Enzyme activities in two sister-species of carnivorous pitcher plants (Nepenthes) with contrasting nutrient sequestration strategies

Abstract

The carnivorous pitcher plants of the genus Nepenthes usually attract, capture and digest arthropod prey to obtain mineral nutrients. But few members of the genus have evolved specialized nutrient sequestration strategies to acquire nitrogen from the faeces and urine of mutualistic mammals, which they attract. Because the plants obtain significant amounts of nitrogen in a more available form, we hypothesized that they have relaxed the production of digestive enzymes. If so, species that digest mammal faeces should show fewer digestive enzymes than closely related species that rely on arthropods. We tested this hypothesis by comparing digestive enzymes in 1) Nepenthes hemsleyana, whose pitchers serve as roosts for the mutualistic woolly bat Kerivoula hardwickii, which also defecate inside the pitchers, and 2) the close relative Nepenthes rafflesiana, a typical arthropod capturing species. To investigate the dynamics of aspartic proteases (nepenthesin I and II) and type III and IV chitinases in both species, we conducted qPCR, western blotting, mass spectrometry, and enzyme activity measurements. We found that mRNA in pitcher tissue and enzyme abundance in the digestive fluid is upregulated in both species in response to faeces and insect feeding. Contrary to our initial hypothesis, the final nepenthesin proteolytic activity in the digestive fluid is higher in response to faeces addition than to insect prey irrespective of Nepenthes species. This indicates that faeces can mimic arthropod prey triggering the production of digestive enzymes and N. hemsleyana retained capacity for production of them.

Keywords: Bat; Carnivorous plant; Chitinase; Digestive enzyme; Nepenthes; Nepenthesin; Pitcher plants; Proteinase; Western blot.