Microhabitat size distribution affects local difference in community structure: Metazoan communities in treeholes

I studied the effect of treehole (microhabitat) size distribution in local habitats on geographic difference in aquatic metazoan community structure by comparing differences between two sites on Iriomote Island, and between Iriomote Island (subtropical) and Tsushima Island (temperate), in southwestern Japan. In treeholes at each local site, the amount of litter, the species richness and total biomass of metazoa were positively correlated with treehole capacity. Between the two sites on Iriomote Island (Shirahama and Komi), the amount of litter, biomass and species number per treehole was greater at Komi where the mean and variance of treehole size were greater, while the dependencies of these parameters on treehole capacity were common to both sites. Total species number was larger at Komi (2 predators and 20 saprophages) than at Shirahama (1 predator and 19 saprophages). Most of the dominant taxa colonized larger treeholes with higher probabilities, although one taxa showed the opposite trend. Treeholes on Tsushima were smaller than those on Iriomote. The metazoan fauna in treeholes consisted of 15 saprophages on Tsushima, being less richer than that on Iriomote Island which had 2 predators and 21 saprophages. However, the dependencies of litter amount and biomass on treehole capacity did not differ significantly between the islands, although treeholes on Iriomote harbored a greater number of species per treehole than those on Tsushima. This study indicated that there are general correlates between community structure within individual treeholes (infracommunity structure) and treehole capacity (microhabitat size). Therefore, microhabitat-size distribution is potentially a significant constraint of local community structure, and its variation may contribute to the variation in local and regional species richness.     

Why are Aedes mosquitoes rare colonisers of Nepenthes pitcher plants?

1. Nepenthes pitcher plants produce fluid‐containing animal traps that are colonised by a variety of specialised arthropods, especially dipterans. However, container‐breeding vector mosquitoes, such as Aedes albopictus Skuse have rarely been recorded from pitchers. Increasing overlap in the geographical ranges of Nepenthes and Ae. albopictus in urban parts of Southeast Asia owing to urbanisation highlights a growing need to investigate the potential role of pitchers as larval habitats for vector mosquitoes. 2. The ability of Ae. albopictus larvae to survive in three common lowland Nepenthes in Peninsular Malaysia that are most likely to co‐occur with Ae. albopictus [viz., Nepenthes ampullaria Jack, Nepenthes gracilis Korth., and Nepenthes mirabilis (Lour.) Druce] was investigated. 3. The larval survival rates of Ae. albopictus in pitcher fluids of the three Nepenthes species were determined, then the effects of low pH, larvicidal agents (such as microbes, predators, and chemical compounds) through manipulative experiments were investigated. 4. It was found that pitchers represent a hostile environment to Ae. albopictus, but that the principal cause of larval mortality varies among Nepenthes species (i.e. low fluid pH in N. gracilis, predation by Toxorhynchites acaudatus Leicester larvae in N. ampullaria, and microbial activity in N. mirabilis). It was concluded that Nepenthes pitchers are generally not suitable larval habitats for Ae. albopictus. However, the pitcher environment of N. ampullaria is worthy of further study, as pitchers that lack predators are nevertheless rarely colonised by Ae. albopictus, indicating that other aspects of the host pitcher environment inhibit oviposition or larval survivorship.     

Bacterial communities associated with the pitcher fluids of three Nepenthes (Nepenthaceae) pitcher plant species growing in the wild

Nepenthes pitcher plants produce modified jug-shaped leaves to attract, trap and digest insect prey. We used 16S rDNA cloning and sequencing to compare bacterial communities in pitcher fluids of each of three species, namely Nepenthes ampullaria, Nepenthes gracilis and Nepenthes mirabilis, growing in the wild. In contrast to previous greenhouse-based studies, we found that both opened and unopened pitchers harbored bacterial DNA. Pitchers of N. mirabilis had higher bacterial diversity as compared to other Nepenthes species. The composition of the bacterial communities could be different between pitcher types for N. mirabilis (ANOSIM: R = 0.340, p < 0.05). Other Nepenthes species had similar bacterial composition between pitcher types. SIMPER showed that more than 50 % of the bacterial taxa identified from the open pitchers of N. mirabilis were not found in other groups. Our study suggests that bacteria in N. mirabilis are divided into native and nonnative groups.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

Local‐ to continental‐scale variation in the richness and composition of an aquatic food web

Aim We investigated patterns of species richness and composition of the aquatic food web found in the liquid‐filled leaves of the North American purple pitcher plant, Sarracenia purpurea (Sarraceniaceae), from local to continental scales. Location We sampled 20 pitcher‐plant communities at each of 39 sites spanning the geographic range of S. purpurea– from northern Florida to Newfoundland and westward to eastern British Columbia. Methods Environmental predictors of variation in species composition and species richness were measured at two different spatial scales: among pitchers within sites and among sites. Hierarchical Bayesian models were used to examine correlates and similarities of species richness and abundance within and among sites. Results Ninety‐two taxa of arthropods, protozoa and bacteria were identified in the 780 pitcher samples. The variation in the species composition of this multi‐trophic level community across the broad geographic range of the host plant was lower than the variation among pitchers within host‐plant populations. Variation among food webs in richness and composition was related to climate, pore‐water chemistry, pitcher‐plant morphology and leaf age. Variation in the abundance of the five most common invertebrates was also strongly related to pitcher morphology and site‐specific climatic and other environmental variables. Main conclusions The surprising result that these communities are more variable within their host‐plant populations than across North America suggests that the food web in S. purpurea leaves consists of two groups of species: (1) a core group of mostly obligate pitcher‐plant residents that have evolved strong requirements for the host plant and that co‐occur consistently across North America, and (2) a larger set of relatively uncommon, generalist taxa that co‐occur patchily.     

Aquatic arthropod communities in Nepenthes pitchers: the role of niche differentiation,aggregation, predation and competition in community organization

Summary The structure and organization of aquatic arthropod communities in Nepenthes ampullaria pitchers were studied at two sites (M in Malacca and K in Kuching) in Malaysia. The communities consisted mainly of aquatic dipteran larvae. Community M was dominated by a filter feeder, Tripteroides tenax, which reached a high density despite a strongly aggregated distribution. Community K had five trophic groups: carrion feeders, filter feeders, detritus feeders, nipping predators and hooking predators, each including multiple species. The summed density of filter feeders in Community K remained much below the level attained by filter feeders in Community M. Niche differentiation within each trophic group with regard to pitcher age and feeding behaviour was not sufficient to allow species coexistence through niche separation alone. Aggregated distributions directly reduced interspecific encounters. Nevertheless, species belonging to the same trophic group commonly shared the same pitcher, because of high occurrence probabilities of dominant species and positive associations between some taxa (due mainly to similar occupancies by pitcher age). Predator coexistence in Community K may have been facilitated by self-limitation of the large predators through intraspecific cannibalism strengthened by aggregation. Prey coexistence, on the other hand, may have relied more on population suppression by predation, especially the selective removal of old instar Tripteroides.     

Pathways for Nutrient Transport in the Pitchers of the Carnivorous Plant Nepenthes alata

Glands of the carnivorous pitcher plant Nepenthesalata are activein transport of materials into and out of the pitcher lumen,indicating dual functions in both secretion and absorption.This study examined the potential for open transport throughthese glands using the ultrastructural tracer lanthanum, whichis restricted to the apoplast, and the fluorescent symplastictracer, 6(5)carboxyfluorescein. Glandular uptake of lanthanumfrom the pitcher fluid occurred through the outer cell wallbetween irregularly spaced cutinized deposits, but was blockedfrom entering the underlying mesophyll cell walls by thick endodermal-likeregions. Similarly, lanthanum localization showed an open apoplasticpathway from the petiole to the endodermal regions in the glandbase. Thus, transport of materials into or out of the glandmust occur through the symplast. 6(5)Carboxyfluorescein showedthat these glands transport fluids directly from the pitcherfluid into vascular endings immediately beneath them via a symplasticroute. When applied to the petiolar vascular system, the fluorescenttracer freely entered immature pitchers, but was blocked fromentering the lumen of the mature pitcher by an endodermal zone.An ultrastructural survey showed infrequent pits with plasmodesmatalconnections to adjoining subepidermal cells. These results indicatethat the function of the gland is developmentally regulated.Prior to maturity, the primary function of the gland appearsto be secretion. However, at maturity, secretion is blockedby an endodermal layer, which limits the function of the glandto absorption. These studies support the theory that the glandsof Nepenthesalata are specialized for the bi-directional transportof materials.Copyright 1999 Annals of Botany Company Apoplastic transport, 6(5)carboxyfluorescein, carnivorous plants, digestive glands, endodermal layer,Nepenthesalata Blanco, lanthanum, pitcher plants.     

Different pitcher shapes and trapping syndromes explain resource partitioning in Nepenthes species

Nepenthes pitcher plants display interspecific diversity in pitcher form and diets. This species‐rich genus might be a conspicuous candidate for an adaptive radiation. However, the pitcher traits of different species have never been quantified in a comparative study, nor have their possible adaptations to the resources they exploit been tested. In this study, we compare the pitcher features and prey composition of the seven Nepenthes taxa that grow in the heath forest of Brunei (Borneo) and investigate whether these species display different trapping syndromes that target different prey. The Nepenthes species are shown to display species‐specific combinations of pitcher shapes, volumes, rewards, attraction and capture traits, and different degrees of ontogenetic pitcher dimorphism. The prey spectra also differ among plant species and between ontogenetic morphotypes in their combinations of ants, flying insects, termites, and noninsect guilds. According to a discriminant analysis, the Nepenthes species collected at the same site differ significantly in prey abundance and composition at the level of order, showing niche segregation but with varying degrees of niche overlap according to pairwise species comparisons. Weakly carnivorous species are first characterized by an absence of attractive traits. Generalist carnivorous species have a sweet odor, a wide pitcher aperture, and an acidic pitcher fluid. Guild specializations are explained by different combinations of morpho‐functional traits. Ant captures increase with extrafloral nectar, fluid acidity, and slippery waxy walls. Termite captures increase with narrowness of pitchers, presence of a rim of edible trichomes, and symbiotic association with ants. The abundance of flying insects is primarily correlated with pitcher conicity, pitcher aperture diameter, and odor presence. Such species‐specific syndromes favoring resource partitioning may result from local character displacement by competition and/or previous adaptations to geographically distinct environments.     

Effects of capacity on resource input and the aquatic metazoan community structure in phytotelmata

Aquatic metazoan communities in treeholes and bamboo stumps were studied on Tsushima Island, Japan, in March and June in relation to the habitat size and the resouce amount. The amount of leaf detritus increased with an increase in the capacity (volume) of the phytotelma. The input rate of leaves depended upon the opening area of the phytotelma. Twenty-one metazoan inhabitant taxa consisted of 17 dipteran saprophages, a helodid beetle larva, a nematoda, a naidid, and a predator,Toxorhynchites towadensis mosquito larva. Treeholes harbored 17 taxa and bamboo stumps 20; 16 taxa were common in both phytotelmata. The number of taxa and the biomass of metazoa per phytotelma was positively correlated with the capacity. The densities of dominant saprophages such as helodid, chironomid, andCulicoides larvae were positively correlated with the capacity of phytotelmata, and the variation in their abundance contributed much to the variation in the species composition in individual phytotelmata. With an increase in capacity, treeholes harbored more helodids andCulicoides, whereas bamboo stumps harbored more chironomids andDasyhelea. Thus, the amount of resource, species richness and biomass per phytotelma largely depended upon the capacity, and the difference in community structure between treeholes and bamboo stumps was attributable to the differential colonization patterns of different taxa.     

Reinigung der Proteinase aus Nepenthes-Kannensaft

Zusammenfassung Der sterile Kannensaft von drei Nepenthes-Arten wurde chromatographisch und elektrophoretisch untersucht. Bei Auftrennungen durch Adsorptionschromatographie an Ecteola-Cellulose und durch Elektrophorese wurde jeweils nur eine proteolytische Enzymfraktion erhalten. In gereinigten, kohlenhydratfreien Enzymfraktionen wurde ein pH-Optimum bei 2,2 und ein Temperaturoptimum bei 50°C gefunden.

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Purification of the proteinase from Nepenthes pitcher secretion

Summary The proteolytic enzymes from the sterile secretion of the pitchers of 3 Nepenthes species were purified to electrophoretic homogenity by chromatography on Ecteola cellulose. The properties of the purified enzymes were investigated.

     

The metazoan food webs from six Bornean Nepenthes species

Abstract.

• 1 Two contrasting hypotheses concerning patterns in food web structure within pitchers of Nepenthes are tested using new information from six species of Nepenthes from Borneo.
• 2 In general, predictions that webs will be more complex, and the food chains they contain will be longer, the closer they are to the centre of Nepenthes species diversity, are supported.
• 3 For Nepenthes albomarginata, a widespread species with a distinctive north Bornean form, a contrasting pattern is evident explicable in terms of the morphology of the pitchers and local habitat preferences.
• 4 General explanations for food web patterns will always be susceptible to exception, reflecting nuances of natural history.

     

Morphological correlates of necromass accumulation in the traps of an Eastern tropical pitcher plant, Nepenthes ampullaria Jack, and observations on the pitcher infauna and its reconstitution following experimental removal

I studied the trap morphology, necromass accumulation rates and pitcher infauna of an eastern tropical pitcher plant, Nepenthes ampullaria, that grew in `kerangas' heath forest in the Sungei Ingei Conservation Area, Brunei. I surveyed 164 pitchers distributed among 35 plants and extracted the necromass and larval infauna from the pitchers and then resampled the pitcher contents after 14 days. Plants varied significantly in the morphology of their pitchers, in their rate of necromass accumulation per pitcher and in the abundance and composition of the pitcher infaunas. On average, pitchers accumulated 11.5 mg dry weight over 14 days, but larger pitchers accumulated more necromass than smaller ones. Pitcher morphology explained 45% of the variation in necromass accumulation among plants. On average, pitchers initially contained 26.3 individual larval inquilines. Collectively, the larval infauna was composed of nine taxa of dipteran larvae and infrequent anuran tadpoles. These ten taxa were never found together in a single pitcher and the mean species richness per pitcher was 4.0. Of the six taxa that could be assessed, all except Toxorhynchites spp. had a contagious distribution among the pitchers. Pitcher morphology and necromass accumulation explained only 15% of the variation in inquiline abundance among plants. I found little evidence for the existence of density-dependent interactions between inquiline species: a partial correlation analysis detected only one statistically significant pairwise relationship between the abundances of inquiline taxa, which was a positive association. Fourteen days after being emptied, pitchers contained an average of 9.6 inquilines. There was no evidence that the species composition of the infauna recolonising each pitcher was related to that of its pre-removal infauna. Received: 2 June 1997 / Accepted: 9 September 1997     

Slippery surfaces of carnivorous plants: composition of epicuticular wax crystals in<Emphasis Type="Italic"> Nepenthes alata</Emphasis> Blanco pitchers

Plants in the genus Nepenthes obtain a substantial nutrient supply by trapping insects in highly modified leaves. A broad zone of the inner surface of these pitchers is densely covered with wax crystals on which most insects lose their footing. This slippery wax surface, capturing prey and preventing its escape from the trap, plays a pivotal role in the carnivorous syndrome. To understand the mechanism of slipperiness, the present investigation aimed at an ultrastructural and physico-chemical characterization of the wax crystals in pitchers of N. alata Blanco. Scanning electron microscopy revealed that entire platelets protruded perpendicularly from the surface. Methods were developed that allowed the mechanical removal of wax crystals from the pitcher surface. It could be shown that the sampling was selective for the epicuticular wax, relevant for plant–insect interactions. The crystals consisted of a mixture of aliphatic compounds dominated by very-long-chain aldehydes. Triacontanal, at 43% the most abundant constituent, was largely responsible for crystal formation. Solubility data indicate that the Nepenthes crystals contained polymeric forms of this aldehyde. The resulting mechanical properties of the polymer crystals and the mechanism of slipperiness are discussed.Abbreviation SEM scanning electron microscopy     

A keystone predator controls bacterial diversity in the pitcher-plant (Sarracenia purpurea) microecosystem

The community of organisms inhabiting the water-filled leaves of the carnivorous pitcher-plant Sarracenia purpurea includes arthropods, protozoa and bacteria, and serves as a model system for studies of food web dynamics. Despite the wealth of data collected by ecologists and zoologists on this food web, very little is known about the bacterial assemblage in this microecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify bacterial diversity within the pitchers as a function of pitcher size, pH of the pitcher fluid and the presence of the keystone predator in this food web, larvae of the pitcher-plant mosquito Wyeomyia smithii. Results were analysed at two spatial scales: within a single bog and across three isolated bogs. Pitchers were sterile before they opened and composition of the bacterial assemblage was more variable between different bogs than within bogs. Measures of bacterial richness and diversity were greater in the presence of W. smithii and increased with increasing pitcher size. Our results suggest that fundamental ecological concepts derived from macroscopic food webs can also be used to predict the bacterial assemblages in pitcher plants.     

Photosynthetic characterization of Australian pitcher plant <Emphasis Type="Italic">Cephalotus follicularis</Emphasis>

Australian carnivorous pitcher plant Cephalotus follicularis Labill. produces two types of leaves. During the spring time, the plant produces a foliage type of noncarnivorous leaf called lamina. Later, the second type of leaf is produced — carnivorous pitcher. Using simultaneous measurements of gas exchange and chlorophyll (Chl) fluorescence photosynthetic efficiency of these two distinct forms of leaves were compared. In addition stomatal density, an important component of gas exchange, and Chl concentration were also determined. Pitcher trap had lower net photosynthetic rate (P _N) in comparison to noncarnivorous lamina, whereas the rate of respiration (R _D) was not significantly different. This was in accordance with lower stomatal density and Chl concentration in the pitcher trap. On the other hand maximum quantum yield of PSII (F_v/F_m) and effective quantum yield of photochemical energy conversion in PSII (Φ_PSII) was not significantly different. Nonphotochemical quenching (NPQ) was significantly higher in the lamina at higher irradiance. These data are in accordance with hypothesis that changing the leaf shape in carnivorous plants to make it a better trap generally makes it less efficient at photosynthesis. However, the pitcher of Cephalotus had much higher P _N than it was expected from the data set of the genus Nepenthes. Because it is not possible to optimize for contrasting function such as photosynthesis and carnivory, it is hypothesized that Cephalotus pitchers are less elaborated for carnivorous function than the pitchers of Nepenthes.     

Chemical composition of epicuticular wax crystals on the slippery zone in pitchers of five <Emphasis Type="Italic">Nepenthes</Emphasis> species and hybrids

Plants of the carnivorous genus Nepenthes efficiently trap insects in leaf pitchers, mostly employing epicuticular wax crystals on the pitcher walls to make them slippery for the prey. In the present study, the compositions and micromorphologies of the wax crystals of five Nepenthes species and hybrids were analysed in order to test whether the chemical principles underlying this ecological function are widespread within the genus. Three wax layers could be distinguished within the Nepenthes pitcher cuticles: (1) the outermost part of the crystals forming the platelets visible in standard scanning electron microscopy, (2) the bottom portion of the epicuticular wax crystals, and (3) an intracuticular wax layer. The composition of the intracuticular wax differed significantly from that of the neighbouring epicuticular layer. The compositions of corresponding wax mixtures from all five Nepenthes species and hybrids were very similar, with almost equal amounts of very long chain aldehydes and primary alcohols. While triacontanal (C₃₀ aldehyde) was prevailing in the epicuticular crystals of Nepenthes albomarginata and Nepenthes x intermedia, Nepenthes x superba and Nepenthes x henriana were found to have especially high percentages of dotriacontanal (C₃₂ aldehyde). Nepenthes “khasiana” had an intermediate aldehyde composition with almost equal amounts of both chain lengths.     

Analysis of feeding mechanism in a pitcher ofNepenthes hybrida

The process of digestion of captured feeds in a pitcher, an insect-trapping organ, ofNepenthes was studied. Changes in bacterial population, pH and NH₄ ⁺ concentrations in pitcher juice were examined. Strong activities of both acid- and alkaline phosphatase, phosphoamidase, esterase C4 and esterase C8 were found in the pitcher juice. Optimum pH of proteases in the juice and those secreted from bacteria showed pH 3.0 and pH 8.0–9.0, respectively. Twenty six strains of bacteria were isolated from 4 pitchers: 10 strains were gram positive, 16 strains were gram negative (10 strains had casein hydrolase activity). A proton excretion was induced by NH₄ ⁺ released from the added solutions, and accordingly, the pH of the solutions fell. As a simulation model of the digestion process of feeds in pitcher juice and polypeptone solution was added into the washed pitcher. A good correlation was found among the NH₄ ⁺ concentration, pH and bacterial cell titer.     

Geographic variation in network structure of a nearctic aquatic food web

Aim The network structure of food webs plays an important role in the maintenance of diversity and ecosystem functioning in ecological communities. Previous research has found that ecosystem size, resource availability, assembly history and biotic interactions can potentially drive food web structure. However, the relative influence of climatic variables that drive broad‐scale biogeographic patterns of species richness and composition has not been explored for food web structure. In this study, we assess the influence of broad‐scale climatic variables in addition to known drivers of food web structure on replicate observations of a single aquatic food web, sampled from the leaves of the pitcher plant (Sarracenia purpurea), at different geographic sites across a broad latitudinal and climatic range. Location Using standardized sampling methods, we conducted an extensive ‘snapshot’ survey of 780 replicated aquatic food webs collected from the leaves of the pitcher plant S. purpurea at 39 sites from northern Florida to Newfoundland and westward to eastern British Columbia. Methods We examined correlations of 15 measures of food web structure at the pitcher and site scales with geographic variation in temperature and precipitation, concentrations of nutrients from atmospheric nitrogen deposition, resource availability, ecosystem size and the abundance of the pitcher plant mosquito (Wyeomyia smithii), a potential keystone species. Results At the scale of a single pitcher plant leaf, linkage density, species richness, measures of chain length and the proportion of omnivores in a web all increased with pitcher volume. Linkage density and species richness were greater at high‐latitude sites, which experience low mean temperatures and precipitation and high annual variation in both of these variables. At the site scale, variation in 8 of the 15 food web metrics decreased at higher latitudes, and variation in measures of chain length increased with the abundance of mosquitoes. Main conclusions Ecosystem size and climatic variables related to latitude were most strongly correlated with network structure of the Sarracenia food web. However, in spite of large sample sizes, thorough standardized sampling and the large geographic extent of the survey, even the best‐fitting models explained less than 40% of the variation in food web structure. In contrast to biogeographic patterns of species richness, food web structure was largely independent of broad‐scale climatic variables. The large proportion of unexplained variance in our analyses suggests that stochastic assembly may be an important determinant of local food web structure.     

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

Background and Aims

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.

Methods

Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma–optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.

Key Results

The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.

Conclusions

The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.