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.

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 decreasing ocean pH seems to adversely affect marine organisms, including crustaceans, which leads to potential threats to seafood safety. The present investigation evaluated the effect of seawater acidification on the edible marine mud crab Scylla serrata instars. The experimental setup was designed using a multi-cell cage based system assembled with 20 pre holed PVC pipes containing 20 individual crabs to avoid cannibalism. The crab instars were exposed to CO₂ driven acidified seawater at pH 7.8 (IPCC forecast pH at the end of the 21ˢᵗ century), 7.6, 7.4, 7.2, and 7.0 for 60 days. The crabs reared in seawater without acidification at pH 8.2 served as control. The present study revealed a notable decrease in survival, feed intake, growth, molting, tissue biochemical constituents, minerals, chitin, and alkaline phosphatase in S. serrata instar reared in acidified seawater, denotes the adverse effect of seawater acidification on crabs. The significant elevations in antioxidants, lipid peroxidation, and metabolic enzymes in all acidified seawater compared to ambient pH indicates the physiological stress of the crabs' instars. The changes in the metabolic enzymes reveal the metabolism of protein and glucose for additional energy required by the crabs to tolerate the acidic stress. Hence, the present study provides insight into the seawater acidification can adversely affect the crab S. serrata.

Morphological tools can assist in the evaluation of effects of insecticides on non-target insects. Pyriproxyfen, a juvenile hormone analog, is known to interfere with growth and metamorphosis of insects. However, there are studies showing indirect effects on natural enemies, including green lacewings. Few prior studies describe morphological effects of pyriproxyfen on target insect organs, especially on natural enemies. Through morphological tools, this study aimed to characterize the midgut and fat body, both important organs of digestion and great metabolic activity respectively, of the predator Ceraeochrysa claveri after chronic exposure to pyriproxyfen. Larvae of C. claveri were fed Diatraea saccharalis egg clusters treated with pyriproxyfen in solution of 50 or 100 mg a.i. L⁻¹ throughout the larval stage. The biological data revealed significant increases in development time, especially in the third instar, and in cumulative mortality from the prepupal into the pupal stage. Morphological analysis of adult midgut (≤24 h old) showed damage including formation of epithelial folds, intercellular spaces, emission of cytoplasmic protrusions. Both fat body regions presented decrease of lipid droplets, vacuolization of trophocytes and mitochondrial injury featuring a multisystemic action. In both organs, pyriproxyfen exposure induced significant oxidative stress by mitochondrial superoxide production. Cytoprotective responses were induced in midgut and fat body cells by augmenting the number of cytoplasmic granules containing calcium and expression of HSP 90. Both organs proved to be efficient in presenting histopathological alterations, showing the sensitivity and applicability of this morphological tool for evaluating other insecticides in non-target organisms.

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 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.

Using Artemia salina (A. salina) cysts (capsulated and decapsulated) and larvae [instar I (0–24 h), II (24–48 h) and III (48–72 h)] as experimental models, developmental toxicity of oxidized multi-walled carbon nanotubes (O-MWCNTs) was evaluated. Results revealed that hatchability of capsulated and decapsulated cysts was significantly decreased (p < 0.01) following exposure to 600 mg/L for 36 h. Mortality rates were 33.8, 55.7 and 40.7% for instar I, II and III larvae in 600 mg/L. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Instar II showed the greatest sensitivity to O-MWCNTs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Effects on hatchability, mortality and swimming were accounted for O-MWCNTs rather than metal catalyst impurities. Body length was decreased with the concentrations increased from 0 to 600 mg/L. O-MWCNTs attached onto the cysts, gill and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were increased following exposure, indicating that the effects were related to oxidative stress. O-MWCNTs were ingested and distributed in phagocyte, lipid vesicle and intestine. Most of the accumulated O-MWCNTs were excreted by A. salina at 72 h, but some still remained in the organism. Data of uptake kinetics showed that O-MWCNTs contents in A. salina were gradually increased from 1 to 48 h and followed by rapidly decreased from 48 to 72 h with a range from 5.5 to 28.1 mg/g. These results so far indicate that O-MWCNTs have the potential to affect aquatic organisms when released into the marine ecosystems.

MeHg biomagnification by the phantom midge Chaoborus in relation to MeHg concentrations in their prey and its migratory behavior was investigated in two Canadian Precambrian Shield lakes. Three Chaoborus species with contrasted migratory behavior were collected in a fishless and a fish-inhabited lake. All species accumulated MeHg through their ontogenic development. In the lake inhabited by fish, all instars of Chaoborus punctipennis displayed a marked migratory behavior and were unable to biomagnify MeHg, whereas in the fishless lake, Chaoborus americanus and Chaoborus trivittatus biomagnified MeHg. Reduced biomagnification capacity of C. trivittatus, the coexisting species living with C. americanus, was also ascribed to a progressive vertical segregation with age. Growth dilution, amount and type of prey items or trophic position could not explain the different patterns of biomagnification. Our findings demonstrate that the most common invertebrate predator of temperate planktonic food webs can biomagnify mercury, contrarily to previous reports.

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.