There's a rhythm to life in temperate and sub-polar areas due to seasonal changes in warmth and food availability. The Colorado potato beetle waits out winter in a low-energy state of suspended animation known as diapause before emerging in the spring to look for food and a mate. Metabolic suppression is the hallmark of diapause, but exactly how animals turn it on and off remains an open question. In an integrative study, Jackie Lebenzon and colleagues from the University of Western Ontario, Canada, used respirometry, high-powered microscopes and a cutting-edge gene silencing technique to show how diapausing beetles use their mitochondria like a metabolic thermostat.
During diapause, the Colorado potato beetle's flight muscles waste away, only to be completely rebuilt in time for spring. As 40% of insect flight muscle is made up of mitochondria, Lebenzon and colleagues hypothesized that these powerhouse organelles have an essential role in controlling diapause. Checking mitochondria freshly extracted from diapausing beetles, the team found that they consumed 90% less oxygen, matching the decrease in metabolic rate observed in living beetles. That the beetle and its isolated mitochondria had such similar metabolic responses confirmed that the mitochondria had a big role in setting the beetles’ low metabolic rate during diapause. But how were the mitochondria keeping the metabolic rate so low?
One clue was that diapausing beetles had a very low activity for citrate synthase, a protein only found deep within the mitochondria that prepares metabolites for the mitochondria to use. Wondering whether low citrate synthase activity translated into fewer mitochondria, the researchers viewed the muscle under a high-powered microscope: it was nearly bereft of the familiar bean-shaped organelles. In addition, the cell's disposal system was bustling, with lysosomes, autophagosomes and autolysosomes – which break down old and damaged parts of the cell – dotted around the field of view. Despite this, diapause muscle had the same number of cells and oxygen-delivery tubes as active muscle; all signs pointed to beetles selectively breaking down their mitochondria, a process known as mitophagy, as a way of bringing their metabolic rate down during diapause.
Mitophagy is a complicated process. Key proteins include Parkin, which tags proteins on damaged mitochondria, and ATG5, which aids uptake of tagged proteins into lysosomes for disposal. Both the Parkin and ATG5 genes were much more active during diapause. Mitophagy is one of two complementary processes, the other being the production of new mitochondria, which animals use to balance mitochondria levels. During diapause, the beetles also bumped up the levels of two transcription factors, PGC1α and NRF1, that promote mitochondria production. Together, these observations told the researchers that a complex interplay between mitochondrial breakdown and production was responsible for the start, maintenance and end of diapause. Early mitochondrial breakdown brought metabolic rates down in the winter and kept them low, only to give way to mitochondrial production as winter thawed to spring.
Finally, to connect gene activation in the muscle with metabolic suppression in the beetle, the researchers injected beetle muscle with custom RNA sequences that turned off the Parkin gene. The injected beetles had 80% less Parkin protein and didn't lose their mitochondria to mitophagy, showing that all the pieces of the pathway worked together in living animals. Interestingly, the injected beetles only recovered about 40% of their metabolic rate during diapause, suggesting that while Parkin is important, it isn't the only regulator of metabolism during diapause.
Colorado potato beetles destroy and regrow their own mitochondria on demand to suppress their metabolism and, in doing so, expertly weather the seasonal rhythms of their environment.
