Symbiont-mediated evasion of plant defenses
Plants counter challenges from herbivores and pathogens through the production of noxious secondary metabolites; some are constitutively expressed (Wittstock and Gershenzon 2002) while others can be induced upon attack (Stotz et al. 2000). Enriched within leaves, plant toxins include metabolites as chemically diverse as alkaloids, cyanogenic glycosides, phenolics, terpenes, benzoxazinoids, and glucosinolates, among others (Fiirstenberg-Hagg, Zagrobelny, and Bak 2013). Their mode of action remains elusive in many cases, but these compounds are often involved in the disruption of gut membranes, hindering metabolism and preventing normal molecular signaling, ion and nutrient transport, as well as triggering the interruption of hormone-controlled physiological processes (Mithofer and Boland 2012). Thus, folivores are under a strong selective pressure to evolve strategies to overcome these compounds. While many of these adaptations are endogenously encoded in a folivore’s metabolic repertoire (Zhu-Salzman, Bi, and Liu 2005; Despres, David, and Gallet 2007), it is now evident that microbial symbionts play a central role in mediating host plant use by interfering with and degrading plant chemical defenses.
Contingent on the nature of the attack, plants induce different defense responses. Following microbial infection, plants activate salicylic acid (SA)-dependent defenses, whereas herbivory induces jasmonic acid (JA) synthesis. Often, these pathways negatively cross talk. Towards feeding on tomato plant (Solanaceae) leaves, the Colorado potato beetle (Leptinotarsa decemlineata) secretes bacteria-containing saliva into the wound. Detecting the threat as microbial instead of herbivorous, the plant mounts a defense response based on SA instead of JA. This deception benefits the insect, which avoids antiherbivore defenses and experiences higher larval growth (Chung et al. 2013). Among the several bacterial taxa present in the oral secretion, only three isolates. Pseudomonas, Enterobacter, and Stenotrophomonas, suppress JA-dependent defenses. In particular, among the different bacterial components in these three isolates, flagellin isolated from Pseudomonas, was identified as one of the effectors that downregulate JA synthesis (Chung et al. 2013). These strategies are conserved in other herbivorous insect lineages. For instance. Aster Yellows (AY) phytoplasmas, a plant pathogen, could be considered a facultative symbiont of its vector leafhopper Macrosteles quadrilineatus. AY excretes an effector protein (SAP11) that modulates host plant (Arabidopsis) defense responses, downregulating the production of JA-derived metabolites to the benefit of the insect, which experiences higher fecundity (Sugio et al. 2011).
While the aforementioned examples feature symbionts protecting their insect host against plant allelochemicals prior to their synthesis, most known examples involve protection against metabolites that have already been produced, either by symbiont-mediated avoidance or breakdown of these compounds. Upon herbivorous attack, soybean plants induce the production of protein inhibitors targeting folivore proteinases, consequently hindering insect digestion (Carlini and Grossi-de-Sa 2002). Despite this, the velvet-bean caterpillar, Anticarsia gemmatalis, represents a major pest of soybeans. Antibiotic treatment of these insects disrupts its gut bacterial community and results in lower caterpillar grow'th and survival, suggesting a beneficial role of microbes. Subsequent enzymatic assays demonstrate that proteolytic and lipolytic activities are significantly affected by symbiont loss (Visotto et al. 2009). Interestingly, some members of the gut microbiome are able to synthesize proteinases that are immune to soybean proteinase inhibitors (Pilon et al. 2013), suggesting that microbial proteases may serve as a secondary set of enzymes that mediate the insect host circumventing plant defenses.
The gypsy moth (Lymantria dispar), a generalist folivore and pest, is well known for population outbreaks that drastically alter forest ecosystems (Liebhold et al. 1994). Given its broad host plant range, L. dispar caterpillars tolerates an equally diverse assortment of plant secondary metabolites. The gypsy moth’s preferred host plant, the quaking aspen (Populus tremuloides), is rich in terpenes, phenolic glycosides (salicinoids), and tannins (Lindroth and St. Clair 2013). Earlier descriptions of L. dispar gut bacterial community found members of the Rhodococus genus; noted for their production of terpene-degrading enzymes (van der Vlugt-Bergmans and van der Werf 2001; Broderick et al. 2004). Salicinoids lower growth and development in gypsy moth caterpillars (Hemming and Lindroth 1995). While L. dispar has evolved some adaptations to overcome plant toxins, such as a highly alkaline midgut, and a battery of detoxification enzymes, these strategies can be overwhelmed by high concentrations of phenolic glycosides. However, gypsy moth larvae harbor gut bacterial communities that can degrade salicinoids, leading to increased larval growth following chemical challenges by the secondary metabolite (Mason. Couture, and Raffa 2014). As described in other lepidopterans, the gut microbiome composition in this species is largely shaped by the microbial community present in ingested leaves, whereas its structure is determined by insect physiology (Mason and Raffa 2014).
Accordingly, caterpillars harboring an aspen-derived community are better adapted to tolerate aspen-derived defenses than those harboring nonaspen associated bacteria (Mason, Couture, and Raffa 2014). It appears that the benefit of these gut symbionts to their insect hosts may be derived from their ability to defend themselves against plant secondary metabolites in their primary environment as leaf-associated microbes.
Among fungus-growing ants, the transition to folivory corresponded with the necessity to contend with a wide diversity of plant secondary metabolites (Berenbaum 1988). Although leaf cutting ants can exploit nearly 75% of all plants present in the New' World tropical forests, they avoid foraging on some species (Howard, Cazin, and Wiemer 1988), presumably those that contain toxins with detrimental effect to themselves and their symbiotic fungal partner (Seaman 1984). The metabolic repertoire of the fungal cultivar, which includes the ability to detoxify some plant toxins, may have been a key factor for leaf-cutting ants in becoming dominant folivores in the New' World. Towards degrading phenols, the fungal cultivar of attini ants produces several phenol-oxidizing enzymes of the laccase family. One of them, LgLccl is highly expressed in the gongylidia and is ingested by ant workers, surviving digestion before being defecated on top of the garden. This mechanism ensures that laccase activity is highest in the garden top layers where new' leaf material is being deposited and detoxification is most needed (De Fine Licht et al. 2013). Additionally, the presence of the laccase in the gut of foragers may aid in detoxifying phenolics ants may drink w'hen cutting and chewing leaves to manure the fungal garden. Analyses of orthologous genes encoding this phenol-oxidizing enzyme demonstrate that LgLccl has been selected for in gongylidia-producing fungal gardens. Consistent with this observation, laccase activity is highest in the nests of leaf-cutting ants than in their noncutting counterparts (De Fine Licht et al. 2013).
Microbial degradation of plant noxious compounds is not unique to folivorous insects. Folivorous vertebrates such as cows, sheep and rats, as well as birds, harbor bacterial communities w'ith the ability to degrade plant secondary metabolites (Garcfa-Amado et al. 2007; Kohl and Dearing 2012; Kohl et al. 2016). Similarly, other herbivorous insects besides folivores also outsource some of their adaptations against plant toxins to microbial symbionts (Barr et al. 2010; Boone et al. 2013; Hammerbacher et al. 2013; Ceja-Navarro et al. 2015; Welte et al. 2016; Berasategui et al. 2017). Thus, there is increasing evidence that symbiotic microbes can mediate host plant use through the manipulation, degradation and inactivation of plant defenses, and is possibly representative of a widespread occurance (Shen and Dowd 1990).
Dietary specialization carries the risk of temporal instability. This is most evident in the challenges faced by folivores in their coevolution with deciduous plants, where resource quality and accessibility are directly influenced by seasonality and abiotic conditions (Giron et al. 2007; Kaiser et al. 2010; Gutzwiller et al. 2015; Zhang et al. 2017). Despite a leafs inevitable developmental progression, from initiation to senescence (Bar and Ori 2014), numerous herbivorous lineages have nonetheless evolved strategies to stall that process to continue exploiting foliage nutritionally well beyond seasonal fall. Endophagous herbivores, such leaf miners and insect gallers, can achieve this by stalling morphogenesis and coopting plant architecture to generate structures that buffer against environmental change, protect from natural enemies, and, ultimately, extend the lifespan of a dietary niche. By coopting their host plant’s metabolism, endophagous herbivores can actively trigger the differentiation of their microhabitat to shape its chemical composition and dietary value in situ. Among leaf-miners, this process is mediated by the bacterial symbiont, Wolbachia.
Responsible for their spectacular induction of “green islands” on yellowing leaves during autumn, the leaf-miner moth Phyllonorycter (Lepidoptera: Gracillariidae) is able to preserve a photosynthetically active patch embedded within an otherwise decaying leaf (Giron et al. 2007). Defined by elevated cytokinins, these patches reflect the continued maintenance of chlorophyll and the inhibited progression of senescence. Infection by Wolbachia directly impacts the ability of Phyllonorycter to induce green islands, since symbiont loss corresponds with the absence of the phenotype in yellowing leaves (Kaiser et al. 2010). Responding to a deteriorating ecological niche, aposymbiotic insects exhibit compensatory feeding and higher levels of mortality relative to Phyllonorycter infected by Wolbachia (Kaiser et al. 2010). By mediating its host’s manipulation leaf tissues vis-a-vis cytokinin production, Wolbachia ensures that the insect’s access to sugar-rich, metabolically active habitat despite a rapidly decaying enviornment (Zhang et al. 2017). While symbiont-induced nutritional homeostasis is estimated to be widespread across the Gracillariidae, the identification of two separate Wolbachia strains suggests several independent origins of green-island induction within this insect family (Gutzwiller et al. 2015).
Symbiont acquisition and replacement are essential processes, coinciding with the integration of novel metabolic features and the ability to exploit previously inaccessible niches (Moran 2007). Among insects exploiting highly specialized diets, microbes serve as an important source of metabolites and supplements towards upgrading their hosts’ nutritional ecology. This is best documented in the evolutionary independent associations between sap-feeding hemipterans and nutritional endosymbionts that supplement the essential amino acids lacking in their diet (Bennett and Moran 2015). Similarly, convergent mutualistic factors are enriched in the primary endosymbionts of blood-feeding insects. Across ticks (Duron et al. 2018), bedbugs (Hosokawa et al. 2010), and tsetse flies (Akman et al. 2002), haematophagy is made possible through symbiont-encoded В vitamin contributions to balance a diet that is highly deficient in these cofactors. This chapter outlines that leaf feeding poses an inherently different set of challenges for obligate folivores. Challenges largely countered by animals engaging in stable symbioses with microbes that contribute towards the degradation of complex polymers, detoxification of noxious compounds, and niche preservation. While many of the examples highlighted here feature taxonomically diverse microbes, the range of services endowed to the insect host are nonetheless conserved. Given that conservation, and the experimental tractability of insect symbioses to pursue novel and emerging questions within the field of microbiome research, we emphasize the suitability of folivorous insects as dynamic study systems to illustrate the metaorganismal basis of adaptation in animals.