Sedentary behavior and altered metabolic activity by AgNPs ingestion in Drosophila melanogaster

Abstract

Among several nanoparticles, silver nanoparticles (AgNPs) are extensively used in a wide variety of consumer products due to its unique antimicrobial property. However, dosage effect of AgNPs on behavior and metabolic activity in an in vivo condition is not well studied. Therefore, to elucidate the impact of AgNPs on behavior and metabolism, systematic and detailed dosages study of AgNPs was performed by rearing Drosophila melanogaster on food without and with AgNPs. We found that dietary intake of AgNPs at early larval stage leads to behavioral abnormalities such as poor crawling and climbing ability of larvae and adults respectively. Interestingly, intake of higher dosage of AgNPs at larval stage significantly altered metabolic activity that includes lipid, carbohydrate and protein levels in adult flies. Further, detailed analysis revealed that AgNPs causes remarkable reduction in the number of lipid droplets (LDs) which are lipid storage organelles in Drosophila. We also observed an increased production of reactive oxygen species (ROS) in AgNPs ingested larval tissues. These results strongly imply that higher dosage of AgNPs ingestion from early larval stage of Drosophila is inimical and thereby draws concern towards the usage of AgNPs in consumer goods.

Figure 1

Compromised behavioral activity of F1 progeny reared on AgNPs-supplemented food. (A) No effect on vertical climbing of AgNPs-supplemented and not supplemented (control) adult flies aged for short 10 and long 20 and 30 days. (B) Dose-dependent impairment in crawling ability of wandering F1 third instar larvae eclosed from different treatment conditions (P − L−/P − L + /P + L−/P + L+). (C) Significant reduction in climbing behavior of F1 adult progeny at an age of day 1 and 7 reared on 25 and 50 mg/L of AgNPs (Please note that P + L− climbing data was comparable to P − L− and P − L+ was comparable to P + L+; therefore only P− L− (0 mg/L) and P − L+ data is shown in the graph). Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test (MSD α_0.05: Crawling (F1) = 0.84; Climbing (F1) = 28.4). ***p < 0.001 (Student’s t-test).

Figure 2

AgNPs ingestion at larval stage causes depigmentation and drop in body weight. (A) notable loss in body pigmentation was seen in F1 flies eclosed from larvae reared on AgNPs-supplemented food. Reduction in (B) fresh body weight, (C) dry weight and (D) water content of F1 flies eclosed from all experimental conditions. Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test (MSD α_0.05: Fresh weight = 163.4; Dry weight = 41.4; Water content = 227.8). ***p < 0.001 (Student’s t-test).

Figure 3

Impedance in feeding behavior upon exposure to AgNPs. (A) Larvae reared on standard and AgNPs-supplemented food were fed with a mixture of yeast and 4% blue dye #1. (B) Larvae reared on higher dose of AgNPs-supplemented food showed poor rate of feeding as compared to control larvae. (C) Adult flies emerged from standard and AgNPs-supplemented food were fed with a mixture of yeast and 2.5% blue dye #1. (D) Rate of food intake in F1 adult progeny. Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test (MSD α_0.05: Adult feeding = 0.12). ***p < 0.001 (Student’s t-test).

Figure 4

Alteration in lipid levels and accumulation by larval AgNPs ingestion. A notable reduction in lipid content of F1 adult flies on (A) Day 0 and (B) Day 15 post eclosion. (C) Lipid droplets in fat body of control and AgNPs ingested flies. (D) Total surface area occupied by lipid droplets. Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test (MSD α_0.05: Lipid content (Day 0) = 54.7; Lipid content (Day 15) = 45; LD = 7862.01). ***p<0.001 (Student’s t-test).

Figure 5

Modulation in trehalose, glycogen and protein content by AgNPs ingestion. AgNPs ingestion in larvae alters (A) trehalose content (B) glycogen content and (C) protein content in F1 adult flies. Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test (MSD α_0.05: Trehalose content = 1.0; Glycogen content = 17.5; Protein content = 41.4). ***p < 0.001 (Student’s t-test).

Figure 6

Higher dosage of AgNPs larval ingestion enhanced ROS production. (A) ROS production in larval fat body and wing imaginal disc. Quantification of the mean fluorescence intensity of DHE staining in (B) larval fat body and (C) wing imaginal disc. Significance was calculated by using an analysis of variance (ANOVA) followed by Tukey-Kramer MSD post hoc test. (MSD α_0.05: Intensity (fat body) = 887.5; Intensity (wing imaginal disc = 757.9). ***p < 0.001 (Student’s t-test).