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.

Identification of limonoids from Walsura yunnanensis and evaluation of their cytotoxicity against cancer cell lines.

Three new limonoids, walsurauias A-C (1-3), along with four known ones, were isolated from the leaves and twigs of Walsura yunnanensis C. Y. Wu. Their structures were determined on the basis of comprehensive spectroscopic data analysis. The new limonoids were screened for their cytotoxic activity (IC50 0.81-5.73 µM) against four human cancer cell lines, including A549, HepG2, HCT116 p21KO and CNE-2. And α,ß-unsaturated ketone moieties in rings A and B are essential for their cytotoxic activity. Selected compounds were further investigated. Compounds 1-3 effectively induced G2/M cell cycle arrest and apoptosis in a dose-dependent manner in cancer cells. In addition, compounds 1-3 inhibited the colony formation and compounds 2 and 3 suppressed the migration of cancer cells.

Effects of sublethal azadirachtin on the immune response and midgut microbiome of Apis cerana cerana (Hymenoptera: Apidae).

As a wildly used plant-derived insecticide, azadirachtin (AZA) is commonly reported as harmless to a range of beneficial insects. However, with the research on the effect of AZA against pollinators in recent years, various negative physiological effects on other Apidae species have been demonstrated. Thus to explore the safety of azadirachtin to Apis cerana cerana, the different physiological effects of sublethal concentration of azadirachtin on worker bees A.c.cerana has been studied. With the exposure of 5 mg·L-1 and 10 mg·L-1 azadirachtin for 5 d, the relative expression of Apidaecin, Abaecin and Lysosome genes in workers has decreased significantly at 1, 2,3 and 5 d, and the mRNA levels of Defensin 2 and Hymenoptaecin were also significantly inhibited by 10 mg·L-1 azadirachtin at each check point. Besides, the activity of midgut antioxidant enzymes Superoxide Dismutase (SOD) and Catalase (CAT) which are the first line of defence in antioxidant systems was not affected by AZA, the activity of Peroxidase (POD) showed a fluctuating pattern at 24 h and 48 h, while the activity of polyphenol oxidase (PPO) has significantly inhibited by AZA. However, through 16sRNA analysis it was observed that 5 mg·L-1 AZA did not affect the midgut microbiome colony composition and relative abundance, as well as its main function. Therefore, to a certain extent, azadirachtin is safe for workers, but we should pay more attention to the sublethal effect of AZA that also detrimental to the healthy development of the honeybee colony.

Using Azadirachtin to Transform Spodoptera frugiperda from Pest to Natural Enemy.

Spodoptera frugiperda and Rhopalosiphum maidis, as main pests, seriously harm the safety of maize. At present, chemical pesticides are mainly used to control these pests. However, due to residue and resistance problems, more green, environmentally benign, simple preventive control technology is needed. In this study, we reported the reason for the antifeedant activity of azadirachtin on S. frugiperda and proposed that S. frugiperda treated with azadirachtin would turn from pest into natural enemy. S. frugiperda showed an obvious antifeeding phenomenon to maize leaf treated with various azadirachtin concentrations (0.5~20 mg/L). It was found that maize leaf treated with 1 mg/L of azadirachtin has a stimulating effect on the antenna and sensillum basiconicum of S. frugiperda, and azadirachtin can affect the feeding behavior of S. frugiperda. Additionally, after treating maize leaves or maize leaves + R. maidis with 1 mg/L of azadirachtin, the predatory behavior of S. frugiperda changed from a preference for eating maize leaves to R. maidis. Moreover, the molting of R. maidis can promote the change of this predatory behavior. Our results, for the first time, propose that the combined control technology of azadirachtin insecticide and biological control could turn S. frugiperda from pest into natural enemy, which can effectively eliminate R. maidis and protect maize. This combined control technology provides a new way for pest management and has good ecological, environmental, and economic benefits.

The characterization, expression and activity analysis of three superoxide dismutases in Eriocheir hepuensis under azadirachtin stress.

Superoxide dismutase (SOD) can effectively eliminate of excess ROS, which causes oxidative damage to lipids, proteins, and DNA. In this study, we cloned the CuZn-SOD, cMn-SOD1, and cMn-SOD2 genes in Eriocheir hepuensis, and found that the coding sequence (CDS) lengths were 627 bp, 861 bp and 1062 bp, which encoded 208, 286, and 353 amino acids, respectively. Phylogenetic analysis indicated that all SOD genes were evolutionarily conserved, while cMn-SOD2 had an extra gap (67 amino acids) in the conserved domain compared with cMn-SOD1 without huge changes in the tertiary structure of the conserved domain, suggesting that cMn-SOD2 may be a duplicate of cMn-SOD1. qRT-PCR showed that the three SOD genes were widely expressed in all the tested tissues, CuZn-SOD and cMn-SOD1 were mostly expressed in the hepatopancreas, while cMn-SOD2 was mostly expressed in thoracic ganglia. Under azadirachtin stress, the oxidation index of surviving individuals, including the T-AOC, SOD activity, and MDA contents increased in the early stage and then remained steady except for a decrease in MDA contents in the later stage. qRT-PCR showed that the three SOD genes displayed the same trends as SOD activity in surviving individuals, and the highest expressions of CuZn-SOD in the hepatopancreas, heart, and gill were 14.16, 1.41, and 30.87 times that of the corresponding control group, respectively. The changes were 1.35, 5.77 and 3.33 fold for cMn-SOD1 and 1.62, 1.71 and 1.79 fold for cMn-SOD2, respectively. However, the activity and expression of SOD genes in dead individuals were lower than that observed in surviving individuals. These results reveal that SOD plays a significant role in the defence against azadirachtin-induced oxidative stress.

Limonin ameliorates acute pancreatitis by suppressing JAK2/STAT3 signaling pathway.

Acute pancreatitis (AP) is one of the most common acute abdomen of digestive system and has the characteristics of dangerous condition and rapid development. Limonin has been confirmed to hold anti-inflammatory and antioxidant effects in various diseases. However, its potential beneficial effect on AP and the concrete mechanisms have never been revealed. Here, two mouse models were used to investigate the protective effects of limonin on AP, the caerulein-induced mild acute pancreatitis (MAP) model and L-arginine-induced severe AP (SAP) model. Firstly, it was found that limonin administration attenuated lipase and serum amylase levels and ameliorated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. Additionally, the amelioration of AP by limonin was associated with reduced levels of inflammation initiators (IL-6, IL-1ß, CCL2, and TNF-α). Mechanistically, we found that limonin suppressed the Janus Activating Kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, as evident by the decreased levels of JAK2 and p-STAT3. And activation of JAK2 using JAK2 activator rescued the protective effects of limonin on AP. Thus, our results demonstrate that limonin can ameliorate AP in two mice models via suppressing JAK2/STAT3 signaling pathway.

Identification of azadirachtin responsive genes in Spodoptera frugiperda larvae based on RNA-seq.

The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous pest with 353 plant species as its hosts, including maize, sorghum, cotton, and rice. Azadirachtin is one of the most effective botanical insecticides. The effect of azadirachtin against S. frugiperda remains to be determined. Here we report strong growth inhibition of azadirachtin on S. frugiperda larvae under either 1.0 or 5.0 µg/g azadirachtin. To explore the relevant mechanisms, the larvae fed with normal artificial diet and with 1.0 µg/g azadirachtin exposure for 3 days were collected as samples for RNA-Seq. RNA-Seq on S. frugiperda larvae under different treatments identified a total of 24,153 unigenes, including 3494 novel genes, were identified. Among them, 1282 genes were affected by 1.0 µg/g azadirachtin exposure, with 672 up-regulated and 610 down-regulated. The impacted genes include 61 coding for detoxification enzymes (31 P450s, 7 GSTs, 11 CarEs, 7 UGTs and 5 ABC transporters), 31 for cuticle proteins, and several proteins involved in insect chitin and hormone biosynthesis. Our results indicated that azadirachtin could regulate the growth of S. frugiperda by affecting insect chitin and hormone biosynthesis pathway. The enhanced expression of detoxification enzymes might be related to detoxifying azadirachtin. These findings provided a foundation for further delineating the molecular mechanism of growth regulation induced by azadirachtin in S. frugiperda larvae.

Treating green pea aphids, Myzus persicae, with azadirachtin affects the predatory ability and protective enzyme activity of harlequin ladybirds, Harmonia axyridis.

As a natural enemy of green peach aphids, harlequin ladybirds, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), are also indirectly affected by azadirachtin. In this study, we evaluated the effects of ladybird exposure to azadirachtin through azadirachtin-treated aphids. About 2 mg/L azadirachtin treated aphid can deliver the azadirachtin to ladybird larvae in 12 and 24 h. And azadirachtin treatment affected the rate at which fourth instar larvae and adult ladybirds preyed on aphids. Furthermore, the antifeedant effect increased with increasing azadirachtin concentrations. Twelve hours after exposing fourth instar ladybird larvae to aphids treated with 10 mg/L azadirachtin, the antifeedant effect was 47.70%. Twelve hours after exposing adult ladybirds to aphids treated with 2 mg/L azadirachtin, the antifeedant effect was 67.49%. Forty-eight hours after exposing ladybird larvae to azadirachtin-treated aphids, their bodyweights were 8.37 ± 0.044 mg (2 mg/L azadirachtin), 3.70 ± 0.491 mg (10 mg/L azadirachtin), and 2.39 ± 0.129 mg (50 mg/L azadirachtin). Treatment with azadirachtin affected the ability of ladybirds to prey on aphids. The results indicated that the instant attack rate of ladybird larvae and adults and the daily maximum predation rate were reduced by azadirachtin treatment. Superoxide dismutase (SOD), peroxidase (POD), and peroxide (CAT) enzyme activities of ladybirds were affected after feeding on aphids treated with azadirachtin. Azadirachtin has certain antifeedant effects on ladybirds and affects the ability of ladybirds to prey on aphids and the activities of SOD, POD, and CAT enzymes, which results in inhibition of normal body development.

Acute toxicity of organophosphate pesticide profenofos, pyrethroid pesticide λ cyhalothrin and biopesticide azadirachtin and their sublethal effects on growth and oxidative stress enzymes in benthic oligochaete worm, Tubifex tubifex.

The present study was aimed to assess the acute toxicity of organophosphate pesticide, profenofos; synthetic pyrethroid pesticide, λ cyhalothrin and biopesticide, azadirachtin and their sublethal effects on growth rate and oxidative stress biomarkers in Tubifex tubifex in vivo. The results showed that 96 h LC50 value of profenofos, λ cyhalothrin and azadirachtin to Tubifex tubifex are 0.59, 0.13 and 82.15 mg L-1 respectively. Pesticide treated worms showed several behavioral abnormalities including increased mucus secretion, erratic movements, wrinkling activity and decreased clumping tendency during acute exposure. The percentage of autotomy increased significantly (p < 0.05) with the increasing concentration of the pesticides at 96 h of exposure. Sublethal concentrations of profenofos (0.059 and 0.118 mg L-1), λ cyhalothrin (0.013 and 0.026 mg L-1) and azadirachtin (8.2 and 16.4 mg L-1) caused significant alterations in growth rate and oxidative stress enzymes in T. tubifex during 14 days exposure period. The growth rate of the pesticide exposed worms decreased significantly (P < 0.05) in a concentration and duration-dependent manner. Superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-s-transferase (GST) and glutathione peroxidase (GPx) demonstrated a noteworthy (p < 0.05) initial induction followed by a subsequent reduction, while catalase (CAT) and malondialdehyde (MDA) exhibited noteworthy induction (p < 0.05) all through the exposure time. Through principal component analysis, correlation matrix, and integrated biomarker response, the effects of profenofos, λ cyhalothrin and azadirachtin on T. tubifex were distinguished. These results indicate that exposure to profenofos, λ cyhalothrin and azadirachtin affect survivability, change the behavioral responses, reduce the growth rate and induce oxidative stress enzymes in T. tubifex.

Cytochrome P450 3A4-Mediated Bioactivation and Its Role in Nomilin-Induced Hepatotoxicity.

Nomilin is a furan-containing triterpenoid isolated from the medicinal plants of citrus. The aim of this study was to investigate the in vitro and in vivo bioactivation of nomilin and the role in nomilin-induced hepatotoxicity. Microsomal incubations of nomilin supplemented with NADPH and GSH or NAL resulted in the detection of six conjugates (M1-M6). The structures of the metabolites were characterized based on LC-HRMS and NMR. Nomilin was bioactivated to a reactive cis-butene-dial (BDA) intermediate dependent on NADPH, and this intermediate suffered from the reaction with the nucleophiles (GSH and NAL) to form stable adducts. M1-M4 were identified as pyrrole derivatives, and M5 and M6 were pyrrolinone derivatives. M1 was further chemically synthesized and characterized by 13C NMR spectroscopy. M1 was the major metabolite detected in mice bile. Pretreatment with ketoconazole significantly reduced the formation of M1 in mice bile, while pretreatment with rifampicin significantly increased the formation of M1. Chemical inhibition together with recombinant human CYP450 phenotyping demonstrated that CYP3A4 was the major enzyme contributing to the bioactivation of nomilin. Toxicity study suggested that nomilin displayed dose-dependent liver injury in mice, while tetrahydro-nomilin was found to be nonhepatotoxic. Pretreatment with ketoconazole prevented mice from nomilin-induced liver injury. The liver injury induced by nomilin was deteriorated when the mice were pretreated with rifampicin. These findings provide evidence that CYP3A4-mediated bioactivation was indispensable in nomilin-induced hepatotoxicity.

Biological Activities of Gedunin-A Limonoid from the Meliaceae Family.

Gedunin is an important limonoid present in several genera of the Meliaceae family, mainly in seeds. Several biological activities have been attributed to gedunin, including antibacterial, insecticidal, antimalarial, antiallergic, anti-inflammatory, anticancer, and neuroprotective effects. The discovery of gedunin as a heat shock protein (Hsp) inhibitor represented a very important landmark for its application as a biological therapeutic agent. The current study is a critical literature review based on the several biological activities so far described for gedunin, its therapeutic effect on some human diseases, and future directions of research for this natural compound.

Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents.

As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms. Previous study have demonstrated that apoptosis occurred in Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. However, the selection of reference genes influences the accuracy of results profoundly. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin; RPL13 and RPL3 for cells treated with camptothecin; EF1α and ß-1-TUB for cells irradiated under ultraviolet; and EF1α and EF2 for combinational analyses of samples. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells.

Limonin: A Review of Its Pharmacology, Toxicity, and Pharmacokinetics.

Limonin is a natural tetracyclic triterpenoid compound, which widely exists in Euodia rutaecarpa (Juss.) Benth., Phellodendron chinense Schneid., and Coptis chinensis Franch. Its extensive pharmacological effects have attracted considerable attention in recent years. However, there is no systematic review focusing on the pharmacology, toxicity, and pharmacokinetics of limonin. Therefore, this review aimed to provide the latest information on the pharmacology, toxicity, and pharmacokinetics of limonin, exploring the therapeutic potential of this compound and looking for ways to improve efficacy and bioavailability. Limonin has a wide spectrum of pharmacological effects, including anti-cancer, anti-inflammatory and analgesic, anti-bacterial and anti-virus, anti-oxidation, liver protection properties. However, limonin has also been shown to lead to hepatotoxicity, renal toxicity, and genetic damage. Moreover, limonin also has complex impacts on hepatic metabolic enzyme. Pharmacokinetic studies have demonstrated that limonin has poor bioavailability, and the reduction, hydrolysis, and methylation are the main metabolic pathways of limonin. We also found that the position and group of the substituents of limonin are key in affecting pharmacological activity and bioavailability. However, some issues still exist, such as the mechanism of antioxidant activity of limonin not being clear. In addition, there are few studies on the toxicity mechanism of limonin, and the effects of limonin concentration on pharmacological effects and toxicity are not clear, and no researchers have reported any ways in which to reduce the toxicity of limonin. Therefore, future research directions include the mechanism of antioxidant activity of limonin, how the concentration of limonin affects pharmacological effects and toxicity, finding ways to reduce the toxicity of limonin, and structural modification of limonin-one of the key methods necessary to enhance pharmacological activity and bioavailability.

Azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae by releasing cathepsin in the midgut.

Azadirachtin, a botanical insecticide with high potential, has been widely used in pest control. Azadirachtin has shown strong biological activity against Bactrocera dorsalis in toxicological reports, but its mechanism remains unclear. This study finds that azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae. The larval weights and body sizes of the azadirachtin-treated group were significantly less than those of the control group in a concentration-dependent manner. Further, pathological sections revealed that azadirachtin destroyed the midgut cell structure and intestinal walls, while TUNEL staining showed that azadirachtin could induce apoptosis of midgut cells, and Western blot analysis indicated that Bcl-XL expression was inhibited and cytochrome c (CytC) released into the cytoplasm. The results also imply azadirachtin-induced structural alterations in the Bactrocera dorsalis larvae midgut by activation of apoptosis. RNA-seq analysis of midgut cells found that 482 and 708 unique genes were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were enriched in apoptotic and lysosomal signaling pathways and included 26 genes of the cathepsin family. qRT-PCR verified the expression patterns of some DEGs, indicating that Cathepsin F was upregulated by 278.47-fold and that Cathepsin L and Cathepsin D were upregulated by 28.06- and 8.97-fold, respectively. Finally, association analysis between DEGs and DEMs (differentially expressed metabolites) revealed that azadirachtin significantly reduced the digestion and absorption of carbohydrates, proteins, fats, vitamins and minerals in the midgut. In conclusion, azadirachtin induces the release of cathepsin from lysosomes, causing apoptosis in the midgut. Ultimately, this leads to reduced digestion and absorption of nutrient metabolites in the midgut and inhibition of the growth and development of Bactrocera dorsalis larvae.

Bio-pesticide applications may impair the pollination services and survival of foragers of honey bee, Apis cerana Fabricius in oilseed brassica.

Honey bees and brassica plants are co-evolved and due to the peculiar floral characters, mustard (Brassica juncea) plants are strongly dependent on bees for survival. Mustard is one of the most important oilseeds across the world. Insect pests often cause huge economic losses in mustard and their management, especially during flowering stage is very crucial to achieve maximum yield, although this step often displays undesirable effects on the foraging bees. Effects of synthetic pesticides on bees are widely documented and extensively reported. Although the numbers of pesticides/bio-pesticides are widely used in oilseed brassica's, the reports are mostly focused on neonicotinoids. To identify the bee-friendly pesticides, the study was conducted in two tier approach (i.e. laboratory and field conditions) and determined the potential impacts of widely used biopesticides on Asiatic honey bees, Apis cerana Fabricius. The LC50, LC90 and LD50 were determined for four destructive pests and honey bees, to assess their risk against honey bees. In laboratory studies, LC50's of pesticides to the honey bee was in the order of Beauveria bassiana 1.5L (4.79%) > Bacillus thuriengiensis 8SP (1.67%) > Azadirachtin 0.03 EC (1.64%) > Annonin 1 EC (1.22%) > Spinosad 2.5 SC (0.006%) > Imidacloprid 17.8SL (0.005%). Based on three essential risk assessment criteria's (viz., Selectivity ratio, Probit substitution method (%) and Hazard Ratio/Risk quotient); the Azadirachtin, Anonnin, B. bassiana and Bt var. k were found selective, and slightly to moderately toxic to the honeybee; whereas Spinosad and Imidacloprid was found non-selective and dangerous to the bees. Entomopathogenic fungus, Nomuraea rileyi was found absolutely harmless to the bees. In field studies, the relative abundance, foraging rate and foraging speed of honey bees was significantly affected in different treatments even up to 2 days of spraying. Among bio-pesticides, deterrence/repellent effect was, however, strongly observed in Annonin and Spinosad treatments. Significantly higher yield was obtained in Azadirachtin (1.43 t/ha) and Anonin (1.22 t/ha) treated plots. Except Spinosad, remaining bio-pesticides were found selective to the foraging bees, nevertheless considering the efficiency in pest control and higher yield, Azadirachtin 0.03 EC and Annonin 1 EC could be efficiently used in Integrated Pest cum Pollinator Management Programme (IPPM) in oilseed brassica's. The spraying of Spinosad may be discouraged, especially at flowering time.

Limonin attenuates the stemness of cervical carcinoma cells by promoting YAP nuclear-cytoplasmic translocation.

This work aimed to investigate the role and related mechanism of limonin in regulating the stemness of cervical carcinoma (CC) cells. In the present study, we constructed adriamycin-resistant CC cells and found that they exhibited greater stemness than parental cells. Additionally, limonin attenuated the stemness of CC cells that were resistant or sensitive to adriamycin, as evidenced by the decreases in spheroid formation capacity, stemness markers expression and ALDH1 activity, whereas limonin did not affect the viability of normal cervical epithelial cells. Furthermore, limonin enhanced adriamycin sensitivity and attenuated adriamycin resistance in CC cells. Mechanistically, the nuclear-cytoplasmic translocation of YAP, not TAZ, was promoted by limonin in CC cells. Additionally, YAP overexpression attenuated the inhibitory effects of limonin on CC cell stemness. Therefore, limonin can attenuate the stemness, and thus the chemoresistance, of CC cells by promoting the nuclear-cytoplasmic translocation of YAP.

Immunosuppressive effects of the limonoid azadirachtin, insights on a nongenotoxic stress botanical, in flesh flies.

The tetranortriterpenoid azadirachtin (Aza) is a well-known insect growth disruptor of plant origin. Although its actions on insects have been extensively studied; fragmentary reports are available from the immunological point of view. Therefore, in the present study, total (THC) and differential hemocyte counts (DHC), nodulation, phenoloxidase (PO) activity, immune-reactive lysozymes and inducible nitric oxide (NO) were assessed, as measures of immune responses, in Sarcophaga argyrostoma 3rd instars challenged individually with M. luteus or Aza, or in combination with both compared to the control larvae. THC was significantly declined after 12 h and 24 h of treatment with Aza. DHC varied considerably; in particular, plasmatocytes were significantly decreased after 36 h and 48 h of treatment with Aza; whereas granulocytes were significantly increased. Nodulation was significantly increased with the increase of time after all treatments. Challenging with M. luteus significantly increased the activity of PO in hemocytes and plasma; whereas such activity was significantly decreased after treatment with Aza or combined Aza and M. luteus. Treatment with Aza or M. luteus alone or in couple significantly increased lysozyme activity of fat body, hemocytes and plasma. However, challenging with M. luteus significantly increased NO concentration in the same tissues. A hypothetical model of Aza as a potential mutagen is presented. However, no genotoxic effect was observed through tracking apoptosis-associated changes in Aza-treated hemocytes via flow cytometry-based apoptosis detection. Our study suggests that the integration of Aza, as an eco-friendly pesticide, with bacterial biopesticides may be a successful approach for controlling insect pests.

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.

Neurophysiological responses in the brain tissues of rainbow trout (Oncorhynchus mykiss) treated with bio-pesticide.

The aim of this study was to investigate neurophysiological responses in rainbow trout brain tissue exposed to natural/botanical pesticides. Fish were exposed to botanical and synthetic pesticides over a 21-day period. At the end of the treatment period, oxidative DNA damage (indicated by 8-OHdG (8-hydroxy-2'-deoxyguanosine), AChE activity (acetylcholinesterase) and transcriptional parameters (gpx (glutathione peroxidase), sod (superoxide dismutase), cat (catalase), HSP70 (heat shock protein 70) and CYP1A (cytochromes P450)) was investigated in control and application groups. Our results indicated that brain AChE activities decreased very significantly in fish treated with both insecticide types when compared with control (p < 0.05). 8-OHdG activity increased in a dose/time-dependent situation in the brain tissues of Oncorhynchus mykiss (p < 0.05). In addition, with regards to gene expression, gpx sod and, cat expressions were down-regulated, whereas CYP1A and HSP70 gene expression were up-regulated in fish treated with both insecticides when compared to the control group (p < 0.05). The data for this study suggests that bio-pesticides can cause neurophysiological changes in fish brain tissue.

Endocrine disruption and chronic effects of plant protection products in bees: Can we better protect our pollinators?

Exposure to plant protection products (PPPs) is one of the causes for the population decline of pollinators. In addition to direct exposure, pollinators are exposed to PPPs by pollen, nectar and honey that often contain residues of multiple PPPs. While in legislation PPPs are regarded mainly for their acute toxicity in bees, other effects such as neurotoxicity, immunotoxicity, behavioural changes, stress responses and chronic effects that may harm different physiologically and ecologically relevant traits are much less or not regarded. Despite the fact that endocrine disruption by PPPs is among key effects weakening survival and thriving of populations, pollinators have been poorly investigated in this regard. Here we summarize known endocrine disruptive effects of PPPs in bees and compare them to other chronic effects. Endocrine disruption in honey bees comprise negative effects on reproductive success of queens and drones and behavioural transition of nurse bees to foragers. Among identified PPPs are insecticides, including neonicotinoids, fipronil, chlorantraniliprole and azadirachtin. So far, there exists no OECD guideline to investigate possible endocrine effects of PPPs. Admittedly, investigation of effects on reproduction success of queens and drones is rarely possible under laboratory conditions. But the behavioural transition of nurse bees to foragers could be a possible endpoint to analyse endocrine effects of PPPs under laboratory conditions. We identified some genes, including vitellogenin, which regulate this transition and which may be used as biomarkers for endocrine disruptive PPPs. We plea for a better implementation of the adverse outcome pathway concept into bee's research and propose a procedure for extending and complementing current assessments, including OECD guidelines, with additional physiological and molecular endpoints. Consequently, assessing potential endocrine disruption in pollinators should receive much more relevance.