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Premise of research. The aspartate protease nepenthesin is the primary constituent of pitcher fluids in the pitcher plant genus Nepenthes and is responsible for the hydrolysis of prey protein. Nepenthes pitchers are inhabited by many inquiline species, such as microbes and dipteran larvae, which have been shown to facilitate nitrogen sequestration in pitchers. Inquiline digestive mutualists and fluid proteases may be functionally redundant, but the effect of inquiline presence—or other extrinsic environmental factors—on pitcher protease secretion has never been investigated before. The aims of this study were thus to identify intrinsic and extrinsic factors that predict the in situ proteolytic activity of Nepenthes pitcher fluids. Methodology. We collected the fluids of 129 Nepenthes gracilis pitchers at six different localities in Singapore. Proteolytic activities of the fluids were determined using a fluorometric protease detection test. Environmental and biotic variables hypothesized to affect nepenthesin secretion rates were also measured at each sampled pitcher. Correlations between fluid proteolytic activity and environmental and biotic variables were then examined using linear mixed effects models. Pivotal results. Fluid proteolytic activity was correlated with fluid turbidity, presence of inquiline dipteran larvae, pitcher size, pitcher age, and amount of prey trapped by pitchers. Importantly, pitchers that were inhabited by dipteran larvae had significantly lower fluid proteolytic activity than those that were not, although this effect was absent in pitchers that also had high concentrations of fluid microbes. Conclusions. Our findings confirm that in situ proteolytic activity of Nepenthes pitcher fluids varies significantly with extrinsic factors and suggest that nepenthesin secretion may be subject to multiple regulation mechanisms. The mutualistic activity of dipteran larvae may allow Nepenthes pitcher plants to reduce the secretion of metabolically costly enzymes and obtain a greater proportion of their nitrogen through the dipteran larvae–facilitated ammonium pathway. [ABSTRACT FROM AUTHOR]