The crab spider-pitcher plant relationship is a nutritional mutualism that is dependent on prey-resource quality.

Nutritional mutualisms are one of the three major categories of mutualisms and involve the provision of limiting nutrients (resources) to one species by another. It was recently shown in laboratory experiments that two species of pitcher-dwelling crab spiders (Thomisidae), Thomisus nepenthiphilus and Misumenops nepenthicola, increased capture rates of flesh flies (Sarcophagidae) for their host, Nepenthes gracilis. The spiders ambushed pitcher-visiting flesh flies and dropped their carcasses into pitchers after consuming them. The consumption of shared prey-resources by crab spiders and pitcher plants presents the possibility of parasitism between them. However, ecologically generalizable mechanisms that predict the context-dependent outcomes of such mutualisms are not known. The effectiveness framework (mutualism effectiveness = quality × quantity) is useful for examining the total effect of mutualisms, but its quality component can be difficult to define. We identify the crab spider-pitcher plant interaction as a type of resource conversion mutualism and propose that the quality component in such interactions is the amount of the underlying resource contained in each unit of resource processed. We then used the crab spider-pitcher plant interaction to test the hypothesis that resource conversion mutualisms are more beneficial to the nutrient recipient when operating through high-quality resources (i.e., large prey, in this interaction). We sampled the prey and inquilines of 107 N. gracilis upper pitches in situ and analysed the differences between pitchers that were inhabited or uninhabited by crab spiders, and the differences between nutritional contents of prey that were consumed by crab spiders or not. Pitchers inhabited by T. nepenthiphilus contained higher numbers of several prey taxa, many of which were flying insects. Consumption by T. nepenthiphilus reduced the nutrient contents in all prey examined. Overall, T. nepenthiphilus-assisted prey capture is likely to result in a net nutrient gain for N. gracilis that is proportional to the size of prey consumed by T. nepenthiphilus. Our results suggest that resource conversion mutualisms are more likely to operate through high-quality resources, since the nutrient-processing species necessarily reduces the quality of the resource it processes while increasing its availability to the nutrient recipient species.

Digestive mutualism in a pitcher plant supports the monotonic rather than hump-shaped stress-gradient hypothesis model.

The stress-gradient hypothesis (SGH) predicts that the strength and frequency of facilitative interactions increase monotonically with increasing environmental stress, but some empirical studies have found this decrease at extreme stress levels, suggesting a hump-shaped SGH instead. However, empirical studies of the SGH are often hindered by confounding resource and non-resource stress gradients. Nepenthes pitcher plants trap animal prey using modified-leaf pitfall traps which are also inhabited by organisms known as inquilines. Inquilines may assist pitchers in the digestion of trapped prey. This interaction is known as a digestive mutualism and is both mutualistic and facilitative by definition. Inquiline species may also facilitate each other via processing chain commensalisms. We used in vitro experiments to examine the isolated effect of resource stress on the outcomes of two facilitative interactions: (i) digestive mutualism-facilitation of pitcher nutrient sequestration by two inquiline dipteran larvae, culicids and phorids; (ii) processing chain commensalism-facilitation between these two inquiline taxa. The net nutritional benefit of phorids on N. gracilis was found to conform more to a monotonic rather than hump-shaped SGH model. However, the effect of culicids on N. gracilis and the effects of culicids and phorids on each other were weak. These findings provide compelling evidence that changes in facilitation along an isolated resource stress gradient conform to the predictions of the monotonic SGH model rather than that of the revised hump-shaped model, and highlight the importance of isolating stress gradients in empirical tests of the SGH.

A dipteran larva-pitcher plant digestive mutualism is dependent on prey resource digestibility.

Positive species interactions tend to be context dependent. However, it is difficult to predict how benefit in a mutualism changes in response to changing contexts. Nepenthes pitcher plants trap animal prey using leaf pitfall traps known as pitchers. Many specialized inquiline organisms inhibit these pitchers, and are known to facilitate the digestion of prey carcasses in them. Nepenthes gracilis traps diverse arthropod prey taxa, which are likely to differ greatly in the ease with which they may be digested, independently of inquilines, by plant enzymes. In this study, we used in vitro experiments to compare the nutritional benefit provided by phorid (scuttle fly) and culicid (mosquito) dipteran larvae to their host, N. gracilis, and to each other. The effects of phorids on N. gracilis nutrient sequestration were very variable, being positive for large prey which have low digestibility, but negative for small prey which are highly digestible. However, the effect of culicids on N. gracilis and the effects of culicids and phorids on each other were not significantly altered by prey type. These results show that a digestive mutualism is highly dependent on the digestibility of the resource-a context dependency that conforms well to the predictions of the stress-gradient hypothesis in facilitation research. Our findings have significant implications for many other digestive mutualisms, and also suggest that greater insights may be gained from the synthesis of concepts between the fields of mutualism and facilitation research.

Novel pitcher plant-spider mutualism is dependent upon environmental resource abundance.

Positive species interactions are ubiquitous and crucial components of communities, but they are still not well incorporated into established ecological theories. The definitions of facilitation and mutualism overlap, and both are often context dependent. Many interactions that are facilitative under stressful conditions become competitive under more benign ones. This is known as the stress-gradient hypothesis, which is a specific case of context dependency. Stress can be further divided into resource and non-resource categories, but a better mechanistic understanding is necessary to improve the theory's predictions. We examined if two pitcher-dwelling crab spiders (Thomisidae), Thomisus nepenthiphilus and Misumenops nepenthicola, can facilitate nitrogen sequestration in their pitcher plant host, Nepenthes gracilis, by ambushing pitcher-visiting flies and dropping their carcasses into pitchers after consumption. This relationship is, by definition, both mutualistic and facilitative. Laboratory experiments found that both crab spiders increased prey-capture rates of N. gracilis. Nutrient analyses showed that both crab spiders also decreased per unit nitrogen yield of prey. Using experiment duration as a proxy of prey-resource availability, we constructed a mechanistic conceptual model of nutritional benefit. The nutritional benefit received by N. gracilis from T. nepenthiphilus decreases with increasing levels of the limiting resource in the environment (i.e., decreasing levels of resource stress). Our findings suggest that any nutritional mutualism that increases the quantity of resource capture (e.g. number of prey individuals) but decreases the quality of the captured resource (e.g. nitrogen content of individual prey) will necessarily conform to the resource-based predictions of the stress gradient hypothesis.

Dipteran larvae and microbes facilitate nutrient sequestration in the Nepenthes gracilis pitcher plant host.

The fluid-containing traps of Nepenthes carnivorous pitcher plants (Nepenthaceae) are often inhabited by organisms known as inquilines. Dipteran larvae are key components of such communities and are thought to facilitate pitcher nitrogen sequestration by converting prey protein into inorganic nitrogen, although this has never been demonstrated in Nepenthes Pitcher fluids are also inhabited by microbes, although the relationship(s) between these and the plant is still unclear. In this study, we examined the hypothesis of digestive mutualism between N. gracilis pitchers and both dipteran larvae and fluid microbes. Using dipteran larvae, prey and fluid volumes mimicking in situ pitcher conditions, we conducted in vitro experiments and measured changes in available fluid nitrogen in response to dipteran larvae and microbe presence. We showed that the presence of dipteran larvae resulted in significantly higher and faster releases of ammonium and soluble protein into fluids in artificial pitchers, and that the presence of fluid microbes did likewise for ammonium. We showed also that niche segregation occurs between phorid and culicid larvae, with the former fragmenting prey carcasses and the latter suppressing fluid microbe levels. These results clarify the relationships between several key pitcher-dwelling organisms, and show that pitcher communities facilitate nutrient sequestration in their host.

The Antibacterial Agent Identified from Acidocella spp. in the Fluid of Nepenthes gracilis Against Multidrug-Resistant Klebsiella pneumoniae: A Functional Metagenomic Approach.

Aims: The fluid of Nepenthes gracilis harbors diverse bacterial taxa that could serve as a gene pool for the discovery of the new genre of antimicrobial agents against multidrug-resistant Klebsiella pneumoniae. The aim of this study was to explore the presence of antibacterial genes in the fluids of N. gracilis growing in the wild. Methods: Using functional metagenomic approach, fosmid clones were isolated and screened for antibacterial activity against three strains of K. pneumoniae. A clone that exhibited the most potent antibacterial activity was sent for sequencing to identify the genes responsible for the observed activity. The secondary metabolites secreted by the selected clone was sequentially extracted using hexane, chloroform, and ethyl acetate. The chemical profiles of a clone (C6) hexane extract were determined by gas chromatography/mass spectrometry (GC-MS). Results: Fosmid clone C6 from the fluid of pitcher plant that exhibited antibacterial activity against three strains of K. pneumoniae was isolated using functional metagenome approach. A majority of the open reading frames detected from C6 were affiliated with the largely understudied Acidocella genus. Among them, the gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway could be involved in the observed antibacterial activity. Based on the GC-MS analysis, the identities of the putative bioactive compounds were 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane. Conclusions: The gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway as well as the secondary metabolites, namely 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane could be the potential antibacterial molecules responsible for the antibacterial activity of C6.

Crystallization of nepenthesin I using a low-pH crystallization screen.

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Šresolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = ß = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = ß = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.