Testing the joint effects of arbuscular mycorrhizal fungi and ants on insect herbivory on potato plants.

MAIN CONCLUSION: Ants, but not mycorrhizae, significantly affected insect leaf-chewing herbivory on potato plants. However, there was no evidence of mutualistic interactive effects on herbivory. Plants associate with both aboveground and belowground mutualists, two prominent examples being ants and arbuscular mycorrhizal fungi (AMF), respectively. While both of these mutualisms have been extensively studied, joint manipulations testing their independent and interactive (non-additive) effects on plants are rare. To address this gap, we conducted a joint test of ant and AMF effects on herbivory by leaf-chewing insects attacking potato (Solanum tuberosum) plants, and further measured plant traits likely mediating mutualist effects on herbivory. In a field experiment, we factorially manipulated the presence of AMF (two levels: control and mycorrhization) and ants (two levels: exclusion and presence) and quantified the concentration of leaf phenolic compounds acting as direct defenses, as well as plant volatile organic compound (VOC) emissions potentially mediating direct (e.g., herbivore repellents) or indirect (e.g., ant attractants) defense. Moreover, we measured ant abundance and performed a dual-choice greenhouse experiment testing for effects of VOC blends (mimicking those emitted by control vs. AMF-inoculated plants) on ant attraction as a mechanism for indirect defense. Ant presence significantly reduced herbivory whereas mycorrhization had no detectable influence on herbivory and mutualist effects operated independently. Plant trait measurements indicated that mycorrhization had no effect on leaf phenolics but significantly increased VOC emissions. However, mycorrhization did not affect ant abundance and there was no evidence of AMF effects on herbivory operating via ant-mediated defense. Consistently, the dual-choice assay showed no effect of AMF-induced volatile blends on ant attraction. Together, these results suggest that herbivory on potato plants responds mainly to top-down (ant-mediated) rather than bottom-up (AMF-mediated) control, an asymmetry in effects which could have precluded mutualist non-additive effects on herbivory. Further research on this, as well as other plant systems, is needed to examine the ecological contexts under which mutualist interactive effects are more or less likely to emerge and their impacts on plant fitness and associated communities.

Test of Specificity in Signalling between Potato Plants in Response to Infection by Fusarium Solani and Phytophthora Infestans.

Plant-plant signalling via volatile organic compounds (VOCs) in response to insect herbivory has been widely studied, but its occurrence and specificity in response to pathogen attack has received much less attention. To fill this gap, we carried out a greenhouse experiment using two fungal pathogens (Fusarium solani and Phytophthora infestans) to test for specificity in VOC induction and signalling between potato plants (Solanum tuberosum). We paired potato plants in plastic cages, one acting as VOC emitter and the other as receiver, and subjected emitters to one of the following treatments: no infection (control), infected by F. solani, or infected by P. infestans. We measured total emission and composition of VOCs released by emitter plants to test for pathogen-specificity in VOC induction, and then conducted a pathogen infection bioassay to assess resistance levels on receiver plants by subjecting half of the receivers of each emitter treatment to F. solani infection and the other half to P. infestans infection. This allowed us to test for specificity in plant VOC signalling by comparing its effects on conspecific and heterospecific sequential infections. Results showed that infection by neither F. solani or P. infestans produced quantitative (total emissions) or qualitative (compositional) changes in VOC emissions. Mirroring these patterns, emitter infection treatment (control vs. pathogen infection) did not produce a significant change in pathogen infection levels on receiver plants in any case (i.e., either for conspecific or heterospecific sequential infections), indicating a lack of signalling effects which precluded pathogen-based specificity in signalling. We discuss possible mechanisms for lack of pathogen effects on VOC emissions and call for future work testing for pathogen specificity in plant-plant signalling and its implications for plant-pathogen interactions under ecologically relevant scenarios involving infections by multiple pathogens.

Volatile-Mediated Signalling Between Potato Plants in Response to Insect Herbivory is not Contingent on Soil Nutrients.

Plant-plant signalling via volatile organic compounds (VOCs) has been studied intensively, but its contingency on abiotic conditions (e.g., soil nutrients, drought, warming) is poorly understood. To address this gap, we carried out a greenhouse experiment testing whether soil nutrients influenced signalling between potato (Solanum tuberosum) plants in response to insect leaf herbivory by the generalist caterpillar Spodoptera exigua. We placed pairs of plants in plastic cages, where one plant acted as a VOC emitter and the other as a receiver. We factorially manipulated soil nutrients for both emitter and receiver plants, namely: unfertilized (baseline soil nutrients) vs. fertilized (augmented nutrients). Then, to test for signalling effects, half of the emitters within each fertilization level were damaged by S. exigua larvae and the other half remained undamaged. Three days after placing larvae, we collected VOCs from emitter plants to test for herbivory and fertilization effects on VOC emissions and placed S. exigua larvae on receivers to test for signalling effects on leaf consumption and larval mass gain as proxies of induced resistance. We found that herbivory increased total VOC emissions and altered VOC composition by emitter plants, but these effects were not contingent on fertilization. In addition, bioassay results showed that receivers exposed to VOCs from herbivore-damaged emitters had lower levels of herbivory compared to receivers exposed to undamaged emitters. However, and consistent with VOC results, fertilization did not influence herbivore-induced signalling effects on receiver resistance to herbivory. In sum, we found evidence of S. exigua-induced signalling effects on resistance to herbivory in potato plants but such effects were not affected by increased soil nutrients. These results call for further work testing signalling effects under broader range of nutrient concentration levels (including nutrient limitation), teasing apart the effects of specific nutrients, and incorporating other abiotic factors likely to interact or covary with soil nutrients.

Effect of herbivore load on VOC-mediated plant communication in potato.

MAIN CONCLUSION: VOC emissions increased with herbivore load, but this did not result in concomitant increases in resistance in neighbouring plants, suggesting that communication occurred independently of herbivore load in emitter plants. Herbivore-damaged plants emit volatile organic compounds (VOCs) that can alert neighbours and boost their resistance. While VOC-mediated plant communication has been shown to be herbivore-specific, we know little about its contingency on variation in herbivore load. To address this knowledge gap, we tested herbivore load effects on VOC-mediated communication between potato plants (Solanum tuberosum) using the generalist herbivore Spodoptera exigua. First, we tested whether herbivore load (three levels: undamaged control, low, and high load) affected total VOC emissions and composition. Second, we matched emitter and receiver plants and subjected emitters to the same herbivore load treatments. Finally, we performed a bioassay with S. exigua on receivers to test for induced resistance due to VOC-mediated communication. We found that herbivory significantly increased total VOC emissions relative to control plants, and that such increase was greater under high herbivore load. In contrast, we found no detectable effect of herbivory, regardless of the load, on VOC composition. The communication experiment showed that VOCs released by herbivore-induced emitters boosted resistance in receivers (i.e., lower leaf damage than receivers exposed to VOCs released by control emitters), but the magnitude of such effect was similar for both levels of emitter herbivore load. These findings suggest that changes in VOCs due to variation in herbivore load do not modify the outcomes of plant communication.

Plant genetic relatedness and volatile-mediated signalling between Solanum tuberosum plants in response to herbivory by Spodoptera exigua.

It has been proposed that plant-plant signalling via herbivore-induced volatile organic compounds (VOCs) should be stronger between closely related than unrelated plants. However, empirical tests remain limited and few studies have provided detailed assessments of induced changes in VOCs emissions across plant genotypes to explain genetic relatedness effects. In this study, we tested whether airborne signalling in response to herbivory between Solanum tuberosum (potato) plants was contingent on plant genetic relatedness, and further investigated genotypic variation in VOCs potentially underlying signalling and its contingency on relatedness. We carried out a greenhouse experiment using 15 S. tuberosum varieties placing pairs of plants in plastic cages, i.e. an emitter and a receiver, where both plants were of the same genotype or different genotype thereby testing for self-recognition, an elemental form genetic relatedness effects. Then, for half of the cages within each level of relatedness the emitter plant was damaged by Spodoptera exigua larvae whereas for the other half the emitter was not damaged. Three days later, we placed S. exigua larvae on receivers to test for emitter VOC effects on leaf consumption and larval weight gain (i.e. induced resistance). In addition, we used a second group of plants subjected to the same induction treatment with the same S. tuberosum varieties to test for herbivore-induced changes in VOC emissions and variation in VOC emissions among these plant genotypes. We found that herbivory drove changes in VOC composition but not total emissions, and also observed quantitative and qualitative variation in constitutive and induced VOC emissions among varieties. Results from the bioassay showed that the amount of leaf area consumed and larval weight gain on receiver plants exposed to damaged emitters were significantly lower compared to mean values on receivers exposed to control emitters. However, and despite genotypic variation in induced VOCs, this signalling effect was not contingent on plant genetic relatedness. These findings provide evidence of VOCs-mediated signalling between S. tuberosum plants in response to S. exigua damage, but no evidence of self-recognition effects in signalling contingent on variation in VOC emissions among S. tuberosum varieties.

Early land plants: Plentiful but neglected nutritional resources for herbivores?

Plants and herbivores have been engaged in a co-evolutionary arms race for millions of years, during which plants evolved various defenses and other traits to cope with herbivores, whereas herbivores evolved traits to overcome the plants' resistance strategies. Herbivores may also avoid certain plants merely because these lack suitable nutrients for their development. Interestingly, the number of herbivores that attack individual early land plants like mosses and ferns is quite low. Among others, poor nutrient quality has been hypothesized to explain the apparent low herbivory pressure on such plants but still waits for scientific evidences. Here, the nutritive suitability of representative mosses and liverworts (bryophytes) and ferns (pteridophytes) for herbivores was investigated using feeding assays combined with quantifications of nutrients (proteins, amino acids, and sugars). Growth and survival of two polyphagous herbivores, a caterpillar and a snail, were monitored when fed on 15 species of bryophytes and pteridophytes, as well as on maize (Zea mays, angiosperm) used as an external indicative nutritional resource. Overall, our results show that the poor performance of the herbivores on the studied early land plants is not correlated with nutritional quality. The growth and performance of snails and caterpillars fed with these plants were highly variable and independent of nutrient content. These findings arguably dismiss the poor nutrient quality hypothesis as the cause of herbivory deficit in bryophytes and pteridophytes. They suggest the possible presence of early resistance traits that have persisted all through the long evolutionary history of plant-herbivore interactions.

Bioturbation by endogeic earthworms facilitates entomopathogenic nematode movement toward herbivore-damaged maize roots.

Entomopathogenic nematodes (EPNs) have been extensively studied as potential biological control agents against root-feeding crop pests. Maize roots under rootworm attack have been shown to release volatile organic compounds, such as (E)-ß-caryophyllene (Eßc) that guide EPNs toward the damaging larvae. As yet, it is unknown how belowground ecosystems engineers, such as earthworms, affect the biological control capacity of EPNs by altering the root Eßc-mediated tritrophic interactions. We here asked whether and how, the presence of endogeic earthworms affects the ability of EPNs to find root-feeding larvae of the beetle Diabrotica balteata. First, we performed a field mesocosm experiment with two diverse cropping systems, and revealed that the presence of earthworms increased the EPN infection potential of larvae near maize roots. Subsequently, using climate-controlled, olfactometer-based bioassays, we confirmed that EPNs response to Eßc alone (released from dispensers) was two-fold higher in earthworm-worked soil than in earthworm-free soil. Together our results indicate that endogeic earthworms, through burrowing and casting activities, not only change soil properties in a way that improves soil fertility but may also enhance the biocontrol potential of EPNs against root feeding pests. For an ecologically-sound pest reduction in crop fields, we advocate agricultural practices that favour earthworm community structure and diversity.

The smell of hunger: Norway rats provision social partners based on odour cues of need.

When individuals exchange helpful acts reciprocally, increasing the benefit of the receiver can enhance its propensity to return a favour, as pay-offs are typically correlated in iterated interactions. Therefore, reciprocally cooperating animals should consider the relative benefit for the receiver when deciding to help a conspecific. Norway rats (Rattus norvegicus) exchange food reciprocally and thereby take into account both the cost of helping and the potential benefit to the receiver. By using a variant of the sequential iterated prisoner's dilemma paradigm, we show that rats may determine the need of another individual by olfactory cues alone. In an experimental food-exchange task, test subjects were provided with odour cues from hungry or satiated conspecifics located in a different room. Our results show that wild-type Norway rats provide help to a stooge quicker when they receive odour cues from a hungry rather than from a satiated conspecific. Using chemical analysis by gas chromatography-mass spectrometry (GC-MS), we identify seven volatile organic compounds that differ in their abundance between hungry and satiated rats. Combined, this "smell of hunger" can apparently serve as a reliable cue of need in reciprocal cooperation, which supports the hypothesis of honest signalling.

The Combined Use of an Attractive and a Repellent Sex Pheromonal Component by a Gregarious Parasitoid.

Gregarious parasitoids usually clump their cocoons together and the adults emerge in a synchronized fashion. This makes it easy for them to find mating partners and most copulations indeed take place at the natal patch. Yet, males should leave such sites when females are no longer receptive. As yet, this decision-making process and the possible involvement of pheromones were poorly understood. Here we report on a remarkable use of attractive and repellent pheromones of the well-studied gregarious parasitoid species Cotesia glomerata (L.) (Hymenoptera: Braconidae). Virgin C. glomerata females were found to release an attractive as well as a repellent compound, which in combination arrest males on the natal patch, but after mating the females stop the production of the attractant and the males are repelled. The repellent compound was identified as heptanal, which was also released by males, probably reducing male-male competition on the natal patch. We also confirmed that the sex ratio of the emerging wasps can vary considerably among patches, depending on the relative quality of hosts and the number of females that parasitize a host. The newly revealed use of attractive and repellent pheromone compounds by C. glomerata possibly helps maximize mating success under these variable conditions.

Environmental gradients and the evolution of tri-trophic interactions.

Long-standing theory predicts herbivores and predators should drive selection for increased plant defences, such as the specific production of volatile organic compounds for attracting predators near the site of damage. Along elevation gradients, a general pattern is that herbivores and predators are abundant at low elevation and progressively diminish at higher elevations. To determine whether plant adaptation along such a gradient influences top-down control of herbivores, we manipulated soil predatory nematodes, root herbivore pressure and plant ecotypes in a reciprocal transplant experiment. Plant survival was significantly higher for low-elevation plants, but only when in the presence of predatory nematodes. Using olfactometer bioassays, we showed correlated differential nematode attraction and plant ecotype-specific variation in volatile production. This study not only provides an assessment of how elevation gradients modulate the strength of trophic cascades, but also demonstrates how habitat specialisation drives variation in the expression of indirect plant defences.

Correction to: Root-colonizing bacteria enhance the levels of (E)-ß-caryophyllene produced by maize roots in response to rootworm feeding.

Unfortunately, family name of author "Xavier Chiriboga M" was incorrectly identified in the original publication and the same is corrected here. The original article has been corrected.

Root-colonizing bacteria enhance the levels of (E)-ß-caryophyllene produced by maize roots in response to rootworm feeding.

When larvae of rootworms feed on maize roots they induce the emission of the sesquiterpene (E)-ß-caryophyllene (EßC). EßC is attractive to entomopathogenic nematodes, which parasitize and rapidly kill the larvae, thereby protecting the roots from further damage. Certain root-colonizing bacteria of the genus Pseudomonas also benefit plants by promoting growth, suppressing pathogens or inducing systemic resistance (ISR), and some strains also have insecticidal activity. It remains unknown how these bacteria influence the emissions of root volatiles. In this study, we evaluated how colonization by the growth-promoting and insecticidal bacteria Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 affects the production of EßC upon feeding by larvae of the banded cucumber beetle, Diabrotica balteata Le Conte (Coleoptera: Chrysomelidae). Using chemical analysis and gene expression measurements, we found that EßC production and the expression of the EßC synthase gene (tps23) were enhanced in Pseudomonas protegens CHA0-colonized roots after 72 h of D. balteata feeding. Undamaged roots colonized by Pseudomonas spp. showed no measurable increase in EßC production, but a slight increase in tps23 expression. Pseudomonas colonization did not affect root biomass, but larvae that fed on roots colonized by P. protegens CHA0 tended to gain more weight than larvae that fed on roots colonized by P. chlororaphis PCL1391. Larvae mortality on Pseudomonas spp. colonized roots was slightly, but not significantly higher than on non-colonized control roots. The observed enhanced production of EßC upon Pseudomonas protegens CHA0 colonization may enhance the roots' attractiveness to entomopathogenic nematodes, but this remains to be tested.

Chicks of the great spotted cuckoo may turn brood parasitism into mutualism by producing a foul-smelling secretion that repels predators.

The great spotted cuckoo (Clamator glandarius) is an important brood parasite of carrion crows (Corvus corone corone) in northern Spain. We recently found that, unlike what is commonly known for cuckoo-host interactions, the great spotted cuckoo has no negative impact on average crow fitness in this region. The explanation for this surprising effect is a repulsive secretion that the cuckoo chicks produce when they are harassed and that may protect the brood against predation. Here, we provide details on the chemical composition of the cuckoo secretion, as well as conclusive evidence that the dominating volatile chemicals in the secretion are highly repellent to model species representative of common predators of the crows. These results support the notion that, in this particular system, the production of a repulsive secretion by the cuckoo chicks has turned a normally parasitic interaction into a mutualistic one.

From parasitism to mutualism: unexpected interactions between a cuckoo and its host.

Avian brood parasites lay eggs in the nests of other birds, which raise the unrelated chicks and typically suffer partial or complete loss of their own brood. However, carrion crows Corvus corone corone can benefit from parasitism by the great spotted cuckoo Clamator glandarius. Parasitized nests have lower rates of predation-induced failure due to production of a repellent secretion by cuckoo chicks, but among nests that are successful, those with cuckoo chicks fledge fewer crows. The outcome of these counterbalancing effects fluctuates between parasitism and mutualism each season, depending on the intensity of predation pressure.

Do induced responses mediate the ecological interactions between the specialist herbivores and phytopathogens of an alpine plant?

Plants are not passive victims of the myriad attackers that rely on them for nutrition. They have a suite of physical and chemical defences, and are even able to take advantage of the enemies of their enemies. These strategies are often only deployed upon attack, so may lead to indirect interactions between herbivores and phytopathogens. In this study we test for induced responses in wild populations of an alpine plant (Adenostyles alliariae) that possesses constitutive chemical defence (pyrrolizidine alkaloids) and specialist natural enemies (two species of leaf beetle, Oreina elongata and Oreina cacaliae, and the phytopathogenic rust Uromyces cacaliae). Plants were induced in the field using chemical elicitors of the jasmonic acid (JA) and salicylic acid (SA) pathways and monitored for one month under natural conditions. There was evidence for induced resistance, with lower probability and later incidence of attack by beetles in JA-induced plants and of rust infection in SA-induced plants. We also demonstrate ecological cross-effects, with reduced fungal attack following JA-induction, and a cost of SA-induction arising from increased beetle attack. As a result, there is the potential for negative indirect effects of the beetles on the rust, while in the field the positive indirect effect of the rust on the beetles appears to be over-ridden by direct effects on plant nutritional quality. Such interactions resulting from induced susceptibility and resistance must be considered if we are to exploit plant defences for crop protection using hormone elicitors or constitutive expression. More generally, the fact that induced defences are even found in species that possess constitutively-expressed chemical defence suggests that they may be ubiquitous in higher plants.

Synergies and trade-offs between insect and pathogen resistance in maize leaves and roots.

Determining links between plant defence strategies is important to understand plant evolution and to optimize crop breeding strategies. Although several examples of synergies and trade-offs between defence traits are known for plants that are under attack by multiple organisms, few studies have attempted to measure correlations of defensive strategies using specific single attackers. Such links are hard to detect in natural populations because they are inherently confounded by the evolutionary history of different ecotypes. We therefore used a range of 20 maize inbred lines with considerable differences in resistance traits to determine if correlations exist between leaf and root resistance against pathogens and insects. Aboveground resistance against insects was positively correlated with the plant's capacity to produce volatiles in response to insect attack. Resistance to herbivores and resistance to a pathogen, on the other hand, were negatively correlated. Our results also give first insights into the intraspecific variability of root volatiles release in maize and its positive correlation with leaf volatile production. We show that the breeding history of the different genotypes (dent versus flint) has influenced several defensive parameters. Taken together, our study demonstrates the importance of genetically determined synergies and trade-offs for plant resistance against insects and pathogens.

Counter-intuitive developmental plasticity induced by host quality.

Adaptation to different hosts plays a central role in the evolution of specialization and speciation in phytophagous insects and parasites, and our ability to experimentally rank hosts by their quality is critical to research to understand these processes. Here we provide a counter-intuitive example in which growth is faster on poor quality hosts. The leaf beetles Oreina elongata and Oreina cacaliae share their host plant with the rust Uromyces cacaliae. Larvae reared on infected Adenostyles alliariae show reduced growth rate, reduced maximum weight and longer development time. However, they normally respond adaptively to the rust's mid-season arrival. When switched during development from healthy to infected leaves, larvae accelerate growth and reduce development time, but pupate at lower body weight. In this novel plant-insect-fungus interaction, infection forms the cue to trade off life-history traits in order to complete development within the brief alpine summer. It represents a novel mode of developmental plasticity, which is likely to be found in other host-parasite systems whenever host quality deteriorates due to multiple infection or ageing. This phenotypic plasticity would modify competition after co-infection and the mutual selection imposed by hosts and parasites, and creates a paradoxical negative correlation between growth rate and environmental quality.