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.

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.

Species of the genus Microcystis are among the most notorious cyanobacteria in eutrophic lakes worldwide, with ability present adverse effects on many aquatic organisms. In the surface sediments, Microcystis can be ingested by benthic macroinvertebrates such as Chironomus. However, the potential negative effects of Microcystis on Chironomus life history traits remain unclear. In the present study, we investigated the effect of different Microcystis diets on specific behaviors (burrowing activity, locomotion ability) and life history traits of Chironomus pallidivittatus (Diptera, Chironomidae). We also studied the interactive effects of microcystin-producing M. aeruginosa and temperature (15, 20, and 25 °C) stress on chironomid larvae. The results showed that the inhibitory effect on the cumulative emergence and burrowing activity of larvae was more severe when they were fed M. aeruginosa among the three Microcystis diets groups. Locomotion ability (i.e., locomotor distance and velocity) and adult dry weight decreased significantly in the group fed M. aeruginosa. Locomotion was significantly inhibited and mortality increased when the larvae were fed a mixture of M. aeruginosa and M. wesenbergii, which may have been the result of additive or synergistic effect of the toxins. Under the stress of lower temperature, C. pallidivittatus larvae exhibited weaker locomotion and growth ability, and the emerging adults were mostly male. At both the lower and higher temperature conditions, M. aeruginosa cause cumulative emergence decreased, and sex ratio imbalance, which inhibited the reproduction of larvae from the population perspective. The fourth-instar larvae showed better adaption to Microcystis than did the other instars. This study thus highlights the adverse effects of microcystin-producing M. aeruginosa on Chironomus. It also provides a novel perspective on how environmental factors may influence the behavior and life history traits of chironomid larvae, and how they may respond to cyanobacterial blooms and global warming.

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.

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.

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 μg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates.

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.

The current study extends our previous findings concerning the sensitivity of Lymantria dispar larvae to cadmium in light of ecotoxicological risk assessment. Here we report the results of the comet assay performed for the first time on this species. We examined the chronic effects of two cadmium concentrations (50 and 100 μg Cd/g dry food) on DNA integrity and haemocyte viability, as well as on fitness-related traits (larval mass and development duration parameters). All parameters were assessed individually and then used to calculate the integrated biomarker response (IBR) index. Egg-masses of L. dispar were collected from two locations in Serbia - the uncontaminated Homolje mountains and a metal-polluted area near Bor copper mines, smelter and refinery. Distinctive patterns in the response of these populations to cadmium exposure were noticed. In haemocytes of larvae from the pollution-free location both cadmium treatments increased the level of DNA damage, although in a similar range. Haemocyte viability and larval mass were reduced, while duration of the fourth instar and total development time were prolonged in a concentration-dependent manner. Cadmium tolerance was noticeable in the population from the metal-contaminated site at all organizational levels. Nevertheless, haemocyte viability in that population was reduced by the stronger treatment. Haemocyte viability was recognized as a promising biomarker due to the evident response of both populations to dietary cadmium. Genotoxicity, fitness-related traits and the IBR index could be used for biomonitoring of sensitive populations not previously exposed to metals.

Midge larvae (Chironomus riparius) were exposed to sediments from a deposition sampled at a site along the Rhône River (France) downstream of an industrial site releasing various perfluorinated chemicals. This sediment is characterized by high concentrations of perfluoroundecanoic acid (PFUnA) and perfluorotridecanoic acid (PFTrDA) and a low perfluorooctane sulfonate (PFOS) concentration. Concentrations of 23 perfluoroalkyl compounds, including C4–C14 carboxylate acids, C4–C10 sulfonates, and seven precursors, were analyzed in overlying and pore water, sediment, and larvae. Midge larvae accumulated carboxylate acids (C11–C14), PFOS, and two precursors (perfluorooctane sulfonamide: FOSA and 6:2 fluorotelomer sulfonic acid, 6:2 FTSA). These substances accumulated mainly during the fourth instar larvae exponential growth phase. Accumulation of 6:2 FTSA, PFUnA, and PFOS occured via trophic and tegumentary routes. Other compounds mainly accumulated from food. Kinetics followed a partition model, from which uptake and elimination constants were derived.