, Nostoc, etc., are documented for >90% removal of different pesticides, mainly through the biodegradation mechanism. The antioxidant enzymes such as ascorbate peroxidase, superoxide dismutase, and catalase, as well as the complex structure of microalgae cell walls, are mainly involved in eliminating pesticides and are also crucial for the defense mechanism of microalgae against reactive oxygen species. However, higher pesticide concentrations may alter the biochemical composition and gene expression associated with microalgal growth and metabolism, which may vary depending on the type of strain, the pesticide type, and the concentration. The final section of this review discussed the challenges and prospects of how microalgae can become a successful tool to remediate pesticides.

Odorant Binding Protein Expressed in Legs Enhances Malathion Tolerance in Bactrocera dorsalis (Hendel).

Bactrocera dorsalis is a highly invasive species and is one of the most destructive agricultural pests worldwide. Organophosphorus insecticides have been widely and chronically used to control it, leading to the escalating development of resistance. Recently, odorant binding proteins (OBPs) have been found to play a role in reducing insecticide susceptibility. In this study, we used RT-qPCR to measure the expression levels of four highly expressed OBP genes in the legs of B. dorsalis at different developmental stages and observed the effect of malathion exposure on their expression patterns. The results showed that OBP28a-2 had a high expression level in 5 day old adults of B. dorsalis, and its expression increased after exposure to malathion. By CRISPR/Cas9 mutagenesis, we generated OBP28a-2-/- null mutants and found that they were more susceptible to malathion than wild-type adults. Furthermore, in vitro direct affinity assays confirmed that OBP28a-2 has a strong affinity for malathion, suggesting that it plays a role in reducing the susceptibility of B. dorsalis to malathion. Our findings enriched our understanding of the function of OBPs. The results highlighted the potential role of OBPs as buffering proteins that help insects survive exposure to insecticides.

Fine-scale monitoring of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Sri Lanka and modeling the phenotypic resistance using rational approximation.

BACKGROUND: The unplanned and intensified use of insecticides to control mosquito-borne diseases has led to an upsurge of resistance to commonly used insecticides. Aedes aegypti, the main vector of dengue, chikungunya, and Zika virus, is primarily controlled through the application of adulticides (pyrethroid insecticides) and larvicides (temephos). Fine spatial-scale analysis of resistance may reveal important resistance-related patterns, and the application of mathematical models to determine the phenotypic resistance status lessens the cost and usage of resources, thus resulting in an enhanced and successful control program. METHODS: The phenotypic resistance for permethrin, deltamethrin, and malathion was monitored in the Ae. aegypti populations using the World Health Organization (WHO) adult bioassay method. Mosquitoes' resistance to permethrin and deltamethrin was evaluated for the commonly occurring base substitutions in the voltage-gated sodium channel (vgsc) gene. Rational functions were used to determine the relationship between the kdr alleles and the phenotypic resistant percentage of Ae. aegypti in Sri Lanka. RESULTS: The results of the bioassays revealed highly resistant Ae. aegypti populations for the two pyrethroid insecticides (permethrin and deltamethrin) tested. All populations were susceptible to 5% malathion insecticide. The study also revealed high frequencies of C1534 and G1016 in all the populations studied. The highest haplotype frequency was detected for the haplotype CC/VV, followed by FC/VV and CC/VG. Of the seven models obtained, this study suggests the prediction models using rational approximation considering the C allele frequencies and the total of C, G, and P allele frequencies and phenotypic resistance as the best fits for the area concerned. CONCLUSIONS: This is the first study to our knowledge to provide a model to predict phenotypic resistance using rational functions considering kdr alleles. The flexible nature of the rational functions has revealed the most suitable association among them. Thus, a general evaluation of kdr alleles prior to insecticide applications would unveil the phenotypic resistance percentage of the wild mosquito population. A site-specific strategy is recommended for monitoring resistance with a mathematical approach and management of insecticide applications for the vector population.

Quantifying resistance to very-long-chain fatty acid-inhibiting herbicides in Amaranthus tuberculatus using a soilless assay.

Resistance to preemergence (PRE) soil-applied herbicides, such as inhibitors of very-long-chain fatty acid (VLCFA) elongases, was documented in two waterhemp [Amaranthus tuberculatus (Moq.) J.D. Sauer] populations (SIR and CHR) from Illinois, USA. To limit the spread of resistant weed populations, rapid detection measures are necessary. Soil-based resistance assays are limited by edaphic factors, application timing, variable seeding depth and rainfall amount. Therefore, cost-effective techniques mitigating effects of edaphic factors that are appropriate for small- to large-scale assays are needed. Our research goal was to identify and quantify resistance to the VLCFA-inhibiting herbicides, S-metolachlor and pyroxasulfone, using a soilless greenhouse assay. Dose-response experiments were conducted under greenhouse conditions with pre-germinated waterhemp seeds planted on the vermiculite surface, which had been saturated with S-metolachlor (0.015-15 µM), pyroxasulfone (0.0005-1.5 µM), or S-metolachlor plus the cytochrome P450 (P450) inhibitor, malathion. Lethal dose estimates of 50% (LD50) and growth reduction of 50% (GR50) were calculated for S-metolachlor and pyroxasulfone PRE and used to determine resistance indices (RI) for resistant populations (CHR and SIR) relative to sensitive populations, SEN and ACR. RI values for S-metolachlor using LD50 values calculated relative to SEN and ACR were 17.2 and 15.2 (CHR) or 11.5 and 10.1 (SIR), while RI values for pyroxasulfone using LD50 values calculated relative to SEN and ACR were 3.8 and 3.1 (CHR) or 4.8 and 3.8 (SIR). Malathion decreased the GR50 of S-metolachlor to a greater degree in CHR compared to ACR, consistent with P450 involvement in S-metolachlor resistance in CHR. Results from these soilless assays are in accord with previous findings in soil-based systems that demonstrate CHR and SIR are resistant to S-metolachlor and pyroxasulfone. This method provides an effective, reproducible alternative to soil-based systems for studying suspected PRE herbicide-resistant populations and will potentially assist in identifying non-target-site resistance mechanisms.

Susceptibility status of Aedes aegypti (Diptera: Culicidae) against insecticides of public health use in Delhi and NCR region, India.

BACKGROUND & OBJECTIVES: Aedes (Stegomyia) aegypti is a primary vector responsible for the transmission of various arboviral diseases in India. Without an effective drug or vaccine against these diseases, chemical insecticide-based vector control supplemented with source reduction remains the best option for their effective management. The development of insecticide resistance due to the continuous use of insecticides might affect the control operations. METHODS: Adults and larvae of Aedes aegypti were collected from different localities in Delhi. Larvae were exposed to discriminating (0.02mg/l) and application (1mg/l) doses of temephos. WHO tube assay was conducted for F1 adults using impregnated insecticide papers of dichlorodiphenyltrichloroethane (DDT), malathion, deltamethrin, permethrin, cyfluthrin, and lambda-cyhalothrin. RESULTS: Larvae of Ae. aegypti were found resistant (76.0%) to the discriminating dose of temephos, whereas suscep-tible (100.0%) to the application dose of the temephos. Adult Aedes (Fl) mosquitoes were resistant to DDT (23.7%), malathion (90.5%), deltamethrin (76.0%), permethrin (96.2 %) cyfluthrin (85.5%), and lambda-cyhalothrin (94.0%). INTERPRETATION & CONCLUSION: Indoor residual spray is not used in Delhi for vector control. Resistance in Aedes might be due to pesticide usage for agricultural activities in peripheral regions of Delhi. There is a need to investigate more on the insecticide resistance mechanisms for indirect resistance development. Understanding the insecticide susceptibility status of urban vectors is critical for planning effective control strategies.

Insecticide resistance: Status and potential mechanisms in Aedes aegypti.

Aedes aegypti, an important vector in the transmission of human diseases, has developed resistance to two commonly used classes of insecticides, pyrethroids and organophosphates, in populations worldwide. This study examined sensitivity/resistance to chlorpyrifos, fenitrothion, malathion, deltamethrin, permethrin, and ß-cyfluthrin, along with possible metabolic detoxification and target site insensitivity, in three Aedes aegypti mosquito strains. The resistant strain (PR) had developed high levels of resistance to all three pyrethroid insecticides compared to a susceptible population, with 6, 500-, 3200- and 17,000-fold resistance to permethrin, ß-cyfluthrin, and deltamethrin, respectively. A newly emerged Ae. aegypti population collected from St. Augustine, Florida (AeStA) showed elevated levels of resistance to malathion (12-fold) and permethrin (25-fold). Synergists DEF (S,S,S,-tributyl phosphorotrithioate) and DEM (diethyl maleate) showed no or minor effects on insecticide resistance in both the AeStA and PRG20strains, but PBO (piperonyl butoxide) completely abolished resistance to both malathion and permethrin in AeStA and partially suppressed resistance in PR. The voltage-gated sodium channel sequences were examined to explore the mechanism that only partially inhibited the suppression of resistance to PBO in PR. Two mutations, V1016G/I and F1534C substitutions, both of which are associated with the development of pyrethroid resistance, were identified in the PRG20 strain but not in AeStA. These results suggest that while cytochrome P450 mediated detoxification may not be solely responsible, it is the major mechanism governing the development of resistance in AeStA. Both P450 mediated detoxification and target site insensitivity through the mutations in the voltage-gated sodium channel contribute to the high levels of resistance in the PRG20 strain.

Cuticular competing endogenous RNAs regulate insecticide penetration and resistance in a major agricultural pest.

BACKGROUND: The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. RESULTS: Here, we find the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. Our investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. Our analyses find an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, we elucidate the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. CONCLUSIONS: We unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.

Learning perturbation-inducible cell states from observability analysis of transcriptome dynamics.

A major challenge in biotechnology and biomanufacturing is the identification of a set of biomarkers for perturbations and metabolites of interest. Here, we develop a data-driven, transcriptome-wide approach to rank perturbation-inducible genes from time-series RNA sequencing data for the discovery of analyte-responsive promoters. This provides a set of biomarkers that act as a proxy for the transcriptional state referred to as cell state. We construct low-dimensional models of gene expression dynamics and rank genes by their ability to capture the perturbation-specific cell state using a novel observability analysis. Using this ranking, we extract 15 analyte-responsive promoters for the organophosphate malathion in the underutilized host organism Pseudomonas fluorescens SBW25. We develop synthetic genetic reporters from each analyte-responsive promoter and characterize their response to malathion. Furthermore, we enhance malathion reporting through the aggregation of the response of individual reporters with a synthetic consortium approach, and we exemplify the library's ability to be useful outside the lab by detecting malathion in the environment. The engineered host cell, a living malathion sensor, can be optimized for use in environmental diagnostics while the developed machine learning tool can be applied to discover perturbation-inducible gene expression systems in the compendium of host organisms.

Identification, expression profiles and involvement in insecticides tolerance and detoxification of carboxylesterase genes in Bactrocera dorsalis.

Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis. Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α-esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes. With the exception of BdCarE21, all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis.

Mutation at the 197 site and P450-mediated metabolic resistance are involved in bensulfuron-methyl resistance in Sagittaria trifolia.

Sagittaria trifolia control is threatened by the emergence of resistance to acetolactate synthase (ALS)-inhibiting herbicides. Hence, we systematically uncovered the molecular mechanism of resistance to the main herbicide (bensulfuron-methyl) in Liaoning Province from target-site and non-target-site resistance perspectives. The suspected resistant population (TR-1) exhibited high-level resistance. A new amino acid substitution (Pro-197-Ala) in resistant Sagittaria trifolia for ALS was detected, and the molecular docking results showed that the spatial structure of ALS changed significantly after the substitution, manifested by an increase in the number of contacted amino acid residues and the disappearance of hydrogen bonds. Dose-response test of transgenic Arabidopsis thaliana further demonstrated that the Pro-197-Ala substitution conferred bensulfuron-methyl resistance. The assays found that the sensitivity of the ALS enzyme in TR-1 to this herbicide was decreased in vitro; and this population had developed resistance to other types of ALS-inhibiting herbicides. Furthermore, the resistance of TR-1 to bensulfuron-methyl was significantly alleviated after co-treatment with a P450-inhibitor (malathion). TR-1 metabolized bensulfuron-methyl significantly faster than sensitive population (TS-1) did, but this gap was narrowed after malathion treatment. Overall, the resistance of Sagittaria trifolia to bensulfuron-methyl was derived from the mutation of the target-site gene and the enhancement of the P450s-mediated detoxification metabolism.

Metabolic Exploration for Cyhalofop-Butyl Antagonism in Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] Following Pretreatment of Malathion.

The present study investigated the effects of broad-spectrum metabolic inhibitors malathion (cytochrome P450 inhibitor) and/or 4-chloro-7-nitrobenzofurazan (NBD-Cl; glutathione S-transferase inhibitor) on the metabolism of cyhalofop-butyl (CyB) in barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes confirmed previously with multiple resistance to two herbicides CyB and florpyrauxifen-benzyl. The metabolic inhibitors were not effective at recovering the sensitivity of resistant barnyardgrass biotypes to CyB treated at the labeled rate (313 g ai ha-1). Rather, treatment with malathion followed by CyB caused antagonism, reducing the efficacy of CyB and promoting the growth of resistant biotypes. Pretreatment with malathion did not influence absorption/translocation of the applied form CyB and its conversion to the active herbicide form cyhalofop-acid (CyA), in both susceptible and resistant biotypes. In contrast, metabolism of the applied form (CyB) decreased 1.5 to 10.5 times by the malathion pretreatment. Taken together, the maintained CyA production against the reduced CyB metabolism could be the mechanism to account for the cause of CyB antagonism observed in barnyardgrass following malathion pretreatment. Additionally, the evolution of CyB resistance in barnyardgrass might be associated with reduced production of CyA in resistant biotypes, independent of activities of cytochrome P450 or GST enzymes.

Lethal and sublethal effects of toxic bait formulations on Doryctobracon areolatus (Hymenoptera: Braconidae) and implications for integrated fruit fly management.

The use of toxic baits has become one of the main methods of management of fruit flies in Brazil. The application of toxic baits may cause side effects on the native parasitoid Doryctobracon areolatus (Hymenoptera: Braconidae). Based on the results, formulations made from the food attractants 3% Biofruit, 1.5% Ceratrap, 1.25% Flyral, 3% Isca Samaritá, 3% Isca Samaritá Tradicional, and 7% sugarcane molasses associated with the Malathion 1000 EC and the ready-to-use toxic bait Gelsura (containing the active ingredient alpha-cypermethrin) were classified as harmful (class 4) to D. areolatus (mortality > 85% at 96 HAE). In contrast, for toxic baits formulated with insecticide phosmet, the mortality ranged from 38% to 72%, classified as slightly harmful or moderately harmful. However, when phosmet was added to the 3% Samaritá Tradicional bait, the mortality was only 3.9% (class 1-harmless), similar to the toxicity observed for the Success 0.02 CB ready-to-use bait (0.24 g a.i. spinosad/l) (<5% mortality). Although toxic baits were formulated with spinosyn-based insecticides, all toxic bait formulations were classified as harmless or slightly harmful (<50% mortality) to D. areolatus, with the exception of 1.5% Ceratrap + spinetoram and 7% Sugarcane molasses + spinosad (≈ 60% mortality-moderately harmful). In addition, these formulations did not show sublethal effects in reducing the parasitism and emergence rate of the F1 generation of D. areolatus in A. fraterculus larvae. The results serve as a basis for the correct use of toxic food baits without affecting the biological control.

Species composition and insecticide resistance in malaria vectors in Ellibou, southern Côte d'Ivoire and first finding of Anopheles arabiensis in Côte d'Ivoire.

BACKGROUND: Knowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d'Ivoire. METHODS: A 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR). RESULTS: Overall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2. CONCLUSION: This is the first documented presence of An. arabiensis in Côte d'Ivoire. Its detection - together with a recent finding further north of the country - confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

Potential key genes involved in metabolic resistance to malathion in the southern house mosquito, Culex quinquefasciatus, and functional validation of CYP325BC1 and CYP9M12 as candidate genes using RNA interference.

BACKGROUND: Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing. RESULTS: We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion. CONCLUSION: We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

Current enzyme-mediated insecticide resistance status of Aedes aegypti populations from a dengue-endemic city in southern Mexico.

OBJECTIVE: To identify the enzyme-mediated insecticide resistance in Aedes aegypti in Tapachula, Mexico. MATERIALS AND METHODS: Biochemical assays were undertaken to determine the enzyme levels in mosquitoes from 22 sites collected in 2018 and 2020 in Tapachula. Results of 2018 were correlated with the resistance to insecticides pub-lished. RESULTS: Mosquitoes had higher levels than those of the susceptible strain in 2018 and 2020 respectively of α-esterases in 15 and 12 sites; ß-esterases in 7 and 6 sites; glutathione-S-transferases in 11 and 19 sites; ρNPA-esterases in 21 and 17 sites; and cytochromes P450 in 20 and 22 sites. In mosquitoes of 2018, there was a moderate correlation between previously documented Malathion resistance ratios and the insensitive acetylcholinesterase (r=0.459, p= 0.03). CONCLUSIONS: The elevated enzyme levels found indicate its contribution to the resistance to pyrethroids and organo-phosphates already published in mosquitoes from Tapachula. Bioassays using enzyme inhibitors resulted in greater mor-tality, confirming that metabolism contributes to resistance.

Response to insecticides and underlying mechanisms of resistance in the field populations of Aedes aegypti Linnaeus (Diptera: Culicidae) in Puducherry, India.

Background & objectives: Mosquito-borne diseases are major threats to human health worldwide. Successful control of vector mosquitoes requires periodic updates on their response to the insecticides that are in use. Different classes of neurotoxic insecticides have been used in vector control programs. Ae. aegypti and Ae. albopictus are the primary vectors of dengue and have developed resistance to organophosphates and synthetic pyrethroids that are used in vector control programs. Monitoring insecticide pressure and studying the underlying mechanisms of resistance in the field populations of Aedes aegypti are important to formulate resistant management strategies for their control programs. Methods: Aedes aegypti were collected from study sites Lawspet and Abishegapakkam and F1 progeny was subject to biochemical assays to determine the enzyme activity. Insecticide susceptibility tests were conducted to determine vector susceptibility/resistance to malathion and deltamethrin. Adult dried mosquitoes were subjected to multiplex PCR to detect point mutation in the VGSC gene. Results: Insecticide susceptibility test results revealed that Aedes aegypti is resistant to malathion and incipient resistance to deltamethrin has emerged. It was observed that ß-esterase and monoxygense activity were significantly higher in Lawspet sample than the laboratory strain, whereas it was comparatively lower in Abishegapakkam sample than laboratory strain. Multiplex PCR assays showed no kdr mutation in all Ae. aegypti strains. Interpretation & conclusion: Monitoring insecticide resistance in Ae. aegypti would help the local health authorities to implement a rationalized approach for insecticide use in vector control.

Insecticide susceptibility status of malaria vectors in Himachal Pradesh, India.

BACKGROUND & OBJECTIVES: The state of Himachal Pradesh is one of the hilly forested states of India. Warming of climate has been evidenced in the state due to the ongoing climate change which may cause the upsurge/introduction of mosquito-borne diseases. To curb disease transmission, an effective vector control strategy will be required. METHODS: Insecticide susceptibility status of available malaria vectors was determined using the standard WHO method in six districts Kangra, Una, Mandi, Bilaspur, Solan and Mandi of the state. An. culicifacies and An. fluviatilis were tested against DDT (4%), malathion (5%) and deltamethrin (0.05%) using WHO insecticide susceptibility kits. RESULTS: Overall, An. culicifacies was found resistant to DDT in all the six districts, susceptible to malathion in all districts except Bilaspur and Solan where it showed possible resistance. It was susceptible to deltamethrin in all the study districts. An. fluviatilis was resistant to DDT and susceptible to malathion and deltamethrin in Kangra and Una districts. INTERPRETATION & CONCLUSION: At present, indoor residual spraying (IRS) is not being undertaken in Himachal Pradesh. However, with the information generated through the present study, the state government can plan evidence-based IRS at least for focal spray in limited foci reporting malaria incidence.

A note on the insecticide susceptibility status of secondary malaria vector An. annularis in Jharkhand state of India.

BACKGROUND & OBJECTIVES: An. annularis van der Wulp (1884) is the secondary malaria vector of importance in India. In Jharkhand state it is present in almost all the districts abundantly and transmits malaria. The development of resistance to Dichlorodipheny ltrichloroethane (DDT) in An. annularis was reported from various parts of India. The main objective of this study was to generate information on insecticide susceptibility status of An. annularis to DDT, malathion, deltamethrin and permethrin in different districts of Jharkhand state. Methods; Adult An. annularis female mosquitoes were collected form villages of six tribal districts Simdega (Kurdeg and Simdega CHC), Khunti (Murhu and Khunti CHCs), Gumla (Bharno and Gumla CHCs), West Singhbhum (Chaibasa and Bada Jamda CHCs), Godda (Poraiyahat and Sunderpahari (CHCs) and Sahibganj (Borio and Rajmahal CHCs). Insecticide susceptibility status was determined by using WHO tube test method against prescribed discriminatory dosages of insecticides, DDT - 4.0%, malathion - 5.0%, deltamethrin - 0.05% and permethrin - 0.75%. RESULTS: An. annularis was reported resistant to DDT in six districts, possible resistant to malathion in districts Gumla, Khuntiand Sahibganj and susceptible to deltamehrin (98% to100% mortality) and permethrin (100% mortality). INTERPRETATION & CONCLUSION: An. annularis, the secondary vector species is associated with the transmission of malaria reported resistant to DDT and susceptible to pyrerthroids deltamethrin and permethrin. In view of large-scale distribution of long-lasting insecticidal nets (LLINs) in all the districts, the response to synthetic pyrethroid needs to be periodically monitored to assess the effectiveness.

Pollinator biological traits and ecological interactions mediate the impacts of mosquito-targeting malathion application.

Mosquito adulticides are perceived by the public as detrimental to nontarget arthropods, contributing to declines of native and beneficial insects. However, the actual impact of adulticides on nontarget arthropods in nature needs to incorporate biological and ecological elements. Here, we investigated the effect of biological/behavioral traits (butterfly roosting at different heights, roosting in sites underneath foliage, bumblebee hive usage) and interactions (parasitism, predation) of pollinators (butterflies and bumblebees) that could mediate the impacts of malathion application in manipulative semi-field experiments in Florida, USA. Roosting height from the spray route had a significant negative relationship with mortality of butterflies treated with ULV malathion, with high survival at the highest roosting height (7 m), but butterflies roosting among vegetation did not have higher survival. Bumblebees with hive access had significantly higher survival than bumblebees without hive access. Host plants treated with ULV malathion significantly reduced parasitism of monarch eggs by Trichogramma platneri, but increased predation of monarch caterpillars by Polistes paper wasps. These data provide insight into the realistic impacts of adulticide applications on pollinators in nature which will enable mosquito control districts to better limit nontarget effects of adulticide treatments and may help to address concerns related to potential nontarget effects.

Multiple resistance mechanisms to penoxsulam in Echinochloa crus-galli from China.

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac­sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods.