The mosquito Aedes aegypti is a primary vector for major arboviruses, and its control is mainly based on the use of insecticides. Caffeine and spent coffee grounds (CG) are potential agents in controlling Ae. aegypti by reducing survival and blocking larval development. In this study, we analyzed the effects of treatment with common CG (CCG: with caffeine), decaffeinated CG (DCG: with low caffeine), and pure caffeine on the survival, behavior, and morphology of the midgut of Ae. aegypti under laboratory conditions. Third instar larvae (L3) were exposed to different concentrations of CCG, DCG, and caffeine. All compounds significantly affected larval survival, and sublethal concentrations reduced larval locomotor activity, delayed development, and reduced adult life span. Damage to the midgut of treated larvae included changes in epithelial morphology, increased number of peroxidase-positive cells (more abundant in DCG-treated larvae), and caspase 3-positive cells (more abundant in CCG-treated larvae), suggesting that the treatments triggered cell damage, leading to activation of cell death. In addition, the treatments reduced the FMRFamide-positive enteroendocrine cells and dividing cells compared to the control. CG and caffeine have larvicidal effects on Ae. aegypti that warrant field testing for their potential to control mosquitoes.

Despite the increased attention for temporal aspects of stressor interactions and for effects of warming in ecotoxicological studies, we lack knowledge on how different exposure durations to warming may affect pesticide sensitivity. We tested how three types of exposure duration to 4 °C warming (acute, developmental and transgenerational exposure to 24 °C vs 20 °C) shape the effect of the pesticide chlorpyrifos on two ecologically relevant fitness-related traits of mosquito larvae: heat tolerance and antipredator behaviour. Transgenerational (from the parental generation) and developmental (from the egg stage) warming appeared energetically more stressful than acute warming (from the final instar), because (i) only the latter resulted in an adaptive increase of heat tolerance, and (ii) especially developmental and transgenerational warming reduced the diving responsiveness and diving time. Exposure to chlorpyrifos decreased the heat tolerance, diving responsiveness and diving time. The impact of chlorpyrifos was lower at 24 °C than at 20 °C indicating that the expected increase in toxicity at 24 °C was overruled by the observed increase in pesticide degradation. Notably, although our results suggest that transgenerational warming was energetically more stressful, it did reduce the chlorpyrifos-induced negative effects at 24 °C on heat tolerance and the alarm escape response compared to acute warming. Our results provide important evidence that the exposure duration to warming may determine the impact of a pesticide under warming, thereby identifying a novel temporal aspect of stressor interactions in risk assessment.

Due to increased use of agrochemicals and growing concerns about ecotoxicology, the development of new insecticides, moving away from those with neurotoxic and broad spectrum effects towards insecticides that are safer for the environment and nontarget beneficial species, has been a research priority. Novaluron stands out among these newer insecticides, is an insect growth regulator that is used for the control of insect pests in crops grown close to mulberry plantations. Mulberry serves as food for the silkworm Bombyx mori, which is a nontarget insect of great economic importance to silk production. We investigated the lethal and sublethal effects of Novaluron on the development of B. mori. Larvae were segregated into experimental groups: the control groups (CGs) and the treatment groups (TGs), which were treated with the Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed: larval mortality, changes in the insect life cicle and cytotoxic effects on the midgut cells. This is the first report about the Novaluron’s effects on B.mori. We detected rupture in the integument, complete cessation of feeding, late development, incomplete ecdysis and production of defective cocoons. After 240 h of exposure, there was 100% mortality in TG larvae exposed in the 3rd instar and 20% mortality from larvae exposed in the 5th instar. Cytotoxic effects was observed, such as dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum in addition to changes in mitochondria, the presence of large digestive vacuoles and intercellular spaces and the presence of active caspase. Novaluron exposure impairs the midgut and may affect the physiological functions of this organ. Novaluron additionally compromises several phases of insect development, indicating the importance of toxicology studies that utilize different life stages of nontarget species to evaluate the safe use of insecticides.

The hydrocarbon phenanthrene is an organic compound commonly found in the environment. In aquatic ecosystems, it is highly toxic to organisms, although little is known about its effects on sediment-dwelling organisms. The purpose of this study was to evaluate phenanthrene effects on biochemical, histological, and ontogenetic levels in larvae of the sediment-dwelling invertebrate Chironomus sancticaroli at acute and chronic exposure. Lethal concentrations were estimated and toxicity (acute-96 h and chronic- 8 d) tests were performed at phenanthrene concentrations from 0.12 to 1.2 mg L⁻¹. At acute and chronic exposure, we evaluated acetylcholinesterase (AChE), alpha esterase (EST-α), and beta esterase (EST-β) activities as well as histological alterations. In the assays with chronic exposure, effects on larval development were estimated using antennae length (instar estimative) and body length (growth estimative). The EST-α showed a significantly increased activity after 48 h at acute exposure to high concentrations of phenanthrene, while EST-β activity was increased after 48 and 72 h at acute exposure at higher concentrations and at 0.12 mg L⁻¹ at chronic exposure. At acute exposure, the midgut showed alterations such as brush border disruption, gastric caeca regression, and lumen area reduction; the fat body showed nuclear alteration in the trophocytes, while the Malpighian tubules showed brush border reduction and the salivary glands were subject to cytoplasm vacuolation. At chronic exposure, the same alterations were observed, in addition to vacuolar coalescence in the trophocytes of the fat body. Regarding larval development, a reduction of body length was observed with increasing phenanthrene concentrations. Similarly, molting was delayed; in the control group, all larvae were in the fourth instar, while at higher phenanthrene concentrations, larvae were predominantly in the third instar. Phenanthrene had toxic effects on this chironomid, indicating risks for natural populations.

Lange's metalmark butterfly, Apodemia mormo langei Comstock, is in danger of extinction due to loss of habitat caused by invasive exotic plants which are eliminating its food, naked stem buckwheat. Herbicides are being used to remove invasive weeds from the dunes; however, little is known about the potential effects of herbicides on butterflies. To address this concern we evaluated potential toxic effects of three herbicides on Behr's metalmark, a close relative of Lange's metalmark. First instars were exposed to recommended field rates of triclopyr, sethoxydim, and imazapyr. Life history parameters were recorded after exposure. These herbicides reduced the number of adults that emerged from pupation (24–36%). Each herbicide has a different mode of action. Therefore, we speculate that effects are due to inert ingredients or indirect effects on food plant quality. If these herbicides act the same in A. mormo langei, they may contribute to the decline of this species.

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) can be transferred from genetically engineered crops to herbivores to natural enemies. For the lady beetle Harmonia axyridis, we investigated potential uptake of Cry proteins from the gut to the body and intergenerational transfer. Third and fourth instar H. axyridis fed with pollen or spider mites from SmartStax maize contained substantial amounts of Cry1A.105, Cry1F, Cry2Ab2, Cry3Bb1, and Cry34Ab1. Cry protein concentrations in lady beetle larvae were typically one order of magnitude lower than in the food. When H. axyridis larvae were fed Bt maize pollen, median amounts of Cry protein in the non-feeding pupae were below the limit of detection except for small amounts of Cry34Ab1. No Cry protein was detected in pupae when spider mites were used as food. Cry protein concentrations decreased quickly after H. axyridis larvae were transferred from pollen or spider mites to Bt-free food. Aphids contained very low or no detectable Cry protein, and no Cry protein was found in H. axyridis larvae fed with aphids, and in pupae. When H. axyridis adults were fed with Bt maize pollen (mixed with Ephestia kuehniella eggs), the median concentrations of Cry proteins in lady beetle eggs were below the limit of detection except for Cry34Ab1 in eggs laid later in adult life. No Bt protein was detected in eggs laid by H. axyridis females fed with aphids from Bt maize. Our results confirm previous observations that Cry proteins are degraded and excreted quickly in the arthropod food web without evidence for bioaccumulation. Despite the fact that small amounts of Cry proteins were detected in some samples of the non-feeding pupal stage of H. axyridis as well as in eggs, we conclude that this route of exposure is unlikely to be significant for predators or parasitoids in a Bt maize field.

The effect of microplastics (MP) exposure on the chironomid species Chironomus riparius Meigen, 1804 was investigated using the OECD sediment and water toxicity test. Chironomid larvae were exposed to an environmentally relevant low microplastics concentration (LC), a high microplastics concentration (HC) and a control (C). The LC was 0.007 g m⁻² on the water surface + 2 g m⁻³ in the water column + 8 g m⁻² in the sediment, and the HC was 10 X higher than this for each exposure. The size of the majority of the manufactured microplastic pellets varied between 20 and 100 μm. The MP mixture consisted of: polyethylene-terephtalate (PET), polystyrene (PS), polyvinyl-chloride (PVC) and polyamide (PA) in a ratio of 45%: 15%: 20%: 20%, respectively, for the sediment exposure; 100% polyethylene for the water column exposure; and 50% polyethylene: 50% polypropylene for the water surface exposure. Different endpoints were monitored, including morphological changes in the mandibles and mentums of 4th instar larvae, morphological changes in the wings, mortality, emergence ratio, and developmental time. A geometric morphometric analysis showed a tendency toward widening of the wings, elongation of the mentums and changing the shape of the mandibles in specimens exposed to both concentrations of microplastics. The development time of C. riparius was significantly prolonged by the MP treatment: 13.8 ± 0.5; 14.4 ± 0.6; and 15.3 ± 0.4 days (mean ± SD) in the C, LC, and HC, respectively. This study indicates that even environmentally relevant concentrations of MP mixture have a negative influence on C. riparius, especially at the larval stage.

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica.

The genus Artemia sp. has been accepted as a reliable model organism for aquatic toxicity and nanotoxicity experiments, as far as the ISO TS 20787 has recently been published to standardize nanotoxicity test with this organism. Experimental and environmental conditions may affect the toxicity of nanomaterials on aquatic organisms including Artemia sp. nauplii. In this study, acute toxicity effects of silver nanoparticles (AgNPs) on the nauplii of Artemia salina was investigated under various conditions (e.g. different lights, salinities, temperatures, volume and agitation of exposure media and instar stages of nauplii). The EC values were calculated using Probit program and all data were analyzed statistically by SPSS software. At all test conditions, the immobilization rate of Artemia nauplii increased in a concentration-dependent manner (P < 0.05). The sensitivity of instar stage II to different concentrations of AgNPs was significantly higher than instar I (P < 0.05). The toxicity effect of AgNPs was affected by alteration of environmental conditions, so that the effective concentration (EC) values for instar I of A. salina decreased with increasing water temperature, decreasing water salinity and in continuous darkness condition. The EC50 value of AgNPs was significantly lower in 100 mL beakers (21.35 ± 5.67 mg L−1) than 10 mL well plates (42.44 ± 11.30 mg L−1). Agitation of exposure media did not affect the toxicity of AgNPs. The results indicated that the experimental and environmental conditions influence on the toxicity of AgNPs in the nauplii of A. salina.

Compatibility assessments between selective insecticides and the natural enemies of pests are essential for integrated-pest-management programs. Chrysoperla externa and Eriopis connexa are two principal Neotropical predators of agricultural pests whose conservation in agroecosystems requires a toxicity evaluation of pesticides to minimize the impact on those beneficial insects on the environment. The objective of this work was to evaluate the toxicity of the insecticides pyriproxyfen and acetamiprid on C. externa and E. connexa eggs exposed to the maximum recommended field concentrations of each along with three successive dilutions. The survival and the immature developmental time were assessed daily until adulthood and the mean survival time calculated over a 10-day period. The cumulative survival of E. connexa was reduced at all concentrations of both insecticides, while that of C. externa was significantly decreased by ≥50 mg L⁻¹ of acetamiprid and ≥37.6 mg L⁻¹ of pyriproxyfen. In both species, the reductions occurred principally on the eggs and first larval instar. Survival curves, in general, differed from those of the controls, with the mean survival time of E. connexa being significantly shorter in insecticides treatments than that of the controls. Certain concentrations of each of the insecticide lengthened the egg and first-larval-instar developmental periods of E. connexa and C. externa, respectively. Also, pyriproxyfen reduced the first-larval-instar period and lengthened the fourth of E. connexa. Acetamiprid was more toxic to E. connexa than to C. externa at the two highest concentrations. Conversely, at those same concentrations of pyriproxyfen, the relative toxicity to the two species was reversed. The present work represents the first investigation on the comparative susceptibility of two relevant Neotropical biological control agents to acetamiprid and pyriproxyfen. Also, it highlights the necessity of assessing long-term effects in the compatibility studies between natural enemies of agricultural pests and insecticides.