Background : The carnivorous plants of the genus Nepenthes, widely distributed in the Asian tropics, rely mostly on nutrients derived from arthropods trapped in their pitcher-shaped leaves and digested by their enzymatic fluid. The genus exhibits a great diversity of prey and pitcher forms and its mechanism of trapping has long intrigued scientists. The slippery inner surfaces of the pitchers, which can be waxy or highly wettable, have so far been considered as the key trapping devices. However, the occurrence of species lacking such epidermal specializations but still effective at trapping insects suggests the possible implication of other mechanisms. Methodology/Principal Findings : Using a combination of insect bioassays, high-speed video and rheological measurements, we show that the digestive fluid of Nepenthes rafflesiana is highly viscoelastic and that this physical property is crucial for the retention of insects in its traps. Trapping efficiency is shown to remain strong even when the fluid is highly diluted by water, as long as the elastic relaxation time of the fluid is higher than the typical time scale of insect movements. Conclusions/Significance : This finding challenges the common classification of Nepenthes pitchers as simple passive traps and is of great adaptive significance for these tropical plants, which are often submitted to high rainfalls and variations in fluid concentration. The viscoelastic trap constitutes a cryptic but potentially widespread adaptation of Nepenthes species and could be a homologous trait shared through common ancestry with the sundew (Drosera) flypaper plants. Such large production of a highly viscoelastic biopolymer fluid in permanent pools is nevertheless unique in the plant kingdom and suggests novel applications for pest control.
Carnivorous plants live in nutrient-poor soils and have circumvented this shortage of resources by deriving most of their nutrients from the digestion of arthropods captured through a variety of trapping mechanisms [1][2][3][4]. The traps are generally formed by highly modified leaves, which can take shapes as diverse [5] as pitfall traps in Sarracenia, Cephalotus and Nepenthes, flypaper-traps in sundews (Drosera) and butterworts (Pinguicula) or even be very sophisticated devices such as the snap traps of Dionaea or the suction bladders of Utricularia. All these carnivorous plants secrete a digestive fluid involved in the process of prey digestion [1][2][3][4]. Only in the flypaper plants is the fluid also involved in insect capture in addition to its digestive role [1][2][3]. In these plants, the fluid is secreted by stalked glands in the form of drops of sticky mucilage, where insects are lured and adhere, most of the time irremediably. On the other hand, in pitcher plants such as Nepenthaceae or Sarraceniaceae, the fluid is secreted in far greater quantities in permanent pools within the pitchers (several tens of ml by pitcher compared to the ml-quantities secreted by leaves of flypaper plants); it is never referred to as mucilage and is commonly believed to have as a unique function, prey digestion [1][2][3].
In Nepenthes pitcher plants, prey capture and retention is mainly thought to be fulfilled by the slippery waxy layer which covers the upper inner part of the pitcher in most species [1,[6][7][8][9][10], or by the peristome or nectar rim of the pitcher (in N. bicalcarata for instance) [11]. However, some Nepenthes species lack such specialized surfaces [12] or lose them later in development [13] suggesting that the trapping mechanism of Nepenthes pitcher plants is more complex than commonly acknowledged. Moreover, reports of secretion of wetting agents [3] or viscous substances [14] in some species point to other potential roles of the digestive fluid.
Here we focus on N. rafflesiana, one of the most widespread species of the genus in northern Borneo [12,15] (Fig. 1a). It is common in heath forests and has one of the richest prey spectra of any species in the genus [13,16]. However, in this species the waxy layer is a variable character and is probably of weak adaptive significance since comparison of waxy traps and non-waxy traps did not show any difference in their amount of prey captured [13]. In contrast, the plant secretes a large amount of slimy fluid, which forms sticky filaments when rubbed between the fingers (pers. observ.). Moreover, field observations on insects fallen in the pitchers reveal that they sink and are easily drawn within the pitchers [13]. This could suggest that the physical properties of the fluid are implicated in insect trapping in this species. A slightly lower surface tension (compared to water) has been observed in the fluid of Sarracenia pitcher plants [17] and was suspected to be part of the trapping in Nepenthes by Juniper and co-authors [3] but to our knowledge, no measure of fluid surface tension has been conducted on any species of Nepenthes pitcher plants. Moreover, up to now, the rheological properties of the fluid, which govern how a fluid moves under forces, and their possible role in insect capture have never been investigated. We thus focused our study on the digestive fluid of N. rafflesiana and first tested whether the fluid alone was able to retain insects by comparing retention of insects thrown into glass vials filled with water or pure digestive fluid. Then, to determinate which physical properties of the digestive fluid was implicated in retention, we compared surface properties (surface tension, static wetting) and rheology (viscosity, elasticity) of pure fluid, water and intermediate dilutions. Our study unveils the peculiar viscoelastic properties of the digestive fluid of N. rafflesiana and its crucial role in prey capture.
The fluid was collected from young and newly opened pitchers of N. rafflesiana. The insects (ant workers and flies) were chosen because they represent non-flying and flying insects and are part of the two insect orders (Hymenoptera and Diptera, respectively) most frequently captured by the pitcher plants [3,16]. We first confirmed that flies (Drosophila melanogaster, Calliphora vomitoria) and ants (Linepithema humile) escaped easily from water (successes: 10/10, 10/10 and 9/10 respectively, see Fig 1b for the two last insects). In water, flies typically succeeded in taking off and flying away in a few seconds (high-speed movies: Video S1, S2), whereas ants succeeded in swimming and climbing up the glass walls of the vials. These observations are in strong contrast with observations of the same insects thrown into the digestive fluid of N. rafflesiana. In this fluid, insects were never able to escape during the 5 minutes observations of the tests (successes: 0/10 for the three insect types, Fig 1b, Video S3, S4). High-speed
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