Enric Frago

Cytokinins as key regulators in plant–microbe–insect interactions: connecting plant growth and defence

Summary Plant hormones play important roles in regulating plant growth and defence by mediating developmental processes and signalling networks involved in plant responses to a wide range of parasitic and mutualistic biotic interactions. Plants are known to rapidly respond to pathogen and herbivore attack by reconfiguring their metabolism to reduce pathogen/herbivore food acquisition. This involves the production of defensive plant secondary compounds, but also an alteration of the plant primary metabolism to fuel the energetic requirements of the direct defence. Cytokinins are plant hormones that play a key role in plant morphology, plant defence, leaf senescence and source–sink relationships. They are involved in numerous plant–biotic interactions. These phytohormones may have been the target of arthropods and pathogens over the course of the evolutionary arms race between plants and their biotic partners to hijack the plant metabolism, control its physiology and/or morphology and successfully invade the plant. In the case of arthropods, cytokinin-induced phenotypes can be mediated by their bacterial symbionts, giving rise to intricate plant–microbe–insect interactions. Cytokinin-mediated effects strongly impact not only plant growth and defence but also the whole community of insect and pathogen species sharing the same plant by facilitating or preventing plant invasion. This suggests that cytokinins (CKs) are key regulators of the plant growth-defence trade-off and highlights the complexity of the finely balanced responses that plants use while facing both invaders and mutualists.

Phytohormone mediation of interactions between herbivores and plant pathogens.

Induced plant defenses against either pathogens or herbivore attackers are regulated by phytohormones. These phytohormones are increasingly recognized as important mediators of interactions between organisms associated with plants. In this review, we discuss the role of plant defense hormones in sequential tri-partite interactions among plants, pathogenic microbes, and herbivorous insects, based on the most recent literature. We discuss the importance of pathogen trophic strategy in the interaction with herbivores that exhibit different feeding modes. Plant resistance mechanisms also affect plant quality in future interactions with attackers. We discuss exemplary evidence for the hypotheses that (i) biotrophic pathogens can facilitate chewing herbivores, unless plants exhibit effector-triggered immunity, but (ii) facilitate or inhibit phloem feeders. (iii) Necrotrophic pathogens, on the other hand, can inhibit both phloem feeders and chewers. We also propose herbivore feeding mode as predictor of effects on pathogens of different trophic strategies, providing evidence for the hypotheses that (iv) phloem feeders inhibit pathogen attack by increasing SA induction, whereas (v) chewing herbivores tend not to affect necrotrophic pathogens, while they may either inhibit or facilitate biotrophic pathogens. Putting these hypotheses to the test will increase our understanding of phytohormonal regulation of plant defense to sequential attack by plant pathogens and insect herbivores. This will provide valuable insight into plant-mediated ecological interactions among members of the plant-associated community.

Early herbivore alert matters: plant-mediated effects of egg deposition on higher trophic levels benefit plant fitness.

Induction of plant defences, specifically in response to herbivore attack, can save costs that would otherwise be needed to maintain defences even in the absence of herbivores. However, plants may suffer considerable damage during the time required to mount these defences against an attacker. This could be resolved if plants could respond to early cues, such as egg deposition, that reliably indicate future herbivory. We tested this hypothesis in a field experiment and found that egg deposition by the butterfly Pieris brassicae on black mustard (Brassica nigra) induced a plant response that negatively affected feeding caterpillars. The effect cascaded up to the third and fourth trophic levels (larval parasitoids and hyperparasitoids) by affecting the parasitisation rate and parasitoid performance. Overall, the defences induced by egg deposition had a positive effect on plant seed production and may therefore play an important role in the evolution of plant resistance to herbivores.

Defensive insect symbiont leads to cascading extinctions and community collapse

Abstract Animals often engage in mutualistic associations with microorganisms that protect them from predation, parasitism or pathogen infection. Studies of these interactions in insects have mostly focussed on the direct effects of symbiont infection on natural enemies without studying community‐wide effects. Here, we explore the effect of a defensive symbiont on population dynamics and species extinctions in an experimental community composed of three aphid species and their associated specialist parasitoids. We found that introducing a bacterial symbiont with a protective (but not a non‐protective) phenotype into one aphid species led to it being able to escape from its natural enemy and increase in density. This changed the relative density of the three aphid species which resulted in the extinction of the two other parasitoid species. Our results show that defensive symbionts can cause extinction cascades in experimental communities and so may play a significant role in the stability of consumer‐herbivore communities in the field.

Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids

Many insect parasitoids are highly specialized and thus develop on only one or a few related host species, yet some hosts are attacked by many different parasitoid species in nature. For this reason, they have been often used to examine the consequences of competitive interactions. Hosts represent limited resources for larval parasitoid development and thus one competitor usually excludes all others. Although parasitoid competition has been debated and studied over the past several decades, understanding the factors that allow for coexistence among species sharing the same host in the field remains elusive. Parasitoids may be able to coexist on the same host species if they partition host resources according to size, age, or stage, or if their dynamics vary at spatial and temporal scales. One area that has thus far received little experimental attention is if competition can alter host usage strategies in parasitoids that in the absence of competitors attack hosts of the same size in the field. Here, we test this hypothesis with two parasitoid species in the genus Aphytis, both of which are specialized on the citrus pest California red scale Aonidiella aurantii. These parasitoids prefer large scales as hosts and yet coexist in sympatry in eastern parts of Spain. Parasitoids and hosts were sampled in 12 replicated orange groves. When host exploitation by the stronger competitor, A. melinus, was high the poorer competitor, A. chrysomphali, changed its foraging strategy to prefer alternative plant substrates where it parasitized hosts of smaller size. Consequently, the inferior parasitoid species shifted both its habitat and host size as a result of competition. Our results suggest that density-dependent size-mediated asymmetric competition is the likely mechanism allowing for the coexistence of these two species, and that the use of suboptimal (small) hosts can be advantageous under conditions imposed by competition where survival in higher quality larger hosts may be greatly reduced.

Symbionts protect aphids from parasitic wasps by attenuating herbivore-induced plant volatiles

Plants respond to insect attack by releasing blends of volatile chemicals that attract their herbivores’ specific natural enemies, while insect herbivores may carry endosymbiotic microorganisms that directly improve herbivore survival after natural enemy attack. Here we demonstrate that the two phenomena can be linked. Plants fed upon by pea aphids release volatiles that attract parasitic wasps, and the pea aphid can carry facultative endosymbiotic bacteria that prevent the development of the parasitic wasp larva and thus markedly improve aphid survival after wasp attack. We show that these endosymbionts also attenuate the systemic release of volatiles by plants after aphid attack, reducing parasitic wasp recruitment and increasing aphid fitness. Our results reveal a novel mechanism through which symbionts can benefit their hosts and emphasise the importance of considering the microbiome in understanding insect ecological interactions.Bacterial symbionts are increasingly known to influence behaviour and fitness in insects. Here, Frago et al. show that plants fed on by aphids with symbionts have altered volatile chemical profiles, leading to reduced parasitoid attack of aphids.

Interactions between parasitoids and higher order natural enemies: Intraguild predation and hyperparasitoids

Parasitoids kill and live at the expense of their hosts, but they also serve as food for intraguild predators and hyperparasitoids. Natural enemy diversity can thus challenge herbivore suppression by parasitoids, but this depends on the ecological niches of the species involved and their functional diversity. The spatial context is another important layer of complexity, particularly in areas with reduced habitat complexity and increased fragmentation. Parasitoids have evolved strategies to locate their host, but this can be affected by risk of intraguild predation or hyperparasitism. To better understand these interactions we need more long-term experiments and trophic-web studies. This will provide fundamental knowledge, improve pest control, and allow ecologists to better predict the impact of human activities on species extinctions.

Nonhost diversity and density reduce the strength of parasitoid-host interactions

Abstract The presence of nonprey or nonhosts is known to reduce the strength of consumer– resource interactions by increasing the consumers effort needed to find its resource. These interference effects can have a stabilizing effect on consumer–resource dynamics, but have also been invoked to explain parasitoid extinctions. To understand how nonhosts affect parasitoids, we manipulated the density and diversity of nonhost aphids using experimental host–parasitoid communities and tested how this affects parasitation efficiency of two aphid parasitoid species. To further study the behavioral response of parasitoids to nonhosts, we tested for changes in parasitoid time allocation in relation to their host‐finding strategies. The proportion of successful attacks (attack rate) in both parasitoid species was reduced by the presence of nonhosts. The parasitoid Aphidius megourae was strongly affected by increasing nonhost diversity with the attack rate dropping from 0.39 without nonhosts to 0.05 with high diversity of nonhosts, while Lysiphlebus fabarum responded less strongly, but in a more pronounced way to an increase in nonhost density. Our experiments further showed that increasing nonhost diversity caused host searching and attacking activity levels to fall in A. megourae, but not in L. fabarum, and that A. megourae changed its behavior after a period of time in the presence of nonhosts by increasing its time spent resting. This study shows that nonhost density and diversity in the environment are crucial determinants for the strength of consumer–resource interactions. Their impact upon a consumers efficiency strongly depends on its host/prey finding strategy as demonstrated by the different responses for the two parasitoid species. We discuss that these trait‐mediated indirect interactions between host and nonhost species are important for community stability, acting either stabilizing or destabilizing depending on the level of nonhost density or diversity present.

Role of Large Cabbage White butterfly male-derived compounds in elicitation of direct and indirect egg-killing defenses in the black mustard

To successfully exert defenses against herbivores and pathogens plants need to recognize reliable cues produced by their attackers. Up to now, few elicitors associated with herbivorous insects have been identified. We have previously shown that accessory reproductive gland secretions associated with eggs of Cabbage White butterflies (Pieris spp.) induce chemical changes in Brussels sprouts plants recruiting egg-killing parasitoids. Only secretions of mated female butterflies contain minute amounts of male-derived anti-aphrodisiac compounds that elicit this indirect plant defense. Here, we used the black mustard (Brassica nigra) to investigate how eggs of the Large Cabbage White butterfly (Pieris brassicae) induce, either an egg-killing direct [i.e., hypersensitive response (HR)-like necrosis] or indirect defense (i.e., oviposition-induced plant volatiles attracting Trichogramma egg parasitoids). Plants induced by P. brassicae egg-associated secretions expressed both traits and previous mating enhanced elicitation. Treatment with the anti-aphrodisiac compound of P. brassicae, benzyl cyanide (BC), induced stronger HR when compared to controls. Expression of the salicylic (SA) pathway- and HR-marker PATHOGENESIS-RELATED GENE1 was induced only in plants showing an HR-like necrosis. Trichogramma wasps were attracted to volatiles induced by secretion of mated P. brassicae females but application of BC did not elicit the parasitoid-attracting volatiles. We conclude that egg-associated secretions of Pieris butterflies contain specific elicitors of the different plant defense traits against eggs in Brassica plants. While in Brussels sprouts plants anti-aphrodisiac compounds in Pieris egg-associated secretions were clearly shown to elicit indirect defense, the wild relative B. nigra, recognizes different herbivore cues that mediate the defensive responses. These results add another level of specificity to the mechanisms by which plants recognize their attackers.

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow‐sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade‐offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.