Mitochondrial adaptation in the green crab hybrid zone of the Gulf of Maine

The mitochondrial genome has historically been relegated to a neutral genetic marker, but new evidence suggests mitochondrial DNA to be a target for adaptation to environmental stress. The invasive European green crab (Carcinus maenas) exemplifies this in the Gulf of Maine’s hybrid zone, where interbreeding populations exhibit thermal tolerances influenced by mitochondrial genotype. To better understand the mechanism behind this phenomenon, the effect of mitochondrial genotype on mitochondrial activity was tested by measuring mtDNA copy number (mtCN) and the activity of complex I, II, and IV of the electron transport system via high-resolution respirometry. Mitochondria isolated from frozen heart tissue were measured at three temperature points—5°C, 25°C, and 37°C—to represent thermal stresses and a control. It was predicted that cold-adapted haplogroups would exhibit both higher mtCN and increased activity for each complex, either across all temperatures or exclusively at 5°C compared to a warm-adapted haplogroup. Initial comparisons of mitochondria from fresh and frozen tissue at 25°C found lower activity for complex II and IV in frozen extracts, but they continued to be used for convenience. No differences were observed across haplogroups for mtCN or high-resolution respirometry, suggesting that mitochondrial activity does not underlie differences in thermal tolerance. However, temperature greatly influenced activity measurements with complex II and IV exhibiting the highest rates at 37°C while complex I exhibited optimal activity at 25°C. This study represents the first of its kind for C. maenas, providing a foundation for future experiments to continue exploring mitochondria in the context of adaptive evolution.