The Impact of Inhaled Pollution on Health Outcomes in Drosophila Melanogaster
Updated: Jun 1
Hamza Arman Lateef, Tony Bright, and Gabriel Ralston
Charles J. Colgan High School, Manassas, Virginia, USA
Air pollution is a serious, global problem that threatens all living organisms. The objective of this project was to determine whether face masks can significantly diminish inhaled particulate matter less than 2.5 microns in diameter (PM 2.5), and improve health outcomes in Drosophila melanogaster (fruit fly). It was hypothesized that if Drosophila is exposed to high levels of PM 2.5, then its lifespan and fertility will decrease. Also, if a Vogmask is used, then it will significantly decrease the amount of inhaled PM and mitigate the impact of PM 2.5 on longevity and fertility compared with a doctor’s mask or no mask. Flies were exposed to pollution in a controlled chamber, using the exhaust from a STIHL Echo-One trimmer. An air pollution monitor was used to record PM 2.5 levels.
Mask Efficacy : Exhaust was run through a tube (uncovered, or covered with either a doctor’s mask or Vogmask) and the transmitted PM 2.5 was recorded.
Longevity and Fertility: Drosophila were placed into 4 experimental groups: 1) No pollution; 2) Pollution and no mask; 3) Pollution and vials covered with doctor’s mask; 4) Pollution and vials covered with Vogmask. After 6 hours of pollution exposure, flies in groups 2, 3, and 4 were transferred to an incubator with unexposed flies, and their lifespan was monitored. To assess fertility, 3 male and 3 female flies were placed into each vial and pupae were counted on day 5.
The results of the experiment showed that the doctor’s mask and Vogmask decreased inhaled PM 2.5 by 71% and 92%, respectively. Pollution decreased Drosophila lifespan by 80%, whereas wearing a Vogmask during pollution exposure decreased lifespan by 53%. The control group had significantly more mean offspring (4) than either the Vogmask (1.5), the doctor’s mask (0.5), or no mask (0.25). Face masks can be quite effective at filtering out particulate pollution; however, they are not the ultimate answer to our environmental emergency. We must remain committed to cleaning up our planet in the first place.
9 out of 10 people in the world now breathe polluted air, resulting in the death of 7 million people each year (WHO, 2016). The burning of fossil fuels and its associated climate change are major causes of air pollution.
There are two main types of air pollution - particulate matter and gases. Particulate matter (PM) includes fine inhalable particles that are generally less than 2.5 micrometers (PM 2.5) and particles less than 10 micrometers in size (PM 10) (EPA, 2018). Most particles form in the atmosphere due to complex reactions of chemicals such as sulfur dioxide and nitrogen oxides, which are pollutants emitted from power plants, industries and automobiles. Evidence suggests that fine particles like PM 2.5 play a prominent role in adverse health outcomes such as heart attacks and asthma (Pope, 2006).
The EPA calculates an Air Quality Index or AQI (Figure 1) for five major air pollutants regulated by the Clean Air Act: ground-level ozone, particle pollution (also known as particulate matter), carbon monoxide, sulfur dioxide, and nitrogen.
In countries such as China and India, many people wear face masks in an effort to protect themselves from air pollution. There are many different masks available on the market today, though their efficacy is largely unknown (Cherrie, 2018). In this project, we evaluated two commonly worn masks. The first is a simple surgical mask that can be found at any doctor’s office and contains a single cloth filter layer that is held onto the face with elastic straps. Although it is commonly worn by people in countries with heavy pollution, it is typically thought to offer very little protection from the PM 2.5 pollutants in the air. Some studies, however, have shown that it can protect against pollutants (Smart Air Filters, 2018). The Vogmask is the second mask we evaluated and uses multiple layers of filter material including microfiber cloth and carbon (Vogmask, 2017). It is considered a respirator because it uses valves to let the person wearing the mask exhale more easily and maintains a good seal. This mask is specifically made to protect against harmful PM 2.5 particles (Vogmask, 2017).
Based on a thorough review of the scientific literature, we posed the following question: Do face masks significantly decrease the amount of inhaled particulate matter and does this translate into improved, measurable health outcomes? We chose to study these questions using a convenient animal model such as Drosophila melanogaster (or the fruit fly) for several reasons. Drosophila has a short, simple reproductive cycle lasting about 8-14 days, contains at least a lung equivalent, and its airway system shows striking similarities with mammals regarding its physiology and reaction towards pathogens (Roeder, 2012). Moreover, flies share 75 percent of disease-causing genes with humans. In our study, we determined the amount of particulate matter registered by the pollution meter with and without the addition of each of the face masks. We engineered a pollution chamber to simulate a polluted environment using exhaust from a gas-powered trimmer motor and measured ambient air quality in the chamber using an air quality monitor. We then exposed groups of Drosophila melanogaster, contained in vials covered with and without air pollution masks.
We hypothesized that: 1) If Drosophila is exposed to high levels of airborne PM 2.5 then its lifespan and fertility will decrease; 2) A Vogmask will significantly decrease the amount of inhaled PM 2.5 compared to a doctor’s mask; 3) If a Vogmask is used to filter inhaled pollution, it will greatly diminish the negative impact of inhaled PM 2.5 on the organism’s longevity and fertility compared with a doctor’s mask or no mask.
METHODS AND MATERIALS
Construction of Pollution Chamber/Environment (see Figure 2):
We constructed a chamber as shown in Figure 2, using plywood sheets, a metal dryer vent and pipe, and sheets of plexiglass. We fixed one end of the dryer vent pipe onto the dryer vent to introduce the polluted air into the chamber. We also purchased an Air Quality Monitor (by EG Air), doctors masks, and Vogmasks (see Figures 3a, b and c).
Experiment 1: Dependent Variable = PM 2.5 Reading, Independent Variable = Mask Type
The purpose of this experiment was to determine how effective the pollution masks are at filtering out PM 2.5 pollutants. Pollution was generated using the exhaust from a STIHL Echo-One garden trimmer. We measured PM 2.5 using an air quality monitor in real time. To determine the amount of pollution given off by the motor, we connected the exhaust to one end of the 4” diameter dryer vent pipe and placed the air quality monitor about 6” from the open end. We ran the motor for 30 seconds and recorded the PM 2.5 level from the meter. Then we turned off the motor, waited 30 seconds, and recorded the PM 2.5 level again. We conducted three trials and used a leaf blower to introduce fresh air through the pipe after each trial. To test the effectiveness of the masks, we repeated the same process with each mask secured over the pipe so that the exhaust had to flow through the mask to get to the pollution meter.
Experiment 2: Dependent Variable = Longevity, Independent Variable = Mask Type
Wild type Drosophila melanogaster were acquired from Carolina Biological Company and maintained at room temperature under normal lighting conditions. Twelve vials of culture media were prepared and labeled, three for each of four experimental conditions: 1) No exposure to pollution; 2) Pollution only, no mask; 3) Pollution + doctor’s mask; 4) Pollution + Vogmask. The control vials were covered with regular plugs, and the “pollution, no mask” vials were covered with cling wrap that had holes pierced in a size that allowed pollutants through but blocked flies. The doctor’s mask and Vogmask were cut into circular pieces that were used to cover the “doctor’s mask” and “Vogmask” vials respectively.
The vials for experimental conditions 2, 3, and 4 were placed in pollution chamber. Exhaust of the trimmer motor was fed into the dryer vent. We recorded PM 2.5 levels every 30 minutes for a total of 6 hours. Comparing the average Drosophila lifespan (21-24 days) to human lifespan (79 years) we extrapolated this exposure to be roughly equivalent to 1 year of human life exposure to these conditions. After the exposure, all 12 vials were covered with regular vial plugs and placed in an incubator with the unexposed vials. The adult flies were transferred to new vials every 3-4 days to avoid including their offspring in the longevity count. During each transfer we recorded the number of dead flies in the old vial and living flies in the new vial, and the percentage of flies remaining alive in each of the experimental groups was calculated. Data were tabulated and graphed on Kaplan-Meier type of survival curves.
Experiment 3: Dependent Variable = Fertility, Independent Variable = Mask Type
Twelve vials of culture media were prepared and labeled, three for each of the four experimental groups as described above. Drosophila Kit from Carolina Biological was obtained where gender is matched to eye color. Flies were carefully anaesthetized using Flynap and separated by sex. Exposure to pollution was conducted as described above, for a total of 6 hours. Mean pupae and standard error were recorded for each group on day 5.
Pollution levels of PM 2.5 for our longevity and fertility experiments are shown in Figures 4a and Figure 4b.
Efficacy of Face Masks
The masks did diminish the amount of transmitted pollution to varying degrees as shown in Table 1 and Figure 4. At 30 seconds, the doctor’s mask and Vogmask allowed transmission of 29% and 7% of PM 2.5 pollutants, and at 60 seconds, only 9% and 5% of pollutants, respectively, were being transmitted.
Table 2 shows a snapshot (days 6-10) of our raw data where we calculated the percentage of surviving flies, under each experimental condition. At each time point studied, the control group did the best in terms of survival and the vial with “pollution + no mask” did the worst. Among the three groups exposed to polluted air, the Vogmask group did best. Using this data, survival curves were plotted using a Kaplan Meir model (Figure 6). We then calculated the time taken for 50% of each experimental group to die (Figure 7), showing that the Drosophila exposed to no pollution lived the longest, while exposed Drosophila, without any mask, died the earliest. Importantly, the group that was exposed to pollution but shielded by the Vogmask did better than the pollution group without a mask but not as well as the controls. This demonstrated that the Vogmask was able to protect the Drosophila from the hazards of pollution to some extent but posed more health risks than being in clean air.
Fertility was measured by counting pupae 5 days after exposure to the polluted environment (Figure 8). Means and standard errors were calculated for each experimental group and data is shown in Figure 8. On day 5 the control group had significantly more offspring (4 pupae) than any of the pollution exposed groups. Among those exposed to pollution, the Drosophila covered by the Vogmask (1.6 pupae) did better than the doctor’s mask (0.6 pupae), while the “no mask” group did the worst (0.3 pupae). Breathing polluted air significantly decreased the reproductive potential of Drosophila melanogaster; however, the Vogmask appeared to offer some protection from this adverse health effect.
The World Health Organization has named air pollution as its top threat to global health for 2019. This complex problem must be tackled from both ends - decreasing pollution as well as learning how best to protect ourselves against it. In this project, we successfully engineered a pollution chamber, recorded the efficacy of 2 masks (the doctor’s mask and Vogmask), and examined whether the masks could mitigate some of the negative effects of pollution of lifespan and fertility of Drosophila. Our hypotheses were partially correct. We showed that particulate pollution significantly decreased the lifespan of Drosophila melanogaster and the Vogmask helped mitigate this effect and did so better than the doctor’s mask. The doctor’s mask, however, did show a significant effect in filtering out pollutants and was comparable to the Vogmask in this aspect.
Our study confirmed prior research that Drosophila melanogaster can be used as an effective model organism to understand the health effects of pollution both on larvae (Santana et al., 2018) and adults flies (Eom et al., 2017). Although flies do not have lungs, their airway systems have enough similarities to the respiratory system of mammals to be useful model organisms for inhalation toxicity. We exposed flies to 6 hours of pollution which was estimated to be approximately 12 months of human life exposure. PM 2.5 levels were mostly in the 150-550- μg/m3 range. In China, PM 2.5 was the primary pollutant for 66.8% of the severely polluted days, and in cities like Chongquing, average indoor PM 2.5 levels range from 68-468 μg/m3 (Lin et al., 2018). Thus, flies in our experiment were exposed to worse but somewhat comparable conditions to the most polluted cities in China.
We found that polluted air significantly decreased Drosophila lifespan by 80%, similar to human studies where PM 2.5 caused a measurable decrease in lifespan due to cardiovascular and pulmonary diseases (Laden et al., 2006). Using the Vogmask caused a decrease in lifespan of 53%. In of the case of fertility, we showed a 92% reduction in number of pupae among Drosophila exposed to pollution without a mask compared to controls. However, it is unclear if this huge reduction was caused in part due to the decreased lifespan as well.
In our study the doctor’s mask blocked about 90% of PM 2.5 pollution which seemed surprisingly high but was comparable to other studies where this type of surgical mask has been shown to block 60-90% of PM 2.5 particles while masks like the Vogmask perform better (Vogmask, 2017). Despite high filtration efficiency, the doctor’s mask was unable to significantly mitigate the adverse health effects of the polluted environment of Drosophila in our study. It is possible that that the exhaust fumes used in our study contained significant noxious gas pollution in addition to PM 2.5 particles which could not be filtered by the masks and this was a variable that we did not measure.
In summary, the Vogmask consistently did better at mitigating the deleterious effects of particulate pollution of Drosophila melanogaster’s lifespan and fertility but was not comparable to lifespan and fertility of Drosophila living in clean air.
Face masks are not the ultimate answer to our environmental emergency; they offer only a partial solution to this very large problem. While we must continue to research more effective strategies to protect our health from environmental pollution, we must be equally committed to cleaning up our planet in the first place.
Cherrie, J. W., Apsley, A., Cowie, H., Steinle, S., Mueller, W., Lin, C., . . . Loh, M. (2018).
Effectiveness of face masks used to protect Beijing residents against particulate air pollution. Occupational and Environmental Medicine, 75(6), 446-452. doi:10.1136/oemed-2017-104765
Environmental Protection Agency. (2018). Particulate Matter (PM) Basics. US EPA.
Eom, H., Liu, Y., Kwak, G., Heo, M., Song, K. S., Chung, Y. D., . . . Choi, J. (2017).
Inhalation toxicity of indoor air pollutants in Drosophila melanogaster using integrated transcriptomics and computational behavior analyses. Scientific Reports, 7, 46473. doi:10.1038/srep46473
Khin, C. (2020, December 31). Surgical Masks Surprisingly Effective Against PM2.5.
Smart Air. https://smartairfilters.com/en/blog/surgical-masks-surprisingly-effective-pm2-5/
Laden, Francine, Joel Schwartz, Frank E Speizer and Douglas W Dockery (2006).
Reduction in Fine Particulate Air Pollution and Mortality: Extended Follow-up of the Harvard Six Cities Study, American Journal of Respiratory and Critical Care Medicine, 173 (6): 667–72.
Lin, Y., Zou, J., Yang, W., & Li, C. (2018, March 2). A Review of Recent Advances
in Research on PM2.5 in China. Retrieved from International Journal of Environmental Research and Public Health www.mdpi.com/1660-4601/15/3/438/pdf
Pope CA 3rd, Dockery DW. Health effects of fine particulate air pollution: lines that
connect. J Air Waste Manag Assoc. 2006 Jun;56(6):709-42. doi:
10.1080/10473289.2006.10464485. PMID: 16805397.
Roeder T, Isermann K, Kallsen K, Uliczka K, Wagner C. A Drosophila asthma model –
what the fly tells us about inflammatory diseases of the lung. Adv Exp Med Biol. 2012;710:37-47. doi: 10.1007/978-1-4419-5638-5_5. PMID: 22127884.
Santana, S. L., Verçosa, C. J., Castro, Í F., Amorim, É M., Silva, A. S., Bastos, T. M., . . .
Rohde, C. (2018). Drosophila melanogaster as model organism for monitoring and analyzing genotoxicity associated with city air pollution. Environmental Science and Pollution Research, 25(32), 32409-32417. doi:10.1007/s11356-018-3186-5
World Health Organization. (n.d.). How air pollution is destroying our health. World
Health Organization: Spotlight. Retrieved October 29, 2018, from
Vogmask. (2017). Why. https://www.vogmask.com/pages/why-vogmask