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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Proteomic Profiling Analysis of Male Infertility in Spodoptera Litura Larvae Challenged with Azadirachtin and its Potential-Regulated Pathways in the Following Stages.

Biopesticides are considered as an alternative to synthetic pesticide with a focus on increasing agricultural productivity as well as maintaining the ecosystem. Prior to application, its potential mechanism should be clearly addressed. Here, the effects of azadirachtin on the reproductive behavior in male Spodoptera litura (Fabricius) are determined. To further explore its molecular mechanism, an iTRAQ (isobaric tags for relative and absolute quantitation) based approach is applied to identify the differentially expressed proteins regulated by azadirachtin at two developmental stages. The results demonstrate that many proteins in the pathway of focal adhesion are regulated to exert influences in detachment of cell attachment, the loss of cell-cell interactions and inducing apoptosis at pupal stage, and many proteins in adenosine monophosphate-activated protein kinase pathway are also changed at the adult stage after azadirachtin-treatment as larvae. Moreover, based on their important roles, it is suggested that some proteins, such as ACTB-G1, ste20-related adaptor protein alpha, and regulatory-associated protein of mTOR (mTORC1) could serve as potential target proteins of azadirachtin to induce male infertility. The results of this study could provide evidence to illuminate the mechanism of male infertility induced by azadirachtin and potential targets for the development of environmentally friendly pesticides.

Kostchyienones A and B, new antiplasmodial and cytotoxicity of limonoids from the roots of Pseudocedrela kotschyi (Schweinf.) Harms.

Two new limonoids, kostchyienones A (1) and B (2), along with 12 known compounds 3-14 were isolated from the roots of Pseudocedrela kostchyi. Compound (7) was isolated for the first time from a natural source. Their structures were elucidated on the basis of spectroscopic evidence. Compounds 1-6 and 13-14 gave IC50 values ranging from 0.75 to 5.62 µg/mL for antiplasmodial activity against chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfINDO) strains of Plasmodium falciparum. Compound 5 showed moderate potential cytotoxicity against the HEK239T cell line with an IC50 value of 22.2±0.89 µg/mL. The antiplasmodial efficacy of the isolated compounds supports the medicinal value of this plant and its potential to provide novel antimalarial drugs.

Subchronic Toxicity Studies of Cortex Dictamni Extracts in Mice and Its Potential Hepatotoxicity Mechanisms in Vitro.

Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages (2.3, 4.6, or 9.2 g/kg/day, p.o.) in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines.

Identification of Meliatoxins in Melia azedarach Extracts Using Mass Spectrometry for Quality Control.

Indiscriminate use of synthetic pesticides can be hazardous to both humans and the environment, but the use of natural products as a source of bio-based products, such as Melia azedarach extracts, is an interesting approach to overcome these hazards. Unfortunately, the limonoids found in M. azedarach with desired insecticidal properties (e.g. azadirachtin) may also be present with limonoids toxic to mammals. The goal of this report was to develop a fast and reliable MS-based experiment to characterize meliatoxins in crude extracts of M. azedarach, in order to provide unequivocal assessment of the safety for extracts for application in the field. MS and MS/MS experiments using MALDI ionization were evaluated as tools for the assignment of characteristic ions produced by each meliatoxin in crude extracts.The use of different experiments in combination, such as the analysis of fragment m/z 557 and [M + Na]+ (adducts ions m/z 681 and m/z 667), MALDI-MS can be used for detection of meliatoxins A1/B1 or A2/B2 in a crude extract and may be used to discriminate meliatoxins A from B, respectively. Subsequent MS/MS experiments can distinguish between the presence of group 1 and/or 2 in each class of meliatoxins classifying the proposed approach as a quick and efficient quality control method of meliatoxins in real M. azedarach samples.

Azadirachtin impact on mate choice, female sexual receptivity and male activity in Drosophila melanogaster (Diptera: Drosophilidae).

Azadirachtin, a neem compound (Azadirachta indica) with medical and anti-insect properties, is one the most successful botanical pesticides in agricultural use. However, its controversial impact on non-targeted species and its mechanism of action need to be clarified. In addition, Azadirachtin impact on pre- and post-mating traits remains largely undocumented. The current study examined the effects of Azadirachtin on Drosophila melanogaster as a non-target and model species. Azadirachtin was applied topically at its LD50 (0.63µg) on the day of adult emergence and its effect was evaluated on several traits of reproductive behavior: mate choice, male activity, female sexual receptivity, sperm storage and female sterility. In choice and no choice conditions, only male treatment reduced mating probability. Female treatment impaired mating probability only when males had the choice. Males' mating ability may have been impaired by an effect of the treatment on their mobility. Such an effect was observed in the actimeter, which revealed that treated males were less active than untreated ones, and this effect persisted over 8days. Azadirachtin treatment had, however, no effect on the nycthemeral rhythm of those males. Even when mating occurred, Azadirachtin treatment impaired post-mating responses especially when females or both sexes were treated: remating probability increases and female fertility (presence of larvae) decreases. No impairment was observed on the efficiency of mating, evaluated by the presence of sperm in the spermatheca or the ventral receptacle. Male treatment only had no significant effect on these post-mating responses. These findings provide clear evidence that Azadirachtin alters the reproductive behavior of both sexes in D. melanogaster via mating and post-mating processes.

Azadirachtin induced larval avoidance and antifeeding by disruption of food intake and digestive enzymes in Drosophila melanogaster (Diptera: Drosophilidae).

Botanical insecticides are a promising alternative to reduce the harmful effects of synthetic chemicals. Among the botanical biopesticides, azadirachtin obtained from the Indian neem tree Azadirachta indica A. Juss. (Meliaceae) is probably the biorational insecticide with greatest agriculture use nowadays due to its broad insecticide activity. The current study, evaluated the lethal and sublethal effects of azadirachtin on larval avoidance, food intake and digestive enzymes of Drosophila melanogaster larvae as biological model. Azadirachtin was applied topically at two doses LD25 (0.28µg) and LD50 (0.67µg) on early third instars larvae. Results evaluated 24h after treatment showed that larvae exhibited significant repellence to azadirachtin and prefer keeping in untreated arenas rather than moving to treated one. In addition, azadirachtin avoidance was more marked in larvae previously treated with this compound as compared with naïf larvae (controls). Moreover, azadirachtin treatment decreased significantly the amount of larval food intake. Finally, azadirachtin reduced significantly the activity of larval α-amylase, chitinase and protease and increased the activity of lipase. This finding showed that azadirachtin induced behavioral and physiological disruption affecting the ability of the insect to digest food. This rapid installation of avoidance and long term antifeedancy might reinforce the action of azadirachtin and provide a new behavioral strategy for integrated pest management programs.

Pyranochromones from Dictyoloma vandellianum A. Juss and Their Cytotoxic Evaluation.

One new chromone 3,3-dimethylallylspatheliachromene methyl ether (1), as well as five known chromones, 6-(3-methylbut-2-enyl) allopteroxylin methyl ether (2), 6-(3-methylbut-2-enyl) allopteroxylin (3), 3,3-dimethylallylspatheliachromene (4), 5-O-methylcneorumchromone K (5) and spatheliabischromene (6), two alkaloids, 8-methoxy-N-methylflindersine (7) and 8-methoxyflindersine (8), and two limonoids, limonin diosphenol (9) and rutaevin (10), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16-F10, HepG2, K562 and HL60 and non-tumor cells PBMC were evaluated for compounds 1 - 6. Compound 1 was the most active showing IC50 values ranging from 6.26 to 14.82 µg/ml in B16-F10 and K562 cell lines, respectively, and presented IC50 value of 11.65 µg/ml in PBMC cell line.

Larval exposure to azadirachtin affects fitness and oviposition site preference of Drosophila melanogaster.

Azadirachtin, a biorational insecticide, is one of the prominent biopesticide commercialized today and represent an alternative to conventional insecticides. The current study examined the lethal and sublethal effects of azadirachtin on Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae) as biological model. Various doses ranging from 0.1 to 2µg were applied topically on early third instar larvae and the cumulative mortality of immature stage was determined. In second series of experiments, azadirachtin was applied at its LD25 (0.28µg) and LD50 (0.67µg) and evaluated on fitness (development duration, fecundity, adult survival) and oviposition site preference with and without choice. Results showed that azadirachtin increased significantly at the two tested doses the duration of larval and pupal development. Moreover, azadirachtin treatment reduced significantly adult's survival of both sex as compared to control. In addition, azadirachtin affected fecundity of flies by a significant reduction of the number of eggs laid. Finally results showed that females present clear preference for oviposition in control medium. Pre-imaginal exposure (L3) to azadirachtin increased aversion to this substance suggesting a memorability of the learned avoidance. The results provide some evidence that larval exposure to azadirachtin altered adult oviposition preference as well as major fitness traits of D. melanogaster. Theses finding may reinforce behavioural avoidance of azadirachtin and contribute as repellent strategies in integrated pest management programmes.

Azadirachtin, a neem-derived biopesticide, impairs behavioral and hematological parameters in carp (Cyprinus carpio).

Azadirachtin (Aza) is a promisor biopesticide used in organic production and aquaculture. Although this compound is apparently safe, there is evidence that it may have deleterious effects on fish. Behavioral and hematological tests are grouped into a set of parameters that may predict potential toxicity of chemical compounds. Here, we investigate the effects of Aza, in the commercial formulation Neenmax™ , on carp (Cyprinus carpio) by defining LC50 (96 h), and testing behavioral and hematological parameters. In our study, LC50 was estimated at 80 µL/L. We exposed carp to Aza at 20, 40, and 60 µL/L, values based on 25, 50, and 75% of LC50 , respectively. At 60 µL/L, Aza promoted significant changes in several parameters, increasing the distance traveled and absolute turn angle. In addition, the same concentration decreased the time spent immobile and the number of immobile episodes. Hematological parameters, such as hematocrit, hemoglobin, hematimetrics index, and red cell distribution, were decreased at 60 µL/L Aza exposure. In conclusion, our study demonstrates that 60 µL/L Aza altered locomotor activity, motor pattern, and hematological parameters, suggesting potential toxicity to carp after acute exposure. In addition, this is the first report that evaluates the actions of a chemical contaminant using automated behavioral tracking of carp, which may be a useful tool for assessing the potential toxicity of biopesticides in conjunction with hematological tests. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1381-1388, 2016.