Essential Oil of Rosmarinus officinalis Ecotypes and Their Major Compounds: Insecticidal and Histological Assessment Against Drosophila suzukii and Their Impact on a Nontarget Parasitoid.

Essential oils (EOs) produced by plants in the Lamiaceae family may provide new insecticidal molecules. Novel control compounds are needed to control Drosophila suzukii (Matsumura), a severe economic invasive pest of thin-skinned fruit crops. Thus, we characterized the main compounds of EOs from three rosemary Rosmarinus officinalis ecotypes (ECOs) and evaluated their toxicity to D. suzukii adults, deterrence of oviposition behavior, and histological alterations in larvae. Additionally, we analyzed the lethal and sublethal effect on the pupal parasitoid Trichopria anastrephae. The main compounds identified in the R. officinalis ECOs were α-pinene, camphor and 1,8-cineole. In bioassays via topical application or ingestion, ECOs and their major compounds showed high toxicity on D. suzukii adults and a lower concentration could kill 50% and 90% of flies compared to spinetoram. The dry residues of a-pinene, 1,8-cineole, and camphor provided a repellent effect by reducing D. suzukii oviposition by ~47% compared to untreated fruit. Histological sections of 3rd instar larval D. suzukii posttreatment revealed damage to the fat body, Malpighian tubules, brain, salivary gland, and midgut, which contributed to high larval and pupal mortality. Survival and parasitism by adult T. anastrephae were not affected. Thus, R. officinalis EO and their compounds have potential for developing novel insecticides to manage D. suzukii.

Use of nontarget organism Chironomus sancticaroli to study the toxic effects of nanoatrazine.

Atrazine was banned by the European Union in 2004, but is still used in many countries. Agricultural research employing nanotechnology has been developed in order to reduce the impacts to the environment and nontarget organisms. Nanoatrazine was developed as a carrier system and have been considered efficient in weed control. However, its toxicity must be verified with nontarget organisms. In this context, the aim of the present study was to investigate ecotoxicological effects of solid lipid nanoparticles (empty and loaded with atrazine) and atrazine on Chironomus sancticaroli larvae, evaluating the endpoints: mortality, mentum deformity, development rate and biochemical biomarkers. The contaminant concentrations used were 2, 470, 950, and 1900 µg L-1 in acute (96 h) and 2 µg L-1 in subchronic (10 days) bioassays. An environmentally relevant concentration of atrazine (2 µg L-1) presented toxic and lethal effects towards the larvae. The nanoparticles loaded with atrazine showed toxic effects similar to free atrazine, causing mortality and biochemical alterations on the larvae. The nanoparticle without atrazine caused biochemical alterations and mortality, indicating a possible toxic effect of the formulation on the larvae. In the acute bioassay, most concentrations of nanoparticles loaded with atrazine were not dose dependent for the endpoint mortality. Only the atrazine concentration of 470 µg L-1 was statistically significant to endpoint mentum deformity. The atrazine and nanoparticles (with and without atrazine) did not affect larval development. The results indicate that Chironomus sancticaroli was sensitive to monitor nanoatrazine, presenting potential to be used in studies of toxicity of nanopesticides.

Molecular and biochemical evaluation of effects of malathion, phenanthrene and cadmium on Chironomus sancticaroli (Diptera: Chironomidae) larvae.

In-vitro effects of sub-lethal concentrations of malathion, phenanthrene (Phe) and cadmium (Cd) were tested on Chironomus sancticaroli larvae in acute bioassays by measuring biochemical and molecular parameters. Malathion was evaluated at 0.001, 0.0564 and 0.1006 mg L-1; Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 0.001, 3.2 and 7.4 mg L-1. The recovery test carried out at the highest concentration of each compound showed that survival of larvae exposed to Phe ranged from 4% to 5%, while the effects of malathion and Cd were irreversible, not allowing the emergence of adults. Results showed that malathion and Cd inhibited AChE, EST-α and ES-ß activities at the two highest concentrations. Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 3.2 and 7.4 mg L-1 inhibited glutathione S-transferase activity. Oxidative stress was exclusively induced by the lowest concentration of malathion considering SOD activity once CAT was unaffected by the stressors. Lipid peroxidation was registered exclusively by malathion at the two highest concentrations, and total hemoglobin content was only reduced by Cd at the two highest concentrations. The relationship among biochemical results, examined using the PCA, evidenced that malathion and Cd concentrations were clustered into two groups, while Phe only formed one group. Four hemoglobin genes of C. sancticaroli were tested for the first time in this species, with Hemoglobin-C being upregulated by malathion. The toxicity ranking was malathion > Phe > Cd, while biochemical and molecular results showed the order malathion > Cd > Phe. Our results highlight the importance of combining different markers to understand the effects of the diverse compounds in aquatic organisms.

Accelerating the Morphogenetic Cycle of the Viral Vector Aedes aegypti Larvae for Faster Larvicidal Bioassays.

Any bioassay to test new chemically synthesized larvicides or phytolarvicides against Culicidae and more harmful mosquito species, such as Aedes aegypti and Aedes albopictus, which specifically transmit dengue, yellow fever, chikungunya viral fevers as well as Zika virus, or Anopheles gambiae, a vector for malaria and philariasis, requires thousands of well-developed larvae, preferably at the fourth instar stage. The natural morphogenetic cycle of Aedes spp., in the field or in the laboratory, may extend to 19 days at room temperature (e.g., 25°C) from the first permanent contact between viable eggs and water and the last stage of larval growth or metamorphosis into flying adults. Thus, accelerated sequential molting is desirable for swifter bioassays of larvicides. We achieved this goal in Aedes aegypti with very limited strategic and low-cost additions to food, such as coconut water, milk or its casein, yeast extract, and to a lesser extent, glycerol. The naturally rich coconut water was excellent for quickly attaining the population of instar IV larvae, the most advanced one before pupation, saving about a week, for subsequent larvicidal bioassays. Diluted milk, as another food source, allowed an even faster final ecdysis and adults are useful for mosquito taxonomical purpose.

Cell damage induced by polybrominated diphenyl ethers on Chironomus sancticaroli (Diptera: Chironomidae)

Abstract Histological effects of polybrominated diphenyl ethers (PBDEs) were observed in Chironomus sancticaroli larvae which underwent acute exposure. 2,2′,4-triBDE (BDE-17), 2,2′,4,4′-tetraBDE (BDE-47) and 2,2′,4,4′,5-pentaBDE (BDE-99) were evaluated at 0.5, 2.0 and 20 μg L-1. Cytoplasm vacuolisation of oenocytes was observed in the larvae exposed to BDE-17 and BDE-47. Cuénot cells were disrupted at the brush border as an effect of the three evaluated congeners highlighting BDE-47 at 2.0 μg L-1; 60% of larvae displayed this disruption. The midgut showed changes in the morphology of apex cells located next to the lumen of region I exposed to BDE-17 and BDE-47, while BDE-99 induced a narrowing of the lumen diameter. Significant cytoplasm vacuolisation of the larvae exposed to BDE-47 and BDE-99 was observed in region II of the midgut. Salivary glands showed acidophilic granules in the cytoplasm exposed to BDE-17 and BDE-47. The results showed that the tissues of C. sancticaroli were sensitive to flame retardants; these histopathologies can compromise the health and physiology of this organism, highlighting the concern with the presence of PBDEs in freshwater sediments.

Data of insecticide effects of natural compounds against third instar larvae of Cochliomyia macellaria.

Morphological biomarkers can be used to establish a diagnosis of fly larvae structural damage and toxicity to target cells by biopesticide candidates. Insecticide activity of natural compounds such as Curcuma longa essential oil (CLLEO) extracted from leaves, and its major constituent α-phellandrene have proven to be a novel biopesticide candidate against third instar larvae (L3) of Cochliomyia macellaria. In this way, groups of 20 L3 were placed on filter paper impregnated with different concentrations of CLLEO, from 0.31 to 2.86 µL/cm2 and α-phellandrene, from 0.29 to 1.47 µL/cm2. The extracts were solubilized in ethanol. Data shown in this article is related to the research article "Can an overlooked by-product from turmeric industry be effective for myiasis control?" Chaaban et al., 2019. Data on L3 toxicity was observed after 6 and 24h of contact with both extracts, as well as a marked reduction of L3 movement, color changes in the cuticle and progressive darkening in their body. Major cuticle damage and L3 mortality were reported.

Enrofloxacin and Roundup® interactive effects on the aquatic macrophyte Elodea canadensis physiology.

The co-occurrence of aquatic contaminants, such as antibiotics and herbicides, has motivated investigations into their interactive effects on aquatic organisms. We examined the combined effects of environmental concentrations of the antibiotic Enrofloxacin (Enro; 0-2.25 µg l-1) and Roundup OriginalDI (Roundup®; 0-0.75 µg active ingredient l-1), a glyphosate based-herbicide, on Elodea canadensis. Enro alone was not toxic, but the plants were highly sensitive to Roundup® whose toxicity is related to the induction of oxidative stress. The metabolism of Enro by plants into Ciprofloxacin (Cipro) was observed, and although former is not phytotoxic, oxidative events associated with Cipro generation were observed. The activity of cytochrome P450 was shown to be involved in Enro degradation in E. canadensis. As a cytochrome P450 inhibitor, Roundup® decreases Enro metabolism in plants. Enro, in turn, increases glyphosate uptake and toxicity, so that Enro and Roundup® have synergistic effects, disrupting the physiological processes of E. canadensis. Our results suggest E. canadensis as a potential candidate for the reclamation of Enro in contaminated waters, but not for Roundup® due to its high sensitivity to that herbicide.

Insecticide activity of Curcuma longa (leaves) essential oil and its major compound α-phellandrene against Lucilia cuprina larvae (Diptera: Calliphoridae): Histological and ultrastructural biomarkers assessment.

Lucilia cuprina, known as the Australian blowfly, is of high medico-sanitary and veterinary importance due to its ability to induce myiasis. Synthetic products are the most frequent form of fly control, but their indiscriminate use has selected for resistant populations and accounted for high levels of residues in animal products. This study aimed to assess the effect of essential oil from leaves of Curcuma longa (CLLEO), and its major compound α-phellandrene against L. cuprina L3. An additional goal was to determine the morphological alterations in target organs/tissues through ultrastructural assessment (SEM) and light microscopy, as well as macroscopic damage to cuticle induced by CLLEO. Groups of 20 L3 were placed on filter paper impregnated with increasing concentrations of CLLEO (0.15 to 2.86 µL/cm2) and α-phellandrene (0.29 to 1.47 µL/cm2). Efficacy was determined by quantifying L3 mortality 6, 24 and 48 h after contact with CLLEO and by measuring the structural damage to L3. CLLEO and α-phellandrene inhibited adult emergence by 96.22 and 100%, respectively. Macroscopic cuticle damage, appeared as diffuse pigment and darkening of larval body, was caused by both extracts. The SEM revealed dryness on the cuticle surface, distortion of the sensorial structures and general degeneration in treated L3. Furthermore, alterations in target organs (digestive tract, fat body and brain) were noticed and shall be used as biomarkers in future attempts to elucidate the mechanism of action of these compounds. The vacuolar degeneration and pyknotic profiles observed in the brain tissue of treated larvae with both extracts and the decreased motility within <6 h after treatment leads us to suggest a neurotoxic activity of the products. This work demonstrates the potential use of CLLEO and α-phellandrene as bioinsecticides to be used against L. cuprina, representing an ecofriendly alternative for myiasis control in humans and animals.

Effects of Ciprofloxacin and Roundup on seed germination and root development of maize.

Their continuous release into the environment, associated with their inherent biological activity, has motivated investigations into the detrimental effects of antibiotics and herbicides in natural and agricultural ecosystems. In this study, the interactive effects of the antibiotic ciprofloxacin (Cipro) and the herbicide Roundup on seed germination and root development were investigated. Although both compounds act as inhibitors of the mitochondrial electron transport chain in seeds, neither Cipro nor Roundup disrupted germinability of maize seeds. However, Cipro accelerated germination by promoting ROS accumulation in seeds, while the stimulatory effect of Roundup on ROS-scavenging enzymes (catalase and ascorbate peroxidase) seems to prevent ROS-signaling, delaying the germination process. Roundup reduced root elongation, possibly due to its interference with auxin production, thereby preventing cell division, while Cipro stimulated root elongation by increasing root oxidative status. Cipro and Roundup showed antagonistic effects on maize seeds and root physiology. The presence of the antibiotic is likely not to disturb plant development; however, its stimulatory effects were not sufficient to overcome the deleterious effects of Roundup. According to our results, glyphosate-based herbicides must be carefully used during maize cropping and although antibiotics such as Cipro may not negatively impact agricultural production, their accumulation by crops must be investigated since this can be a pathway of antibiotic-insertion into the food chain.

Cuticular damage of Lucilia cuprina larvae exposed to Curcuma longa leaves essential oil and its major compound α-phellandrene.

Morphological biomarkers as the histopathological assessment and scanning electron microscopy can be used to establish a diagnosis of structure damage and intoxication of target cells by new biopesticide candidate. In this sense, cuticle damage caused by active substances in larvae exposed to biopesticides can help to elucidate the mode action. Thus, insecticide activity analysis of essential oil of Curcuma longa leaves and its major compound α-phellandrene have proven to be a new biopesticide candidate against third instar larvae (L3) of the Australian blowfly Lucilia cuprina. In this way, groups of 20 L3 were placed on filter paper, impregnated with ranging concentrations (from 0.15 to 2.86 µL/cm2) of C. longa leaves EO and (0.29-1.47 µL/cm2) to α-phellandrene. The extracts were solubilized in ethanol. Progressive darkening in the body of L3, marked reduction of movement, color changes in larval cuticle and dead were observed 6 and 24 h after contact with both extracts.

Morpho-histological characterization of immature of the bioindicator midge Chironomus sancticaroli Strixino and Strixino (Diptera, Chironomidae)

ABSTRACTChironomidae immature are used as bioindicators of sediment quality in aquatic ecosystems and ecotoxicological assays. Histological descriptions for this family are outdated and limited and there are no studies with Neotropical species. The aim of this study was to describe the tissue architecture of several organs of the larva of Chironomus sancticaroli. For the description of the histological pattern, the larvae were fixed in Duboscq solution for insects at 56 °C, followed by routine histologic processing, infiltration in paraffin, and the sections were stained with Hematoxylin–Eosin. After examining the slides, the tube digestive, salivary gland, excretory, nervous, endocrine, circulatory, and integumentary systems and fat body were histologically characterized. The histology allows evaluation of cell morphology, and for being not expensive and easily accessible can be routinely used in biomonitoring. In addition, is a useful tool in ecotoxicological assays and allow to evaluate biomarkers at tissue and cell levels.