Toxicity assessment of polyethylene microplastics in combination with a mix of emerging pollutants on Physalaemus cuvieri tadpoles.

Studies in recent years have shown that aquatic pollution by microplastics (MPs) can be considered to pose additional stress to amphibian populations. However, our knowledge of how MPs affect amphibians is very rudimentary, and even more limited is our understanding of their effects in combination with other emerging pollutants. Thus, we aimed to evaluate the possible toxicity of polyethylene MPs (PE-MPs) (alone or in combination with a mix of pollutants) on the health of Physalaemus cuvieri tadpoles. After 30 days of exposure, multiple biomarkers were measured, including morphological, biometric, and developmental indices, behavioral parameters, mutagenicity, cytotoxicity, antioxidant and cholinesterase responses, as well as the uptake and accumulation of PE-MPs in animals. Based on the results, there was no significant change in any of the parameters measured in tadpoles exposed to treatments, but induced stress was observed in tadpoles exposed to PE-MPs combined with the mixture of pollutants, reflecting significant changes in physiological and biochemical responses. Through principal component analysis (PCA) and integrated biomarker response (IBR) assessment, effects induced by pollutants in each test group were distinguished, confirming that the exposure of P. cuvieri tadpoles to the PE-MPs in combination with a mix of emerging pollutants induces an enhanced stress response, although the uptake and accumulation of PE-MPs in these animals was reduced. Thus, our study provides new insight into the danger to amphibians of MPs coexisting with other pollutants in aquatic environments.

Impact of Glyphosate-Roundup® in the Ileal Structure of Male and Female Rats: A Morphological and Immunohistochemical Study.

The current study was aimed to evaluate the effects of variable doses of the weedicide glyphosate on the ileal (the final section of the small intestine) structure of rats of both sexes, using histological, histochemical, and ultrastructural methods. Forty animals were classified into four groups of 10 animals per group (five males and five females). The first group acted as a control, and the remaining groups were treated with glyphosate-Roundup® 25, 50, and 100 mg/kg body weight daily for 15 days. The results indicated extinct histopathological changes manifested in the deformation of villi, foci of leukocytic infiltration in the core of villi, and hyperplasia of goblet cells. Histochemical examination (Alcian blue and Periodic acid-Schiff stain) revealed a strong positive reaction of goblet cells and an increase in their number in all treated groups. In addition, the immunohistochemical investigation revealed the immunoreactivity of matrix metalloproteinase-9 expression. Furthermore, electron microscopic alternations were represented by the deformation of nuclei, destruction of microvilli, and deposition of lipid droplets. Collectively, the present findings indicate that treatment with glyphosate results in extensive morphological alternations to the ileal structure of rats of both sexes and that female rats are more affected than male rats are.

Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito.

Studying insecticide resistance in mosquitoes has attracted the attention of many scientists to elucidate the pathways of resistance development and to design novel strategies in order to prevent or minimize the spread and evolution of resistance. Here, we tested the synergistic action of piperonyl butoxide (PBO) and two octopamine receptor (OR) agonists, amitraz (AMZ) and chlordimeform (CDM) on selected novel insecticides to increase their lethal action on the fourth instar larvae of Aedes aegypti L. However, chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively) tested. Further, PBO synergized all insecticides and the most toxic combinatorial insecticide was nitenpyram even after 48 and 72 h exposure. In addition, OR agonists significantly synergized most of the selected insecticides especially after 48 and 72 h exposure. The results imply that the synergistic effects of amitraz are a promising approach in increasing the potency of certain insecticides in controlling the dengue vector Ae. aegypti mosquito.

Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae).

We recently reported that formamidine pesticides such as amitraz and chlordimeform effectively synergize toxic actions of certain pyrethroid and neonicotinoid insecticides in some insect species on the 4th instar larvae of Aedes aegypti. Here we studied the biochemical basis of the synergistic actions of the formamidines in amplifying the toxicity of neonicotinoids and pyrethroids such as dinotefuran and thiamethoxam, as well as deltamethrin-fenvalerate type of pyrethroids. We tested the hypothesis that their synergistic actions are mediated by the octopamine receptor, and that the major consequence of octopamine receptor activation is induction of trehalase to increase glucose levels in the hemolymph. The results show that formamidines cause a significant up-regulation of the octopamine receptor and trehalase mRNA expressions. Furthermore, formamidines significantly elevate levels of free glucose when co-treated with dinotefuran, deltamethrin and fenvalerate, but not with permethrin or fenitrothion, which showed no synergistic toxic effects with formamidines. These results support the conclusion that the main mode of synergism is based on the ability to activate the octopamine receptor, which is particularly effective with insecticides causing hyperexcitation-induced glucose release and consequently leading to quick energy exhaustion.

Short exposure to nitenpyram pesticide induces effects on reproduction, development and metabolic gene expression profiles in Drosophila melanogaster (Diptera: Drosophilidae).

Although the toxicity of neonicotinoid insecticides has been demonstrated in several studies, the information on metabolism, behavior, and health risk remains limited and has raised concerns about its potential toxicity. Thus, in this study we assessed the effects of nitenpyram using different sublethal concentrations (one-third and one-tenth of the acute LC50 values) on various developmental and metabolic parameters from gene expression regulation in Drosophila melanogaster (model system used worldwide in ecotoxicological studies). As a result, nitenpyram sublethal concentrations prolonged the developmental time for both pupation and eclosion. Additionally, nitenpyram sublethal concentrations significantly decreased the lifespan, pupation rate, eclosion rate, and production of eggs of D. melanogaster. Moreover, the mRNA expression of genes relevant for development and metabolism was significantly elevated after exposure. Mixed function oxidase enzymes (Cyp12d1), (Cyp9f2), and (Cyp4ae1), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated, whereas lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These findings support a solid basis for further research to determine the hazardous effects of nitenpyram on health and the environment.

Gene expression profiling in liver of zebrafish exposed to ethylhexyl methoxycinnamate and its photoproducts.

In recent decades, the ecotoxicological potential of organic ultraviolet filters (OU-VFs) has received growing attention. However, the toxicity of its photoproducts or transformation products on freshwater vertebrates has been little explored. Therefore, the aim of the present study is to evaluate the possible adverse effects of ethylhexyl methoxycinnamate (EHMC) and its photoproducts [2-ethylhexanol (2-EH) and 4-methoxybenzaldehyde (4-MBA)] on the expression of stress-responsive and antioxidant genes. For this, zebrafish (Danio rerio) adults were exposed to pollutants at an environmentally relevant concentration (3 µg/L) and evaluated after 7, 14, and 21 days of exposure. The results of the principal component analysis (PCA) and two-way repeated measures (RM) ANOVA revealed that EHMC, 2-EH, and 4-MBA exposure caused significant downregulation of the genes hsp70, nrf2, cyp1a, ahr, sod1, sod2, cat, gstp1, gpx1a, gss, and gsr (on all trial days) in the liver of the animals. On the other hand, taken together, our data did not show significant differences between the effects induced by EHMC and its photoproducts. The genes evaluated in the present study play a major role in regulating the defensive antioxidant response against EHMC and its photoproducts. Additionally, our study provides an insight into the mechanisms of those OU-VFs in freshwater fish.

Novel methodology for identification and quantification of microplastics in biological samples.

Currently, the evidence of the ingestion of microplastics (MPs) by organisms or the accumulation in different environmental compartments has been achieved using several methodological procedures. However, its uses have not been standardized across studies. In this study, we aim to assess and validate a protocol that can be useful for optimizing the identification and quantification procedures of polyethylene microplastics (PE MPs) in biological samples. Initially, considering that numerous studies filter samples previously digested in cellulosic membranes for isolation and analysis of MPs, we evaluated whether washing these membranes with different reagents could contribute to the complete detachment of particles, as well as to their dispersion in the obtained solutions. However, none of the tested reagents (dimethyl sulfoxide, acetone, ethyl alcohol and chloroform), including purified water, was able to completely remove the MPs adhered to the membranes or facilitate their dispersion in the solutions. On the other hand, we observed that the digestion of the membranes by acetonitrile constituted a procedure that prevents the loss of particles due to adherence, in addition to promoting good dispersion of MPs. Subsequently, we evaluated the use of Neubauer chambers for the quantification of MPs, having observed a good recovery rate (>92%) and results with insignificant variation, in PE MPs solutions with different concentrations (0.15; 0.075 and 0.0375 mg/mL). Ultimately, the validation of the proposed procedures took place from the evaluation of the accumulation of PE MPs in Astyanax spp. juveniles, having demonstrated the efficiency and sensitivity of the method proposed for this purpose. Subsequently, our study provides a methodological alternative that can optimize MPs quantifications in biological samples and reduce the generation of biased or unreliable results.

Can spike fragments of SARS-CoV-2 induce genomic instability and DNA damage in the guppy, Poecilia reticulate? An unexpected effect of the COVID-19 pandemic.

The identification of SARS-CoV-2 particles in wastewater and freshwater ecosystems has raised concerns about its possible impacts on non-target aquatic organisms. In this particular, our knowledge of such impacts is still limited, and little attention has been given to this issue. Hence, in our study, we aimed to evaluate the possible induction of mutagenic (via micronucleus test) and genotoxic (via single cell gel electrophoresis assay, comet assay) effects in Poecilia reticulata adults exposed to fragments of the Spike protein of the new coronavirus at the level of 40 µg/L, denominated PSPD-2002. As a result, after 10 days of exposure, we have found that animals exposed to the peptides demonstrated an increase in the frequency of erythrocytic nuclear alteration (ENA) and all parameters assessed in the comet assay (length tail, %DNA in tail and Olive tail moment), suggesting that PSPD-2002 peptides were able to cause genomic instability and erythrocyte DNA damage. Besides, these effects were significantly correlated with the increase in lipid peroxidation processes [inferred by the high levels of malondialdehyde (MDA)] reported in the brain and liver of P. reticulata and with the reduction of the superoxide dismutase (SOD) and catalase (CAT) activity. Thus, our study constitutes a new insight and promising investigation into the toxicity associated with the dispersal of SARS-CoV-2 peptide fragments in freshwater environments.

Hazardous effects of road-side soils on the redox and cholinesterasic homeostasis of mound-building termite (Cornitermes cumulans).

For our knowledge, the roadside soils end up being the deposit of various residues discarded by drivers or passengers, plus, that coming from the runoff of rainwater. Basically, we do not know the impacts that this pollution causes on animals which inhabit these environments. Thus, in this study, our objective was to evaluate how the presence of plastic microfibers (MPFs), organic compounds and heavy metals affect the redox and cholinesterase homeostasis of mound-building termite [Cornitermes cumulans (workers) adults]. As a result, we noticed that MPFs were present in all sampled areas, being higher in road area (RA). Regardless of the presence of these pollutants, animals sampled in the RA were those in which we observed greater production of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and nitric oxide (NO) (via nitrite), whose higher activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), was not able to counterbalance the oxidative stress suggested by the evaluated biomarkers. Moreover, we observed increase in acetylcholinesterase (AChE) activity in these same animals, which suggests a cholinesterasic effect. Such alterations were positively correlated with the contamination of soil samples by Cd, Pb, Zn, Fe and Cu, as well as with the presence of the 11,10-guaiane-type sesquiterpenoid compound, identified only in the RA. Thus, our unique study reveals that the contamination of roadside soils constitutes an additional environmental stressor to populations of C. cumulans, which reinforces the need for greater attention and further investigation to be given to the pollution of these environments.

Toxicity evaluation of the combination of emerging pollutants with polyethylene microplastics in zebrafish: Perspective study of genotoxicity, mutagenicity, and redox unbalance.

Despite the toxicity of microplastics (MPs) in freshwater fish has been demonstrated in previous studies, their effects when mixed with other pollutants (organic and inorganic) are poorly understood. Thus, we aimed to test the hypothesis that the association of polyethylene MPs (PE-MPs) to a mix of emerging pollutants induces more adverse genotoxic, mutagenic, and redox unbalance effects in adult zebrafish (Danio rerio), after 15 days of exposure. Although the accumulation of MPs in animals was greater in animals exposed to PE-MPs alone, erythrocyte DNA damage (comet assay) and the frequency of erythrocytic nuclear abnormalities (ENAs) evidenced in zebrafish exposed to PE-MPs alone were as pronounced as those observed in animals exposed to the mix of pollutant (alone or in combination with MPs), which constitutes the big picture of the current study. Moreover, we noticed that such effects were associated with an imbalance between pro-and antioxidant metabolism in animals, whose activity of superoxide dismutase (SOD) and catalase (CAT) was assessed in different organs which were not sufficient to counterbalance the production of reactive oxygen species [hydrogen peroxide (H2O2)] and nitrogen [nitric oxide (NO)] evaluated. The principal component analysis (PCA) also revealed that while the antioxidant activity was more pronounced in the brain and liver of animals, the highest production of H2O2 was perceived in the gills and muscles, suggesting that the biochemical response of the animals was organ-dependent. Thus, the present study did not demonstrate antagonistic, synergistic, or additive effects on animals exposed to the combination between PE-MPs and a mix of pollutants in the zebrafish, which reinforces the theory that interactions between pollutants in aquatic ecosystems may be as complex as their effects on freshwater ichthyofauna.

From carrion-eaters to plastic material plunderers: Toxicological impacts of plastic ingestion on black vultures, Coragyps atratus (Cathartiformes: Cathartidae).

Despite plastic ingestion has already been reported in several bird species, its physiological impacts have been little inspected, especially in representatives of the Cathartidae family. Thus, in this study, we aimed to identify, characterize, and evaluate the effects arising from the ingestion of plastic materials by Coragyps atratus adults, that captured in landfill areas. Herein, a total of 51 individuals were captured, the frequency of plastic intake being higher than 40%. The plastic materials consisted mainly of low-density polyethylene and film-type polystyrene, as well as presenting irregular shapes and diameters between 10 and 30 mm. Biochemically, we observed in animals that contained plastics in the stomach ("plastic" group) high production of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and malondialdehyde (MDA) - especially in the intestine, muscle and brain - whose activity of catalase (CAT) and superoxide dismutase (SOD) was not sufficient to counteract the oxidative stress. Moreover, in the liver of these same animals, we observed high production of nitrite and nitrate, suggesting a hepatic nitrosative stress. Plus, we observed a cholinesterase effect in animals from the "plastic" group, marked by increased activity of butyrylcholinesterase (BChE) (in the brain) and muscle and cerebral acetylcholinesterase (AChE). On the other hand, the biochemical changes perceived were not significantly correlated with the identified plastic material concentrations (2.808 ± 0.598 items/g of stomach content and 0.276 ± 0.070 items/g of stomach - fresh weight), body condition of the animals, size, and shape of the identified plastic materials. Hence, our study sheds the light on the toxicity of plastics deposited in landfills and their ingestion by C. atratus, which reinforces the hypothesis that these materials are harming the health of these birds and, consequently, the dynamics of their populations.

Steel wools microfibers causes iron overload and induces biochemical changes in Gallus gallus domesticus chicks (Galliformes: Phasianidae).

Steel wool (SW) has a broad-spectrum of applicability, particularly as abrasives, cleaning household utensils and surfaces in general. However, when present in the natural environment, they can be ingested by animals, such as birds, and may represent a risk to the survival of individuals. Accordingly, in this study, we attempted the hypothesis that the ingestion of SW microfibers (SWMs) by Gallus gallus domesticus chicks (model system used) alters growth/development, induces redox imbalance and cholinesterasic effect, as well as promotes iron overload in different organs. For this, the animals received SWMs twice (within a 24-h interval) in an amount corresponding to 12% of their total stomach volume. At the end of the experiment, we observed less weight gain and less head growth, increased production of hydrogen peroxide (in the brain, liver, crop, and gizzard), nitrite (liver, crop, proventriculus and gizzard), malondialdehyde (brain, liver, muscle, proventriculus, and gizzard), along with increased superoxide dismutase activity in the liver, muscle and crop of animals exposed to SWMs. Such results were associated with iron overload observed in different organs, especially in liver, crop, and gizzard. Furthermore, we evidenced an anti-cholinesterasic effect in birds that ingested the SWMs, marked by a reduction in the acetylcholinesterase activity (in brain). Thus, our study sheds light on the (eco)toxicological potential of SWMs in avifauna, conceding us to associate their ingestion (despite ephemeral and occasional) with damage to the health of individuals, requiring a greater attention spotted to disposal of these materials in ecosystems.

Toxicological impact of SARS-CoV-2 on the health of the neotropical fish, Poecilia reticulata.

There have been significant impacts of the current COVID-19 pandemic on society including high health and economic costs. However, little is known about the potential ecological risks of this virus despite its presence in freshwater systems. In this study, we aimed to evaluate the exposure of Poecilia reticulata juveniles to two peptides derived from Spike protein of SARS-CoV-2, which was synthesized in the laboratory (named PSPD-2002 and PSPD-2003). For this, the animals were exposed for 35 days to the peptides at a concentration of 40 µg/L and different toxicity biomarkers were assessed. Our data indicated that the peptides were able to induce anxiety-like behavior in the open field test and increased acetylcholinesterase (AChE) activity. The biometric evaluation also revealed that the animals exposed to the peptides displayed alterations in the pattern of growth/development. Furthermore, the increased activity of superoxide dismutase (SOD) and catalase (CAT) enzymes were accompanied by increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and hydrogen peroxide (H2O2), which suggests a redox imbalance induced by SARS-CoV-2 spike protein peptides. Moreover, molecular docking analysis suggested a strong interaction of the peptides with the enzymes AChE, SOD and CAT, allowing us to infer that the observed effects are related to the direct action of the peptides on the functionality of these enzymes. Consequently, our study provided evidence that the presence of SARS-CoV-2 viral particles in the freshwater ecosystems offer a health risk to fish and other aquatic organisms.

Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish.

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 µg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems.

Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 µg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences.

The synergistic effect of octopamine receptor agonists on selected insect growth regulators on Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes.

Synergistic effects of octopamine receptor agonists (OR agonists) have attracted many scientists based on their potent effects on mosquitoes. Herein, we determined the toxicity of selected insect growth regulators (IGRs) on fourth instar larvae of Culex quinquefasciatus. We evaluated the synergistic action of OR agonists on the toxicity of IGR insecticides to achieve a better understanding of their mode of action. As a result, pyriproxyfen was the most potent IGR insecticide (EC50 = 0.049 ng/ml) followed by lufenuron, novaluron, and diflubenzuron according to the IGR bioassay. Further, based on the acute bioassay, lufenuron was the most toxic IGR insecticide (LC50 = 44 ng/ml) after 24-h post treatment followed by pyriproxyfen, novaluron, and diflubenzuron (LC50 = 137, 263, and 1127 ng/ml, respectively). Similar tendency was observed after 48 and 72-h post treatment. Furthermore, OR agonists that combined with pyriproxyfen was the most significant effects after 48 and 72-h of exposure. The synergism with amitraz (AMZ) was more significant when co-treated with IGR insecticides compared to chlordimeform (CDM). These findings suggest that OR agonists are promising tools and are important alternative strategies as synergistic compounds in preventing and controlling Culex quinquefasciatus mosquitoes.

Hazardous effects of octopamine receptor agonists on altering metabolism-related genes and behavior of Drosophila melanogaster.

Recent reports demonstrate that octopamine receptor (OR) agonists such as formamidine pesticides cause reproductive and developmental toxicity through endocrine disrupting effects in both humans and animals. Herein, we studied the effects of different sublethal concentrations of OR agonists, Amitraz and Chlordimeform, on growth, development, and reproduction of D. melanogaster from a genotype perspective view. As a result, the sublethal concentrations for both OR agonists delayed the developmental time including pupation and eclosion. It significantly reduced the lifespan, eclosion rate, and production of eggs. The mRNA expression of genes relevant for development and metabolism was significantly changed after exposure to sublethal concentrations of both OR agonists. Octopamine receptor in mushroom bodies (Oamb), trehalase enzyme (Treh), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated whereas, trehalose sugar (Tret1-1), mixed function oxidase enzyme (Cyp9f2), lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These results support the conclusion that OR agonists activate the octopamine receptor in D. melanogaster leading to an increase of trehalase enzyme activity and degradation of trehalose sugar into free glucose which results in rapid energy exhaustion, hyperexcitation, and disturbing of the octopaminergic system in D. melanogaster.

Toxicological Evaluation of Novel Butenolide Pesticide Flupyradifurone Against Culex quinquefasciatus (Diptera: Culicidae) Mosquitoes.

The impact of increasing resistance of mosquitoes to conventional pesticides has led to investigate various unique tools and pest control strategies. Herein, we assessed the potency of flupyradifurone, a novel pesticide, on fourth instar larvae of Culex quinquefasciatus Say. Further, we evaluated the synergistic action of piperonyl butoxide (PBO) and the octopamine receptor agonists (OR agonists) chlordimeform (CDM) and amitraz (AMZ) on the toxicity of flupyradifurone in comparison with sulfoxaflor and nitenpyram to increase their toxicity on Cx. quinquefasciatus. Results demonstrated that flupyradifurone was the most potent pesticide followed by sulfoxaflor and nitenpyram. Further, the synergetic effect of PBO, CDM, and AMZ was significant for all selected pesticides especially flupyradifurone. However, AMZ had the most significant effect in combination with the selected pesticides followed by CDM and PBO. The toxicity of the pesticides was time-dependent and increased over time from 24, 48, to 72 h of exposure in all experiments. The results indicate that flupyradifurone is a promising component in future mosquito control programs.

Piperonyl Butoxide Enhances the Insecticidal Toxicity of Nanoformulation of Imidacloprid on Culex pipiens (Diptera: Culicidae) Mosquito.

The use of conventional pesticides becomes a complicated issue as more species of insect pests become resistant to them. Nanopesticides suit new approaches in pest control. Herein, we tested the toxicological efficacy of imidacloprid compared with three of its nanoformulations (IMD01, IMD02, and IMD03) on second and fourth instar of Culex pipiens larvae. Furthermore, we assessed the synergistic actions of piperonyl butoxide (PBO) on imidacloprid and its nanoformulations against second and fourth instar of C. pipiens. The nanoformulation (IMD03) was the most potent insecticide (LC50 = 14, 6, and 2 ng/mL after 24, 48, and 72 h of exposure, respectively), whereas the lowest toxic nanoformulation was IMD01. However, imidacloprid had the lowest toxicity among the tested compounds (LC50 = 1015, 705, and 621 ng/mL after 24, 48, and 72 h of exposure, respectively). PBO significantly synergized imidacloprid and its nanoformulations. However, the most synergistic effects were on IMD03 and the lowest was imidacloprid itself. Based on our results, nanopesticides are currently the most promising tool to control C. pipiens mosquitoes. However, further semifield and field studies should be done to illustrate the efficacy of imidacloprid and its nanoformulations on C. pipiens mosquitoes.

The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito.

The synergistic action of octopamine receptor agonists (OR agonists) on many insecticide classes (e.g., organophosphorus, pyrethroids, and neonicotinoids) on Aedes aegypti L. has been reported recently. An investigation of OR agonist's effect on insect growth regulators (IGRs) was undertaken to provide a better understanding of the mechanism of action. Based on the IGR bioassay, pyriproxyfen was the most potent IGR insecticide tested (EC50=0.0019ng/ml). However, the lethal toxicity results indicate that diafenthiuron was the most potent insecticide (LC50=56ng/cm(2)) on A. aegypti adults after 24h of exposure. The same trend was true after 48 and 72h of exposure. Further, the synergistic effects of OR agonists plus amitraz (AMZ) or chlordimeform (CDM) was significant on adults. Among the tested synergists, AMZ increased the potency of the selected IGRs on adults the greatest. As results, OR agonists were largely synergistic with the selected IGRs. OR agonists enhanced the lethal toxicity of IGRs, which is a valuable new tool in the field of A. aegypti control. However, further field experiments need to be done to understand the unique potential role of OR agonists and their synergistic action on IGRs.