Investigating discriminating concentrations for monitoring susceptibility to broflanilide and cross resistance to other insecticide classes in Anopheles gambiae sensu lato, using the new WHO bottle bioassay method.

BACKGROUND: Broflanilide is a new insecticide being developed for malaria vector control. As new insecticide chemistries become available, strategies to preserve the susceptibility of local malaria vectors and extend their useful life need to be considered before large scale deployment. This requires the development of appropriate testing procedures and identification of suitable discriminating concentrations for monitoring susceptibility in wild vector populations to facilitate decision making by control programmes. METHODS: Dose-response WHO bottle bioassays were conducted using the insecticide-susceptible Anopheles gambiae s.s. Kisumu strain to determine a discriminating concentration of broflanilide. Bioassays were performed without the adjuvant Mero® and with two concentrations of Mero® (500 ppm and 800 ppm) to investigate its impact on the discriminating concentration of the insecticide. Probit analysis was used to determine the lethal doses at 50% (LC50) and 99% (LC99) at 24-, 48- and 72-hours post-exposure. Cross-resistance to broflanilide and pyrethroids, DDT, dieldrin and carbamates, was investigated using An. gambiae s.l. Covè and An. coluzzii Akron strains. The susceptibility of wild pyrethroid-resistant mosquitoes from communities in Southern Benin to broflanilide was assessed using the estimated discriminating concentrations. RESULTS: Broflanilide induced a dose-dependent and delayed mortality effect. Mortality rates in bottles treated without Mero® were <80% using the range of broflanilide doses tested (0-100 µg/bottle) leading to high and unreliable estimates of LC99 values. The discriminating concentrations defined as 2XLC99 at 72h post exposure were estimated to be 2.2 µg/bottle with 800 ppm of Mero® and 6.0 µg/bottle with 500 ppm of Mero®. Very low resistance ratios (0.6-1.2) were determined with the insecticide resistant An. gambiae s.l. Covè and An. coluzzii Akron strains suggesting the absence of cross-resistance via the mechanisms of resistance to pyrethroids, DDT, dieldrin and carbamates they possess. Bottle bioassays performed with broflanilide at both discriminating concentrations of 6 µg/bottle with 500 ppm of Mero® and 2.2 µg/bottle with 800 ppm of Mero®, showed susceptibility of wild highly pyrethroid-resistant An. gambiae s.l. from villages in Southern Benin. CONCLUSION: We determined discriminating concentrations for monitoring susceptibility to broflanilide in bottle bioassays, using susceptible An. gambiae vectors. Using the estimated discriminating concentrations, we showed that wild pyrethroid-resistant populations of An. gambiae s.l. from southern Benin were fully susceptible to the insecticide. Broflanilide also shows potential to be highly effective against An. gambiae s.l. vector populations that have developed resistance to other public health insecticides.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126-54.00 µg/ml or LC95 × 3 = 0.7437-17.82 µg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Fitness costs associated with a GABA receptor mutation conferring dieldrin resistance in Aedes albopictus.

Understanding the dynamics of insecticide resistance genes in mosquito populations is pivotal for a sustainable use of insecticides. Dieldrin resistance in Aedes albopictus is conferred by the alanine to serine substitution (A302S or RdlR allele) in the γ-aminobutyric acid (GABA) receptor encoded by the Rdl gene. On Reunion Island, dieldrin resistance was initially reported in natural Ae. albopictus populations sampled in 2008 despite the ban of dieldrin since 1994. To monitor insecticide resistance in Ae. albopictus on the island and to identify its drivers, we measured (i) the frequency of resistance alleles in 19 distinct natural populations collected between 2016 and 2017, (ii) fitness costs associated with dieldrin resistance in laboratory-controlled experiments, and (iii) the resistance conferred by RdlR to fipronil, an insecticide widely used on the island and reported to cross-react with RdlR. The results show a persistence of RdlR in Ae. albopictus natural populations at low frequencies. Among the measured life history traits, mortality in pre-imaginal stages, adults' survival as well as the proportion of egg-laying females were significantly affected in resistant mosquitoes. Finally, bioassays revealed resistance of RdlR mosquitoes to fipronil, suggesting that the use of fipronil in natura could select for the RdlR allele. This study shows that dieldrin resistance is persistent in natural mosquito populations likely as a result of combined effects between fitness costs associated with RdlR and selection exerted by cross-reacting environmental insecticides such as fipronil.

Muscarinic acetylcholine receptor activation synergizes the knockdown and toxicity of GABA-gated chloride channel insecticides.

BACKGROUND: Pest management requires continual identification of new physiological targets and strategies to control pests affecting agriculture and public/animal health. We propose the muscarinic system as a target for agrochemicals because of its physiological importance. Unlike the muscarinic system, gamma-amino butyric acid (GABA) receptors are an established insecticide target. Here, we investigated target-site synergism using small molecule probes (agonist and antagonist) against the muscarinic system and their ability to enhance the toxicity of GABAergic insecticides in Drosophila melanogaster (Meigen). RESULTS: Oral delivery of pilocarpine (muscarinic agonist) enhanced the toxicity of dieldrin, fipronil, and lindane, resulting in synergist ratios (SRs) between 4-32-fold (orally delivered) or between 2-67-fold when insecticides were topically applied. The synergism between pilocarpine and the GABA-insecticides was greater than the synergism observed with atropine (muscarinic antagonist), and was greater, or comparable, to the synergism observed with the metabolic inhibitor piperonyl butoxide. In addition to lethality, pilocarpine increased the knockdown of lindane. The mechanism of synergism was also investigated in the central nervous system using extracellular electrophysiology, where pilocarpine (3 µmo/L) lowered the half-maximal inhibitory concentration (IC50 ) of lindane from 1.3 (0.86-1.98) µmol/L to 0.17 (0.14-0.21) µmol/L and fipronil's IC50 from 2.2 (1.54-3.29) µmol/L to 0.56 (0.40-0.77) µmol/L. CONCLUSION: Convergence of the cellular function between the muscarinic and GABAergic systems enhanced the insecticidal activity of GABA receptor blocking insecticides through the modulation of the central nervous system (CNS). The future impact of the findings could be the reduction of the active ingredient needed in a formulation with the development of muscarinic synergists. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The effect of DDE and dieldrin and their parental substances (DDT and aldrin) on PGE2 and PGF2α release from bovine endometrial explants collected during 120 to 180 days of the gestational period.

Dieldrin and DDE are environmental metabolites of the organochlorine pesticides aldrin and DDT, respectively. During pregnancy, these chemicals can quickly infiltrate through the placental barrier, accumulate in amniotic fluid and fetus, and act as endocrine disruptors (EDs). The aim of this study was to investigate the effect of DDE and dieldrin and their parental substances at concentrations of 1 and 10 ng/ml on secretion of PGE2 and PGF2α from bovine endometrial explants (120-150 and 151-180 days of pregnancy) after 24 hr of incubation with EDs. The mRNA expression of COX2, PGES and PGFS and the concentrations of PGE2 and PGF2α were measured. EDs did not affect (p>0.05) COX2 gene expression, but DDT and DDE decreased (p⟨0.05) PGES expression and PGE2 secretion in the explants from 120-150 days of pregnancy. Depending on the dose, DDT and DDE increased (p⟨0.05) PGFS expression and PGF2α secretion from the explants from 120-150 days and decreased PGF2α secretion (p⟨0.05) from the explants from 151-180 days of pregnancy. Aldrin and dieldrin decreased (p⟨0.05) PGFS expression and PGF2α secretion from all explants. In summary, EDs disrupt the secretion of PGE2 and PGF2α by influencing the gene expression of PGES and PGFS.

Investigating the molecular basis of multiple insecticide resistance in a major malaria vector Anopheles funestus (sensu stricto) from Akaka-Remo, Ogun State, Nigeria.

BACKGROUND: Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated. METHODS: Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method. RESULTS: Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance. CONCLUSIONS: The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

RDL mutations in Guangxi Anopheles sinensis populations along the China-Vietnam border: distribution frequency and evolutionary origin of A296S resistance allele.

BACKGROUND: Malaria is a deadly vector-borne disease in tropical and subtropical regions. Although indigenous malaria has been eliminated in Guangxi of China, 473 confirmed cases were reported in the Northern region of neighbouring Vietnam in 2014. Considering that frequent population movement occurs across the China-Vietnam border and insecticide resistance is a major obstacle in disease vector control, there is a need to know the genotype and frequency of insecticide resistance alleles in Anopheles sinensis populations along the China-Vietnam border and to take action to prevent the possible migration of insecticide resistance alleles across the border. METHODS: Two hundred and eight adults of An. sinensis collected from seven locations in Guangxi along the China-Vietnam border were used in the investigation of individual genotypes of the AsRDL gene, which encodes the RDL gamma-aminobutyric acid (GABA) receptor subunit in An. sinensis. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis was deployed to genotype codon 345, while direct sequencing of PCR products was conducted to clarify the genotypes for codons 296 and 327 of the AsRDL gene. The genealogical relation of AsRDL haplotypes was analyzed using Network 5.0. RESULTS: Three putative insecticide resistance related mutations (A296S, V327I and T345S) were detected in all the seven populations of An. sinensis in Guangxi along the China-Vietnam border. The resistance-conferring A296S mutation was found to be widely distributed and present at notably high frequencies (78.8% to 100%). Relatively lower frequencies of mutations V327I (26.9% to 53.2%) and T345S (0% to 28.8%) were observed. The V327I or T345S always occurred in the presence of A296S. Evolutionary analysis of 21 AsRDL haplotypes indicated multiple origins of the A296S and V327I mutations. CONCLUSION: The resistance A296S allele was present at high frequencies in the An. sinensis populations along the China-Vietnam border, indicating a risk of resistance to insecticides targeting RDL. The double mutations (A296S + V327I) may have evolved from alleles carrying the A296S mutation by scaffolding the additional mutation V327I, and A296S allele may have multiple evolutionary origins. These findings will help inform strategies for vector control and malaria prevention.

Malaria and Dengue Mosquito Vectors from Lao PDR Show a Lack of the rdl Mutant Allele Responsible for Cyclodiene Insecticide Resistance.

The gamma-aminobutyric acid (GABA) receptor, RDL, plays important roles in neuronal signaling and is the target of highly effective insecticides. A mutation in RDL, commonly A296S, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here, we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where mosquito-borne diseases such as malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from 12 provinces in Laos. Adult bioassays on Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) showed that all the populations tested were susceptible to dieldrin (4%) following WHO protocols. Exon 7 from a total of 791 mosquitoes was sequenced to identify the amino acid encoded for at 296 of RDL. Only one of these mosquitoes, Anopheles maculatus rampae Harbach and Somboon (Diptera: Culicidae) from Attapeu, carried the mutant allele being heterozygous for A296S. We therefore found a general lack of the Rdl mutant allele indicating that mosquitoes from Laos are not exposed to insecticides that act on the GABA receptor compared to mosquitoes in several other countries. Identifying the prevalence of the Rdl mutation may help inform the potential use of alternative insecticides that act on the GABA receptor should there be a need to replace pyrethroids in order to prevent/manage resistance.

RDL A301S alone does not confer high levels of resistance to cyclodiene organochlorine or phenyl pyrazole insecticides in Plutella xylostella.

Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.

Reduced Insecticide Susceptibility in Aedes vexans (Diptera: Culicidae) Where Agricultural Pest Management Overlaps With Mosquito Abatement.

Mosquito abatement programs in Midwestern communities frequently exist within landscapes dominated by agriculture. Although separately managed, both agricultural pests and mosquitoes are targeted by similar classes of insecticides. As a result, there is the potential for unintended insecticide exposure to mosquito populations from agricultural pest management. To determine the impact that agricultural management practices have on mosquito insecticide susceptibility we compared the mortality of Aedes vexans (Meigen; Diptera: Culicidae) between populations sampled from locations with and without mosquito abatement in South Dakota, a region dominated by agricultural production. Collection locations were either within towns with mosquito abatement programs (n = 2; Brookings and Sioux Falls, SD) or located > 16 km from towns with mosquito abatement programs (n = 2; areas near Harrold and Willow Lake, SD). WHO bioassays were used to test susceptibly of adults to differing insecticide classes relative to their respective controls; 1) an organochlorine (dieldrin 4%), 2) an organophosphate (malathion 5%), and 3) a pyrethroid (lambda-cyhalothrin 0.05%). Corrected mortality did not significantly differ between locations with or without abatement; however, when locations were analized by proportion of developed land within the surrounding landscape pyrethroid mortality was significantly lower where crop production dominated the surrounding landscape and mosquito abatement was present. These data suggest that agricultural pest management may incidentally contribute to reduced mosquito susceptibility where overlap between agricultural pest management and mosquito abatement exists. Decoupling insecticide classes used by both agricultural and public health pest management programs may be necessary to ensure continued efficacy of pest management tools.

Development of a genetic sexing strain of Anopheles arabiensis for KwaZulu-Natal, South Africa.

An efficient sexing system is important for the release of sterile males for any control programme using the sterile insect technique. This study describes the development and characterization of a new genetic sexing strain from South Africa (GMK), needed for the planned implementation of such a programme in northern KwaZulu-Natal Province. The base colony used was a locally modified laboratory strain of Anopheles arabiensis containing a sex-linked gene conferring dieldrin resistance to male mosquitoes. Female A. arabiensis mosquitoes from northern KwaZulu-Natal were mated with these males and backcrossed to introduce the dieldrin resistance gene to the Y chromosome. The resulting strain therefore had an overall genotype representing the local population but with the Y chromosome containing the dieldrin resistance gene. Life-history characteristics, stability of the sex-linked resistance marker, and reduction in dieldrin waste were investigated. The strain showed semi-sterility exhibited by low egg hatch rates, faster development in the immature stages and longer adult survivorship compared with the parental strains. While the GMK strain carrying the dieldrin-resistant gene was successfully established, the stability of the gene is limited, requiring periodic purification. Dieldrin waste can be limited by treating many more eggs than currently recommended.

RDL mutations predict multiple insecticide resistance in Anopheles sinensis in Guangxi, China.

BACKGROUND: Anopheles sinensis is a major vector of malaria in China. The gamma-aminobutyric acid (GABA)-gated chloride channel, encoded by the RDL (Resistant to dieldrin) gene, is the important target for insecticides of widely varied structures. The use of various insecticides in agriculture and vector control has inevitably led to the development of insecticide resistance, which may reduce the control effectiveness. Therefore, it is important to investigate the presence and distribution frequency of the resistance related mutation(s) in An. sinensis RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil. METHODS: Two hundred and forty adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. Two fragments of An. sinensis RDL (AsRDL) gene, covering the putative insecticide resistance related sites, were sequenced respectively. The haplotypes of each individual were reconstructed by the PHASE2.1 software, and confirmed by clone sequencing. The phylogenetic tree was built using maximum-likelihood and Bayesian inference methods. Genealogical relations among different haplotypes were also analysed using Network 5.0. RESULTS: The coding region of AsRDL gene was 1674 bp long, encoding a protein of 557 amino acids. AsRDL had 98.0% amino acid identity to that from Anopheles funestus, and shared common structural features of Cys-loop ligand-gated ion channels. Three resistance-related amino acid substitutions (A296S, V327I and T345S) were detected in all the nine populations of An. sinensis in Guangxi, with the 296S mutation being the most abundant (77-100%), followed by 345S (22-47%) and 327I (8-60%). 38 AsRDL haplotypes were identified from 240 individuals at frequencies ranging from 0.2 to 34.8%. Genealogical analysis suggested multiple origins of the 345S mutation in AsRDL. CONCLUSIONS: The near fixation of the 296S mutation and the occurrence of the 327I and 345S mutations in addition to 296S, in all the nine tested An. sinensis populations in Guangxi, strongly indicate a risk of multiple insecticide resistance. The haplotype diversity plus genetic heterogeneities in the geographical distribution, and multiple origins of AsRDL alleles call for a location-customized strategy for monitoring and management of insecticide resistance.

Dieldrin-induced neurotoxicity involves impaired mitochondrial bioenergetics and an endoplasmic reticulum stress response in rat dopaminergic cells.

Mitochondria are sensitive targets of environmental chemicals. Dieldrin (DLD) is an organochlorine pesticide that remains a human health concern due to high lipid bioaccumulation, and it has been epidemiologically associated to an increased risk for Parkinson's disease (PD). As mitochondrial dysfunction is involved in the etiology of PD, this study aimed to determine whether DLD impaired mitochondrial bioenergetics in dopaminergic cells. Rat immortalized dopaminergic N27 cells were treated for 24 or 48h with one dose of either a solvent control, 2.5, 25, or 250µM DLD. Dopaminergic cells treated with 250µM DLD showed increased Casp3/7 activity at 24 and 48h. DLD also caused a dose dependent reduction in cell viability of ∼25-30% over 24h. No significant effects on cell viability, apoptosis, nor cytotoxicity were detected at 24 or 48h with 2.5µM DLD. Following a 24h exposure to 2.5 and 25µM DLD, viable cells were subjected to a mitochondrial stress test using the Seahorse XFe24 Extracellular Flux Analyzer. Following three independent experiments conducted for rigor, dopaminergic cells that were treated with 2.5 and 25µM DLD consistently showed a reduction in maximum respiration and spare capacity compared to the control group. Molecular responses were measured to determine mechanisms of DLD-induced mitochondrial dysfunction. There were no changes in transcripts associated with mitochondrial membrane potential and permeability (e.g. Ant, Hk1, Tspo, Vdac), nor PI3 K/Akt/mTor signaling or mitochondrial-associated apoptotic factors (Bax, Bcl2, Casp3). However, transcript levels for Chop/Gadd153 (DNA Damage Inducible Transcript 3), an apoptotic gene activated following endoplasmic reticulum (ER) stress, were 3-fold higher in N27 cells treated with DLD, suggesting that DLD-induced mitochondrial dysfunction is related to ER stress. Dopamine cells were also assessed for changes in tyrosine hydroxylase (TH) protein, which did not differ among treatments. This study demonstrates that DLD impairs oxidative respiration in dopamine cells, and ER stress is hypothesized to be associated with the DLD-induced mitochondrial dysfunction. This is important as ER stress is also linked to PD. This study presents mechanistic insight into pesticide-induced mitochondrial dysfunction using a chemical that is reported to be associated to a higher risk for neurodegenerative disease.

Dieldrin Augments mTOR Signaling and Regulates Genes Associated with Cardiovascular Disease in the Adult Zebrafish Heart (Danio rerio).

Dieldrin is a legacy organochlorine pesticide that is persistent in the environment, despite being discontinued from use in North America since the 1970s. Some epidemiologic studies suggest that exposure to dieldrin is associated with increased risks of neurodegenerative disease and breast cancer by inducing inflammatory responses in tissues as well as oxidative stress. However, the direct effects of organochlorine pesticides on the heart have not been adequately addressed to date given that these chemicals are detectable in human serum and are environmentally persistent; thus, individuals may show latent adverse effects in the cardiovascular system due to long-term, low-dose exposure over time. Our objective was to determine whether low-level exposure to dieldrin at an environmentally relevant dose results in aberrant molecular signaling in the vertebrate heart. Using transcriptomic profiling and immunoblotting, we determined the global gene and targeted protein expression response to dieldrin treatment and show that dieldrin affects gene networks in the heart that are associated with processes related to cardiovascular disease, specifically cardiac arrest and ventricular fibrillation. We report that genes regulating inflammatory responses, a significant risk factor for cardiovascular disease, are upregulated by dieldrin whereas transcripts related to lysosomal function are significantly downregulated. To verify these findings, proteins in these pathways were examined with immunoblotting, and our results demonstrate that dieldrin constitutively activates Akt/mTOR signaling and downregulates lysosomal genes, participating in autophagy. Our data demonstrate that dieldrin induces genes associated with cardiovascular dysfunction and compromised lysosomal physiology, thereby identifying a novel mechanism for pesticide-induced cardiotoxicity.

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

Evidence of a multiple insecticide resistance in the malaria vector Anopheles funestus in South West Nigeria.

BACKGROUND: Knowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria. RESULTS: Plasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F0 (77%) and F1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F0 and F1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F0; for F1, 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population. CONCLUSION: The study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.

Protective effects of quercetin on dieldrin-induced endoplasmic reticulum stress and apoptosis in dopaminergic neuronal cells.

Dieldrin, an organochlorine pesticide still used in several developing countries, has been proposed as a risk factor for Parkinson's disease. Quercetin is one of the potent bioactive flavonoids present in numerous plants. In this study, we investigated the protective effects of quercetin on neurotoxicity induced by dieldrin in cultured dopaminergic SN4741 cells. Our initial experiments showed that quercetin (10-40 µM) dose dependently prevented dieldrin (20 µM)-induced cytotoxicity in SN4741 cells. Pretreatment for 1 h with quercetin before dieldrin application could significantly suppress dieldrin-induced apoptotic characteristics, including nuclear condensation, DNA fragmentation, and caspase-3/7 activation. Results showed that dieldrin-induced markers of endoplasmic reticulum (ER) stress response such as chaperone GRP78, heme oxygenase-1, and phosphorylation of the α subunit of eukaryotic initiation factor 2. In addition, dieldrin reduced antiapoptotic Bcl-2 expression, but significantly elevated a proapoptotic transcription factor CHOP. Furthermore, RNA interference to CHOP almost completely repressed dieldrin-induced apoptotic cell death. Interestingly, quercetin prevented the changes in dieldrin-induced ER stress markers. These results suggest that quercetin may suppress the ER stress-CHOP pathway and dieldrin-induced apoptosis in dopaminergic neurons.

First molecular genotyping of A302S mutation in the gamma aminobutyric acid (GABA) receptor in Aedes albopictus from Malaysia.

Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first field evolved instance of A302S mutation in Malaysian insect species.

Identification of California Condor Estrogen Receptors 1 and 2 and Their Activation by Endocrine Disrupting Chemicals.

Recently, California condors (Gymnogyps californianus) have been reintroduced to coastal regions of California where they feed on marine mammal carcasses. There is evidence that coastal-dwelling condors experience reproductive issues, such as eggshell thinning, likely resulting from exposure to endocrine-disrupting chemicals (EDCs). To address this problem, we have identified and cloned condor estrogen receptors (ESRs) 1 and 2 and characterized their activation by EDCs present in the coastal habitats where condors reside. Dichlorodiphenyltrichloroethane (DDT) and its metabolites all activated ESR1 and ESR2, although their relative potency differed between the receptors. Bisphenol A, dieldrin, trans-nonachlor, and polychlorinated biphenyl 52 (PCB52) moderately activated both ESRs, whereas PCB138 and PCB153 stimulated little to no activation. Overall, EDC activation of condor ESR2, which is the first ESR2 cloned from a raptor species, was greater than that of ESR1. Significant activation of both condor ESRs by EDCs occurred at high concentrations (≥1µM), which are within the range of plasma levels of certain EDCs (eg, dichlorodiphenyldichloroethylene [p'p-DDE]) in coastal-dwelling condors. Finally, phylogenetic analyses of ESRs of 41 avian species identified a single amino acid position in ESR2 under positive selection. Mutation of this amino acid affected receptor activation by EDCs, suggesting the identity of this amino acid may influence EDC sensitivity of avian species. Together, these findings broaden our understanding of EDC interactions with ESRs in avian species. For condors specifically, these data could be used to evaluate EDC exposure risk at future release sites to identify those least likely to compromise the continued recovery of this species.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.