Nearly all lab experiments studying malaria infection in mosquitoes are conducted under highly controlled constant temperature conditions. Yet in nature, temperatures cool down at night and warm up in the day, meaning that that mosquitoes feeding in the evening experience different temperature dynamics to those that feed in the morning. In our latest paper in NEE1 we ask whether this variation might make a difference for malaria transmission?
Temperature fluctuation experiments can get complex, and things soon got big. Experiments tested different times of day of blood feeding under constant vs fluctuating temperatures and with two species of mosquitoes to ensure our data were robust.
Eunho is happy that yet another giant feeding experiment is going well.
Upwards of 20,000 mosquitoes were reared per week to support these experiments (thanks to the amazing Janet Teeple, our Insectary Manager). Batches of mosquitoes were separated into different incubators to offset their body clocks so that when they were blood fed together, some were feeding as if it was evening (6pm), some were feeding at midnight (12am), and others as if it were early morning (6am). Thousands of mosquitoes were carefully fed blood meals infected with the human malaria parasite, Plasmodium falciparum. In order to balance the timing of blood meals for the different time-shifted mosquito batches, feeds were often conducted at 2am in real time under red light. So, even though mosquito circadian rhythms were not disrupted, ours often were!
With many repeat experiments and replication across two mosquito species, months were filled with infectious feeds followed by the painstaking task of dissecting the guts and salivary glands from thousands of mosquitoes to determine whether they had picked up the malaria parasite and could potentially transmit it. Everyone in the group pitched in to help, whether to share space, time, or lend moral support. At long last we had a gorgeous collection of repeatable data, with a clear result: Mosquitoes having 6am blood meal were 98% less likely to become infectious compared to those fed the same meal at 6pm! Midnight feeders were in between, and mosquitoes held at constant temperatures mimicked the high infections in the 6pm fluctuating treatment, suggesting a lot of lab experiments likely overestimate infection prevalence compared to nature.
We then conducted further experiments to work out why we see these patterns and whether they are likely robust to mosquito behavior. This required more infectious feeds, more dissections, and hours spent watching how mosquitoes responded to different temperatures. The results suggested that initial stages of parasite establishment can be disrupted by high temperatures. So, when mosquitoes feed in the evening the parasite has a longer period to establish before temperatures get too warm, compared to when mosquitoes feed in the morning. Importantly, the mosquitoes themselves appeared unresponsive to these warmer temperatures, meaning that parasites would likely be exposed to critical temperatures in natural fluctuating environments.
Eunho then met Ellie at an ASTMH meeting and got chatting about some exciting modeling she was doing to explore how biting time might affect the impact of bed nets 2. Combining forces we were able to show that mosquitoes feeding in the evening potentially contribute more to transmission then those feeding at other times of the night, and that any shifts towards evening feeding could greatly undermine the effectiveness of bed nets.
We believe the study has provided insights that are both novel and important for better understanding the ecology of malaria transmission. Getting this far took lots of hard work, some sleepless nights, team spirit, chance encounter and dogged determination. We now look forward to extending this work to some real world field settings that hopefully provide a more glamorous outlook than the windowless insectary.
The Insectary Building at Penn State, as beautiful as it ever gets. Housing thousands of mosquitoes in tropical conditions inside, while outside there’s a foot of snow and below freezing.
Thanks to Eunho Suh and Matthew Thomas for their feedback on this blog post!
1. Suh, E. et al. The influence of feeding behaviour and temperature on the capacity of mosquitoes to transmit malaria. Nature Ecology and Evolution 1–14 (2020). doi:https://doi.org/10.1038/s41559...
2. Sherrard-Smith, E. et al. Mosquito feeding behavior and how it influences residual malaria transmission across Africa. Proc. Natl. Acad. Sci. U.S.A. 116, 15086–15095 (2019).