In Situ Enzyme Activity in the Dissolved and Particulate Fraction of the Fluid from Four Pitcher Plant Species of the Genus Nepenthes

Abstract

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×106 to 2.2×108 cells ml−1. We measured the activity of three common enzymes in the fluid: acid phosphatases, β-d-glucosidases, and β-d-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

“…The fluid pH was then altered to either more or less acidic once the pitchers opened. NGR and NAN had highly acidic pitcher fluids, in which the former were comparable to that previously reported for the fluids of Nepenthes from the Lambir Hills National Park, Malaysia [8]. For the fluid samples of NAM, the acidic fluids consistently found in this work were similar to those found in a previous study [8].…”

“…NGR and NAN had highly acidic pitcher fluids, in which the former were comparable to that previously reported for the fluids of Nepenthes from the Lambir Hills National Park, Malaysia [8]. For the fluid samples of NAM, the acidic fluids consistently found in this work were similar to those found in a previous study [8]. It has been reported that the pH fluctuations of the pitcher fluids may be induced by prey captured, e.g., from pH 5.5 to 3.0 in Nepenthes [35] and also may be caused by H + secretion from living epidermis cells [36,37].…”

“…The fluid is composed of a mixture of enzymes generated by the plants themselves including aspartic proteases called nepenthesins [6,7] and those produced from inhabitant microorganisms [8]. In addition to proteases, the pitcher fluids of Nepenthes also contain esterases, phosphatases, ribonucleases, and chitinases [9][10][11][12][13] as well as different glycosyl hydrolases, e.g., β-D-glucosidases and β-D-glucosaminidases [8] which together act cooperatively on digesting the insect bodies. Starting from native microflora in unopened pitchers [14,15], the hydrolytic microcosms in pitcher fluid are hypothesized to be enriched by airborne microbes and those previously colonized on the insect carcasses.…”

“…This results in variation of nutrient and oxygen availability in different zones of the pitchers [16]. The fluid has been reported to harbor a high density of bacteria (i.e., up to ∼10 8 cells/mL) in mature pitchers [8]. It is suggested that these microbes contribute significantly to the quantities of enzymes produced and might play an indispensible role in prey digestion and nutrient mineralization [8].…”

Dipteran larvae and microbes facilitate nutrient sequestration in theNepenthes gracilispitcher plant host

“…Many researchers believe that microbes benefit the pitcher by assisting in prey decomposition and nitrogen sequestration [3,13], or even prevent prey escape by reducing the surface tension of pitcher fluid [14]. Although indirect evidence for the former are available for Nepenthes [15,16], these studies are not without criticism, and do not demonstrate unequivocally the benefit derived from microbial colonization of its pitchers. By contrast, a growing number of studies in more recent years have shown that pitcher secretions are anti-microbial, an observation that seems to point in the direction of an antagonistic relationship between plant and fluid microbes [17][18][19].…”