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Azadirachtin acting as a hazardous compound to induce multiple detrimental effects in Drosophila melanogaster
Journal of Hazardous Materials 359 (2018) 338–347
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Azadirachtin acting as a hazardous compound to induce multiple detrimental effects in Drosophila melanogaster
T ⁎
Jing Zhanga,b, Tao Suna,b, Zhipeng Suna,b, Haiyi Lia,b, Xiaoxian Qia,b, Guohua Zhonga,b, , ⁎ Xin Yia,b, a b
Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, South China Agricultural University, Guangzhou, China Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Azadirachtin Sub-lethal effects Toxicity Drosophila melanogaster
Azadirachtin, a tetranortriterpenoid botanical insecticide, has varied sub-lethal effects against many insect pests, including antifeedant, repellent, and growth regulatory. Despite extensive studies of the mechanisms that underline these physiological effects, little attention has been given to multiple toxic effects of azadirachtin under a coherent concentration, and there is no definitive overarching consensus on its toxicity. Here, we investigated multiple sub-lethal effects induced by 4 mg L−1 of azadirachtin, which did not elicit antifeedant behavior in Drosophila melanogaster, on metrics of longevity, development, compound eyes and reproduction. Exposure to < 20 mg L−1 azadirachtin did not induce mortality, and 4 mg L−1 of azadirachtin could shorten lifespan, expression of detoxification genes and activities of related detoxification enzymes were higher. The lower activity of chitinase and higher content of chitin in fruit fly exposed to 4 mg L−1 azadirachtin could be important in developmental inhibition effects, and ovarian abnormalities and lower fecundity could have resulted from azadirachtin-mediated influences on juvenile hormone and ecdysone that disrupted the endocrine system. Caspase-3, head involution defective and reaper-dependent apoptosis genes may have been responsible for compound eye abnormalities in flies exposed to azadirachtin. Our findings provide important insights to the potential mechanisms of sub-lethal effects of azadirachtin.
⁎ Corresponding authors at: Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, 510642, China. E-mail addresses: [email protected] (G. Zhong), [email protected] (X. Yi).
https://doi.org/10.1016/j.jhazmat.2018.07.057 Received 26 February 2018; Received in revised form 21 June 2018; Accepted 12 July 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 359 (2018) 338–347
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1. Introduction
7 days of exposure.
The use of chemical pesticides in agricultural production is one of the most important strategies to increase yields in agriculture through the controlling of pests, and diseases [1]. While approximately 4 million tons of pesticides are applied annually to crops to ensure food production [2], well-documented negative effects of the inappropriate use of chemical pesticides comprise pest resistance, environmental pollution, and acute and chronic health problems. Increasing public concern over environmental and health risks of pesticide use has led to the ban of many traditional pesticides and replacement with lowertoxicity compounds [3], especially, those extracted from plants. Azadirachtin is derived from the neem tree Azadirachta indica A. Juss (Meliaceae), and is known to be toxic to many insect pests from a range of taxonomic orders [4], where studies have shown it acts on many physiological functions through the disruption of juvenile hormone and 20-hydroxyecdysone pathways in endocrine system that results in incomplete larval development, sterile eggs, and reduced fecundity [5–7]. Toxicity of azadirachtin varies among insect species and application method. For example, oral ingestion of 5 ppm azadirachtin led to 100% mortality after 72 h in the pest Tobacco whitefly Bemisia tabaci (Hemiptera) [8], whereas, mortality of microcolonies of the pollinator Bombus terrestris (Hymenoptera) exposed to azadirachtin via treated sugar water ranged between 32 and 100% at concentrations between 3.2 and 320 mg L−1 after 11 weeks [9]. Moreover, sublethal effects of azadirachtin have been reported in a number of species, including incomplete development in the Tobacco hornworm Manduca sexta (Lepidoptera) [4], and changes in mating and post-mating behavior [10], and decreases in the rate of fecundity in D.melanogaster [11]. These multiple activities of azadirachtin are likely involve several molecular targets and pathways, however, previous studies have tended to focus on single physiological effects of azadirachtin, with little attention on multiple and combined toxicity effects or possible interactions. Prior to large-scale application of this novel insecticide for the control of insect pests, it is important to confirm its multiple sublethal effects in non-target insects, as part of an effective pesticide risk assessment. Drosophila is a versatile model for toxicological assessments of xenobiotics [12], and is useful in the evaluation of the effects of azadirachtin effects on non-target insects. In this study, we studied metrics of survival, growth, and reproduction in D. melanogaster treated with dose of azadirachtin that did not induce significant antifeedant effect in fruit fly. The results in this study could provide reference to establish comprehensive understanding of azadirachtin toxicity.
2.3. Determination of non-antifeedant concentration of azadirachtin We determined the concentration of azadirachtin that induced antifeeding behavior, so that this effect could be removed from the experiment. Amount of ingestion was measured using the method described previously [13], where 0.5% erioglaucine disodium salt and different concentrations of azadirachtin were added into the standard medium. Next, three replicates of 20 2-day post-emergence adult flies (10 female, 10 male) or 20 larvae were placed into a vial, and replicated for three times. After treatment, the flies were homogenized and centrifuged at 13,000 g for 15 min in distilled water. Absorbance was determined by spectrophotometer and the group without dyed food was set as baseline, while the group without azadirachtin was the control. To ensure the results, for adults, ingestion of grouped adult flies was also measured using the capillary feeder method [14], where different concentrations of azadirachtin were added into liquid food (5% sucrose, 5%yeast extract) that was introduced into a capillary and covered with an oil layer to minimize evaporation. The capillary was then held by pipette tip and inserted into the vial containing 10 flies (female : male = 1:1, two days after emergence), and the volume of liquid in the capillary was examined every 24 h for 5 days. The liquid food without azadirachtin was the control group, and one group without fly was set as blank group, and the amount of ingestion by a single fly was calculated. 2.4. Metrics of longevity 2.4.1. Examination of longevity Based on the antifeedant bioassay, we selected 4 mg L−1 as a constant concentration, since it did not induce antifeeding behavior. Effects of azadirachtin on longevity was tested by placing fly eggs in vials that contained either azadirachtin or control diets. Following emergence, the adult flies were transferred to the standard medium (without azadirachtin) that was renewed every 3 d until all flies had died. The newly emerged flies were separated to three replicate groups of 50 flies, and time to 50% mortality and mean longevity were calculated. 2.4.2. Oxidative stress enzymes We assessed the activity and expression of several enzyme indicators of oxidative stress induced by exposure to azadirachtin (section 2.4.1) in 3rd instar larvae, pupae, and adults (15 d after emergence). For each analysis, three samples of 50 flies of different developmental stages (whole body) were homogenized in 0.9% phosphate buffer normal saline (PBS) (2.964 g NaH2PO4.2H2O, 29.011 g Na2HPO4·12H2O, 9.000 g NaCl, and dissolved to 1000 mL), and centrifuged at 10,000 g for 20 min. and the supernatants were stored prior to analysis. Reactive oxygen species (ROS) content was measured using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method, following the manufacturer’s protocol (Jiancheng, Nanjing, Co., Ltd), where supernatant was centrifuged at 1000 g for 10 min at 4 °C and then incubated for 30 min after the addition of the 10 μM of DCFH-DA; fluorescence intensity at 530 nm was recorded. Malondialdehyde (MDA) content was assayed as previously described [15], where supernatant was centrifuged at 2000 g for 15 min and, then incubated for 45 min at 95 °C, following the addition of thibabituric acid; absobance at 532 nm was recorded. Activity of catalase (CAT) was calculated following the manufacturer’s protocol (Jiancheng, Nanjing, China), where the supernatant was diluted in 470 μl of cold buffer (50 mM potassium phosphate, pH 7.0), before the addition of 500 μl of cold 20 mM H2O2; the reaction was terminated by adding dichromate-acetic acid reagent, and absorbance was recorded at 570 nm. Superoxide dismutase (SOD) activity was measured following the manufacturer’s protocol (Jiancheng, Nanjing, China, Co., Ltd) and the method previously described [16], where reagents in the kit were added to the sample supernatant,
2. Experimental 2.1. Insect material D.melanogaster (Oregon R and y[1]w[1118]) was reared at the South China Agricultural University on standard drosophila medium, comprising corn meal, agar, sucrose, yeast, nipagin and propionic acid, at 25 °C and a relative humidity of 70%, with 12h:12 h (light:dark) photoperiod. For gene expression analysis, D.melanogaster RNA was isolated and quantified using an RNA isolation kit (Omega, USA) according to the instructions of the manufacturer, and cDNA was transcribed using the isolated RNA as a template and oligo (dT)18 as a primer. The cDNA was stored at −20 °C prior to analysis. 2.2. Survival analysis of adults Each experiment contained three biological replicates, and for every replicate, 20 (equal sex ratio) newly emerged adult flies (< 24 h after emergence) were placed into a vial containing different concentrations of azadirachtin (Before emergence, the flies were raised under normal condition without azadirachtin). Mortality was assessed after 3, 5, and 339
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2.5.4. Caspase-3 activity Caspase-3 activity was determined using the Caspase-3 activity Kit (Shenggong, Shanghai, China) following the manufacturer’s instructions, where samples were collected in a cold lysis buffer, incubated on ice for 30 min, and then centrifuged at 12,000 g for 20 min. Then, the supernatant was collected and proteins were quantified using the BCA method [24,25]. After adding samples to the reaction buffer to incubate for 1 h at room temperature, caspase-3 activity was measured at an excitation wavelength of 380 nm and an emission wavelength range of 420–460 nm [26].
and incubated for 40 min at 37 °C; following incubating with the cold developing reagent for 10 min, absorbance at 550 nm wavelengths was recorded. Activity of glutathione S-transferase (GST) was determined using the method described previously [17]. For every sample, 50 flies were examined, and three biological replicates were performed. And in the process of measurement, we carried out three technical replicates. Enzyme activity is expressed as μg protein−1. 2.4.3. The expression levels of oxidative genes For detecting the effect azadirachtin on antioxidant system, the expression level of sod, catalase, GST, sirtuin 2 (sir2), methuselah (mth) and forkhead transcription factor (dFOXO) genes as indicators of lifespan using RT-PCR with rp49 as the reference gene, from three replicate samples. We followed the protocol previously reported [18], where, the reaction was performed by using primers (Supplementary Table 1) in the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR green dye (Taraka, China) to bind double-strand DNA at the end of each elongation cycle. Each sample, we performed three technical replicates to improve accuracy, and relative gene expression data was analyzed using the 2-△△CT method, described by Livak [19].
2.6. Metrics of female reproduction 2.6.1. Determination of fecundity We placed three replicates of 20 (female : male = 1:1) newly emerged adult, which had been reared from eggs under control conditions, into a vial containing either standard media plus 4 mg L−1 azadirachtin or untreated standard media as a control. After 10 h, the number of eggs was recorded to calculate fecundity female fly-1 as previously described [27]. 2.6.2. Caspase-3 activity We analyzed the activity of caspase-3 from ovaries of three replicates of 20 female flies using the method described in section 2.5.4.
2.5. Metrics of development 2.5.1. Morphology As previously described, 4 mg/L azadirachtin was added into the media and the azadirachtin-treatment was conducted from eggs till emergence. After the flies were emerged, they were transferred to standard media without azadirachtin. Three replicates of 10 flies from each developmental stage were dissected and sectioned for observation under a transmission electron mircroscope (TEM) and optical microscope. For TEM, specimens were dissected and fixed in 2.5% glutaraldehyde for 4 h, and then they were washed three times in PBS, and post-fixed in 1% osmium tetraoxide at room temperature for 1 h. The dehydration process was carried out using a series of alcohol concentrations, before specimens were embedded in spur resin. Specimens were sectioned and stained with 0.5% uranyl acetate and lead citrate, and the morphology was observed under TEM (TECHAI G2 12, Netherlands).
2.6.3. Determination of the hormone titer Ovaries from three replicates of 20 treated and control female flies were collected and homogenized. Then, the centrifuged supernatant was used to assay the titers of insulin, ecdysone, and juvenile hormone (JH) using an insect insulin, ecdysone, and juvenile enzyme-linked immune sorbent assay (ELISA) kit (Shanghai MLBIO Biotechnology Co., Ltd) following to the manufacturer’s instructions and according to previous studies [28,29]. 2.7. Metrics of compound eyes 2.7.1. Morphological characteristics of compound eyes Eggs (Oregon R) were placed into vials containing standard media (with 4 mg/L azadirachtin or without azadirachtin) till emergence. Following emergence of adults, the compound eyes of 20 flies from each treatment were dissected and morphological changes were observed using scanning electron microscopy (SEM). The procedure of SEM was similar to that described previously [30], where dissected samples were fixed in 2.5% glutaraldehyde with PBS at 4 °C for 24 h and then subjected to post-fixation in 1% osmium tetroxide for 2 h. After washing three times with double-distilled H2O, the samples were dehydrated in a graded alcohol series of 30, 50, 70, 80, and 90% absolute ethanol for 15 min at each concentration. This dehydration process was followed by drying in a critical point dryer and the samples were subsequently mounted on double-sided carbon sticky tape. Specimens were coated with gold prior to examination using an FEI-XL30 SEM operated at 15 kV.
2.5.2. Chitin Three replicate samples (30 flies for each group), including 2nd instar larvae, 3rd instar larvae, pre-pupae, pupae, mature pupae and adult, were assess for chitin, following the protocol described previously [20,21]. After the weight of each sample had been quantified, 50 ml of 4 M potassium hydroxide was added, and the samples were incubated at 160 °C in glycerol to allow deacetylation of chitin by alkaline digestion. The sample mixture was filtered and washed repeatedly using distillate water, before washing with a series of alcohol concentrations and drying in an oven at 60 °C for 24 h for calculation of chitin dry weight.
2.7.2. Evidence of eye apoptosis Previous work has revealed that eye primordia of flies begin to develop and form into compound eyes at the 3rd larval instar [31], so we used immunofluorescent labeling with anti-elav as primary antibody and Alexa Fluor 555 conjugated secondary antibody, to visualize apoptosis of eye primordial in 10 of the 3rd instar larvae from each treatment (treated as egg, 10 flies were dissected for each group), as indicated by protein localization. Following dissection, eye tissue was washed three times with PBS, fixed in 4% paraformaldehyde (pH 7.2) for 30 min, and then washed several times in PBS. Next, the samples were incubated for 10 min in PBS, which contained 3% bovine serum albumin and 0.3% Triton-X100, washed three times with PBS and permeabilized with 0.075% saponin in PBS for 10 min. Incubation with
2.5.3. Chitinase activity The activity of chitinase was measured using the dinitrosalicylic acid (DNS) method described previously [22,23], where three replicates 15 mg samples of flies from the six developmental stages (section 2.5.2) were collected and homogenized in ice-cold citratephosphate buffer (pH = 5.0). After centrifugation at 8000 g for 10 min, the supernatant was used as enzyme source; 1 ml of colloidal chitin was added to 200 μl of the enzyme solution, which was then incubated at 37 °C for 2 h. Next, 500 μl of the supernatant was mixed with 375 μl of DNS buffer, centrifuged at 12,000 g for 5 min and then incubated in boiling water for 5 min. Absorbance of the cooled mixture was measured at 540 nm and activity was calculated from a standard curve using the series concentrations of N-acetylglucosamine. 340
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1–5 mg L−1 of azadirachtin decreased food consumption, but in the following days, there were no effects on feeding amounts of concentration < 4 mg L−1 of azadirachtin. Therefore, we selected 4 mg L−1 of azadirachtin as the highest dose under which normal feeding behavior was maintained for the assays on survival, development, reproduction, and compound eyes. Treatment duration have less impacts compared with exposure to different concentrations of azadirachtin (Fig. S1), and there is no effects on feeding amonts of 4 mg L−1 of azadirachtin on larvae (Fig. 3).
the primary antibody was carried out overnight at 4 °C, then primary antibodies were detected using a secondary antibody and incubated for 2 h. Cover slips were placed immediately over glass slides for observation and, photography using a fluorescence microscope (FM) (Olympus BX51, Olympus, Japan) or laser scanning congfocal microscope (TCS SP2, Leica, Germany). We also used acridine orange (AO) staining to identify apoptotic cells, where dissected eyes tissue was washed three times with PBS and then the suspension was dyed with AO staining and incubated for 15 min in the darkness. Cover slips were placed over the suspension on glass slides and immediately observed and photographed using FM. Apoptotic nuclei were detected using TdT-mediated dUTP nick-end labeling (TUNEL), where compound eye tissue from the azadirachtintreatment and control group was treated with 0.3% (v/v) hydrogen peroxide in methanol for 30 min at room temperature. Then, the eye tissue samples were washed three times with PBS and incubated for 30 min with 0.1% (v/v) Triton X-100 in 1% (w/v) sodium citrate. DeadEnd Fluorometric TUNEL System (DeadEnd, Promega, Madison, WI, USA) was used to detect apoptotic nuclei, following the manufacturer’s instructions, and green-stained cells within the nucleus were counted as TUNEL positive cells. Expression levels of two apoptotic symbolic genes (head involution defective-hid and reaper-rpr) were quantified using qRT-PCR with rp49 as the reference gene, and their expression patterns were also examined using immunofluorescence, with anti-hid and anti-rpr as primary antibodies, and donkey anti-goat IgG-TR (1:100) as the secondary antibody.
3.3. Effect of azadirachtin on lifespan Azadirachtin decreased mean longevity (Fig. 4a), and led to increases in MDA and ROS content in 3rd instar larvae, pupae and adults (Fig. 4b), and increases in the activities of total SOD, CAT, and GST (Fig. 4c) and expression levels of SOD, Catalase, and GST genes were also increased after treatment with azadirachtin (Fig. 3d). Levels of sir2 and mth did not differ in any of the developmental stages following treatment with azadirachtin, however, expression of dFOXO was increased in 3rd instar larvae (Fig. 4e). 3.4. Effect of azadirachtin on development
3. Results
Exposure to azadirachtin showed inhibited effects on development (Fig. 5a & Table S2). Flies exposed to azadirachtin were smaller than control flies at all stages of development (Fig. 5a), and the cuticle of larvae became fragile and thinner, with severe shrinkage at the pupal stage (Fig. 5a).The chitin layer in treated larvae was approximately half as thick (2nd instar: 2.3 ± 0.2 μm; 3rd instar: 2.0 ± 0.4 μm) as that in control larvae (2nd instar: 4.3 ± 0.3 μm; 3rd instar: 5.6 ± 0.3 μm) (Fig. 5b &Table S3), and chitin content in 3rd instar larvae, pre-pupae, pupae, mature pupae, newly-emerged adults, and mature adults was lower (decreased by 41.62%, 44.63%, 42.99%, 38.69%, 44.03% and 39.45%, respectively) (Fig. 5c). Activity of chitinase and caspase-3 in these developmental stages were increased (Fig. 5c).
3.1. Mortality effects of azadirachtin
3.5. The effects of azadirachtin on reproduction
2.8. Statistical analysis Effect of exposure to azadirachtin was tested using one-way analysis of variance, two-way analysis of variance or t –tests in SPSS 17.0 for Windows (SPSS Inc, Chicago, IL, USA), and data are presented as means ± SEM.
Egg production in treated flies decreased by 59.05% (Fig. 6a), where there was a greater occurrence of abnormal ovaries (15.00%) compared with control group (1.70%) (Fig. 6b&c). The relative titer of JH decreased after 10 d in treated egg and pupae, but there were no difference at the other developmental stages. There was an increase in ecdysone in eggs exposed to azadirachtin for 7 d (Fig. 6d), but there were no differences in titer of insulin and the activity of caspase-3 in the ovary at any of the development stages (Data not shown).
We found there was no mortality after 7-d exposure at concentrations < 10 mg L−1, but after treated for 5 days, 20 mg L−1 of azadirachtin treatment could induce significant increase in mortality (Fig. 1). 3.2. Antifeedant effect of azadirachtin Azadirachtin at concentrations ≤ 4 mg L−1 did not induce differences in amount of food ingestion (Fig. 2A&B). At 2 d post-treatment,
3.6. Effect of azadirachtin on compound eyes In adult fruit fly, the compound eye consists of 800 uniform crystalline-like arrays of ommatidia along with even distribution of the mechanosensory bristles in-between [32,33]. There were a large number of abnormal grooves and furrows on the surface of compound eyes in adult fruit flies exposed to azadirachtin (Fig.7a), and as the 3rd instar larvae was the stage of compound eyes forming by primordial, therefore, the morphology of compound eye at the 3rd instar larvae was observed by SEM. There were abnormal-shaped and smooth ocular primordium without any folds and grooves on the surface in the 3rd instar larvae (Fig.7a, white arrow). At the late stage of 3rd instar larvae, there was no evidence of development of the ectropion in the ocular primordium of azadirachtin treated flies (Fig. 7a, yellow arrow). Flies in the control group had a complement of photoreceptor cells and the differentiated photoreceptor neurons were neatly arranged [34], while in the azadirachtin-treated group, the array was disordered and lacked differentiation (Fig. 7b). AO staining and TUNEL positive signals confirmed apoptosis, where increased intensity of green florescence
Fig. 1. Effects of different concentrations of azadirachtin on Drosophila mortality. We tested for differences in mortality by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent. Different letters indicate significant differences in level of expression at p < 0.05. Three biological replicates were performed. 341
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Fig. 2. Antifeedant effects of azadirachtin on Drosophila. The antifeedant effects of azadirachtin measured with dyeing standard medium by erioglaucine disodium salt [13] (a) and capillary method [14] (b). We tested for differences in food intake by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent. Different letters indicate significant differences in level of expression at p < 0.05. Three biological replicates were performed.
untreated flies (Fig.7h).
4. Discussion The antifeedant effects of azadirachtin on a range of insect taxa has previous been reported in a number of studies [9,35]. In this study, data from the two methods we used to measure food intake, which showed ≤ 4 mg L−1 of azadirachtin did not induce antifeedant behavior in adult fruit fly in both measurements. Decreased activity in the neuron transmission of synaptic signals in the subesophageal ganglion is responsible for antifeedant behavior in the fruit fly [36]. We also examined antifeedant effects in fruit fly larvae, which showed 4 mg L−1 azadirachtin did not trigger aversive feeding behavior in larvae by erioglaucine disodium salt method. These results were consistent with a previous study, where larvae treated with 5 u M azadirachtin grew at the same rate as untreated larvae, without body weight loss [37]. Since we aimed to isolate non-induced aversive feeding behavior in the study of sub-lethal impacts of azadirachtin, we used 4 mg L−1 of azadirachtin that we found to be the highest dose that did not trigger antifeeding in the flies. We assessed acute toxicity of azadirachtin to D. melanogaster, and found that it induced mortality at a dose of 10 mg L−1 after 3–7 d exposure and after 5 d of continuous exposure at a dose of 20 mg L−1. Azadirachtin appears to be less toxic (LD50 = 630 mg L−1, LD50 = 670 mg L−1) when applied topically to adult and larvae fruit
Fig. 3. Antifeedant effects of azadirachtin on Drosophila larvae. We tested for differences in food intake by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent.
indicated late stages of apoptosis (Fig. 7c&d). Level of expression of caspase-3 was higher in azadirachtin-treated flies and there were differences in transcript levels of rpr and hid in the compound eyes of 3rd instar larvae compared with control (Fig. 7e&f). Bright colored points of anti-rpr labeling were visible in treated flies only (Fig. 7g), and anti-hid labeling in treated flies contrasted with the even and regular pattern in 342
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Fig. 4. Effects of azadirachtin treatment on Drosophila lifespan. (a) The effect of 4 mg/L azadiraction treatment on lifespan of D.melanogaster. (b) The effect of azadiractin on the activity of ROS and MDA contents. (c) The effect of azadiractin on enzymes activities of oxidative stress system. (d) The effect of azadirachtin on the mRNA expression levels of genes in oxidative stress system. (e) The effect of azadirachtin on the mRNA expression level of lifespan related genes. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
flies [27,38], indicating that susceptibility to azadirachtin varies with application method and duration of exposure. Previous reports have suggested that azadirachtin is more toxic to B.tabaci than to Drosophila and other insect species, where mortality from a dose of 1 mg L−1 was recorded after 24 h and had reached 77.5% after 72 h exposure [8]. This is perhaps unsurprising, since the toxicity of azadirachtin has been shown to vary among insect orders due to different penetration rates and activities of detoxifying enzymes. The molecular mechanisms of acute of toxicity of ingested azadirachtin is unclear, but studies in D. melanogaster larvae have demonstrated that azadirachtin appears to mainly affect post-transcriptional enzyme regulation, proteins involved in cytoskeleton development, and transcription, translation, and regulation of hormones, and energy metabolism [8,37]. We noted that azadirachtin reduced the lifespan of D.melanogaster. Oxidative stress resistance and longevity are mechanistically and phenotypically linked, so we suggest the effect of azadirachtin on lifespan is generally related to the capacity of insects to catalyze oxidative reactions, leading to the generation of ROS, however, insects may use a range of antioxidant enzymes, such as SOD, CAT, and GST, to protect against oxidative damage caused by elevated and accumulated ROS [39] [40]. In this study, variation in gene expression of SOD, CAT, and GST was similar to the profiles of the respective enzyme activities following exposure to azadirachtin that indicate up-regulation of antioxidant enzymes could eliminate excessive ROS triggered by a stress response to azadirachtin. As the period of exposure extended, SOD and CAT may be unable to block the increasing lipid peroxidation process, and when ROS and MDA accumulate faster than they can be detoxified, oxidative stress occurs, which could cause lipid peroxidation of cell membranes, modification of proteins, DNA mutations or fragmentation, and potential cell death [41]. Oxidative stress induced by azadirachtin may contribute to detrimental effects on longevity [42], and in our study, the
genes linked to lifespan did not appear to be closely associated with azadirachtin treatment, since there were no significant changes in transcript expression during exposure. Previous studies have shown that azadirachtin induced robust developmental delays in the larva-to-pupae transition, where insects treated with 20 u M azadirachtin displayed a molting defect [11] and concentrations higher than 0.1 and 1.0 μg of azadirachtin blocked development in 3rd instar Lutzomyia longipalpis (Diperta) [43]. The ability of azadirachtin to induce deleterious metamorphic effects, such as delays in pupation time and larva-to-pupa transition, has been reported in many insect species [8,37,44]. In our study, adult and larval fruit flies treated with azadirachtin had a smaller body size compared with untreated flies, indicating that the growth of fruit fly was significantly inhibited after treatment. Insect body size and developmental time are affected by external and internal factors [45]. Since we demonstrated that 4 mg L−1 azadirachtin did not induce antifeeding behavior, aversion to feeding cannot be a main explanation for growth inhibition; instead, we suggest that growth inhibition was related to other effects of azadirachtin that have been reported elsewhere that include: inhibited release of hormones, interference of chitin synthesis, and induced apoptosis. Azadirachtin is known to directly affect the production of ecdysone 20-monooxygenase that is responsible for metamorphosis [46]. Since we found there was no difference in wholebody titer of hormones (including 20-hydricyecdysone, data not shown) following exposure to azadirachtin, we suggested that such changes induced by azadirachtin may occur at specific organs, rather than at the scale of the whole insect. Developmental inhibition effects of azadirachin have been reported to be regulated by expression of the chitin synthase gene [47] that is vital in the shedding and regrowth of the chitin exoskeleton to increase size or remodel organs to undergo complete metamorphosis [47] [48]. We found that chitin content was 343
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Fig. 5. Effects of azadirachtin on development. (a) 2nd instar larvae, 3rd instar larvae, pre-pupae, pupae, mature pupae, and adult observed by optical microscope. (b) The effect of 4 mg/L azadirachtin on development at stages of 2nd instar larvae and 3rd instar larvae observed by transmission electron microscope. The red arrow indicated the chitinous layer in the control and treatment group. (c) The chitin content, the activity of chintinase and the activity of caspase in control and treatment group. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
lower, but level of chitinase was higher in flies exposed to azadirachtin, indicating growth inhibition induced by azadirachtin may be closely associated with chitin content. We noted that following exposure to azadirachtin, activity of caspase-3 in the entire insect was higher, which indicated that caspase-dependent apoptosis may also be involved in developmental inhibition in fruit flies caused by azadirachtin via its influences on the digestion and absorption of nutrients in midgut cells, as demonstrated by our previous study in Spodoptera litura [49]. This inhibition of development in fruit fly could be attributed to the influence of azadirachtin on chitin synthesis and induced apoptosis, but more evidence is needed to support this hypothesis. Oviposition is another physiological behavior regulated by endogenous hormones, such as ecdysone and juvenile hormone that play roles in the regulating of female sex pheromone production, mating, and courtship in D. melanogaster [50]. Our results showed that fecundity in fruit flies exposed to azadirachtin was lower than in untreated flies and this effect was consistent with a previous study, which showed 6-week exposure to 3.2 mg L−1 of azadirachtin treatment inhibited egg-laying and consequently the production of drones [51]. We found that the morphology of ovaries and the size of the oocyte were adversely affected by azadirachtin treatment that probably led to a decrease in fecundity. Previously, azadirachtin was found to decrease the number and size of oocytes [51] due to impacts on the follicle cells, germinal vesicle [52] and alteration to mitochondria [53]. In this study and previous studies, azadirachtin has been shown to affect the titer of JH and ecdysone that likely impact oocyte development in D.melanogaster [10], as highlighted here, JH stimulates vitellogenesis in developing oocytes and, together with 20E and insulin-signaling pathways, controls the nutrient-sensitive checkpoint in oogenesis [51]. The impact
of azadirachtin on female reproduction could be explained by this interference of JH and ecdysteroids, and eventually lead to abnormal ovaries and an associated decrease in fecundity. In our study, exposure to azadirachtin triggered abnormal phenotypic changes to compound eyes structure, including deformity, bristle loss, and an irregular arrangement of ommatidium. The disorganized ommatidial units in adult flies originated from evidence of abnormality and apoptotic masses in the eye imaginal discs of treated larvae. Consistent with our results, a previous study demonstrated that the optic lobe of the brain and compound eye were poorly developed and the ommatidia were entirely damaged following exposure to azadirachtin in Desert Locust Schistocerca gregaria Forskål [54]. Since apoptosis was observed in the compound eyes following exposure to azadirachtin treatment, we suggest azadirachtin-induced apoptosis was responsible for the morphological damage to the compound eyes. Azadirachtin-induced apoptosis causes general disruption to organs, including muscles, fat body and gut epithelial cells [49], and previous study showed that mitochondria-dependent apoptosis contributed to the rough eye phenotype in fruit fly due to delay in S-phase progression and activation of compensatory proliferation in the eye imaginal discs [33]. In this study, immunofluorescent labeling and variations in mRNA levels indicated that the rpr and hid may play roles in azadirachtininduced apoptosis. Indeed, the roles of these genes in induction of apoptosis have been confirmed in a previous study that showed products of the rpr and hid genes act in concert in post-embryonic neurons to induce apoptosis [55], and the eye-specific expression of rpr could cause eye ablation as a result of apoptosis [56,57]. It is likely that azadirachtin-induced ablation in compound eyes in this study could be attributed to apoptosis from activation of ectopic expression of hid and 344
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Fig. 6. Effects of azadirachtin on oviposition. (a) The effect of azadirachtin on fecundity. (b) The occurrence of abnormal ovary in control and treated group. (c) The morphological characteristics of normal and abnormal ovary. (d) The relative liter of hormone after treated by 4 mg/L azadirachtin. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
compound eyes due to interference with different endocrinological and physiological functions, but not as a result of antifeeding behavior. The quantification of sub-lethal effects of a pesticide to non-target organisms prior to large-scale application is particularly important, because it allows a better understanding of the potential toxicity and physiological response mechanisms to the compound; consequently, pesticides choice and use, either as a single or combined treatment, in integrated pest
rpr genes during development in the larvae stage. 5. Conclusions Our laboratory experiments demonstrated that ingestion of 4 mg L−1 azadirachtin elicited a range of sub-lethal effects in the fruit fly D.melanogaster, including longevity, development, reproduction and 345
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Fig. 7. The characteristics of compound eyes of adult D.melanogaster after treated by 4 mg/L azadirachtin. (a) Morphological changes of compound eyes of adult flies after treated by 4 mg/L azadirachtin. Red arrow indicated grooves and furrows in the treated eyes. White arrow indicated the development process of ocular primordium in control and treated group. Yellow arrow indicated the ectropion of the ocular primordium in control and treated group. (b) Immunofluorescence by using anti-elav as antibody and observed by confocal at 510–590 nm at the stage of the 3rd instar larvae after treated as egg. (c) AO staining in control and treated group observed by fluorescence microscope at the stage of the 3rd instar larvae after treated as egg. (d) TUNEL assays in control and treated group observed by fluorescence microscope at the stage of the 3rd instar larvae after treated as egg. (e) Caspase activities in control and treated group at the stage of the 3rd instar larvae after treated as egg. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni). (f) Relative expression levels of rpr and hid gene at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni). (g) Immunofluorescence by using anti-rpr as primary antibody at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment as egg. (h) Immunofluorescence by using anti-rpr as primary antibody at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment as egg.
management may be facilitated.
online version, at doi:https://doi.org/10.1016/j.jhazmat.2018.07.057.
Acknowledgements
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This work was supported by the grants from National Natural Science Foundation of China (No. 31701812&31572335), Guangdong Province Natural Science Foundation (No. 2014A030313461), and Science and Technology Planning Project of Guangdong Province (No. 2016A020210090 and No. 2017A010105023). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 346
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Azadirachtin acting as a hazardous compound to induce multiple detrimental effects in Drosophila melanogaster
T ⁎
Jing Zhanga,b, Tao Suna,b, Zhipeng Suna,b, Haiyi Lia,b, Xiaoxian Qia,b, Guohua Zhonga,b, , ⁎ Xin Yia,b, a b
Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, South China Agricultural University, Guangzhou, China Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Azadirachtin Sub-lethal effects Toxicity Drosophila melanogaster
Azadirachtin, a tetranortriterpenoid botanical insecticide, has varied sub-lethal effects against many insect pests, including antifeedant, repellent, and growth regulatory. Despite extensive studies of the mechanisms that underline these physiological effects, little attention has been given to multiple toxic effects of azadirachtin under a coherent concentration, and there is no definitive overarching consensus on its toxicity. Here, we investigated multiple sub-lethal effects induced by 4 mg L−1 of azadirachtin, which did not elicit antifeedant behavior in Drosophila melanogaster, on metrics of longevity, development, compound eyes and reproduction. Exposure to < 20 mg L−1 azadirachtin did not induce mortality, and 4 mg L−1 of azadirachtin could shorten lifespan, expression of detoxification genes and activities of related detoxification enzymes were higher. The lower activity of chitinase and higher content of chitin in fruit fly exposed to 4 mg L−1 azadirachtin could be important in developmental inhibition effects, and ovarian abnormalities and lower fecundity could have resulted from azadirachtin-mediated influences on juvenile hormone and ecdysone that disrupted the endocrine system. Caspase-3, head involution defective and reaper-dependent apoptosis genes may have been responsible for compound eye abnormalities in flies exposed to azadirachtin. Our findings provide important insights to the potential mechanisms of sub-lethal effects of azadirachtin.
⁎ Corresponding authors at: Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, 510642, China. E-mail addresses: [email protected] (G. Zhong), [email protected] (X. Yi).
https://doi.org/10.1016/j.jhazmat.2018.07.057 Received 26 February 2018; Received in revised form 21 June 2018; Accepted 12 July 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
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1. Introduction
7 days of exposure.
The use of chemical pesticides in agricultural production is one of the most important strategies to increase yields in agriculture through the controlling of pests, and diseases [1]. While approximately 4 million tons of pesticides are applied annually to crops to ensure food production [2], well-documented negative effects of the inappropriate use of chemical pesticides comprise pest resistance, environmental pollution, and acute and chronic health problems. Increasing public concern over environmental and health risks of pesticide use has led to the ban of many traditional pesticides and replacement with lowertoxicity compounds [3], especially, those extracted from plants. Azadirachtin is derived from the neem tree Azadirachta indica A. Juss (Meliaceae), and is known to be toxic to many insect pests from a range of taxonomic orders [4], where studies have shown it acts on many physiological functions through the disruption of juvenile hormone and 20-hydroxyecdysone pathways in endocrine system that results in incomplete larval development, sterile eggs, and reduced fecundity [5–7]. Toxicity of azadirachtin varies among insect species and application method. For example, oral ingestion of 5 ppm azadirachtin led to 100% mortality after 72 h in the pest Tobacco whitefly Bemisia tabaci (Hemiptera) [8], whereas, mortality of microcolonies of the pollinator Bombus terrestris (Hymenoptera) exposed to azadirachtin via treated sugar water ranged between 32 and 100% at concentrations between 3.2 and 320 mg L−1 after 11 weeks [9]. Moreover, sublethal effects of azadirachtin have been reported in a number of species, including incomplete development in the Tobacco hornworm Manduca sexta (Lepidoptera) [4], and changes in mating and post-mating behavior [10], and decreases in the rate of fecundity in D.melanogaster [11]. These multiple activities of azadirachtin are likely involve several molecular targets and pathways, however, previous studies have tended to focus on single physiological effects of azadirachtin, with little attention on multiple and combined toxicity effects or possible interactions. Prior to large-scale application of this novel insecticide for the control of insect pests, it is important to confirm its multiple sublethal effects in non-target insects, as part of an effective pesticide risk assessment. Drosophila is a versatile model for toxicological assessments of xenobiotics [12], and is useful in the evaluation of the effects of azadirachtin effects on non-target insects. In this study, we studied metrics of survival, growth, and reproduction in D. melanogaster treated with dose of azadirachtin that did not induce significant antifeedant effect in fruit fly. The results in this study could provide reference to establish comprehensive understanding of azadirachtin toxicity.
2.3. Determination of non-antifeedant concentration of azadirachtin We determined the concentration of azadirachtin that induced antifeeding behavior, so that this effect could be removed from the experiment. Amount of ingestion was measured using the method described previously [13], where 0.5% erioglaucine disodium salt and different concentrations of azadirachtin were added into the standard medium. Next, three replicates of 20 2-day post-emergence adult flies (10 female, 10 male) or 20 larvae were placed into a vial, and replicated for three times. After treatment, the flies were homogenized and centrifuged at 13,000 g for 15 min in distilled water. Absorbance was determined by spectrophotometer and the group without dyed food was set as baseline, while the group without azadirachtin was the control. To ensure the results, for adults, ingestion of grouped adult flies was also measured using the capillary feeder method [14], where different concentrations of azadirachtin were added into liquid food (5% sucrose, 5%yeast extract) that was introduced into a capillary and covered with an oil layer to minimize evaporation. The capillary was then held by pipette tip and inserted into the vial containing 10 flies (female : male = 1:1, two days after emergence), and the volume of liquid in the capillary was examined every 24 h for 5 days. The liquid food without azadirachtin was the control group, and one group without fly was set as blank group, and the amount of ingestion by a single fly was calculated. 2.4. Metrics of longevity 2.4.1. Examination of longevity Based on the antifeedant bioassay, we selected 4 mg L−1 as a constant concentration, since it did not induce antifeeding behavior. Effects of azadirachtin on longevity was tested by placing fly eggs in vials that contained either azadirachtin or control diets. Following emergence, the adult flies were transferred to the standard medium (without azadirachtin) that was renewed every 3 d until all flies had died. The newly emerged flies were separated to three replicate groups of 50 flies, and time to 50% mortality and mean longevity were calculated. 2.4.2. Oxidative stress enzymes We assessed the activity and expression of several enzyme indicators of oxidative stress induced by exposure to azadirachtin (section 2.4.1) in 3rd instar larvae, pupae, and adults (15 d after emergence). For each analysis, three samples of 50 flies of different developmental stages (whole body) were homogenized in 0.9% phosphate buffer normal saline (PBS) (2.964 g NaH2PO4.2H2O, 29.011 g Na2HPO4·12H2O, 9.000 g NaCl, and dissolved to 1000 mL), and centrifuged at 10,000 g for 20 min. and the supernatants were stored prior to analysis. Reactive oxygen species (ROS) content was measured using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method, following the manufacturer’s protocol (Jiancheng, Nanjing, Co., Ltd), where supernatant was centrifuged at 1000 g for 10 min at 4 °C and then incubated for 30 min after the addition of the 10 μM of DCFH-DA; fluorescence intensity at 530 nm was recorded. Malondialdehyde (MDA) content was assayed as previously described [15], where supernatant was centrifuged at 2000 g for 15 min and, then incubated for 45 min at 95 °C, following the addition of thibabituric acid; absobance at 532 nm was recorded. Activity of catalase (CAT) was calculated following the manufacturer’s protocol (Jiancheng, Nanjing, China), where the supernatant was diluted in 470 μl of cold buffer (50 mM potassium phosphate, pH 7.0), before the addition of 500 μl of cold 20 mM H2O2; the reaction was terminated by adding dichromate-acetic acid reagent, and absorbance was recorded at 570 nm. Superoxide dismutase (SOD) activity was measured following the manufacturer’s protocol (Jiancheng, Nanjing, China, Co., Ltd) and the method previously described [16], where reagents in the kit were added to the sample supernatant,
2. Experimental 2.1. Insect material D.melanogaster (Oregon R and y[1]w[1118]) was reared at the South China Agricultural University on standard drosophila medium, comprising corn meal, agar, sucrose, yeast, nipagin and propionic acid, at 25 °C and a relative humidity of 70%, with 12h:12 h (light:dark) photoperiod. For gene expression analysis, D.melanogaster RNA was isolated and quantified using an RNA isolation kit (Omega, USA) according to the instructions of the manufacturer, and cDNA was transcribed using the isolated RNA as a template and oligo (dT)18 as a primer. The cDNA was stored at −20 °C prior to analysis. 2.2. Survival analysis of adults Each experiment contained three biological replicates, and for every replicate, 20 (equal sex ratio) newly emerged adult flies (< 24 h after emergence) were placed into a vial containing different concentrations of azadirachtin (Before emergence, the flies were raised under normal condition without azadirachtin). Mortality was assessed after 3, 5, and 339
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2.5.4. Caspase-3 activity Caspase-3 activity was determined using the Caspase-3 activity Kit (Shenggong, Shanghai, China) following the manufacturer’s instructions, where samples were collected in a cold lysis buffer, incubated on ice for 30 min, and then centrifuged at 12,000 g for 20 min. Then, the supernatant was collected and proteins were quantified using the BCA method [24,25]. After adding samples to the reaction buffer to incubate for 1 h at room temperature, caspase-3 activity was measured at an excitation wavelength of 380 nm and an emission wavelength range of 420–460 nm [26].
and incubated for 40 min at 37 °C; following incubating with the cold developing reagent for 10 min, absorbance at 550 nm wavelengths was recorded. Activity of glutathione S-transferase (GST) was determined using the method described previously [17]. For every sample, 50 flies were examined, and three biological replicates were performed. And in the process of measurement, we carried out three technical replicates. Enzyme activity is expressed as μg protein−1. 2.4.3. The expression levels of oxidative genes For detecting the effect azadirachtin on antioxidant system, the expression level of sod, catalase, GST, sirtuin 2 (sir2), methuselah (mth) and forkhead transcription factor (dFOXO) genes as indicators of lifespan using RT-PCR with rp49 as the reference gene, from three replicate samples. We followed the protocol previously reported [18], where, the reaction was performed by using primers (Supplementary Table 1) in the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR green dye (Taraka, China) to bind double-strand DNA at the end of each elongation cycle. Each sample, we performed three technical replicates to improve accuracy, and relative gene expression data was analyzed using the 2-△△CT method, described by Livak [19].
2.6. Metrics of female reproduction 2.6.1. Determination of fecundity We placed three replicates of 20 (female : male = 1:1) newly emerged adult, which had been reared from eggs under control conditions, into a vial containing either standard media plus 4 mg L−1 azadirachtin or untreated standard media as a control. After 10 h, the number of eggs was recorded to calculate fecundity female fly-1 as previously described [27]. 2.6.2. Caspase-3 activity We analyzed the activity of caspase-3 from ovaries of three replicates of 20 female flies using the method described in section 2.5.4.
2.5. Metrics of development 2.5.1. Morphology As previously described, 4 mg/L azadirachtin was added into the media and the azadirachtin-treatment was conducted from eggs till emergence. After the flies were emerged, they were transferred to standard media without azadirachtin. Three replicates of 10 flies from each developmental stage were dissected and sectioned for observation under a transmission electron mircroscope (TEM) and optical microscope. For TEM, specimens were dissected and fixed in 2.5% glutaraldehyde for 4 h, and then they were washed three times in PBS, and post-fixed in 1% osmium tetraoxide at room temperature for 1 h. The dehydration process was carried out using a series of alcohol concentrations, before specimens were embedded in spur resin. Specimens were sectioned and stained with 0.5% uranyl acetate and lead citrate, and the morphology was observed under TEM (TECHAI G2 12, Netherlands).
2.6.3. Determination of the hormone titer Ovaries from three replicates of 20 treated and control female flies were collected and homogenized. Then, the centrifuged supernatant was used to assay the titers of insulin, ecdysone, and juvenile hormone (JH) using an insect insulin, ecdysone, and juvenile enzyme-linked immune sorbent assay (ELISA) kit (Shanghai MLBIO Biotechnology Co., Ltd) following to the manufacturer’s instructions and according to previous studies [28,29]. 2.7. Metrics of compound eyes 2.7.1. Morphological characteristics of compound eyes Eggs (Oregon R) were placed into vials containing standard media (with 4 mg/L azadirachtin or without azadirachtin) till emergence. Following emergence of adults, the compound eyes of 20 flies from each treatment were dissected and morphological changes were observed using scanning electron microscopy (SEM). The procedure of SEM was similar to that described previously [30], where dissected samples were fixed in 2.5% glutaraldehyde with PBS at 4 °C for 24 h and then subjected to post-fixation in 1% osmium tetroxide for 2 h. After washing three times with double-distilled H2O, the samples were dehydrated in a graded alcohol series of 30, 50, 70, 80, and 90% absolute ethanol for 15 min at each concentration. This dehydration process was followed by drying in a critical point dryer and the samples were subsequently mounted on double-sided carbon sticky tape. Specimens were coated with gold prior to examination using an FEI-XL30 SEM operated at 15 kV.
2.5.2. Chitin Three replicate samples (30 flies for each group), including 2nd instar larvae, 3rd instar larvae, pre-pupae, pupae, mature pupae and adult, were assess for chitin, following the protocol described previously [20,21]. After the weight of each sample had been quantified, 50 ml of 4 M potassium hydroxide was added, and the samples were incubated at 160 °C in glycerol to allow deacetylation of chitin by alkaline digestion. The sample mixture was filtered and washed repeatedly using distillate water, before washing with a series of alcohol concentrations and drying in an oven at 60 °C for 24 h for calculation of chitin dry weight.
2.7.2. Evidence of eye apoptosis Previous work has revealed that eye primordia of flies begin to develop and form into compound eyes at the 3rd larval instar [31], so we used immunofluorescent labeling with anti-elav as primary antibody and Alexa Fluor 555 conjugated secondary antibody, to visualize apoptosis of eye primordial in 10 of the 3rd instar larvae from each treatment (treated as egg, 10 flies were dissected for each group), as indicated by protein localization. Following dissection, eye tissue was washed three times with PBS, fixed in 4% paraformaldehyde (pH 7.2) for 30 min, and then washed several times in PBS. Next, the samples were incubated for 10 min in PBS, which contained 3% bovine serum albumin and 0.3% Triton-X100, washed three times with PBS and permeabilized with 0.075% saponin in PBS for 10 min. Incubation with
2.5.3. Chitinase activity The activity of chitinase was measured using the dinitrosalicylic acid (DNS) method described previously [22,23], where three replicates 15 mg samples of flies from the six developmental stages (section 2.5.2) were collected and homogenized in ice-cold citratephosphate buffer (pH = 5.0). After centrifugation at 8000 g for 10 min, the supernatant was used as enzyme source; 1 ml of colloidal chitin was added to 200 μl of the enzyme solution, which was then incubated at 37 °C for 2 h. Next, 500 μl of the supernatant was mixed with 375 μl of DNS buffer, centrifuged at 12,000 g for 5 min and then incubated in boiling water for 5 min. Absorbance of the cooled mixture was measured at 540 nm and activity was calculated from a standard curve using the series concentrations of N-acetylglucosamine. 340
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1–5 mg L−1 of azadirachtin decreased food consumption, but in the following days, there were no effects on feeding amounts of concentration < 4 mg L−1 of azadirachtin. Therefore, we selected 4 mg L−1 of azadirachtin as the highest dose under which normal feeding behavior was maintained for the assays on survival, development, reproduction, and compound eyes. Treatment duration have less impacts compared with exposure to different concentrations of azadirachtin (Fig. S1), and there is no effects on feeding amonts of 4 mg L−1 of azadirachtin on larvae (Fig. 3).
the primary antibody was carried out overnight at 4 °C, then primary antibodies were detected using a secondary antibody and incubated for 2 h. Cover slips were placed immediately over glass slides for observation and, photography using a fluorescence microscope (FM) (Olympus BX51, Olympus, Japan) or laser scanning congfocal microscope (TCS SP2, Leica, Germany). We also used acridine orange (AO) staining to identify apoptotic cells, where dissected eyes tissue was washed three times with PBS and then the suspension was dyed with AO staining and incubated for 15 min in the darkness. Cover slips were placed over the suspension on glass slides and immediately observed and photographed using FM. Apoptotic nuclei were detected using TdT-mediated dUTP nick-end labeling (TUNEL), where compound eye tissue from the azadirachtintreatment and control group was treated with 0.3% (v/v) hydrogen peroxide in methanol for 30 min at room temperature. Then, the eye tissue samples were washed three times with PBS and incubated for 30 min with 0.1% (v/v) Triton X-100 in 1% (w/v) sodium citrate. DeadEnd Fluorometric TUNEL System (DeadEnd, Promega, Madison, WI, USA) was used to detect apoptotic nuclei, following the manufacturer’s instructions, and green-stained cells within the nucleus were counted as TUNEL positive cells. Expression levels of two apoptotic symbolic genes (head involution defective-hid and reaper-rpr) were quantified using qRT-PCR with rp49 as the reference gene, and their expression patterns were also examined using immunofluorescence, with anti-hid and anti-rpr as primary antibodies, and donkey anti-goat IgG-TR (1:100) as the secondary antibody.
3.3. Effect of azadirachtin on lifespan Azadirachtin decreased mean longevity (Fig. 4a), and led to increases in MDA and ROS content in 3rd instar larvae, pupae and adults (Fig. 4b), and increases in the activities of total SOD, CAT, and GST (Fig. 4c) and expression levels of SOD, Catalase, and GST genes were also increased after treatment with azadirachtin (Fig. 3d). Levels of sir2 and mth did not differ in any of the developmental stages following treatment with azadirachtin, however, expression of dFOXO was increased in 3rd instar larvae (Fig. 4e). 3.4. Effect of azadirachtin on development
3. Results
Exposure to azadirachtin showed inhibited effects on development (Fig. 5a & Table S2). Flies exposed to azadirachtin were smaller than control flies at all stages of development (Fig. 5a), and the cuticle of larvae became fragile and thinner, with severe shrinkage at the pupal stage (Fig. 5a).The chitin layer in treated larvae was approximately half as thick (2nd instar: 2.3 ± 0.2 μm; 3rd instar: 2.0 ± 0.4 μm) as that in control larvae (2nd instar: 4.3 ± 0.3 μm; 3rd instar: 5.6 ± 0.3 μm) (Fig. 5b &Table S3), and chitin content in 3rd instar larvae, pre-pupae, pupae, mature pupae, newly-emerged adults, and mature adults was lower (decreased by 41.62%, 44.63%, 42.99%, 38.69%, 44.03% and 39.45%, respectively) (Fig. 5c). Activity of chitinase and caspase-3 in these developmental stages were increased (Fig. 5c).
3.1. Mortality effects of azadirachtin
3.5. The effects of azadirachtin on reproduction
2.8. Statistical analysis Effect of exposure to azadirachtin was tested using one-way analysis of variance, two-way analysis of variance or t –tests in SPSS 17.0 for Windows (SPSS Inc, Chicago, IL, USA), and data are presented as means ± SEM.
Egg production in treated flies decreased by 59.05% (Fig. 6a), where there was a greater occurrence of abnormal ovaries (15.00%) compared with control group (1.70%) (Fig. 6b&c). The relative titer of JH decreased after 10 d in treated egg and pupae, but there were no difference at the other developmental stages. There was an increase in ecdysone in eggs exposed to azadirachtin for 7 d (Fig. 6d), but there were no differences in titer of insulin and the activity of caspase-3 in the ovary at any of the development stages (Data not shown).
We found there was no mortality after 7-d exposure at concentrations < 10 mg L−1, but after treated for 5 days, 20 mg L−1 of azadirachtin treatment could induce significant increase in mortality (Fig. 1). 3.2. Antifeedant effect of azadirachtin Azadirachtin at concentrations ≤ 4 mg L−1 did not induce differences in amount of food ingestion (Fig. 2A&B). At 2 d post-treatment,
3.6. Effect of azadirachtin on compound eyes In adult fruit fly, the compound eye consists of 800 uniform crystalline-like arrays of ommatidia along with even distribution of the mechanosensory bristles in-between [32,33]. There were a large number of abnormal grooves and furrows on the surface of compound eyes in adult fruit flies exposed to azadirachtin (Fig.7a), and as the 3rd instar larvae was the stage of compound eyes forming by primordial, therefore, the morphology of compound eye at the 3rd instar larvae was observed by SEM. There were abnormal-shaped and smooth ocular primordium without any folds and grooves on the surface in the 3rd instar larvae (Fig.7a, white arrow). At the late stage of 3rd instar larvae, there was no evidence of development of the ectropion in the ocular primordium of azadirachtin treated flies (Fig. 7a, yellow arrow). Flies in the control group had a complement of photoreceptor cells and the differentiated photoreceptor neurons were neatly arranged [34], while in the azadirachtin-treated group, the array was disordered and lacked differentiation (Fig. 7b). AO staining and TUNEL positive signals confirmed apoptosis, where increased intensity of green florescence
Fig. 1. Effects of different concentrations of azadirachtin on Drosophila mortality. We tested for differences in mortality by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent. Different letters indicate significant differences in level of expression at p < 0.05. Three biological replicates were performed. 341
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Fig. 2. Antifeedant effects of azadirachtin on Drosophila. The antifeedant effects of azadirachtin measured with dyeing standard medium by erioglaucine disodium salt [13] (a) and capillary method [14] (b). We tested for differences in food intake by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent. Different letters indicate significant differences in level of expression at p < 0.05. Three biological replicates were performed.
untreated flies (Fig.7h).
4. Discussion The antifeedant effects of azadirachtin on a range of insect taxa has previous been reported in a number of studies [9,35]. In this study, data from the two methods we used to measure food intake, which showed ≤ 4 mg L−1 of azadirachtin did not induce antifeedant behavior in adult fruit fly in both measurements. Decreased activity in the neuron transmission of synaptic signals in the subesophageal ganglion is responsible for antifeedant behavior in the fruit fly [36]. We also examined antifeedant effects in fruit fly larvae, which showed 4 mg L−1 azadirachtin did not trigger aversive feeding behavior in larvae by erioglaucine disodium salt method. These results were consistent with a previous study, where larvae treated with 5 u M azadirachtin grew at the same rate as untreated larvae, without body weight loss [37]. Since we aimed to isolate non-induced aversive feeding behavior in the study of sub-lethal impacts of azadirachtin, we used 4 mg L−1 of azadirachtin that we found to be the highest dose that did not trigger antifeeding in the flies. We assessed acute toxicity of azadirachtin to D. melanogaster, and found that it induced mortality at a dose of 10 mg L−1 after 3–7 d exposure and after 5 d of continuous exposure at a dose of 20 mg L−1. Azadirachtin appears to be less toxic (LD50 = 630 mg L−1, LD50 = 670 mg L−1) when applied topically to adult and larvae fruit
Fig. 3. Antifeedant effects of azadirachtin on Drosophila larvae. We tested for differences in food intake by using two-way analysis of variance (ANOVA, Bonferroni posttests). Data are means ± S.E.M of three independent.
indicated late stages of apoptosis (Fig. 7c&d). Level of expression of caspase-3 was higher in azadirachtin-treated flies and there were differences in transcript levels of rpr and hid in the compound eyes of 3rd instar larvae compared with control (Fig. 7e&f). Bright colored points of anti-rpr labeling were visible in treated flies only (Fig. 7g), and anti-hid labeling in treated flies contrasted with the even and regular pattern in 342
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Fig. 4. Effects of azadirachtin treatment on Drosophila lifespan. (a) The effect of 4 mg/L azadiraction treatment on lifespan of D.melanogaster. (b) The effect of azadiractin on the activity of ROS and MDA contents. (c) The effect of azadiractin on enzymes activities of oxidative stress system. (d) The effect of azadirachtin on the mRNA expression levels of genes in oxidative stress system. (e) The effect of azadirachtin on the mRNA expression level of lifespan related genes. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
flies [27,38], indicating that susceptibility to azadirachtin varies with application method and duration of exposure. Previous reports have suggested that azadirachtin is more toxic to B.tabaci than to Drosophila and other insect species, where mortality from a dose of 1 mg L−1 was recorded after 24 h and had reached 77.5% after 72 h exposure [8]. This is perhaps unsurprising, since the toxicity of azadirachtin has been shown to vary among insect orders due to different penetration rates and activities of detoxifying enzymes. The molecular mechanisms of acute of toxicity of ingested azadirachtin is unclear, but studies in D. melanogaster larvae have demonstrated that azadirachtin appears to mainly affect post-transcriptional enzyme regulation, proteins involved in cytoskeleton development, and transcription, translation, and regulation of hormones, and energy metabolism [8,37]. We noted that azadirachtin reduced the lifespan of D.melanogaster. Oxidative stress resistance and longevity are mechanistically and phenotypically linked, so we suggest the effect of azadirachtin on lifespan is generally related to the capacity of insects to catalyze oxidative reactions, leading to the generation of ROS, however, insects may use a range of antioxidant enzymes, such as SOD, CAT, and GST, to protect against oxidative damage caused by elevated and accumulated ROS [39] [40]. In this study, variation in gene expression of SOD, CAT, and GST was similar to the profiles of the respective enzyme activities following exposure to azadirachtin that indicate up-regulation of antioxidant enzymes could eliminate excessive ROS triggered by a stress response to azadirachtin. As the period of exposure extended, SOD and CAT may be unable to block the increasing lipid peroxidation process, and when ROS and MDA accumulate faster than they can be detoxified, oxidative stress occurs, which could cause lipid peroxidation of cell membranes, modification of proteins, DNA mutations or fragmentation, and potential cell death [41]. Oxidative stress induced by azadirachtin may contribute to detrimental effects on longevity [42], and in our study, the
genes linked to lifespan did not appear to be closely associated with azadirachtin treatment, since there were no significant changes in transcript expression during exposure. Previous studies have shown that azadirachtin induced robust developmental delays in the larva-to-pupae transition, where insects treated with 20 u M azadirachtin displayed a molting defect [11] and concentrations higher than 0.1 and 1.0 μg of azadirachtin blocked development in 3rd instar Lutzomyia longipalpis (Diperta) [43]. The ability of azadirachtin to induce deleterious metamorphic effects, such as delays in pupation time and larva-to-pupa transition, has been reported in many insect species [8,37,44]. In our study, adult and larval fruit flies treated with azadirachtin had a smaller body size compared with untreated flies, indicating that the growth of fruit fly was significantly inhibited after treatment. Insect body size and developmental time are affected by external and internal factors [45]. Since we demonstrated that 4 mg L−1 azadirachtin did not induce antifeeding behavior, aversion to feeding cannot be a main explanation for growth inhibition; instead, we suggest that growth inhibition was related to other effects of azadirachtin that have been reported elsewhere that include: inhibited release of hormones, interference of chitin synthesis, and induced apoptosis. Azadirachtin is known to directly affect the production of ecdysone 20-monooxygenase that is responsible for metamorphosis [46]. Since we found there was no difference in wholebody titer of hormones (including 20-hydricyecdysone, data not shown) following exposure to azadirachtin, we suggested that such changes induced by azadirachtin may occur at specific organs, rather than at the scale of the whole insect. Developmental inhibition effects of azadirachin have been reported to be regulated by expression of the chitin synthase gene [47] that is vital in the shedding and regrowth of the chitin exoskeleton to increase size or remodel organs to undergo complete metamorphosis [47] [48]. We found that chitin content was 343
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Fig. 5. Effects of azadirachtin on development. (a) 2nd instar larvae, 3rd instar larvae, pre-pupae, pupae, mature pupae, and adult observed by optical microscope. (b) The effect of 4 mg/L azadirachtin on development at stages of 2nd instar larvae and 3rd instar larvae observed by transmission electron microscope. The red arrow indicated the chitinous layer in the control and treatment group. (c) The chitin content, the activity of chintinase and the activity of caspase in control and treatment group. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
lower, but level of chitinase was higher in flies exposed to azadirachtin, indicating growth inhibition induced by azadirachtin may be closely associated with chitin content. We noted that following exposure to azadirachtin, activity of caspase-3 in the entire insect was higher, which indicated that caspase-dependent apoptosis may also be involved in developmental inhibition in fruit flies caused by azadirachtin via its influences on the digestion and absorption of nutrients in midgut cells, as demonstrated by our previous study in Spodoptera litura [49]. This inhibition of development in fruit fly could be attributed to the influence of azadirachtin on chitin synthesis and induced apoptosis, but more evidence is needed to support this hypothesis. Oviposition is another physiological behavior regulated by endogenous hormones, such as ecdysone and juvenile hormone that play roles in the regulating of female sex pheromone production, mating, and courtship in D. melanogaster [50]. Our results showed that fecundity in fruit flies exposed to azadirachtin was lower than in untreated flies and this effect was consistent with a previous study, which showed 6-week exposure to 3.2 mg L−1 of azadirachtin treatment inhibited egg-laying and consequently the production of drones [51]. We found that the morphology of ovaries and the size of the oocyte were adversely affected by azadirachtin treatment that probably led to a decrease in fecundity. Previously, azadirachtin was found to decrease the number and size of oocytes [51] due to impacts on the follicle cells, germinal vesicle [52] and alteration to mitochondria [53]. In this study and previous studies, azadirachtin has been shown to affect the titer of JH and ecdysone that likely impact oocyte development in D.melanogaster [10], as highlighted here, JH stimulates vitellogenesis in developing oocytes and, together with 20E and insulin-signaling pathways, controls the nutrient-sensitive checkpoint in oogenesis [51]. The impact
of azadirachtin on female reproduction could be explained by this interference of JH and ecdysteroids, and eventually lead to abnormal ovaries and an associated decrease in fecundity. In our study, exposure to azadirachtin triggered abnormal phenotypic changes to compound eyes structure, including deformity, bristle loss, and an irregular arrangement of ommatidium. The disorganized ommatidial units in adult flies originated from evidence of abnormality and apoptotic masses in the eye imaginal discs of treated larvae. Consistent with our results, a previous study demonstrated that the optic lobe of the brain and compound eye were poorly developed and the ommatidia were entirely damaged following exposure to azadirachtin in Desert Locust Schistocerca gregaria Forskål [54]. Since apoptosis was observed in the compound eyes following exposure to azadirachtin treatment, we suggest azadirachtin-induced apoptosis was responsible for the morphological damage to the compound eyes. Azadirachtin-induced apoptosis causes general disruption to organs, including muscles, fat body and gut epithelial cells [49], and previous study showed that mitochondria-dependent apoptosis contributed to the rough eye phenotype in fruit fly due to delay in S-phase progression and activation of compensatory proliferation in the eye imaginal discs [33]. In this study, immunofluorescent labeling and variations in mRNA levels indicated that the rpr and hid may play roles in azadirachtininduced apoptosis. Indeed, the roles of these genes in induction of apoptosis have been confirmed in a previous study that showed products of the rpr and hid genes act in concert in post-embryonic neurons to induce apoptosis [55], and the eye-specific expression of rpr could cause eye ablation as a result of apoptosis [56,57]. It is likely that azadirachtin-induced ablation in compound eyes in this study could be attributed to apoptosis from activation of ectopic expression of hid and 344
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Fig. 6. Effects of azadirachtin on oviposition. (a) The effect of azadirachtin on fecundity. (b) The occurrence of abnormal ovary in control and treated group. (c) The morphological characteristics of normal and abnormal ovary. (d) The relative liter of hormone after treated by 4 mg/L azadirachtin. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni).
compound eyes due to interference with different endocrinological and physiological functions, but not as a result of antifeeding behavior. The quantification of sub-lethal effects of a pesticide to non-target organisms prior to large-scale application is particularly important, because it allows a better understanding of the potential toxicity and physiological response mechanisms to the compound; consequently, pesticides choice and use, either as a single or combined treatment, in integrated pest
rpr genes during development in the larvae stage. 5. Conclusions Our laboratory experiments demonstrated that ingestion of 4 mg L−1 azadirachtin elicited a range of sub-lethal effects in the fruit fly D.melanogaster, including longevity, development, reproduction and 345
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Fig. 7. The characteristics of compound eyes of adult D.melanogaster after treated by 4 mg/L azadirachtin. (a) Morphological changes of compound eyes of adult flies after treated by 4 mg/L azadirachtin. Red arrow indicated grooves and furrows in the treated eyes. White arrow indicated the development process of ocular primordium in control and treated group. Yellow arrow indicated the ectropion of the ocular primordium in control and treated group. (b) Immunofluorescence by using anti-elav as antibody and observed by confocal at 510–590 nm at the stage of the 3rd instar larvae after treated as egg. (c) AO staining in control and treated group observed by fluorescence microscope at the stage of the 3rd instar larvae after treated as egg. (d) TUNEL assays in control and treated group observed by fluorescence microscope at the stage of the 3rd instar larvae after treated as egg. (e) Caspase activities in control and treated group at the stage of the 3rd instar larvae after treated as egg. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni). (f) Relative expression levels of rpr and hid gene at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment. The data represent the mean ± S.E.M of three replicates (*** p < 0.001, **p < 0.01, *p < 0.05, ANOVA, Bonferroni). (g) Immunofluorescence by using anti-rpr as primary antibody at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment as egg. (h) Immunofluorescence by using anti-rpr as primary antibody at the stage of the 3rd instar larvae after 4 mg/L azadirachtin treatment as egg.
management may be facilitated.
online version, at doi:https://doi.org/10.1016/j.jhazmat.2018.07.057.
Acknowledgements
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This work was supported by the grants from National Natural Science Foundation of China (No. 31701812&31572335), Guangdong Province Natural Science Foundation (No. 2014A030313461), and Science and Technology Planning Project of Guangdong Province (No. 2016A020210090 and No. 2017A010105023). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 346
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