2.1 Reagents and fly culture

10, 50, and 100 nm TiO₂ NPs were purchased from Beijing Dk Nano Technology Company. They were dissolved in double distilled water and strongly sonicated with a BILON-R1200 sonicator before mixing with agar media or fly food. w1118 flies were raised at 25 ℃ and 60% relative humidity on cornmeal-yeast food. Each fly tube contained fifteen newly laid embryos or newly eclosed adult flies.

2.2 Selective media for bacteria culture

To test the antibacterial activities of TiO₂ NPs in vitro, commensal bacteria cultured in liquid selective media were plated onto selective plates with or without pre-mixed TiO₂ NPs. To test the antibacterial activities of TiO₂ NPs in vivo, fly larvae or adults were raised on food with or without pre-mixed TiO₂ NPs. Dissected midguts were homogenized in PBS and plated onto selective plates. Selective media were generated as below [24]. Acetobacteria: 25 g/l D-mannitol, 5 g/l yeast extract, 3 g/l peptone, and 15 g/l agar (for plates). Enterobacteria: 10 g/l Tryptone, 1.5 g/l yeast extract, 10 g/l glucose, 5 g/l sodium chloride, and 12 g/l agar (for plates). Lactobacilli: 70 g/l BD Difco Lactobacilli MRS media and 12 g/l agar (for plates). Plates were incubated at 30 ℃ for 36 h–48 h and bacterial colonies were counted. Colony-forming units (CFU) represented the average number from four plates.

2.3 Measurement of Drosophila weight, lipid and locomotor activity

Drosophila weight was measured with a Mettler Toledo XP6 analytic balance. Triacylglyceride levels were measured as previously described [25]. Briefly, eight third instar larvae or adults were suspended in 400 μL chilled lysis buffer (0.01 mM KH₂PO₄, 1 mM EDTA, protease inhibitors cocktail, 0.05% Tween 20), lysed by Tissuelyser II (Qiagen). The lysate was extracted with 1 ml chloroform: methanol (2:1). The organic phase was transfered to a new tube and air-dried over night at room temperature. The dried lipid was re-dissolved in 300 μL alcohol with 1% Trition-X100, and assayed with Free Glycerol Reagent (Sigma F6428) and Triglyceride Reagent (Sigma T2449). Lipid levels were normalized to total protein levels. Locomotor activity was measured according to the previous report [26, 27]. Forty-five adult flies were dispersed into three plastic vials and gently tapped to the bottom of the vials. The climbing distance of each fly was measured after 5 s. The experiment was repeated three times.

3.1 Effects of TiO₂ NPs on Drosophila commensal bacteria in vitro

Previous studies propose that TiO₂ NPs inhibit bacterial growth via their photocatalytic activity, which creates ROS and hydrogen peroxide through UV radiation [13]. However, whether they possess other activities that affect bacterial growth is unclear. In addition, whether they can affect commensal bacteria and pathogenic bacteria in a similar way is largely unknown. To test these possibilities, we cultured Drosophila commensal bacteria on plates containing TiO₂ NPs. These bacteria were plated in a darkroom (with red light) and incubated in darkness to avoid UV radiation. To quantify each symbiotic bacterial species, we measured colony-forming units (CFU) using selective plates to identify Acetobacteria, Enterobacteria, and Lactobacilli, three major forms of Drosophila commensal bacteria.

Here we tested TiO₂ NPs with three different sizes (referred to as TiO₂-10 nm, TiO₂-50 nm, and TiO₂-100 nm). The surface of these TiO₂ NPs was modified with hydroxyl groups. Dynamic light scattering showed that they possessed similar zeta potential: -27.3 mV for TiO₂-10 nm, -26.6 mV for TiO₂-50 nm, and -31.6 mV for TiO₂-100 nm. On selective plates containing 200 μg/ml TiO₂ NPs, growth of Acetobacteria was not affected by any type of TiO₂ NPs. When the concentration of TiO₂ NPs was increased to 2 mg/ml, all types of TiO₂ NPs could inhibit the growth of Acetobacteria to a similar extent (Fig. 1(a) and 1(d)). Enterobacteria seemed to be more sensitive to TiO₂ NPs than Acetobacteria. The growth of Enterobacteria was significantly inhibited by 200 μg/ml TiO₂ NPs, and was further inhibited by 2 mg/ml TiO₂ NPs. Similar to the effects on Acetobacteria, the different sizes of TiO₂ NPs did not cause different antibacterial activities on Enterobacteria (Fig. 1(b) and 1(e)). In contrast, the size of the TiO₂ NPs affected Lactobacilli. At concentrations of both 200 μg/ml and 2 mg/ml, TiO₂-50 nm and TiO₂-100 nm inhibited Lactobacilli growth more severely than TiO₂-10 nm (Fig. 1(c) and 1(f). These results suggest that TiO₂ NPs can inhibit the growth of commensal bacteria in vitro, in a photocatalytic activity-independent manner. Different genuses of commensal bacteria had different sensitivities to the dosage or the size of the TiO₂ NPs.

Fig. 1.Full-screen View

(Color online) Effects of TiO₂ NPs on Drosophila commensal bacteria in vitro. (a–c) Statistics of colony-forming units (CFU) of in vitro cultured Acetobacteria (a), Enterobacteria (b), and Lactobacilli (c) on selective plates containing 200 μg/ml or 2 mg/ml TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm. n.s.: not significant. *P < 0.05; **P < 0.01; ***P<0.001. Representative results of the effect of 2 mg/ml TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm on the growth of Acetobacteria (d), Enterobacteria (e), and Lactobacilli (f). 100 μl medium was plated on selective plates. The number of colonies on the plates was indicated on the lower right of each panel.

3.2 Effects of TiO₂ NPs on Drosophila commensal bacteria in vivo

Next we examined the effects of TiO₂ NPs on commensal bacteria in vivo. We dispersed TiO₂ NPs into fly food and fed them to Drosophila larvae or adults. As male larvae or male adults showed a large variance in the number of commensal bacteria among individuals, we decided to focus on female flies. Female larvae were collected before pupation and female adults were collected after 5 days for gut dissection and quantification of symbiotic bacteria. Under this condition, we found that the growth of Acetobacteria, Enterobacteria, or Lactobacilli in larvae was not affected by three types of TiO₂ NPs, even though the dosage was the same as that used in vitro (Fig. 2). Similarly, either 1 mg/ml or 2 mg/ml TiO₂ NPs of three different sizes failed to significantly inhibit the growth of Acetobacteria, Enterobacteria, or Lactobacilli in adult flies (Fig. 3).

Fig. 2.Full-screen View

Effects of TiO₂ NPs on commensal bacteria in Drosophila female larvae. Statistics of colony-forming units (CFU) of Acetobacteria (a), Lactobacilli (b), and Enterobacteria (c) from homogenized Drosophila larval guts. Larvae were raised on food containing 200 μg/ml or 2 mg/ml TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm until they reached the stop-wondering stage. Female larvae were picked for dissection. n.s.: not significant.

Fig. 3.Full-screen View

Effects of TiO₂ NPs on commensal bacteria in Drosophila female adults. Statistics of colony-forming units (CFU) of Acetobacteria (a), Lactobacilli (b), and Enterobacteria (c) from homogenized Drosophila adult guts. Newly eclosed female flies were raised on food containing 200 μg/ml or 2 mg/ml TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm for five days before dissection. n.s.: not significant.

3.3 Effects of TiO₂ NPs on Drosophila development and metabolism

The results above were not consistent with those from in vitro. To confirm that our observation indeed reflected a discrepancy between in vitro and in vivo, we further analyzed commensal bacterial-associated developmental and metabolic functions in flies fed with TiO₂ NPs. Gut commensal bacteria play crucial roles in host development, metabolic homeostasis, immune status, and aging [15-21]. Therefore we would expect to observe defects in developmental cycle, energy store, or locomotor activity in Drosophila if their gut commensal bacteria were inhibited by dietary TiO₂ NPs.

We first examined the effect of dietary TiO₂ NPs on Drosophila larval development. In the control group, about 90% of female larvae formed pupae 120 h after egg laying. Either 200 μg/ml or 2 mg/ml dietary TiO₂ NPs of three different sizes did not affect the pupation cycle of the Drosophila larvae (Fig. 4(a)). In addition, the weight and lipid level of the female larvae were not altered by any type of TiO₂ NPs (Fig. 4(b) and 4(c)).

Fig. 4.Full-screen View

Effects of TiO₂ NPs on Drosophila larval development and metabolism. The effects of 200 μg/ml or 2 mg/ml dietary TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm on pupation rate (a), weight (b), and lipid level (c) of female larvae were shown. n.s.: not significant.

We then examined five-day-old female adult flies raised on food containing 1 mg/ml or 2 mg/ml TiO₂ NPs. Similar to our observation in larvae, the weight and lipid level in female adults were comparable to those in the control group (Fig. 5(a) and 5(b)). We also measured the climbing index (i.e. average climbing speed of flies on the wall of the fly tubes) of these flies and observed no significant changes with dietary TiO₂ NPs (Fig. 5(c)). Together, these results imply that the in vivo antibacterial activity of TiO₂ NPs on Drosophila gut commensal bacteria differs from in vitro activity.

Fig. 5.Full-screen View

Effects of TiO₂ NPs on metabolism and locomotor activity in Drosophila female adults. The effects of 200 μg/ml or 2 mg/ml dietary TiO₂-10 nm, TiO₂-50 nm, or TiO₂-100 nm on weight (a), lipid level (b), and climbing index (c) of five-day-old female adults were shown. n.s.: not significant.