Incense burning successfully generated PM2.5 at concentrations that simulated real haze episodes

The World Health Organization air quality guidelines recommend concentrations of PM2.5 to be kept below 25 µg m−3, over a 24 h period, in order to prevent damage to human health45. The Singapore National Environment Agency considers air quality unhealthy when PM2.5 rises above 55 µg m−3 over a 24 h period46. The high amount of PM2.5 in the smoke cages (117 µg m−3) suggested that the burning of coil incense was effective in producing unhealthy polluted air conditions, comparable to particular matter counts taken during the last haze events in Southeast Asia (SEA) countries. Haze episodes are recurrent in SEA, happening every 1 to 3 years since 198247,48. In Singapore, the 2013 and 2015 episodes lasted around one and 2 months respectively, with [PM2.5] higher than 100 µg m−3 for 4 to 12 consecutive days, including 3 consecutive days when the concentration exceeded 230 µg m−3 in 201349,50,51. In countries closer to the actual fires, the [PM2.5] remained dangerously high for long periods of time. For example, in 1997, the annual mean PM2.5 concentration reached 200 μg m−3 in Southeast Sumatra and Southern Borneo12. The September-October 2015 average [PM2.5] was above 300 μg m−3 in east Sumatra and south Kalimantan islands, Indonesia52. Human health and wildlife damages attributed to the haze are therefore expected to be extremely severe in these areas12.

Aside from differences in smoke particles measured across the two cage treatments, there were also likely differences in air composition across the two cages that were not quantified. Ambient temperature and humidity, however, were the same across all cages, so these environmental factors are unlikely to have contributed to differences in larval fitness observed across treatments.

The smoke and the smoke-induced plant toxicity impaired the development of larvae

Caterpillars that grew subjected to continuous smoke, but not the ones that were fed with smoked-plants, suffered higher mortality in both experiments 1 and 2. The individuals reared in smoke in experiment 2 also had longer development time and lower pupal mass when compared to those that grew in clean air conditions. Individuals that were fed with smoked plants also had increased development time and reduced weight, although the effect sizes were smaller than in experiment 1. Therefore, it seems that the smoke alone induces caterpillar mortality, and that both the smoke and the plant toxicity induced by the smoke increased larval development time and reduced pupal weight, two important components of fitness. These results are in line with a meta-analysis which concluded that arthropods from areas with air pollution were generally smaller than those from control sites53. As smoke-treated larvae, and larvae fed with smoked plants have a reduced pupal mass, they likely resulted in smaller butterflies with reduced fecundity compared to the control-reared ones54,55. Since PM2.5 were absent from the tracheal system of the dead caterpillars reared in the smoke cage, we cannot conclude that the impaired development was caused by particles blocking the insect’s airways. We suggest, instead, that individuals, upon detection of poor air quality or air particles, closed their spiracle valves for longer time durations, decreasing O 2 intake and reducing metabolism. Alternatively, toxic incense smoke components might have been responsible for the higher larval mortality, longer development time, and the lower pupal mass.

The individuals reared in the smoke environment and on smoked plants may have suffered from food stress

Food deprivation was previously shown to lengthen the larval development time and produce smaller butterflies56, and may have originated from larvae having difficulty in locating the corn leaves, or from poor leaf quality. Pollutants such as nitrogen oxides, emitted in high quantities by burning incense and during forest fires10,32, enhance the formation of ground-level ozone by reacting with floral or foliar volatile compounds57,58. Ozone was shown to alter the plant volatiles that herbivores and pollinators use as cues to locate their food source, inducing them to search more and to forage less (e.g.58,59). In addition, ozone and sulfur components can both induce necrotic patches on the plant leaf surfaces and reduce the herbivore food quality60,61,62. Silkworm larvae, for instance, have non-uniform growth and delayed cocooning when they are on host plants exposed to SO 2 63. Junonia coenia caterpillars raised on foliage exposed to high-CO 2 grew more slowly and experienced greater mortality, especially in early instars, than those raised on foliage exposed to low-CO 2 , probably due to the reduced foliar water and nitrogen concentrations of those plants64. Thus, the B. anynana caterpillars reared with incense may have had difficulties locating the corn leaves, may have breathed toxic compounds and ingested more toxic food than their control counterparts, leading to slower development and smaller pupal weight. In support of the first interpretation we also observed that larvae from the smoke treatment group had larger pieces of corn uneaten after each day of feeding, than larvae in the control treatment.

The consumption of smoked plants impacted larval development time and pupal weight less than the joint effect of the smoke and plant toxicity. Thus, in addition to food stress, the larvae from experiments 1 and 2 might have also suffered from chronic inflammation. As air exchange in insects occurs directly via diffusion through tracheoles to the surrounding tissues, acidic SO 2 and NO 2 from the smoke may directly cause tissue damage in the larvae, initiating an inflammation. Under certain stress factors, immune response signals can reduce larval growth rates and delay molting65. In mice, SO 2 , NO 2 , and PM2.5 induced inflammatory and endothelial dysfunction in the heart66.

Finally, the caterpillars from experiment 1 and 2 might have suffered from hypoxia, a deficiency in the amount of oxygen necessary for the tissue to work properly67. Although the proportion of gazes was not measured in our experiments, oxygen levels may have been lower in the smoke cages due to incense combustion. The air blown through the HEPA filter might have not been sufficient to renew the oxygen inside the smoke cage. Hypoxia often results in the reduction of insect survival, growth rate, body size, and reproduction, while inducing the damage of macromolecules and inflammation67,68. Insects can respond to hypoxia by decreasing their activities, such as feeding or moving68, increasing the risk of food stress. Insects can also compensate for a reduction of O 2 in their environment by opening their spiracles and increasing abdominal pumping of air68, processes that also increase the intake of toxic gases. Testing these inter-linked hypotheses to identify the precise cause of increased mortality and development time in caterpillars growing in smoke environments remains open for future studies.

Smoke did not impact pupal mortality

The similar pupal mortality observed in both treatments, and across the 3 experiments, suggests that smoke and the smoke-induced plant toxicity had little impact on pupal development. This is intriguing as there is high metabolic activity and oxygen demands during the late pupal stage especially67, and the air exchange increases24, including the intake of more toxic gas. In experiments 1 and 2, an insufficient sample size at the pupal stage caused by high mortality up to that point may have impaired the discovery of significant effects of smoke on pupal development. Alternatively, the low mortality at this stage might indicate that the effect of gases on food location and food quality are more important factors in reducing larval fitness than direct air toxicity, as pupae do not feed.

Different types of food stress might have contributed to the different patterns of mortality and development time observed in experiments 1 and 2

Smoke-treated larvae fed in cups (experiment 1) suffered relatively less mortality compared to the ones fed on whole plants in experiment 2 (compare Fig. 2a with e). This difference cannot be explained by food toxicity since larval mortality in experiment 3 was low. However, locating the food might have been hard for the larvae in experiment 2, especially after the plants were replaced, because individuals were placed at the bottom of the pots rather than on top of the cut leaves (as in exp. 1). In addition, both caterpillar groups had longer larval development times in experiment 1 (~35 days) relative to experiment 2 (23 and 28 days) and 3 (~22 and 24 days). This increased development in experiment 1 might have led to overall smaller pupae and to higher pupal mortality. While we did not measure the weight of pupae in experiment 1, we observed that the time to pupation and pupal weight were inversely correlated in animals of experiment 2 and experiment 3. Feeding larvae in containers also led to an overall higher pupal mortality of both groups relative to the one measured in experiment 2 and 3 (compare Fig. 2b with f). This high mortality happened as most larvae entered into the pupation process, but failed to complete it, forming an unfinished pupa that would die after one day. The longer development time and higher pupal mortality in experiment 1 were likely due to food deprivation, as the corn amount allocated to the animals was rationed. Late larval stages of both treatment groups ate all the leaves allocated to them on a daily basis. This was not the case in experiment 2 and 3 where the animals were fed ad libitum on a live plant. These results are similar to earlier studies showing that starvation at the larval stage extended B. anynana larval development time, reduced the larval growth rate, increased the pupal mortality and decreased the butterfly body size, fecundity and reproductive investment54,56.

The spiracles might be effective barriers against particulate matter (PM) and toxic gases