Local adaptation of Daphnia to protease inhibitors in cyanobacteria
Final Report Abstract
Global warming and ongoing eutrophication of lakes and ponds will continue to foster cyanobacterial mass developments, which in general lead to deterioration of water bodies. The latter is partly caused by the failure of the most important phytoplankton consumers, representatives of the genus Daphnia, to efficiently consume cyanobacterial production. This largely is attributed to low food quality of cyanobacteria for Daphnia, which results in low Daphnia abundances during cyanobacterial mass developments. Three factors have been identified to cause this low food quality: (i) formation of noningestible colonies and filaments, (ii) deficiency in lipids that are essential for Daphnia, and (iii) cyanobacterial inhibitors and toxins. With respect to the latter, it is known that Daphnia can locally adapt to the co-existence with toxic cyanobacteria, although the molecular mechanisms have not yet been investigated. This project capitalized on Daphnia magna populations with different histories of co-existence with cyanobacteria and on the finding that an experienced population seemed to be locally adapted to cyanobacteria that produce protease inhibitors (PIs), which target digestive chymotrypsins (CTs) in D. magna. In the frame of this project the molecular basis of this putative local adaptation of D. magna to cyanobacterial PIs should be investigated on the population level. It addressed these questions: 1) Does increased transcription of digestive proteases contributes to enhanced tolerance to PIs? 2) Are nonsynonymous changes in CTs among Daphnia populations adaptations to cyanobacterial PIs? 3) Do such non-synonymous changes in CTs contribute to differences in tolerance to cyanobacterial PIs? The results show that that sensitivity to cyanobacterial CT inhibitors is related with the cyanobacterial history of a Daphnia population. In the presence of cyanobacterial PIs, more sensitive populations showed higher relative gene expression of CT448, and in these populations higher copy numbers of CT448 were found. Elevated gene copy numbers thus represent a means to compensate the higher sensitivity of CTs by elevated expression. Detailed population genetic investigations of the D. magna population that is locally adapted to cyanobacterial CT inhibitors, provided strong evidences that on CT loci positive selection has occurred. This strongly indicates that these digestive proteases have most probably been targets of selection. This is the first study that demonstrates that cyanobacterial inhibitors exert selection on their target proteins in natural Daphnia populations. Using heterologous expression of CT448 zymogen in insect cells, subsequent purification and activation by trypsin yielded active protease that allowed for qualitative characterization, which showed that it is a chymotrypsin-like elastase. In the further course of the project it turned out that activation by trypsin as well led to degradation of CT448. Hence, a new construct for heterologous expression with subsequent activation by factor Xa was developed. In conclusion, the overall results have considerably improved our understanding of how Daphnia populations adapt to cyanobacterial PIs.
Publications
- 2017. Copy number variation of a protease gene of Daphnia: Its role in population tolerance. Journal of Experimental Zoology. Part A, Ecological and integrative physiology 327 (2-3), 119–126
Schwarzenberger, A., Keith, N.R., Jackson, C.E., Elert, E. von
(See online at https://doi.org/10.1002/jez.2077) - 2018. Heterologous expression and characterization of a novel serine protease from Daphnia magna: A possible role in susceptibility to toxic cyanobacteria. Aquatic Toxicology 205, 140–147
Lange, J., Demir, F., Huesgen, P.F., Baumann, U., von Elert, E., Pichlo, C.
(See online at https://doi.org/10.1016/j.aquatox.2018.09.013) - 2020. Positive selection of digestive proteases in Daphnia: A mechanism for local adaptation to cyanobacterial protease inhibitors. Molecular Ecology 29 (5), 912–919
Schwarzenberger, A., Hasselmann, M., von Elert, E.
(See online at https://doi.org/10.1111/mec.15375)
