The liquid from carnivorous marsh pitcher plants (Heliamphora sp.) contains endogenous enzymes for prey digestion.

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conclusionDespite doubts that have lasted for centuries, the carnivorous marsh pitcher plants of the genus Heliamphora can secrete their own hydrolytic digestive enzymes similar to those of other genera of carnivorous plants. The marsh pitcher plant (Heliamphora sp.) is a genus of carnivorous plant in which the production of endogenous enzymes for prey digestion is still dubious. In this study, we tried to elucidate the ability of prey digestion by the plant's own hydrolytic enzymes in H. parva, H. neblinae, and the hybrid H. heterodoxa × minor. Three Heliamphora taxa were fed on fruit flies, and pitcher fluids were analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), Western blots, and enzyme activity measurements for the presence of endogenous hydrolytic enzymes. Production of the enzymes in pitchers through ontogeny was also investigated. Asian pitcher plant Nepenthes truncata was used as a reference carnivorous plant. All investigated taxa produced plant-derived hydrolytic enzymes similar to those found in other carnivorous plant genera. The most important are aspartic protease, subtilisin-like protease, type III chitinase, β-1,3-glucanase, purple acid phosphatase, and GDSL esterase/lipase. Although some variability exists among studied taxa, in general, the activity of phosphatase and proteolytic enzymes was low and increased over time, irrespective of feeding status. Immunoblotting of aspartic protease showed that the abundance of the enzyme in pitcher fluid can accumulate developmentally without any prey stimuli. The microbial and prey-derived proteins were almost exclusively detected in fed samples. In conclusion, our study suggests that the species of the genus Heliamphora can be considered as holocarnivorous plants with the ability to produce their own endogenous hydrolytic enzymes, which, however, does not rule out the contribution of microbial digestion in their natural habitat. Moreover, their own digestive potential can be strongly diminished in nature due to the absence of a protective lid and high rainfall in some parts of the year, diluting the pitcher fluids.