Association of growth-regulating insecticides and limonoid-based formulations: physicochemical compatibility and toxicity against Spodoptera frugiperda (Lepidoptera: Noctuidae).

The objective of this study was to evaluate the physicochemical compatibility of mixtures of synthetic and botanical limonoid-based insecticides, as well as the toxicity of these associations, in the management of Spodoptera frugiperda (J.E. Smith) under laboratory and field conditions. For this, the associations of 4 commercial botanical insecticides based on neem registered in Brazil (Azamax, Agroneem, Azact CE, and Fitoneem) were tested with synthetic insecticides from the group of growth regulators (IGRs [triflumuron, lufenuron, methoxyfenozide and tebufenozide]). When mixed, all combinations caused a significant reduction in the pH of the mixture and a significant increase in electrical conductivity. However, all tested combinations showed similar stability behavior to the negative control (distilled water), which demonstrated their physicochemical compatibility. Furthermore, in laboratory and field bioassays, mixtures of IRGs with limonoid-based formulations provided satisfactory effects in the management of S. frugiperda. However, binary mixtures of insecticide Intrepid 240 SC with Azamax or Azact CE (at LC25 previously estimated) showed the highest toxicities on S. frugiperda larvae in laboratory bioassays and damage reduction caused by S. frugiperda in a 2-yr field experiments. Therefore, mixtures of IGRs with limonoid-based botanical insecticides are promising alternatives for the management of S. frugiperda and important component of integrated pest management and insect resistance management programs.

Lethal and Sublethal Toxicity of Insecticides to the Lacewing Ceraeochrysa Cubana.

The lethal and sublethal effects of 11 insecticides on the predator Ceraeochrysa cubana (Hagen) were assessed under laboratory conditions. First-instar larvae and adults ≤ 48 h old were sprayed with the highest insecticides doses allowed to control Diaphorina citri Kuwayama in the citrus crop. The survival and duration rates of the different development stages, sex ratio, pre-oviposition period, fecundity, and fertility of the insects were evaluated. In the larval bioassay, chlorpyrifos and malathion had lethal effect which none larvae survived. Azadirachtin, lambda-cyhalothrin + chlorantraniliprole, lambda-cyhalothrin + thiamethoxam, and thiamethoxam had lethal and sublethal effects that did not allow to estimate the life table parameters because the low number of couples formed. Esfenvalerate, imidacloprid WG and SC, phosmet, and pyriproxyfen had sublethal effects which were reflected in the net reproductive rate and in the intrinsic rate of natural increase. In bioassay using adults, none of the individuals survived in the chlorpyrifos, lambda-cyhalothrin + chlorantraniliprole, lambda-cyhalothrin + thiamethoxam, malathion, or thiamethoxam treatments, and the azadirachtin, esfenvalerate, imidacloprid WG and SC, phosmet, and pyriproxyfen treatments were significantly lower compared to the control. None of the insecticides was harmless to first-instar larvae and adults of C. cubana under laboratory conditions showing their potential to reduce the efficiency of this predator.

The reduced-risk insecticide azadirachtin poses a toxicological hazard to stingless bee Partamona helleri (Friese, 1900) queens.

Large-scale pesticide application poses a major threat to bee biodiversity by causing a decline in bee populations that, in turn, compromises ecosystem maintenance and agricultural productivity. Biopesticides are considered an alternative to synthetic pesticides with a focus on reducing potential detrimental effects to beneficial organisms such as bees. The production of healthy queen stingless bees is essential for the survival and reproduction of hives, although it remains unknown whether biopesticides influence stingless bee reproduction. In the present study, we investigated the effects of the biopesticide azadirachtin on the survival, behavior, morphology, development, and reproduction of queens of the stingless bee Partamona helleri (Friese, 1900). The neonicotinoid imidacloprid was used as a toxic reference standard. Queens were orally exposed in vitro to a contaminated diet (containing azadirachtin and imidacloprid) during development. Azadirachtin resulted in reduced survival, similarly to imidacloprid, altered development time, caused deformations, and reduced the size of the queens' reproductive organs. All of these factors could potentially compromise colony survival. Results from the present study showed azadirachtin posed a toxicological hazard to P. helleri queens.

Survival and Locomotory Behavior of Earwigs After Exposure to Reduced-Risk Insecticides.

The conservation of natural enemies is an important tactic to promote biological control of arthropod pests. The earwig Doru luteipes (Sccuder) is the most important predator of the fall armyworm Spodoptera frugiperda (J.E. Smith) in corn fields. One way of conserving these predators in the field is by using only selective insecticides when the pest population reaches the economic threshold. Some recent insecticides such as azadirachtin, chlorantraniliprole, and novaluron have been claimed to pose reduced risk for natural enemies. Nevertheless, there is a dearth of information regarding the selectivity of these insecticides upon earwigs in specific. In this study, we carried out a series of laboratory assays to examine the survivorship and locomotory behavior of D. luteipes after exposure to fresh dry residue of azadirachtin, chlorantraniliprole, and novaluron. Our results show a significant survival reduction for D. luteipes nymphs exposed to fresh residues of chlorantraniliprole and novaluron. In the behavioral studies, adults of D. luteipes stopped more often, spent more time resting (inactive), and moved more slowly immediately after exposure to chlorantraniliprole residue. These results suggest that chlorantraniliprole may mediate an impaired movement and a behavior arrestment of earwigs after contact with this insecticide fresh residue. This could translate into reduced foraging efficiency, and increase exposure and insecticide uptake. Although chlorantraniliprole and novaluron showed a potential to undermine the biological control provided by earwigs, it is yet essential to conduct field trials in order to confirm our laboratory results.

Identification of Meliatoxins in Melia azedarach Extracts Using Mass Spectrometry for Quality Control.

Indiscriminate use of synthetic pesticides can be hazardous to both humans and the environment, but the use of natural products as a source of bio-based products, such as Melia azedarach extracts, is an interesting approach to overcome these hazards. Unfortunately, the limonoids found in M. azedarach with desired insecticidal properties (e.g. azadirachtin) may also be present with limonoids toxic to mammals. The goal of this report was to develop a fast and reliable MS-based experiment to characterize meliatoxins in crude extracts of M. azedarach, in order to provide unequivocal assessment of the safety for extracts for application in the field. MS and MS/MS experiments using MALDI ionization were evaluated as tools for the assignment of characteristic ions produced by each meliatoxin in crude extracts.The use of different experiments in combination, such as the analysis of fragment m/z 557 and [M + Na]+ (adducts ions m/z 681 and m/z 667), MALDI-MS can be used for detection of meliatoxins A1/B1 or A2/B2 in a crude extract and may be used to discriminate meliatoxins A from B, respectively. Subsequent MS/MS experiments can distinguish between the presence of group 1 and/or 2 in each class of meliatoxins classifying the proposed approach as a quick and efficient quality control method of meliatoxins in real M. azedarach samples.

Pyranochromones from Dictyoloma vandellianum A. Juss and Their Cytotoxic Evaluation.

One new chromone 3,3-dimethylallylspatheliachromene methyl ether (1), as well as five known chromones, 6-(3-methylbut-2-enyl) allopteroxylin methyl ether (2), 6-(3-methylbut-2-enyl) allopteroxylin (3), 3,3-dimethylallylspatheliachromene (4), 5-O-methylcneorumchromone K (5) and spatheliabischromene (6), two alkaloids, 8-methoxy-N-methylflindersine (7) and 8-methoxyflindersine (8), and two limonoids, limonin diosphenol (9) and rutaevin (10), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16-F10, HepG2, K562 and HL60 and non-tumor cells PBMC were evaluated for compounds 1 - 6. Compound 1 was the most active showing IC50 values ranging from 6.26 to 14.82 µg/ml in B16-F10 and K562 cell lines, respectively, and presented IC50 value of 11.65 µg/ml in PBMC cell line.

Population-level effects of abamectin, azadirachtin and fenpyroximate on the predatory mite Neoseiulus baraki.

The coconut production system, in which the coconut mite Aceria guerreronis is considered a key pest, provides an interesting model for integration of biological and chemical control. In Brazil, the most promising biological control agent for the coconut mite is the phytoseiid predator Neoseiulus baraki. However, acaricides are widely used to control the coconut mite, although they frequently produce unsatisfactory results. In this study, we evaluated the simultaneous direct effect of dry residue contact and contaminated prey ingestion of the main acaricides used on coconut palms (i.e., abamectin, azadirachtin and fenpyroximate) on life-history traits of N. baraki and their offspring. These acaricides are registered, recommended and widely used against A. guerreronis in Brazil, and they were tested at their label rates. The offspring of the exposed predators was also evaluated by estimating the instantaneous rate of population increase (r i ). Abamectin compromised female performance, whereas fenpyroximate did not affect the exposed females (F0). Nonetheless, fenpyroximate strongly compromised the offspring (F1) net reproductive rate (R0), intrinsic rate of population growth (r i ), and doubling time (DT). In contrast, fenpyroximate did not have such effects on the 2nd generation (F2) of predators with acaricide-exposed grandparents. Azadirachtin did not affect the predators, suggesting that this acaricide can be used in association with biological control by this predatory species. In contrast, the use of abamectin and fenpyroximate is likely to lead to adverse consequences in the biological control of A. guerreronis using N. baraki.

Acute exposure to the biopesticide azadirachtin affects parameters in the gills of common carp (Cyprinus carpio).

The biopesticide, azadirachtin (Aza) is less hazardous to the environment, but may cause several toxic effects in aquatic organisms. The Cyprinus carpio (n=12, for all concentrations) after 10days of acclimation under controlled conditions, were exposed at 20, 40, and 60µL/L of Aza during 96h. After this period, fish were anesthetized and euthanized then mucus layer and gills collected. In this study, the effects of exposure to different Aza concentrations were analysed through a set of biomarkers: Na(+)/K(+-)ATPase, lipid peroxidation (TBARS), protein carbonyl (PC), superoxide dismutase (SOD), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx), non-protein thiols (NPSH), ascorbic acid (AsA) and histological parameters and, yet, protein and glucose concentration in the surface area of mucous layer. Na(+)K(+-)ATPase was inhibited at 40 and 60µL/L compared to control. TBARS decreased at 40µL/L compared to control. PC, SOD and GST increased at 60µL/L in comparison to control. CAT increased at 20 and 60µL/L, and GPx increased in all Aza concentrations compared to control. NPSH decreased and AsA increased in all concentrations in comparison to control. Histological analyses demonstrated an increase in the intensity of the damage with increasing Aza concentration. Alterations in histological examination were elevation and hypertrophy of the epithelial cells of the secondary filament, hypertrophy and hyperplasia of the mucous and chlorate cells and lamellar aneurism. Glucose and protein concentrations in mucus layer increased at 60µL/L compared to control. In general, we suggest that 60µL/L Aza concentration affected several parameters causing disruptions carp metabolism.

Azadirachtin, a neem-derived biopesticide, impairs behavioral and hematological parameters in carp (Cyprinus carpio).

Azadirachtin (Aza) is a promisor biopesticide used in organic production and aquaculture. Although this compound is apparently safe, there is evidence that it may have deleterious effects on fish. Behavioral and hematological tests are grouped into a set of parameters that may predict potential toxicity of chemical compounds. Here, we investigate the effects of Aza, in the commercial formulation Neenmax™ , on carp (Cyprinus carpio) by defining LC50 (96 h), and testing behavioral and hematological parameters. In our study, LC50 was estimated at 80 µL/L. We exposed carp to Aza at 20, 40, and 60 µL/L, values based on 25, 50, and 75% of LC50 , respectively. At 60 µL/L, Aza promoted significant changes in several parameters, increasing the distance traveled and absolute turn angle. In addition, the same concentration decreased the time spent immobile and the number of immobile episodes. Hematological parameters, such as hematocrit, hemoglobin, hematimetrics index, and red cell distribution, were decreased at 60 µL/L Aza exposure. In conclusion, our study demonstrates that 60 µL/L Aza altered locomotor activity, motor pattern, and hematological parameters, suggesting potential toxicity to carp after acute exposure. In addition, this is the first report that evaluates the actions of a chemical contaminant using automated behavioral tracking of carp, which may be a useful tool for assessing the potential toxicity of biopesticides in conjunction with hematological tests. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1381-1388, 2016.

Biopesticide-induced behavioral and morphological alterations in the stingless bee Melipona quadrifasciata.

Because of their natural origin, biopesticides are assumed to be less harmful to beneficial insects, including bees, and therefore their use has been widely encouraged for crop protection. There is little evidence, however, to support this ingrained notion of biopesticide safety to pollinators. Because larval exposure is still largely unexplored in ecotoxicology and risk assessment on bees, an investigation was performed on the lethal and sublethal effects of a diet treated with 2 bioinsecticides, azadirachtin and spinosad, on the stingless bee, Melipona quadrifasciata, which is one of the most important pollinators in the Neotropics. Survival of stingless bee larvae was significantly compromised at doses above 210 ng a.i./bee for azadirachtin and 114 ng a.i./bee for spinosad. No sublethal effect was observed on larvae developmental time, but doses of both compounds negatively affected pupal body mass. Azadirachtin produced deformed pupae and adults as a result of its insect growth regulator properties, but spinosad was more harmful and produced greater numbers of deformed individuals. Only spinosad compromised walking activity of the adult workers at doses as low as 2.29 ng a.i./bee, which is 1/5000 of the maximum field recommended rate. In conclusion, the results demonstrated that bioinsecticides can pose significant risks to native pollinators with lethal and sublethal effects; future investigations are needed on the likelihood of such effects under field conditions.

Antiplasmodial activity of the andiroba (Carapa guianensis Aubl., Meliaceae) oil and its limonoid-rich fraction.

ETHNOPHARMACOLOGICAL RELEVANCE: From seeds of Carapa guianensis the Amazon native people extracts the andiroba oil, which is traditionally used as febrifuge, anti-malarial, insecticidal and repellant. The non-saponifiable fraction separated from the oil is rich in limonoids, which assigns its pharmacological effects. MATERIALS AND METHODS: The andiroba oil and its limonoid-rich fraction were submitted to in vitro antiplasmodial bioassay using W(2) and Dd(2) strains of Plasmodium falciparum. The acute toxicity of andiroba oil was evaluated. The limonoid-rich fraction was subjected to fractionation and identified its major constituents. RESULTS: Andiroba oil and its limonoid-rich fraction inhibited the growth of W(2) clone in 100%, between 24 and 72 h, at concentrations of 8.2 µg/mL and 3.1 µg/mL, respectively. Under the same conditions, the parasitaemia of Dd(2) clone provoked by the andiroba oil showed inhibition of 31% (IC(50) >82 µg/mL) with a time-dependent relationship of 24h and inhibition of 88% (IC(50) 8.4 µg/mL) after 72 h, while for the limonoid-rich fraction the inhibition of Dd(2) clone was 56% (IC(50) 2.8µg/mL) at 24h and 82% (IC(50) 0.4 µg/mL) after 72 h. Andiroba oil in acute toxicity test with a fixed dose (LD(50) >2000 mg/kg) was not toxic The limonoids identified in the oil were gedunin, 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, 7-deacetylgedunin, 1,2-dihydro-3ß-hydroxy-7-deacetoxy-7-oxogedunin and andirobin. Gedunin and derivatives has been reputed as anti-malarials. CONCLUSION: The results support the traditional use of andiroba oil as antiplasmodial, which additionally proved not to be toxic in bioassays conducted with mice.

Modulation of T lymphocyte and eosinophil functions in vitro by natural tetranortriterpenoids isolated from Carapa guianensis Aublet.

We have previously described the anti-allergic activities of a pooled fraction of tetranortriterpenoids (TNTPs) containing 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, andirobin and methyl angolensate isolated from the seeds of Carapa guianensis. In the present study, we performed in vitro studies in order to elucidate the mechanisms by which TNTPs present their anti-allergic effects and to identify the bioactive compound(s) present in such fraction. Here, we show that in vitro incubation of eosinophils with the pooled TNTP fraction, as well as with each one of the five isolated tetranortriterpenoids, impaired the adhesion of eosinophils to tumor necrosis factor-α (TNF-α)-primed tEND.1 endothelial cells. Furthermore, the individual or pooled TNTPs impaired CCL11/eotaxin-mediated chemotaxis. By contrast, pooled TNTPs failed to inhibit adhesion and chemotaxis of T lymphocytes. However, TNTPs were able to impair anti-CD3 monoclonal antibody-induced T cell proliferation and the expression of CD25 and CD69. These data suggest that TNTPs prevent T cell activation. Pretreatment of splenocytes with the pooled TNTP fraction, as well as with each one of the five isolated TNTPs, inhibited ovalbumin (OVA)-induced in vitro production of interleukin-2, chemokine (C-C motif) ligand 11 (CCL11) and regulated on activation normal T cell expressed and secreted (RANTES, also known as CCL5). TNTPs (except 6α-acetoxygedunin) also impaired nuclear factor-κB (NFκB) nuclear translocation in OVA-challenged splenocytes. Taken together, these results demonstrate that the anti-allergic effects of TNTPs isolated from C. guianensis might rely on their ability to inhibit eosinophil migration, as well as the activation of T lymphocytes, which is shared by the five isolated TNTPs.

Insecticide survival and behavioral avoidance in the lacewings Chrysoperla externa and Ceraeochrysa cubana.

Insecticide impact on non-target species, such as insect predators and parasitoids, is an ever-growing concern in agriculture and recent studies have been shifting focus from lethal to sub-lethal effects since they may prevail in field conditions, although more difficult to assess. Synthetic insecticides are the main concern, but the recent spread of biopesticide use in agriculture draws attention, particularly the main botanical insecticide currently in use - azadirachtin. Here we assessed the lethal and behavioral sub-lethal response of predatory larvae of the lacewing species Chrysoperla externa and Ceraeochrysa cubana to two frequently used synthetic insecticides, malathion and permethrin, and to the bioinsecticide azadirachtin. The recommended field concentration of the synthetic insecticides led to low survival time of lacewing larvae from both species, in contrast with azadirachtin. However, all three compounds led to 100% mortality of the lacewing larvae from both species. Insecticide repellence (i.e., avoidance without contact) was similar for both synthetic insecticides in both species, but azadirachtin was a stronger repellent for C. externa, but not C. cubana. In addition, insecticide irritability (i.e., avoidance after contact) occurred in both lacewing species to all three insecticides tested. The notion that natural compounds are safer than synthetic compounds to non-target species is refuted in the present study, which also detected significant irritability to all of the insecticides regardless of their origin, and species-specific repellence elicited particularly by azadirachtin. Therefore, bioinsecticides should not be exempted from risk assessment, and non-target sub-lethal effects should not be neglected when considering potential insecticide use in agriculture.

Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world.

Botanical insecticides have long been touted as attractive alternatives to synthetic chemical insecticides for pest management because botanicals reputedly pose little threat to the environment or to human health. The body of scientific literature documenting bioactivity of plant derivatives to arthropod pests continues to expand, yet only a handful of botanicals are currently used in agriculture in the industrialized world, and there are few prospects for commercial development of new botanical products. Pyrethrum and neem are well established commercially, pesticides based on plant essential oils have recently entered the marketplace, and the use of rotenone appears to be waning. A number of plant substances have been considered for use as insect antifeedants or repellents, but apart from some natural mosquito repellents, little commercial success has ensued for plant substances that modify arthropod behavior. Several factors appear to limit the success of botanicals, most notably regulatory barriers and the availability of competing products (newer synthetics, fermentation products, microbials) that are cost-effective and relatively safe compared with their predecessors. In the context of agricultural pest management, botanical insecticides are best suited for use in organic food production in industrialized countries but can play a much greater role in the production and postharvest protection of food in developing countries.