Investigation of titania nanoparticles on behaviour ...

Physiology & Behavior 167 (2016) 76–85

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Investigation of titania nanoparticles on behaviour and mechanosensory organ of Drosophila melanogaster Debabrat Sabat a, Abhinandan Patnaik a, Basanti Ekka b, Priyabrat Dash b, Monalisa Mishra a,⁎ a b

Department of Life Science, National Institute of Technology, Rourkela, Odisha 769008, India Department of Chemistry, National Institute of Technology, Rourkela, Odisha 769008, India

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Titania NPs exposure N 200 mg·L− 1 delays life cycle and decrease pupation. • Abnormal behaviour is observed in larval crawling and climbing behaviour assay at higher TiO2 concentration. • Titania NPs affects the development, mechanosensory bristles and wing venation.

a r t i c l e

i n f o

Article history: Received 13 April 2016 Received in revised form 6 August 2016 Accepted 30 August 2016 Available online 05 September 2016 Keywords: Titania nanoparticles Drosophila melanogaster Toxicity Behaviour

a b s t r a c t Titania nanoparticles are used in food, cosmetic, medicine, paint and many more domestic items. Its extensive use has raised the threat to the physiological system and thus the functioning of the body. In the current study, the toxicity of TiO2 is checked by adding it in food and using Drosophila melanogaster as a model organism. Various concentrations of TiO2 (50, 100, 200, 250 mg·L−1) toxicity was assessed via oral route exposure. Survivability, life-cycle, mechanosensory behaviour and structure of various mechanosensory organs were monitored as a read out of nanoparticle toxicity. TiO2 NPs generate reactive oxygen species which can modify multiple signalling pathways and thus can alter the development and behavioural pattern of the fly. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The nanoparticles (NPs) have broad application and have extensive use in construction, aviation, health sector, robotics and domestic products. Its nano size (b 100 nm) and acquired characteristic properties help to meet several challenges. The positive aspect of NP is in the growth, drug targeting and treatment of various plants or animals. ⁎ Corresponding author. E-mail address: [email protected] (M. Mishra).

http://dx.doi.org/10.1016/j.physbeh.2016.08.032 0031-9384/© 2016 Elsevier Inc. All rights reserved.

Various other nanoparticles such as silver (Ag) and gold (Au) are used as antimicrobials, imaging and tumour treatment [1]. Similarly, magnetic NPs are used for targeted drug delivery [2] while zinc oxide, hydroxyapatite or silica NPs can be used as nano-fertilizers [3]. This also provides an opportunity for the NPs to enter into the food chain without being noticed or undetected due to its nano-form. Among all the synthesised NPs, titania (TiO2) has distinguished chemical properties producing white pigment due to high refractive index. It is highly stable and used in self-cleaning tiles, windows, textiles, and anti-fogging car mirrors. Its anticorrosive and photocatalytic

D. Sabat et al. / Physiology & Behavior 167 (2016) 76–85

property increases catalytic activity due to its increased surface area. [4]. In the field of nanomedicine, TiO2 has application in photodynamic therapy (PDT), photosensitizers, imaging, nano-therapeutics, prosthetic implants of hip and knee [5–7]. Products like cake, candies, chewing gum, sweet and drugs contained a higher amount of TiO2 [8,9]. For example, chewing gum on an average has 2.4–7.5 mg of TiO2 NPs and the intake of TiO2 increase in a time-dependent manner. In a chewing gum, the majority of TiO2 NPs (~93%) is of size N200 nm and rest are with b100 nm range [10]. Similarly, several food product additives contain TiO2 at a level of 0.02 to 9.0 mg/g and out of which 15% are b100 nm in size [9]. Although cosmetic and toothpaste contains some amount of TiO2 NPs, so far its toxic effect on the body is not yet reported [11]. The toxicity in NP depends on the size, surface chemistry, route of administration and exposure route of NPs which is usually through inhalation, oral or dermal exposure. The food and drug administration (U.S.) have approved TiO2 b 1% by body weight while Occupational Safety & Health Administration (OSHA) approved TiO2 b 15 mg/m3. Besides its wide application, its toxic effect classifies it to be as a group 2B carcinogen to humans [12]. The toxicity also depends on the grade of TiO2 used [8]. The industrial grade shows toxicity up to 0.2 mg mL− 1 while food grade does not show toxicity up to 2 mg mL− 1. A recent report suggested the oral route intake of TiO2, induced cytotoxicity on mid-gut, imaginal disc and increased DNA damage in Drosophila haemocytes [13]. The dietary intake of TiO2 NPs increases the stress gene catalase and superoxide dismutase (SOD) activity [14]. However, dietary intake of (0.1–10 mM) anatase nano-TiO2 did not induce genotoxicity in the wing spot test of D. melanogaster larvae [15] as it is well-studied in other model organisms [16,17]. In the rat model, TiO2 NPs impair central nervous system when administered intranasally [18]. TiO2 exposure produces free radical and damages the brain microglia, affects cell cycle of neurons, induces apoptosis in the neuronal cell [19]. The toxicity of TiO2 on the various physiological systems including nervous system is alarming and warrants a model organism to investigate the molecular mechanism and its toxicity. Drosophila serves to be an excellent model to study disease mechanism, drug testing, pathogen infection and also nanoparticle toxicity [20–23]. The advantage for selecting this model is due to its short lifespan, cost effectiveness, the whole genome sequenced, and 75% diseased genes in humans share homology with Drosophila [24–26]. The current study focuses on testing the toxicity of TiO2 NPs (via oral route) in the Drosophila model by checking its behavioural, survival and phenotypical changes that the fly undergoes on exposure of TiO2 NPs at various concentrations.

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2.3. Characterisation of synthesised NP The chemically synthesised NPs was characterised by using XRD, FESEM, TEM and FTIR techniques. 2.3.1. X-ray diffractometer (XRD) analysis The powdered sample was subjected to XRD analysis. The crystal structure of the synthesised nanoparticle was determined by X-ray diffractometer (XRD) of RIGAKU JAPAN/ULTIMA-IV with CuKα radiation (λ = 0.154 nm), 2θ in the range of 10°–80° and a scan rate of 2° per minute. 2.3.2. Field emission scanning electron microscopy and transmission electron microscopy The size, morphology and structure of the nanoparticle were investigated by field emission scanning electron microscopy (FESEM), performed in Nova NANOSEM/FEI. The sample was prepared by dispersing TiO2 powder in methanol. Then one drop of the suspension was spared over ITO glass slides for analysis The Transmission Electron micrographs (TEM) of the TiO2 NPs were recorded using PHILIPS CM 200 equipment using carbon coated copper grids. The HRTEM and SAED pattern of the NPs was also analysed. 2.3.3. Zeta potential The TiO2 NPs were dispersed in Milli-Q water and sonicated for 20 min than the zeta potential was measured using Malvern NANOZS-90. 2.3.4. FTIR analysis The FT-IR spectroscopy (Perkin-Elmer) was used to determine the functional groups in TiO2 NPs. The spectrum was scanned from 4000 cm−1 to 400 cm−1. Nearly 3–4 mg of the sample was mixed thoroughly with 30 mg of dried KBr and made into pallets. The pallets were stored in vacuum desiccators and exposed to IR lamp for 1 min before the IR measurement. 2.4. Fly stock Oregon-R (OR) flies were obtained from the fly facility, C-CAMP Bangalore, India. The flies are grown on standard corn meal media with sucrose, agar agar type-I and yeast. The flies are kept in 25 °C, a 60% relative humidity (RH) incubator with 12 h of Light/Dark cycle. 2.5. Preparation of titanium dioxide stock solution

2. Materials and methods 2.1. Materials Titanium isopropoxide (Ti(OCH(CH3)2)4) (analytical grade) was obtained from Sigma-Aldrich. Methanol, trypan blue, agarose and molecular sieves were obtained from Hi-media. The chemicals were used as obtained and without any further purification. 2.2. Synthesis of TiO2 nanoparticles For the synthesis of TiO2 NPs, 5 mL of methanol was taken in a beaker and 0.25 mL (5 vol.%) of water was added to it and stirred. 2.02 mL of titanium isopropoxide (Ti(OCH(CH3)2)4) taken as the titanium precursor, was added to 5 mL of hot methanol freshly dried using molecular sieves. This was then added to the beaker immediately, and a gel was formed in 5–10 min. This was left to age overnight, followed by drying at a temperature of 150–200 °C for 1–2 h. The dried gel was then powdered using a mortar and pestle and calcined in air at a temperature of 550 °C for 3 h [27,28].

The 1000 mg L−1 stock solution of Titanium dioxide (TiO2) NP was prepared by mixing 50 milligrams (mg) of TiO2 NPs in 50 mL of distilled water. The prepared mixture was sonicated using for 20–30 min under the ice to maintain a homogenised distribution of the NPs throughout the solution. The homogenised TiO2 nanoparticles were mixed in the food to achieve a final concentration of 50 mg L− 1, 100 mg L−1, 200 mg L−1, and 250 mg L−1. The food containing different concentrations of TiO2 NPs were equally distributed for 3 sets of the replica. Later, an equal number of wild type (WT) Oregon-R male and female flies were transferred to each vial. 2.6. Survivability study (toxic effect on lifecycle) The toxicity assay was measured as reported by Ales Panacek et al. [29]. The flies fed with different concentrations of TiO2 NPs (50 mg·L−1, 100 mg·L−1, 200 mg·L−1, 250 mg·L−1) lay their eggs on food. The eggs developing to pupal stages were marked on each vial, and the number of flies hatching from each vial was recorded every day. The graph is plotted by the percentage of flies emerging from each concentration.

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2.7. Trypan blue staining

2.12. Climbing behaviour assay

Trypan blue staining was performed on third instar larvae by following a reported protocol with slight modification [30]. Five third instar larvae were analysed for three times in triplicates from each TiO2 treatment including the control. Larvae were collected and washed with PBS (1×) solution thoroughly to remove any food traces prior experiment. Larvae were then transferred to trypan blue stain [31] and kept in shaking condition for 30 min. With the onset of time, the larvae were washed thoroughly with PBS solution again for 15 more minutes to remove any extra dye that may be present on its surface or that might have been ingested. The larvae were then observed under a stereomicroscope and imaged to check for any cell damage.

The 30 hatched flies were counted and transferred from the treated food vials to a graduated 100 mL measuring cylinder for performing the climbing assay. The mouth of the measuring cylinder was then closed with a cotton plug. The measuring cylinder was tapped two to three times to drop the flies at the bottom of the cylinder, after which the flies were allowed to move upwards. This movement was recorded for 30 s after which the number of flies below the 10 cm mark was counted, and the percentage of flies below and above the threshold was calculated.

2.8. Larva crawling behaviour assay

After hatching from pupae the adult flies from each vial were carefully observed under a stereomicroscope to check for any abnormal fly phenotypes. Various body parts especially head, eye, thorax and wings were imaged. For phenotypical analysis of bristle and wings, 100 flies were used from each treatment. All analysis was made from triplicate set of results.

Larvae crawling behaviour was monitored by following an earlier reported protocol [32]. The crawling behaviour of larva for different concentrations was checked by making them walk on 2% agarose plates because they leave trail marks on agarose. Third instar larvae were collected from each concentration and placed at the centre of the agar plates from their respective treated concentration. Graph papers can be used to make a representative scale, for distance calculation and then analyse the speed in cm/min. The larva crawling pattern was observed and the trail pattern is marked. This behaviour assay is monitored for three times for three different sets of larvae in triplicates for each concentration. 2.9. Nitroblue tetrazolium assay for reactive oxygen species (ROS) Nitroblue tetrazolium (NBT) reduction assay helps to measure the amount of reactive oxygen species [33]. In this assay, reduction of yellow water soluble powder turns to blue insoluble formazan crystals on reduction. Both treated and control larva cells (extracted haemolymph) were incubated with NBT for 1 h in the incubation medium and finally the reaction was stopped by adding one volume of acetic acid. The insoluble formazan crystals formed are solubilized by adding 150 μL 50% acetic acid followed by 5 min vortexing. The absorbance of the final solution was now determined at 595 nm in a plate reader. 2.10. Comet assay It is also known as Single Cell Gel Electrophoresis assay (SCGE), performed to qualitatively or quantitatively identify the DNA breakage. Thirty third instar larvae were collected from different treatments and haemocyte was collected by puncturing their cuticle using fine forceps. Cold PBS is added to the extracted haemolymph and kept in ice in order to prevent melanisation. The comet assay of Drosophila haemocyte was performed following Carmona et al. [34]. The collected sample is resuspended in 0.75% LMA (low melting agarose) and mounted on 1% NMA (normal melting agarose) pre-coated slides (24 h dried at room temperature). The slides were kept in cold lysing solution (100 mM Na2 EDTA, 10 mM Tris, 2.5 M NaCl and 1% Triton X-100 set at pH 10) for 2 h under dark condition at 4 °C to prevent additional DNA damage. The slides were placed in chilled electrophoresis buffer (300 mM NaOH and 1 mM Na2 EDTA, pH 13) for 15 min before running it for 20 min at 300 mA, 25 V. The slides were than neutralised by dripping 0.4 M Tris buffer for 5 min and gel was stained with EtBr solution. The imaging of the gel was done using florescence microscope (Olympus IX71). 2.11. Body weight The body weight of the flies was measured by taking 30 newly hatched adult flies from each concentration, and the weight was compared with the control.

2.13. Phenotype observation

2.14. Statistical analysis The experiment was designed with three replicates for each concentration and with an equal number of flies distributed in the same sex ratio. The results of the graphs were plotted with Graph Pad Prism 6.0 (Graph Pad Software Inc., San Diego, CA) with (mean ± S.D.) and statistical analysis was done using unpaired two tailed Student's t-test between the control and the treatment groups. 3. Results 3.1. Characterisation of titania nanoparticles 3.1.1. XRD analysis The XRD patterns of the TiO2 NPs clearly show the anatase phase of TiO2 (Fig. 1A). The diffraction pattern having a peak at 2θ values of 25.3°, 37.8°, 48.0°, 53.9°, 55.5°, 62.9°, 68.9°, 70.1°, and 75.2° correspond to [101], [004], [200], [105], [211], [204], [220], and [215] crystal planes which can be indexed to the anatase phase of TiO2 which matches well to the reported JCPDS data (Card No. 21-1272). 3.1.2. Field emission scanning electron microscope (FESEM) and transmission electron microscopy The detailed size, morphology, internal structure and crystallinity of the TiO2 nanoparticles were investigated by FESEM, TEM and HRTEM. Fig. 1B shows the spherical particles of TiO2 nanoparticles with rough surface indicating the agglomeration of the synthesised nanoparticles. From (Fig. 1C) TEM image, it is confirmed that the several individual TiO2 particles seems to be agglomerated to form a bigger particle size around 105 ± 5 nm (Fig. 1D). The size of the individual TiO2 nanoparticles are of 30 ± 5 nm which can be clearly seen from Fig. 1E. Furthermore, it forms lattice fringes with a d-spacing of 0.351 nm, corresponding to the (101) plane of anatase TiO2. The ring patterns can be seen from SAED image (Fig. 1F) of TiO2 nanoparticles, confirming the polycrystalline nature of the samples and the patterns could be indexed to the tetragonal phase of TiO2, thus validating XRD data (Fig. 1A). 3.1.3. Zeta potential The TiO2 NPs were suspended in the Milli-Q water and sonicated for 20 min. The zeta potential is found to be − 3.60 mV (Fig. 1G), thus explaining the cause of the agglomeration and its instability in water medium which has also been reported by Carmona et al. [34].


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Fig. 1. Characterisation of TiO2 NPs: (A) XRD pattern of TiO2 nanoparticle. (B) FE-SEM image of TiO2 NPs with spherical shape. (C) TEM image of TiO2 NPs. (D) Size distribution (Histogram) of TiO2 NPs. (E) HRTEM image showing d = 0.351 (101). (F) SAED pattern of TiO2 nanoparticles. (G) Zeta potential of TiO2 NPs, dispersed in water medium used during the food preparation. (H) FT-IR spectra of TiO2 nanoparticles.

3.1.4. FT-IR spectroscopy FT-IR spectrum reveals the presence of various functional groups in the currently used TiO2 nanoparticles (Fig. 1H). Two absorption bands at 3400 cm−1 and 1630 cm−1 are found, which are characteristic peak of O\\H bending modes of adsorbed water and hydroxyl groups respectively. Besides this, the spectrum also has strong absorption bands at 670 cm−1, which indicates the presence of Ti-O-Ti bond in TiO2 nanoparticles [35].

3.2. Analysis of NPs on Drosophila The flies laid their eggs in each vial of different treatment. The development time was monitored in each vial. Normal development was observed in the control, 50 and 100 mg·L−1 but there was a marked delay in development found at 200 and 250 mg·L−1 TiO2 treated flies. The developmental delay starts appearing during the 2nd instar larval stages,

as the larva started feeding and significant internal changes are still in progress. The delay is consistent till the adult. The pupation stages in 200 and 250 mg·L−1 concentrations had extended by 50 h in comparison to the control (Fig. 2).

3.3. Survivability study (toxic effect on lifecycle) This assay is used to determine the number of flies that successfully undergo eclosion from the pupal stages (Fig. 3). About 98.66 ± 0.25% of flies successfully hatched from pupae in the control vial. There was a significant difference found in 50 mg·L−1 and 100 mg·L−1 concentration where only 93.87 ± 1.70% and 82.30 ± 0.57% of flies hatched, respectively. Higher concentration of 200 mg·L−1 decrease hatching in flies by 63.14 ± 1.93%. The maximum concentration of 250 mg·L−1 marked severe decreases up to 56.55 ± 0.62%, reducing the flies to nearly half. There was also the incidence of larvae and pupae blackening at

Fig. 2. Life cycle of Drosophila treated with different concentrations of TiO2. In 200 mg·L−1 and 250 mg·L−1 significant developmental delay was observed.

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3.5. Larva crawling Various larvae crawling behavioural pattern were observed for different TiO2 treatment groups (Fig. 4A–E). In the control plate larvae walk straight, with minimal pause and making light turns with a speed of ~6–7 cm/min (Fig. 4F). In 50 mg·L−1 and 100 mg·L−1 concentration, crawling speed decreases to an average rate of ~5 cm/min however the path covered remains more or less the same. At maximum concentration (250 mg·L−1) the speed is b 3 cm/min with intervals of pause and increase in zigzag turns. Higher concentration of TiO2 (200 mg·L−1 and 250 mg·L−1) in larvae seemed to make their movement sluggish (Fig. 4F). 3.6. Nitroblue tetrazolium assay for reactive oxygen species

Fig. 3. Toxicity assay: The percentage (%) of flies hatching from the pupae at different concentrations of TiO2. The results significant as compared to the control are marked with asterisks (* for P-value b 0.05, **** for P-value b 0.001).

200 mg·L−1 and 250 mg·L−1 with dead larvae at 250 mg·L−1 concentration (Fig. 5D, E).

3.4. Trypan blue staining Trypan blue staining distinguishes the viable cells from dead cells. Positive trypan blue staining is observed only in 250 mg·L−1 concentration indicating the damage of the gut inner layer. Among the gut, only the anterior part of the gut seems to be affected (Fig. 5A–C, C′) as the larval stages of Drosophila is in voracious feeding stage, and higher concentration TiO2 appears to have affected its anterior region majorly.

Nitroblue tetrazolium (NBT) binds with free radicals and turns the soluble yellow compound to insoluble formazan crystals. As the concentration increases the amount of free radicals increases for which the absorbance at 595 nm increases. 200 mg·L−1 and 250 mg·L−1 (Fig. 6A) showed significant increase in absorbance reading indicating that TiO2 NPs exposure generates ROS. 3.7. Comet assay The DNA damage caused by the exposure of TiO2 NPs reported in comet assay (Fig. 6B). The tail length is very prominent in 200 mg·L−1 and 250 mg·L−1 concentration. 3.8. Body weight A decrease in body weight was observed in the newly hatched adult flies. The higher concentrations treatment (200 and 250 mg·L− 1) of TiO2 NPs showed a decrease in weight by 2–5 mg for a total of 30 flies (Fig. 7).

Fig. 4. Larva crawling behaviour of 3rd instar larva after treatment with various concentrations of nanoparticles: (A) Wild type larvae follows a straight path. (B–E) TiO2 treated flies shows deviation from their path. (F) Graphical representation of the distances travelled by the larvae in different concentrations of TiO2 NPs (mean ± S.D.) with the significance values against the control are represented as asterisk (n = 27; * for P-value b 0.05, ** for P-value b 0.004, **** for P-value b 0.001).


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Fig. 5. Trypan blue staining in 3rd instar larva and larva, pupae damage: Effect of titania nanoparticles at 3rd instar larval stage (trypan blue being used as marker for dead cells, also detects presence of any tissue damage). (A) Larvae from the control, shows no sign of cell or tissue damage. (B) 250 mg·L−1 treatment larvae showed internal gut damage. (C) In 250 mg·L−1 treatment, the dissected gut was dipped in trypan blue and the brain ganglia region seemed to be affected (a magnified image in C′ inset). (D) Dead larva found in 250 mg·L−1. (E) Control pupae and dead pupae found in 250 mg·L−1.

3.9. Climbing assay Climbing assay divulges significant behavioural variations on graviception, and agility in the fly (Fig. 8). In the control group, 92.66 ± 2.08% flies can climb the threshold of 10 cm mark within 10 s. Treated flies showed a gradual decrease in climbing with an increase in TiO2 concentration. In 50 mg·L−1 vials 87.04 ± 1.4% flies and in 100 mg·L−1 vials 82.23 ± 0.68% flies could cross the 10 cm mark. The climbing rate decreases N20% at a higher concentration in comparison to the control. A further decrease can be noted in 200 mg·L−1 where 70.00 ± 3.0% flies were capable of climbing the mark. The number of flies further dropped to 57.38 ± 1.47% at 250 mg·L−1 treatment.

various regions of Drosophila like thorax (bristles arrangement) and wings (venation). The variations observed in different concentrations were not the same. 3.10.1. Wing phenotype The wing venation pattern in wild type is divided into 6 parts L1 to L6. L1 as vein L1, L2-radial vein, L3-medial vein, L4-cubital vein, L5-distal vein and L6-vein (represented in Fig. 9A). L3–L4 veins were joined by anterior cross vein (a-cv), and L4–L5 is joined by posterior cross vein (p-cv) [36]. With treatment of different concentrations of TiO2 NPs different wing phenotypes were observed (Fig. 9B–F). NPs affect the wing phenotype either completely by changing the margin of the wing of or by altering the veins presents in the wing. The percentage of flies that show affected wing phenotype are shown in Fig. S1(A).

3.10. Phenotype observation To score the structural changes in the body phenotypic variations were searched under the microscope. Variations were marked in the

3.10.2. Bristle phenotype The wild-type flies have 13 pairs of bristles on the thorax (Fig. 10A). The bristles are divided in between anterior scutum and posterior

Fig. 6. Reactive oxygen species causing oxidative damage: (A) NBT assay shows significant increase in ROS in 200 and 250 mg·L−1. (B) Comet assay showing percentage of DNA tail damage with increasing concentration.

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Fig. 7. Body weight: The body weight observed from the adult flies after hatching. Significant results as compared with the control are marked with asterisks (* for Pvalue b 0.05, ns = non-significant).

scutellum. The bristles are named as reported earlier. In wild-type, aDC marked anterior dorso central, pDC-posterior dorso central, aSCanterior scutellar, pSC-posterior scutellar, pPA-posterior post-alar, aPA-anterior post-alar, pSA-posterior supra-alar (Fig. 10A), aSA-anterior supra-alar, Hu-humeral (2 in number), aNP-anterior notopleural, pNP-posterior notopleural, and PS represents for presutural (not shown in the figure) [36]. The general arrangement of bristles remains unaffected however the bristle number varies in almost all the concentration (Fig. 10B–H). The bristle variation does not follow in a concentration-dependent manner. The percentage of flies that showed affected bristle phenotype are represented in Fig. S1(B). 4. Discussion NPs are already enlisted to pose a toxic threat by causing damage to the cell membrane, nuclear material and induce apoptotic cell death [37–39]. The mechanism reported so far, the cell death is caused by the generation of free radical or by upregulated cytokine, growth factor (TNF α, Interlukin-1, 6, 8) and nuclear factor-kappa B (NF-κB) [40]. NPs do also affect various physiological systems including neurons. It affects the neurons by distressing nerve growth factor, the voltage-gated channel of hippocampal neurons, blood-brain barrier (BBB) and reportedly causes neuronal degeneration [41–43]. Silver NPs have caused a more

Fig. 8. Climbing assay: The percentage of flies climbing the 10 cm mark of the 100 mL measuring cylinder in order to test their behaviour against negative geotropism hatching from the pupa at different concentrations of TiO2 (mean ± S.D.). Significance for the results as compared with the control are marked with asterisks (* for P-value of 0.0179, ** for P-value = 0.0012, *** for P-value of 0.0004, **** for P-value of b0.0001).

negative impact on neurons. Other NPs like ferrous, manganese, zincoxide, and silica also known to affect the neurons either by suppressing neuronal activity via dopamine depletion, heat-stress aggravation and degeneration of BBB or edema formation [42]. Copper NPs affect the somatosensory neurons from dorsal root ganglia exposure which occurs with increased dose, concentration and size [44]. In the current study, TiO2 affects the mechanosensory neurons of Drosophila at different developmental stages of the life cycle which have been identified by larva crawling and climbing assay. Although there are several modes of administration of NPs, the current study evaluates the toxicity of TiO2 through the oral intake route. Questioning as, why the oral administration method is better than the other method of exposure? The oral route of administration is a natural way to which the engulfed NPs will have a chance to eliminate from the body, detoxify its effect and stimulate the oxidative response of the body. The NPs pass through Drosophila digestive system (foregut, midgut, and hindgut) of varying pH. Thus, the fate of NPs (pass/cellular uptake) within the gut is decided based upon its surface charge [45]. It has been proposed that the negatively charged NPs are harmful to the body as it can alter the pH of the digestive system and thus its functionality which now stands true in our case. The zeta potential of currently used TiO2 NPs is found to be − 3.60 mV. Negative zeta potential of TiO2 and its toxic effect were reported in Drosophila by Carmona et al. [34]. With increasing NPs concentration, flies survivability is observed to be affected. Survival assay is a standard test to check the effect of various chemicals [46,47] and recently, this test was used to screen for nanomaterial toxicity. TiO2 NPs has earlier been reported to affect the survivability and growth of the organism via generating reactive oxygen species (ROS) [34,48,49]. How TiO2 affect the survivability of the fly? One possible mechanism is interaction of eggs with TiO2 present within the food. If NPs can damage the eggs due to its toxic stress, then the egg do not proceed further towards its next developmental stages. A similar mechanism was reported in magnetite NPs [50] where NPs, penetrate the eggs, producing reactive oxygen species (ROS) within them and thus affect the survivability [51]. Since TiO2 generates reactive oxygen species (ROS) [52] a known causative factor for oxidative stress a similar mechanism is anticipated [53]. Oxidative stress is further known to be associated with developmental delay of various insects including D. melanogaster [54]. The eggs changing to the larvae (a voracious eater) have a greater chance of exposure to NPs due to accumulation of NPs in the gut. The higher concentration (250 mg·L−1) NPs in the gut is toxic for the larvae resulting early death producing blackening in many larvae. Loss of eggs and larvae at various developmental stage resulted in a decrease in the percentage of adult flies in higher concentration. The toxicity of chemically synthesised TiO2 found in the current study is less as compared to reported work from Philbrook [48] but higher when compared to the food grade TiO2 [14]. The toxicity of NPs is known to be scaled to the size of NPs. The variations observed in the current study are probably due to variations of NPs used in both the study. Based on the surface chemistry, TiO2 NPs are majorly of two different types: (1) anatase form and (2) rutile form. Both the forms have a different level of toxicity, out of which anatase form is more capable of generating ROS [55]. The anatase form absorbs water molecules and dissociates it to H+ and OH−. The anatase form can convert OH− ions into OH•. OH• is highly reactive and capable of generating ROS by interaction with other biomolecules. Anatase form of TiO2 can induce oxidative stress even without UV exposure since the conduction band lies close to the biological redox potential which is (− 4.12 eV to −4.84 eV) and less energy is required for the e− transfer. This causes an exchange of e-between NPs, cells and vice-versa [56]. This non-photocatalytic toxic property of TiO2 results by 1) adsorption of extracellular NPs and inducing intracellular ROS (2) interaction with the biomolecule and cause cell damage (3) free radical generation even without irradiation [57]. The cytotoxicity of anatase form is already


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Fig. 9. Wing phenotypes from a different set of treatment groups. (A) The control shows the regular arrangement of veins and cross veins in the wings. Various veins are marked as follows. L1-vein L1; L2-radial vein; L3-medial vein; L4-cubital vein; L5-distal vein; L6-vein L6; a-cv anterior cross-vein; p-cv posterior cross-vein. (B–C) In 50 mg·L−1 treatment, the L5 vein remains incomplete (in B and C) and extended L6 vein found in B. (D–F) In 100 mg·L−1 treatment the p-cv affect (either incomplete, multiple number appear) (in D–G). In 200 mg·L−1 treatment margin of wing gets affected. (H) 250 mg·L−1 treatment margin gets affected.

proved in cell culture media [55]. Since the currently used TiO2 is of anatase form, we anticipate similar surface chemistry mechanism behind the toxicity of TiO2 NPs used in the current study. Does TiO2 have a toxic effect on the larvae neurons which survived in the vial? Larva crawling assay, a well-known method was used to identify the neuronal damage at an early developmental stage [58]. In the current study, WT larvae can crawl with short pause, turns and cover a certain distance with an average speed of 6–7 cm/min which is in agreement with the previous report [32] but not in the treated larvae where the average crawling rate decreases to almost half with increased number of turn, unusual twist and pause. The higher concentration TiO2 exposed larvae were sluggish and showed unusual twist and turn. The twist and turn behaviour of the larva signifies the toxic stress on mechanosensory neuron with improper brain coordination and defective sub-oesophageal ganglion [59]. The sluggishness further indicates cholinergic neurons are affected resulting abnormal muscle contraction and crawling [59].

In the larvae, gut damage observed at higher concentration (250 mg·L−1) in the current study is in agreement with the earlier report of Shakya et al. [60]. NPs does induce cell death mostly by membrane peroxidation or oxidative stress produced by ROS-induced mechanism which is observed with NBT and comet assay results [19]. The larvae after metamorphosis transform to adult. To check whether the neuronal defect of larvae persists in the adults, behavioural assay (climbing assay) was performed [61]. TiO2 treated flies shows decreased climbing ability indicating the damage during the larval stage could not be repaired during metamorphosis and resulted in abnormal behavioural phenotype in the adult. During metamorphosis, most of the embryonic, larval tissue imaginal disc are transformed to adult organs [62]. It has been reported that in D. melanogaster nanoparticles predominately accumulate in the fat body of larva and not in imaginal discs [63]. However, various wing phenotypes observed in the current study indicates the wing imaginal disc must be affected during the developmental stage. One possible mechanism could be a small amount of NPs probably

Fig. 10. Bristle phenotypes. (A) The control shows the general arrangement of bristles over the thorax region. Various bristles are marked as follows. aDC-anterior dorso central; pDCposterior dorso central; aSC-anterior scutellar; pSC-posterior scutellar; pPA-posterior post-alar; aPA-anterior post-alar; aSA-anterior supra alar; pSA-posterior supra alar. (B–C) In 50 mg·L−1 concentration loss of both pSC region is observed (D–E). 100 mg·L−1 shows loss in the right-left pSC bristle in D and left pSC bristle missing, short aSC and bent right aSC in E–F. 200 mg·L−1 showed missing right pSC and short right pPA (G–H). 250 mg·L−1 treatment missing right pSC and right pPA in G and missing right pSC bristle in (H). The arrow marks the bristle affected.

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interferes with the signalling pathway during the metamorphosis and thus causes a phenotypic defect in the wing. Our result shows deviation from earlier TiO2 toxicity assessment reports; this study found that even a small dose exposure (50 mg·L−1) could induce phenotypic alterations in the adult. The phenotypic defect observed suggests signalling pathways being affected in the due course of development. The deviation achieved in the current study could be due to different forms (anatase and rutile) of TiO2 NPs, used in either study as the toxicity of TiO2 NPs is further scaled with the physicochemical properties [34]. Wing development from imaginal disc to adult involves various signalling pathways [64]. Notch signalling is induced in the third instar larval imaginal disc and causes reciprocal activation of Delta and Serrate ligands. The spacing between L3–L4 is regulated by hedgehog signalling, determined at the second and third instar larval stages [65]. Similarly, the L2–L3 and L4–L5 spacing is due to Bone Morphogenetic Protein (BMP) signalling [66]. The margin formed between the dorsal and ventral is by the Notch signalling [67]. Any disruption of signalling pathway or ligands results in deformed wing pattern. The various wings deformities observed in TiO2 treatment flies are likely because of TiO2 toxicity that affects various signalling pathway during metamorphosis. Analogous to the wing phenotype, bristle phenotype is also controlled by a plethora of signalling and patterning factors [68]. The TiO2 toxicity affects the bristle number indicating mechanosensory bristle development is affected. The TiO2 treated fly phenotype is analogous with scutoid mutants where the posterior scutellar macrochaete are missing. Bristle development begins with 4 cells from two successive divisions of single organ precursor (SOP) cells. The SOP daughter cells differentiate into bristle, socket and glial cells [69]. The bristle morphogenesis involves three stages [69]. The first stage is the separation of the disc of pro-neuronal clusters i.e. transformation of 20–30 cells to SOP cells. Next is the determination and localization of cells inside proneural cluster [69] which is under the control of selector genes [70]. The process of cell determination morphogenesis of the proneural cluster cells depends on the intracellular regulation of the proneural gene achaete-scute (AS-C) activity and intercellular events of EGFR and Notch signalling pathway [69]. Alteration in any of the pathway resulted in the complete or partial loss of macrocheate. Loss of bristles indicates the TiO2 NPs can affect the signalling pathway involved in bristle formation. 5. Summary The current article reveals the effect of TiO2 NPs on the development of Drosophila melanogaster. The TiO2 NPs are administered through the food immediately after hatching from the egg the larva starts feeding treated food. Once these NPs enters into the gut, it interferes with the developmental process resulting abnormal larval behaviour and generating abnormal phenotypes. Larvae shows neuronal defect when fed with TiO2 NPs. If the damage induced by TiO2 is severe, then the larvae lethality is observed. If larvae strive to survive the stress during larval stages, the defect appears to be noticed in the adult stage with interrupted developmental process during metamorphosis. Being a foreign particle, it disturbs various signalling pathways resulting defective wing, bristle and behavioural phenotype for which further research is required. The toxic effect of NPs on neurons is alarming and needs to be considered before the extensive use of chemically synthesised NPs in food and medicine. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.physbeh.2016.08.032. Acknowledgments We are thankful to Pragalbha Jaysingh, Ruchika Nayak, and Samyogita Sathpathy for their help in bristle counting and analysis of fly phenotype used in the current study. We are also thankful to the technical assistants of NIT Rourkela SEM, TEM facility and Prof. Santanu

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