Drastic Genome Reduction in an Herbivore's Pectinolytic Symbiont.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

You are what you eat-ecological niche and microhabitat influence venom activity and composition in aquatic bugs.

True water bugs (Nepomorpha) are mostly predacious insects that live in aquatic habitats. They use their piercing-sucking mouthparts to inject venomous saliva that facilitates the capture and extra-oral digestion of prey animals, but their venom can also be deployed for defence. In Central Europe, nepomorph species representing different families coexist in the same habitat. However, their feeding ecology, including venom composition and deployment, has not been investigated in detail. We used an integrated proteotranscriptomic and bioactivity-based approach to test whether venom composition and activity differ between four water bug species sharing the same habitat but occupying different ecological niches. We found considerable species-dependent differences in the composition of digestive enzymes and venom components that probably evolved as adaptations to particular food sources, foraging strategies and/or microhabitats. The venom of Corixa punctata differed substantially from that of the three strictly predatory species (Ilyocoris cimicoides, Notonecta glauca and Nepa cinerea), and the abundance of herbivory-associated proteins confirms a mostly plant-based diet. Our findings reveal independent adaptations of the digestive and defensive enzyme repertoires accompanied by the evolution of distinct feeding strategies in aquatic bugs.

Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline.

Herbivorous insects have evolved counteradaptations to overcome the chemical defences of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established, little is known about their microevolutionary dynamics in the field. In particular, it is not known whether and how host plant diversity shapes diversity in insect counteradaptations. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralogue major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defence system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. Both P. melete and P. napi revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defence.

The genomic basis of circadian and circalunar timing adaptations in a midge.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Genomic analysis of novel Yarrowia-like yeast symbionts associated with the carrion-feeding burying beetle Nicrophorus vespilloides.

BACKGROUND: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica. RESULTS: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations. CONCLUSIONS: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.

A Whole-Genome Scan for Association with Invasion Success in the Fruit Fly Drosophila suzukii Using Contrasts of Allele Frequencies Corrected for Population Structure.

Evidence is accumulating that evolutionary changes are not only common during biological invasions but may also contribute directly to invasion success. The genomic basis of such changes is still largely unexplored. Yet, understanding the genomic response to invasion may help to predict the conditions under which invasiveness can be enhanced or suppressed. Here, we characterized the genome response of the spotted wing drosophila Drosophila suzukii during the worldwide invasion of this pest insect species, by conducting a genome-wide association study to identify genes involved in adaptive processes during invasion. Genomic data from 22 population samples were analyzed to detect genetic variants associated with the status (invasive versus native) of the sampled populations based on a newly developed statistic, we called C2, that contrasts allele frequencies corrected for population structure. We evaluated this new statistical framework using simulated data sets and implemented it in an upgraded version of the program BayPass. We identified a relatively small set of single-nucleotide polymorphisms that show a highly significant association with the invasive status of D. suzukii populations. In particular, two genes, RhoGEF64C and cpo, contained single-nucleotide polymorphisms significantly associated with the invasive status in the two separate main invasion routes of D. suzukii. Our methodological approaches can be applied to any other invasive species, and more generally to any evolutionary model for species characterized by nonequilibrium demographic conditions for which binary covariables of interest can be defined at the population level.

Poplar protease inhibitor expression differs in an herbivore specific manner.

BACKGROUND: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. RESULTS: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. CONCLUSIONS: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Transcriptomics Reveal the Survival Strategies of Enterococcus mundtii in the Gut of Spodoptera littoralis.

The complex interaction between a higher organism and its resident gut flora is a subject of immense interest in the field of symbiosis. Many insects harbor a complex community of microorganisms in their gut. Larvae of Spodoptera littoralis, a lepidopteran pest, house a bacterial community that varies both spatially (along the length of the gut) and temporally (during the insect's life cycle). To monitor the rapid adaptation of microbes to conditions in the gut, a GFP-tagged reporter strain of E. mundtii, a major player in the gut community, was constructed. After early-instar S. littoralis larvae were fed with the tagged microbes, these were recovered from the larval fore- and hindgut by flow cytometry. The fluorescent reporter confirmed the persistence of E. mundtii in the gut. RNA-sequencing of the sorted bacteria highlighted various strategies of the symbiont's survival, including upregulated pathways for tolerating alkaline stress, forming biofilms and two-component signaling systems for quorum sensing, and resisting oxidative stress. Although these symbionts depend on the host for amino acid and fatty acids, differential regulation among various metabolic pathways points to an enriched lysine synthesis pathway of E. mundtii in the hindgut of the larvae.

Consumption of gossypol increases fatty acid-amino acid conjugates in the cotton pests Helicoverpa armigera and Heliothis virescens.

Gossypol is a toxic sesquiterpene dimer produced by cotton plants which deters herbivory by insects and vertebrates. Two highly reactive aldehyde groups contribute to gossypol toxicity by cross-linking herbivore proteins. We identified another consequence of consuming gossypol in two insect pests of cotton: increased amounts of fatty acid-amino acid conjugates (FACs). Eight different FACs in the feces of larval Helicoverpa armigera and Heliothis virescens increased when larvae consumed artificial diet containing gossypol, but not a gossypol derivative lacking free aldehyde groups (SB-gossypol). FACs are produced by joining plant-derived fatty acids with amino acids of insect origin in the larval midgut tissue by an unknown conjugase, and translocated into the gut lumen by an unknown transporter. FACs are hydrolyzed back into fatty acids and amino acids by an aminoacylase (L-ACY-1) in the gut lumen. The equilibrium level of FACs in the lumen is determined by a balance between conjugation and hydrolysis, which may differ among species. When heterologously expressed, L-ACY-1 of H. armigera but not H. virescens was inhibited by gossypol; consistent with the excretion of more FACs in the feces by H. armigera. FACs are known to benefit the plant host by inducing anti-herbivore defensive responses, and have been hypothesized to benefit the herbivore by acting as a surfactant and increasing nitrogen uptake efficiency. Thus in addition to its direct toxic effects, gossypol may negatively impact insect nitrogen uptake efficiency and amplify the signal used by the plant to elicit release of volatile compounds that attract parasitoids.

Microbiome-assisted carrion preservation aids larval development in a burying beetle.

The ability to feed on a wide range of diets has enabled insects to diversify and colonize specialized niches. Carrion, for example, is highly susceptible to microbial decomposers, but is kept palatable several days after an animal's death by carrion-feeding insects. Here we show that the burying beetle Nicrophorus vespilloides preserves carrion by preventing the microbial succession associated with carrion decomposition, thus ensuring a high-quality resource for their developing larvae. Beetle-tended carcasses showed no signs of degradation and hosted a microbial community containing the beetles' gut microbiota, including the yeast Yarrowia In contrast, untended carcasses showed visual and olfactory signs of putrefaction, and their microbial community consisted of endogenous and soil-originating microbial decomposers. This regulation of the carcass' bacterial and fungal community and transcriptomic profile was associated with lower concentrations of putrescine and cadaverine (toxic polyamines associated with carcass putrefaction) and altered levels of proteases, lipases, and free amino acids. Beetle-tended carcasses develop a biofilm-like matrix housing the yeast, which, when experimentally removed, leads to reduced larval growth. Thus, tended carcasses hosted a mutualistic microbial community that promotes optimal larval development, likely through symbiont-mediated extraintestinal digestion and detoxification of carrion nutrients. The adaptive preservation of carrion coordinated by the beetles and their symbionts demonstrates a specialized resource-management strategy through which insects modify their habitats to enhance fitness.

Symbiotic polydnavirus and venom reveal parasitoid to its hyperparasitoids.

Symbiotic relationships may provide organisms with key innovations that aid in the establishment of new niches. For example, during oviposition, some species of parasitoid wasps, whose larvae develop inside the bodies of other insects, inject polydnaviruses into their hosts. These symbiotic viruses disrupt host immune responses, allowing the parasitoid's progeny to survive. Here we show that symbiotic polydnaviruses also have a downside to the parasitoid's progeny by initiating a multitrophic chain of interactions that reveals the parasitoid larvae to their enemies. These enemies are hyperparasitoids that use the parasitoid progeny as host for their own offspring. We found that the virus and venom injected by the parasitoid during oviposition, but not the parasitoid progeny itself, affected hyperparasitoid attraction toward plant volatiles induced by feeding of parasitized caterpillars. We identified activity of virus-related genes in the caterpillar salivary gland. Moreover, the virus affected the activity of elicitors of salivary origin that induce plant responses to caterpillar feeding. The changes in caterpillar saliva were critical in inducing plant volatiles that are used by hyperparasitoids to locate parasitized caterpillars. Our results show that symbiotic organisms may be key drivers of multitrophic ecological interactions. We anticipate that this phenomenon is widespread in nature, because of the abundance of symbiotic microorganisms across trophic levels in ecological communities. Their role should be more prominently integrated in community ecology to understand organization of natural and managed ecosystems, as well as adaptations of individual organisms that are part of these communities.

An Integrated-Omics/Chemistry Approach Unravels Enzymatic and Spontaneous Steps to Form Flavoalkaloidal Nudicaulin Pigments in Flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

Functional insights from the GC-poor genomes of two aphid parasitoids, Aphidius ervi and Lysiphlebus fabarum.

BACKGROUND: Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts. RESULTS: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes. CONCLUSIONS: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.

An Insect Counteradaptation against Host Plant Defenses Evolved through Concerted Neofunctionalization.

Antagonistic chemical interactions between herbivorous insects and their host plants are often thought to coevolve in a stepwise process, with an evolutionary innovation on one side being countered by a corresponding advance on the other. Glucosinolate sulfatase (GSS) enzyme activity is essential for the Diamondback moth, Plutella xylostella, to overcome a highly diversified secondary metabolite-based host defense system in the Brassicales. GSS genes are located in an ancient cluster of arylsulfataselike genes, but the exact roles of gene copies and their evolutionary trajectories are unknown. Here, we combine a functional investigation of duplicated insect arylsulfatases with an analysis of associated nucleotide substitution patterns. We show that the Diamondback moth genome encodes three GSSs with distinct substrate spectra and distinct expression patterns in response to glucosinolates. Contrary to our expectations, early functional diversification of gene copies was not indicative of a coevolutionary arms race between host and herbivore. Instead, both copies of a duplicated arylsulfatase gene evolved concertedly in the context of an insect host shift to acquire novel detoxifying functions under positive selection, a pattern of duplicate gene retention that we call "concerted neofunctionalization."

Seasonal phenotype-specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana.

The European map butterfly (Araschnia levana) is a well-known example of seasonal polyphenism because the spring and summer imagoes exhibit distinct morphological phenotypes. The day length and temperature during larval and prepupal development determine whether spring or summer imagoes emerge after metamorphosis. Inspired by the fundamentally different transcriptomic profiles in prepupae developing from larvae exposed to long days or short days, we postulate that posttranscriptional epigenetic regulators such as microRNAs (miRNAs) may contribute to the epigenetic control of seasonal polyphenism in A. levana. To test this hypothesis, we used microarrays containing over 2,000 insect miRNAs to identify candidate regulators that are differentially expressed in last-instar larvae or pupae developing under long-day or short-day conditions. We used our transcriptomic database to identify potential 3'-untranslated regions of messenger RNAs to predict miRNA targets by considering both base pair complementarity and minimum free energy hybridization. This approach resulted in the identification of multiple targets of miRNAs that were differentially regulated in polyphenic morphs of A. levana including a candidate (miR-2856-3p) regulating the previously identified diapause bioclock protein gene. In conclusion, the expression profiling of miRNAs provided insights into their possible involvement in seasonal polyphenism of A. levana and offer an important resource for further studies.

Molecular signatures of selection associated with host plant differences in Pieris butterflies.

Adaptive traits that enable organisms to conquer novel niches and experience subsequent diversification are ecologically and evolutionarily important. The larvae of Pieris butterflies express nitrile-specifier proteins (NSPs), a key innovation for overcoming the glucosinolate (GLS)-myrosinase-based defence system of their Brassicales host plants. Nitrile-specifier proteins are a member of the NSP-like gene family, which includes the major allergen (MA) protein, a paralog of NSP with a GLS-disarming function, and a single domain major allergen (SDMA) protein, whose function is unknown. The arms-race between GLS-based defences and the NSP-like gene family is suggested to mediate diversification in both Pierid butterflies and Brassicales plants. Here, we tested whether the expected strong selection on NSP-like gene family correlates with shifts in host plant spectra among Pierid butterflies. We combined feeding experiments using 25 Brassicaceae plants and five Pieris species with larval transcriptome data to investigate the patterns of selection acting on NSP-like gene family members. Although we observed significantly elevated nonsynonymous to synonymous substitution rate ratios in NSPs on branches associated with changes in patterns of host plant usage, no such pattern was observed in MAs or SDMAs. Furthermore, we found evidence for positive selection of NSP at a phylogenetic branch which reflects different host plant spectra. Our data indicate that the NSP-related gene members have evolved differently: NSPs have accumulated more amino acid changes in response to shifting preferences for host plants, whereas MAs and SDMAs appear to be more conserved. Further detailed functional assays of these genes would provide important insights to understand their role in the chemical arms-race between Pieris butterflies and their Brassicales host plants.

A Latex Metabolite Benefits Plant Fitness under Root Herbivore Attack.

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major native insect root herbivore, the larvae of the common cockchafer (Melolontha melolontha), and benefit plant vegetative and reproductive fitness under M. melolontha attack. Across 17 T. officinale genotypes screened by gas and liquid chromatography, latex concentrations of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) were negatively associated with M. melolontha larval growth. Adding purified TA-G to artificial diet at ecologically relevant concentrations reduced larval feeding. Silencing the germacrene A synthase ToGAS1, an enzyme that was identified to catalyze the first committed step of TA-G biosynthesis, resulted in a 90% reduction of TA-G levels and a pronounced increase in M. melolontha feeding. Transgenic, TA-G-deficient lines were preferred by M. melolontha and suffered three times more root biomass reduction than control lines. In a common garden experiment involving over 2,000 T. officinale individuals belonging to 17 different genotypes, high TA-G concentrations were associated with the maintenance of high vegetative and reproductive fitness under M. melolontha attack. Taken together, our study demonstrates that a latex secondary metabolite benefits plants under herbivore attack, a result that provides a mechanistic framework for root herbivore driven natural selection and evolution of plant defenses below ground.

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.