Chemical contents of Macaranga food bodies: adaptations to their role in ant attraction and nutrition

1. Macaranga (Euphorbiaceae) is a paleotropical tree genus comprising myrmecophytic and non-myrmecophytic species. All species are presumed to possess food bodies (FBs) to maintain or attract ants as anti-herbivore defence.
2. The hypothesis was tested that Macaranga species differing in their mode of association with ants would produce FBs differing in their chemical composition. We investigated contents of carbohydrates, proteins and lipids in FBs of four myrmecophytic and one non-myrmecophytic Macaranga as well as one Parthenocissus (Vitaceae) species.
3. On a dry weight basis, FBs of myrmecophytes contained relatively higher amounts of proteins compared to carbohydrates than those of non-myrmecophytes. Soluble carbohydrates showed species-specific patterns and were found in especially high amounts in both non-myrmecophytes. Furthermore, Parthenocissus FBs contained higher amounts of soluble compared to polymerous substances not only in carbohydrates but also in proteins.
4. FBs seem to be specifically adapted to their respective role in ant attraction and nutrition, with myrmecophytes providing ants with high amounts of lipids and proteins and non-myrmecophytes mainly offering carbohydrates in the form of common soluble sugars.     

Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.Plants secrete nectar to attract mutualistic animals, which mainly function as pollinators in the case of floral nectar or as defenders against herbivores in the case of extrafloral nectar (EFN; Simpson and Neff, 1981; Heil, 2008; González-Teuber and Heil, 2009a). Because nectars usually represent aqueous solutions of monosaccharides and disaccharides together with amino acids, they are prone to infestation by microbial organisms. When present in the nectar, fungi (González-Teuber et al., 2009) and yeast (Herrera et al., 2009) in particular can alter the chemical composition of the nectar and thereby reduce its suitability for the plant''s animal mutualists (Herrera et al., 2008). Moreover, several phytopathogenic organisms may use the nectar-secreting tissues as entries to infect other plant organs (Bubán et al., 2003; Farkas et al., 2007). Therefore, being an excellent growing medium for yeast, fungi, and bacteria, nectar requires an efficient antimicrobial protection.Unfortunately, our knowledge of the means by which plants protect nectar from microorganisms is extremely limited. Although the first reports on nectar proteins date back to the 1960s and 1970s (Lüttge, 1961; Baker and Baker, 1975), most studies that considered the defensive function of nectar focused on secondary compounds such as alkaloids and phenols. These metabolites commonly protect nectar from consumption by nectar robbers (animals that feed on nectar without providing a mutualistic service to the plant [Stephenson, 1981; Johnson et al., 2006]) or limit the duration of pollinator visits (Kessler et al., 2008). Only during the last decade did a series of studies discover defensive proteins in the floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae; Carter et al., 1999). In this species, floral nectar contains a limited array of proteins termed “nectarins.” Nectarins serve the protection from microbial infestation through a biochemical pathway called the nectar redox cycle (Carter and Thornburg, 2004a), in which mainly three of the five nectarins are involved: NEC1, NEC3, and NEC5. NEC1 was characterized as a manganese superoxide dismutase (Carter and Thornburg, 2000), NEC3 has carbonic anhydrase and monodehydroascorbate reductase activity (Carter and Thornburg, 2004b), and NEC5 is a Glc oxidase that functions together with NEC1 in the production of high peroxide levels (Carter and Thornburg, 2004c): nectar of ornamental tobacco can accumulate up to 4 mm hydrogen peroxide, concentrations that are clearly high enough to exhibit toxicity on microorganisms. Thus, the floral nectar of ornamental tobacco is kept free of microbes mainly via the production of small reactive oxygen species.By contrast, a proteomic study on EFN of the ant-plant, Acacia cornigera, revealed the presence of several pathogenesis-related (PR) proteins (González-Teuber et al., 2009). Myrmecophytes (ant-plants) are constitutively inhabited by specialized ant species, which serve as a very efficient indirect defense against herbivores (Heil, 2008). In the most specialized cases, both the ant and the plant depend on this interaction, which thus represents an obligate mutualism. In the EFN of A. cornigera, activities of chitinase, β -1,3-glucanase, and peroxidase were detected together with proteins similar to PR-1, osmotin-like proteins, and thaumatin-like proteins (González-Teuber et al., 2009). Most of these proteins, however, were only investigated by tandem mass spectrometry (MS/MS) and characterized via MS-BLAST searches. Because no activity assays had been performed, the presence of these proteins could not be causally linked to the protection of EFN from microorganisms.This study was conducted to determine whether the antimicrobial protection of Acacia EFN can be directly and exclusively allotted to the enzymatic activity of its protein fraction, which would contrast the protective strategy of this nectar from the one that has been described by Carter, Thornburg, and colleagues (Carter et al., 1999; Carter and Thornburg, 2004a). We also aimed at investigating whether Acacia EFN inhibits the growth of phytopathogens and thus can serve in the protection from infection by pathogens that may use nectaries to enter the plant (Bubán et al., 2003). We used four sympatric Acacia species and a closely related Prosopis species, which exhibit different types of ant-plant mutualism and therefore differ in their EFN secretion schemes (Heil et al., 2004) and composition (Heil et al., 2005; González-Teuber and Heil, 2009b). The obligate myrmecophytes among Central American Acacia species secrete EFN constitutively at high rates, and the EFN of these species possesses a much higher level of proteins and of antimicrobial defense than the EFN of congeneric nonmyrmecophytes (González-Teuber et al., 2009). The nonmyrmecophytes, by contrast, secrete EFN at lower rates and only transiently in response to leaf damage; this EFN contains few proteins but high levels of Suc (Heil et al., 2005; González-Teuber et al., 2009).We studied the EFN of the obligate myrmecophytes A. cornigera, Acacia hindsii, and Acacia collinsii and of the two nonmyrmecophytes Acacia farnesiana and Prosopis juliflora. Bioassays were employed to detect inhibitory activities of the nectars against phytopathogens, and in-gel assays were used to determine the presence and functionality of basic and acidic chitinases and glucanases. Size exclusion filtration and heating of the EFN was used to investigate whether the antimicrobial activity of EFN is exclusively caused by the protein fraction. The results demonstrate that the antimicrobial protection of Acacia EFN is caused by the fraction of enzymatically active PR proteins and independent of small, soluble molecules, an observation that represents, to our knowledge, a new strategy by which plants can protect nectar from infestation by potentially deleterious microorganisms.     

Pseudomyrmex ants and Acacia host plants join efforts to protect their mutualism from microbial threats

Plants express numerous ‘pathogenesis-related’ (PR) proteins to defend themselves against pathogen infection. We recently discovered that PR-proteins such as chitinases, glucanases, peroxidases and thaumatin-like proteins are also functioning in the protection of extra-floral nectar (EFN) of Mexican Acacia myrmecophytes. These plants produce EFN, cellular food bodies and nesting space to house defending ant species of the genus Pseudomyrmex. More than 50 PR-proteins were discovered in this EFN and bioassays demonstrated that they actively can inhibit the growth of fungi and other phytopathogens. Although the plants can, thus, express PR-proteins and secrete them into the nectar, the leaves of these plants exhibit reduced activities of chitinases as compared to non-myrmecophytic plants and their antimicrobial protection depends on the mutualistic ants. When we deprived plants of their resident ants we observed higher microbial loads in the leaves and even in the tissue of the nectaries, as compared to plants that were inhabited by ants. The indirect defence that is achieved through an ant-plant mutualism can protect plants also from infections. Future studies will have to investigate the chemical nature of this mechanism in order to understand why plants depend on ants for their antimicrobial defence.Key words: ant-plant mutualism, indirect defense, pathogen resistance, pathogenesis-related proteinPlants fall victim to multiple attackers from various kingdoms and have evolved numerous strategies to defend themselves against herbivores and microbial pathogens. Pathogen resistance is usually based on particular cell wall properties, secondary compounds (phytoalexins) and on pathogenesis-related (PR) proteins.^1,2 Plant resistance to pathogens is thus, mainly based on plant traits that interact with the pathogen and therewith functions as a “direct” defence mechanism. Resistance to herbivores, by contrast, can also be “indirect”: plants house or attract carnivores, which fulfil the defensive function.³ Defensive ant-plant interactions are common mutualisms in which plants provide to ants an array of rewards that comprises both food rewards and domatia (nesting space).^3,4 Ants are attracted or nourished by plant-derived food rewards and defend the plants against herbivores.^3,5 In obligate interactions, myrmecophytes are inhabited by specialised ants during major parts of their life⁴ and the ants are entirely dependent on the food rewards and nesting space that are provided by their host. These ants, in return, defend their host efficiently and aggressively against herbivores and encroaching vegetation. Only recently we found that this protective service might also cover the defence against phytopathogens.In the Mexican Acacia-Pseudomyrmex mutualism, extrafloral nectar (EFN) represents an important plant trait to nourish symbiotic ant colonies. EFN is secreted by special glands called extrafloral nectaries and is rich in mono- and disaccharides and amino acids.⁶ Due to its high content in primary metabolites, EFN appears also attractive to exploiters, which make use of the nectar resource without providing a service to the plant.⁷ For example, EFN can serve as a suitable medium for pathogen growth and is highly prone to microbial infestations, which can have negative consequences on nectar composition.^8,9We recently found that Acacia EFN is biochemically protected from microbial infections.^10,11 More than 50 proteins could be distinguished in EFN of the myrmecophytes, A. hindsii, A. cornigera and A. collinsii, and most of these proteins were annotated as PR-proteins such as chitinases, β-1,3 glucanases, thaumatin-like proteins and peroxidases. In fact, chitinases and glucanases represented more than 50% of the total amount of proteins in EFN of myrmecophytic Acacia plants.^10,11 This dominance of PR-proteins clearly distinguishes the anti-microbial strategy of Acacia-EFN from the strategy of floral nectar of ornamental tobacco. The protection of the latter nectar is mainly based on small metabolites, such as hydrogen peroxide, which are produced by five proteins forming the ‘nectar redox cycle’.^12–14 By contrast, PR-proteins have also been reported from pollination droplets of gymnosperms,¹⁵ although their biological functions remain to be studied. For the Acacia EFN, biotests demonstrated that the chitinases and glucanases are active and that EFN successfully can inhibit the growth of various fungi and oomycetes.¹¹Nevertheless, and although the microorganisms used in these assays were phytopathogens, we have now observed that the protection from microbial infection of the Acacia plant itself depends on certain characteristics of its ant inhabitants. The occurrence of fungi and bacteria in EFN and in the tissue of leaves and nectaries was investigated under natural growing conditions. We collected samples of A. cornigera and A. hindsii plants, which were inhabited by ants or which had been deprived of ants experimentally two weeks before. The tissues were extracted and analyzed for microbial infection by cultivating the extracts on malt agar plates, in order to quantify the abundances of life fungi and bacteria as the numbers of colony-forming units (CFU). Leaves of two species of myrmecophytic Acacia plants showed a significant increase in their bacterial load when they were deprived of the mutualistic P. ferrugineus ants (Fig. 1A). This observation is redolent of an earlier observation made on Macaranga myrmecophytes in Malaysia: lesions of these plants could easily be infected with fungi when the mutualistic Crematogaster ants were absent, but not in the presence of the ants.¹⁶ Similarly, myrmecophytic Piper plants depend, at least in part, on their ant inhabitants to obtain an efficient protection from fungal pathogens.¹⁷ Myrmecophytic Maracanga plants possessed reduced activities of chitinases in their leaves¹⁶ and the same phenomenon was found in Acacia myrmecophytes.¹⁸ Thus, the leaves of the obligate ant-plants of both genera, Acacia and Macaranga, appear not to express PR-proteins at sufficient activities as to protect themselves from pathogens and symbiotic ants are required for this defensive function. The effect is specific for the defending ants, because no significant differences between plants with and without ants could be detected in the case of plants that were inhabited by the non-defending parasite, P. gracilis¹⁹ (Fig. 1B). Most interestingly, even the nectary tissue appears to require ant-mediated protection from phytopathogens, whereas the EFN itself does not: no fungi can usually be isolated from freshly collected nectar of Acacia myrmecophytes under field conditions¹⁰ (Fig. 2A), but the tissue of the nectaries became heavily infected when branches were kept free of the defending ants (Fig. 2B).Open in a separate windowFigure 1Effects of the presence and absence of the symbiotic ant P. ferrugineus and the parasitic ant P. gracilis on the abundance of bacteria [CFU mg dry leaf mass⁻¹] in leaf samples of A. cornigera (A) and A. hindsii (B). Significance levels are indicated: ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001. (Two-factorial ANOVA applied separately for each plant species; independent variables: ant presence and ant species.)Open in a separate windowFigure 2Effects of presence and absence of the symbiotic ant P. ferrugineus on fungal abundance in EFN (A) and nectar tissue (B) of A. cornigera and A. hindsii. Significance levels are indicated: ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001. (Two-factorial ANOVA; independent variables: ant presence and plant species).Our new findings highlight the joint efforts of both ant and plant that are required for an efficient protection from pathogens of the myrmecophyte host plants, and, thus, for the prevention of this mutualism from exploitation by microorganisms. It remains open why the myrmecophytic Acacia plants exhibit reduced chitinase activities in their leaves¹⁸ and thus depend on ants for their antimicrobial protection, although they are fully equipped to express functioning PR-proteins and secrete them into their EFN.^10,11 We could assume that an antimicrobial protection by ants is less costly for the plant than its own biochemical defence mechanisms. Alternatively, plant pathogens, which express multiple effectors in order to suppress their host resistance strategies,¹ might be less capable to cope with ant-derived resistance mechanisms. Every attempt of an explanation, however, remains speculative as long as we do not understand how the ants can protect the leaves of their host plant from infection. The chemical mechanisms remain to be analyzed and may comprise both an ant-mediated resistance induction in the plant and directly ant-derived antimicrobial compounds. Most importantly, the joined efforts of both plant host and ant inhabitant are required to keep leaves, nectaries and nectar free of microbial infections and microbial pathogens have been identified as a further target of the indirect defence, which plants can obtain when they establish an obligate mutualism with ants.     

The network structure of myrmecophilic interactions

1. Ants establish mutualistic interactions involving a wide range of protective relationships (myrmecophily), in which they provide defence against enemies and partners provide food rewards and/or refuge. Although similar in the general outcome, myrmecophilic interactions differ in some characteristics such as quantity and quality of rewards offered by partners which may lead to different specialisation levels and, consequently, to different network properties. 2. The aim of this study was to identify structural patterns in myrmecophilic interaction networks, focusing on aspects related to specialisation: network modularity, nestedness and taxonomic relatedness of interaction ranges. To achieve this, a database of networks was compiled, including the following interactions: ants and domatia‐bearing plants (myrmecophytes); ants and extrafloral nectary‐bearing plants (EFNs); ants and floral nectary‐bearing plants (FNs); ants and Lepidoptera caterpillars; and ants and Hemiptera. 3. Myrmecophilic networks differed in their topology, with ant–myrmecophyte and ant–Lepidoptera networks being similar in their structural properties. A continuum was found, ranging from highly modular networks and phylogenetically structured interaction ranges in ant–myrmecophyte followed by ant–Lepidoptera networks to low modularity and taxonomically unrelated interaction ranges in ant–Hemiptera, EFN and FN networks. 4. These results suggest that different network topologies may be found across communities of species with similar interaction types, but also, that similar network topologies can be achieved through different mechanisms such as those between ants and myrmecophytes or Lepidoptera larvae. This study contributes to a generalisation of myrmecophilic network patterns and a better understanding of the relationship between specialisation and network topology.     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

Seed predators are undeterred by nectar-feeding ants on Chamaecrista nictitans (Caesalpineaceae)

There are many examples of mutualistic interactions between ants and plants bearing extrafloral nectaries (EFN). The annual legume Chamaecrista nictitans (Caesalpineaceae) secretes nectar from EFN, specialized structures that attract ants, spiders, and other arthropods. The effects of manipulated C. nictitans patch size and location on plant-ant interactions were tested. Defense from herbivores was not detected; plants with ants did not set significantly more fruit or seed than plants with ants excluded. On the contrary, in one year, plants without ants set more fruit and seed than C. nictitans with ants. The cause of this was not determined. Furthermore, insect herbivore damage was low during three years of observations. Sennius cruentatus (Bruchidae), a specialist seed predator beetle, escaped ant defense despite the presence of numerous ants. Beetle progeny are protected during development by living inside maturing C. nictitans fruit and preventing fruits from dehiscing before emerging as adults. Although ants reduced percent of infestation in 1995, the total number of S. cruentatus per plant was not affected by ants in years of infestation. Overall, larger experimental C. nictitans patches attracted more ants, parasitoid wasps, and percent infestation by S. cruentatus while insect herbivores declined with increasing patch size. Location of patches within fields, however, did not affect numbers of arthropod visitors. Similar to manipulated populations, very little insect herbivory occurred in four reference populations. In contrast to the experimental populations, no S. cruentatus were recovered in reference populations of C. nictitans. Herbivory by insects may not always depress seed set by C. nictitans or may not exceed a threshold level. Thus, herbivory-reduction by ants may not have been detectable in these results. Seed predation may be more influential on C. nictitans reproduction. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adaptations to biotic and abiotic stress: Macaranga-ant plants optimize investment in biotic defence

Obligate ant plants (myrmecophytes) in the genus Macaranga produce energy- and nutrient-rich food bodies (FBs) to nourish mutualistic ants which live inside the plants. These defend their host against biotic stress caused by herbivores and pathogens. Facultative, 'myrmecophilic' interactions are based on the provision of FBs and/or extrafloral nectar (EFN) to defending insects that are attracted from the vicinity. FB production by the myrmecophyte, M. triloba, was limited by soil nutrient content under field conditions and was regulated according to the presence or absence of an ant colony. However, increased FB production promoted growth of the ant colonies living in the plants. Ant colony size is an important defensive trait and is negatively correlated to a plant's leaf damage. Similar regulatory patterns occurred in the EFN production of the myrmecophilic M. tanarius. Nectar accumulation resulting from the absence of consumers strongly decreased nectar flow, which increased again when consumers had access to the plant. EFN flow could be induced via the octadecanoid pathway. Leaf damage increased levels of endogenous jasmonic acid (JA), and both leaf damage and exogenous JA application increased EFN flow. Higher numbers of nectary visiting insects and lower numbers of herbivores were present on JA-treated plants. In the long run, this decreased leaf damage significantly. Ant food production is controlled by different regulatory mechanisms which ensure that costs are only incurred when counterbalanced by defensive effects of mutualistic insects.     

Extrafloral nectaries as a deterrent mechanism against seed predators in the chemically protected weed Crotalaria pallida (Leguminosae)

Abstract In several plants, extrafloral nectaries (EFN) are located close to the reproductive structures, suggesting that ants may act as a defence against specialized seed predators that overcome chemical defences. Alternatively, ants may also deter herbivores in a generalized manner, thereby protecting the whole plant. In this work, we examined the relationship between the chemically protected weed Crotalaria pallida Ait. (Leguminosae) that bears EFN, its specialized seed predator, the larvae of the arctiid moth Utetheisa ornatrix L. (Arctiidae) and ants. We tested two hypotheses related to the type of deterrence caused by ants. The Seed Predator Deterrence Hypothesis predicts that ant deterrence is directed primarily towards herbivores that destroy seeds and other reproductive structures, without attacking herbivores on vegetative structures. The General Deterrence Hypothesis states that ants are general in their effects, equally deterring herbivores in vegetative and reproductive structures. Our results supported the predictions of the Seed Predator Deterrence Hypothesis, namely, that (i) ant activity on EFN was related to the vulnerability of reproductive structures to attack by U. ornatrix; (ii) ant patrolling was restricted almost entirely to racemes; (iii) ants removed termites used as baits more frequently on racemes than on leaves; and (iv) U. ornatrix larvae were often expulsed from the racemes. These results indicate that EFN can act as another deterrent mechanism in chemically protected plants by promoting the expulsion of specialist seed predators.     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

Increased Host Investment in Extrafloral Nectar (EFN) Improves the Efficiency of a Mutualistic Defensive Service

Extrafloral nectar (EFN) plays an important role as plant indirect defence through the attraction of defending ants. Like all rewards produced in the context of a mutualism, however, EFN is in danger of being exploited by non-ant consumers that do not defend the plant against herbivores. Here we asked whether plants, by investing more in EFN, can improve their indirect defence, or rather increase the risk of losing this investment to EFN thieves. We used the obligate plant-ant Acacia-Pseudomyrmex system and examined experimentally in the field during the dry and the rainy seasons how variations in EFN secretion are related to (i) ant activity, to (ii) the ant-mediated defence against herbivores and (iii) the exploitation of EFN by non-ant consumers. Extrafloral investment enhanced ant recruitment and was positively related to the ant mediated defence against herbivores. The ant-mediated protection from exploiters also increased in proportion to the nectar sugar concentration. Although the daily peak of EFN production coincided with the highest activity of EFN thieves, Pseudomyrmex ferrugineus ants protected this resource effectively from exploiters. Nevertheless, the defensive effects by ants differed among seasons. During the dry season, plants grew slower and secreted more EFN than in the rainy season, and thus, experienced a higher level of ant-mediated indirect defence. Our results show that an increased plant investment in an indirect defence trait can improve the resulting defensive service against both herbivores and exploiters. EFN secretion by obligate ant-plants represents a defensive trait for which the level of investment correlates positively with the beneficial effects obtained.     

Aphid infestation induces PR-proteins differently in barley susceptible or resistant to the birdcherry-oat aphid (Rhopalosiphum padi)

The effect of infestation by the birdcherry-oat aphid ( Rhopalosiphum padi L.), on induction of PR-proteins was investigated in barley ( Hordeum vulgare L.), using barley lines susceptible or resistant to R. padi. The PR-proteins PR-1a (unknown function), PR-5a (acidic thaumatin) and peroxidase (EC 1.11.1.7) were not affected, whereas one chitinase (EC 3.2.1.14) and 4 β -1,3-glucanases (EC 3.2.1.39) were induced by the aphid treatment. In the resistant breeding line CI 16145, but not in the susceptible cultivar Golf, accumulation of one basic chitinase and two acidic β -1,3-glucanases increased with time from 2 until 11 days after infestation, as determined by western blots, with antibodies raised against purified chitinase (PR-3a) and β -1,3-glucanase (PR-2a) from barley. By isoelectric focusing, two additional basic β -1,3-glucanases were detected, which increased after infestation in both the resistant and the susceptible barley. The basic chitinase was only detected at days 7 and 11 in the susceptible cultivar, but already at day 2 in the resistant line. The induction was localized to the infested leaf. The PR-proteins PR-3a and PR-2a were also induced by the fungal pathogen ( Blumeria [syn. Erysiphe ] graminis f. sp. hordei ), methyl salicylate and, to a lower extent, by wounding with tweezers and methyl jasmonate (MeJA). Needle wounding performed to mimic aphid stylet penetration did not induce chitinase or β -1,3-glucanase. It is concluded that the fungal pathogen and the aphid infestation induce both similar and different responses, and that the aphid induction is not due to wounding only. The different responses in resistant and susceptible lines indicate that the induced enzymes may play a role in the resistance against aphid infestation.     

Geographical variation in an ant–plant interaction correlates with domatia occupancy,local ant diversity,and interlopers

Interactions between potentially mutualistic partners can vary over geographic areas. Myrmecophytes, which are plants harbouring ants, often do not exhibit sufficient intraspecific variability to permit comparative studies of myrmecophytic traits over space or time. Humboldtia brunonis (Fabaceae), a dominant, endemic myrmecophyte of the Indian Western Ghats, is unique in exhibiting considerable variability in myrmecophytic traits, e.g. domatia presence, as well as domatia occupancy and associated ant diversity throughout its geographic range. Although its caulinary domatia are occupied by at least 16 ant species throughout its distribution, young leaves and floral buds producing extrafloral nectar (EFN) are protected by ants from herbivory only in the southernmost region, where Technomyrmex albipes (Dolichoderinae) is the most abundant ant species. The extent of protection by ants was positively related to local species richness of ants and their occupancy of domatia. On the other hand, the highest abundance of interlopers in the domatia, including non‐protective ants, the arboreal earthworm Perionyx pullus, and other invertebrates, occurred in sites with the least protection from herbivory by ants. Whereas domatia morphometry did not vary between sites, domatia occupied by protective ants and invertebrate interlopers were longer and broader than empty ones at all sites. The lowest percentage of empty domatia was found at the southernmost site. There was a progressive decline in ant species richness from that found at the sites, to that feeding on H. brunonis EFN, to that occupying domatia, possibly indicating constraints in the interactions with the plants at various levels. Our study of this dominant myrmecophyte emphasizes the impact of local factors such as the availability of suitable ant partners, domatia occupancy, and the presence of interlopers on the emergence of a protection mutualism between ants and plants. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 538–551.     

Behavioural ecology of defence in a risky environment: caterpillars versus ants in a Neotropical savanna

1. Predatory ants may reduce infestation by herbivorous insects, and slow‐moving Lepidopteran larvae are often vulnerable on foliage. We investigate whether caterpillars with morphological or behavioural defences have decreased risk of falling prey to ants, and if defence traits mediate host plant use in ant‐rich cerrado savanna. 2. Caterpillars were surveyed in four cerrado localities in southeast Brazil (70–460 km apart). The efficacy of caterpillar defensive traits against predation by two common ant species (Camponotus crassus, C. renggeri) was assessed through experimental trials using caterpillars of different species and captive ant colonies. 3. Although ant presence can reduce caterpillar infestation, the ants' predatory effects depend on caterpillar defence traits. Shelter construction and morphological defences can prevent ant attacks (primary defence), but once exposed or discovered by ants, caterpillars rely on their size and/or behaviour to survive (secondary defence). 4. Defence efficiency depends on ant identity: C. renggeri was more aggressive and lethal to caterpillars than C. crassus. Caterpillars without morphological defences or inside open shelters were found on plants with decreased ant numbers. No unsheltered caterpillar was found on plants with extrafloral nectaries (EFNs). Caterpillars using EFN‐bearing plants lived in closed shelters or presented morphological defences (hairs, spines), and were less frequently attacked by ants during trials. 5. The efficiency of defences against ants is thus crucial for caterpillar survival and determines host plant use by lepidopterans in cerrado. Our study highlights the effect of EFN‐mediated ant‐plant interactions on host plant use by insect herbivores, emphasizing the importance of a tritrophic viewpoint in risky environments.     

Host‐plant use by two Orthomeria (Phasmida: Aschiphasmatini) species feeding on Macaranga myrmecophytes

Myrmecophytes depend on symbiotic ants (plant‐ants) to defend against herbivores. Although these defensive mechanisms are highly effective, some herbivorous insects can use myrmecophytes as their host‐plants. The feeding habits of these phytophages on myrmecophytes and the impacts of the plant‐ants on their feeding behavior have been poorly studied. We examined two phasmid species, Orthomeria alexis and O. cuprinus, which are known to feed on Macaranga (Euphorbiaceae) myrmecophytes in a Bornean primary forest. Our observations revealed that: (i) each phasmid species relied on two closely‐related myrmecophytic Macaranga species for its host‐plants in spite of their normal plant‐ant symbioses; and (ii) there was little overlap between their host‐plant preferences. More O. cuprinus adults and nymphs were found on new leaves, which were attended by more plant‐ants than mature leaves, while most adults and nymphs of O. alexis tended to avoid new leaves. In a feeding choice experiment under ant‐excluded conditions, O. alexis adults chose a non‐host Macaranga myrmecophyte that was more intensively defended by plant‐ants and was more palatable than their usual host‐plants almost as frequently as their usual host‐plant, suggesting that the host‐plant range of O. alexis was restricted by the presence of plant‐ants on non‐host‐plants. Phasmid behavior that appeared to minimize plant‐ant attacks is described.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Pea aphids (Acyrthosiphon pisum Harris) reduce secretion of extrafloral nectar in broad bean (Vicia faba)

1. Herbivores sometimes suppress plant defences. This study tested whether the presence of pea aphids (Acyrthosiphon pisum Harris) on broad bean (Vicia faba) led to decreased secretion of extrafloral nectar (EFN) which functions as an indirect plant defence against herbivores. 2. To determine effects of aphid infestation on EFN secretion, a comparison was done between EFN secretion in uninfested plants and that in plants infested by A. pisum and another aphid species (Aphis craccivora Koch). 3. When broad bean plants were infested by A. pisum, they secreted significantly smaller amounts of EFN than did uninfested plants and A. craccivora‐infested plants. There was no significant difference in EFN secretion between uninfested plants and A. craccivora‐infested plants. The number of extrafloral nectaries did not differ among the three treatments. 4. These results suggest that A. pisum reduced EFN production in broad bean plants.     

Plant lock and ant key: pairwise coevolution of an exclusion filter in an ant-plant mutualism

Although observations suggest pairwise coevolution in specific ant-plant symbioses, coevolutionary processes have rarely been demonstrated. We report on, what is to the authors' knowledge, the strongest evidence yet for reciprocal adaptation of morphological characters in a species-specific ant-plant mutualism. The plant character is the prostoma, which is a small unlignified organ at the apex of the domatia in which symbiotic ants excavate an entrance hole. Each myrmecophyte in the genus Leonardoxa has evolved a prostoma with a different shape. By performing precise measurements on the prostomata of three related myrmecophytes, on their specific associated ants and on the entrance holes excavated by symbiotic ants at the prostomata, we showed that correspondence of the plant and ant traits forms a morphological and behavioural filter. We have strong evidence for coevolution between the dimensions and shape of the symbiotic ants and the prostoma in one of the three ant-Leonardoxa associations.     

Conspicuous extra-floral nectaries are inducible in Vicia faba

Mutualistic interactions are dynamic associations that vary depending on the costs and benefits to each of the interacting parties. Phenotypic plasticity in mutualistic interactions allows organisms to produce rewards to attract mutualists when the benefits of their presence outweigh the costs of producing the rewards. In ant–plant defensive mutualisms, defences are indirect as plants produce extra‐floral nectaries (EFN) to attract predatory ants to deter herbivores. Here we demonstrate that in broad bean, Vicia faba, the overall number of EFNs on a plant increases dramatically following leaf damage. In two damage treatments, removal of: (1) one‐third of one leaf in a single leaf pair or (2) one‐third of both halves of a single leaf pair, resulted in a 59 and 106% increase in the number of EFNs on the plants, respectively, over 1 week. We suggest that the increased production of visually conspicuous EFNs is an adaptive inducible response, to attract predatory arthropods when risk of herbivory increases.