Biology, Ecology, and Management of Flea Beetles in Brassica Crops.

Brassica vegetable and oilseed crops are attacked by several different flea beetle species (Chrysomelidae: Alticini). Over the past decades, most research has focused on two Phyllotreta species, Phyllotreta striolata and Phyllotreta cruciferae, which are major pests of oilseed rape in North America. More recently, and especially after the ban of neonicotinoids in the European Union, the cabbage stem flea beetle, Psylliodes chrysocephala, has become greatly important and is now considered to be the major pest of winter oilseed rape in Europe. The major challenges to flea beetle control are the prediction of population dynamics in the field, differential susceptibility to insecticides, and the lack of resistant plant cultivars and other economically viable alternative management strategies. At the same time, many fundamental aspects of flea beetle biology and ecology, which may be relevant for the development of sustainable control strategies, are not well understood. This review focuses on the interactions between flea beetles and plants and summarizes the literature on current management strategies with an emphasis on the potential for biological control in flea beetle management.

A radiation of Psylliodes flea beetles on Brassicaceae is associated with the evolution of specific detoxification enzymes.

Flea beetles of the genus Psylliodes have evolved specialized interactions with plant species belonging to several distantly related families, mainly Brassicaceae, Solanaceae, and Fagaceae. This diverse host use indicates that Psylliodes flea beetles are able to cope with different chemical defense metabolites, including glucosinolates, the characteristic defense metabolites of Brassicaceae. Here we investigated the evolution of host use and the emergence of a glucosinolate-specific detoxification mechanism in Psylliodes flea beetles. In phylogenetic analyses, Psylliodes species clustered into four major clades, three of which contained mainly species specialized on either Brassicaceae, Solanaceae, or Fagaceae. Most members of the fourth clade have broader host use, including Brassicaceae and Poaceae as major host plant families. Ancestral state reconstructions suggest that Psylliodes flea beetles were initially associated with Brassicaceae and then either shifted to Solanaceae or Fagaceae, or expanded their host repertoire to Poaceae. Despite a putative ancestral association with Brassicaceae, we found evidence that the evolution of glucosinolate-specific detoxification enzymes coincides with the radiation of Psylliodes on Brassicaceae, suggesting that these are not required for using Brassicaceae as hosts but could improve the efficiency of host use by specialized Psylliodes species.

Different myrosinases activate sequestered glucosinolates in larvae and adults of the horseradish flea beetle.

ß-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous ß-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches.

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.

Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Rapid and Selective Absorption of Plant Defense Compounds From the Gut of a Sequestering Insect.

Many herbivorous insects exploit defense compounds produced by their host plants for protection against predators. Ingested plant defense compounds are absorbed via the gut epithelium and stored in the body, a physiological process that is currently not well understood. Here, we investigated the absorption of plant defense compounds from the gut in the horseradish flea beetle, Phyllotreta armoraciae, a specialist herbivore known to selectively sequester glucosinolates from its brassicaceous host plants. Feeding experiments using a mixture of glucosinolates and other glucosides not found in the host plants showed a rapid and selective uptake of glucosinolates in adult beetles. In addition, we provide evidence that this uptake mainly takes place in the foregut, whereas the endodermal midgut is the normal region of absorption. Absorption via the foregut epithelium is surprising as the apical membrane is covered by a chitinous intima. However, we could show that this cuticular layer differs in its structure and overall thickness between P. armoraciae and a non-sequestering leaf beetle. In P. armoraciae, we observed a thinner cuticle with a less dense chitinous matrix, which might facilitate glucosinolate absorption. Our results show that a selective and rapid uptake of glucosinolates from the anterior region of the gut contributes to the selective sequestration of glucosinolates in P. armoraciae.

Sequestration of Plant Defense Compounds by Insects: From Mechanisms to Insect-Plant Coevolution.

Plant defense compounds play a key role in the evolution of insect-plant associations by selecting for behavioral, morphological, and physiological insect adaptations. Sequestration, the ability of herbivorous insects to accumulate plant defense compounds to gain a fitness advantage, represents a complex syndrome of adaptations that has evolved in all major lineages of herbivorous insects and involves various classes of plant defense compounds. In this article, we review progress in understanding how insects selectively accumulate plant defense metabolites and how the evolution of specific resistance mechanisms to these defense compounds enables sequestration. These mechanistic considerations are further integrated into the concept of insect-plant coevolution. Comparative genome and transcriptome analyses, combined with approaches based on analytical chemistry that are centered in phylogenetic frameworks, will help to reveal adaptations underlying the sequestration syndrome, which is essential to understanding the influence of sequestration on insect-plant coevolution.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases.

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the ß-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle's energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

Sugar transporters enable a leaf beetle to accumulate plant defense compounds.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

Gut microbiota degrades toxic isothiocyanates in a flea beetle pest.

Microbial symbionts of herbivorous insects have been suggested to aid in the detoxification of plant defense compounds; however, quantitative studies on microbial contribution to plant toxin degradation remain scarce. Here, we demonstrate microbiome-mediated degradation of plant-derived toxic isothiocyanates in the cabbage stem flea beetle Psylliodes chrysocephala, a major pest of oilseed rape. Suppression of microbiota in antibiotic-fed beetles resulted in up to 11.3-fold higher levels of unmetabolized isothiocyanates compared to control beetles but did not affect other known detoxification pathways in P. chrysocephala. We characterized the microbiome of laboratory-reared and field-collected insects using 16S rRNA amplicon sequencing and isolated bacteria belonging to the three core genera Pantoea, Acinetobacter and Pseudomonas. Only Pantoea isolates rapidly degraded isothiocyanates in vitro, and restored isothiocyanate degradation in vivo when reintroduced in antibiotic-fed beetles. Pantoea was consistently present across beetle life stages and in field and lab populations. In addition, Pantoea was detected in undamaged tissues of the host plant Brassica rapa, indicating that P. chrysocephala could possibly acquire an isothiocyanate detoxifying bacterium through their diet. Our results demonstrate that both insect endogenous mechanisms and the microbiota can contribute to the detoxification of plant defense compounds and together they can better account for the fate of ingested plant metabolites.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle.

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

Identification and evolution of glucosinolate sulfatases in a specialist flea beetle.

Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. Several insect herbivores, including the cabbage stem flea beetle (Psylliodes chrysocephala), prevent glucosinolate activation by forming desulfo-glucosinolates. Here we investigated the molecular basis of glucosinolate desulfation in P. chrysocephala, an important pest of oilseed rape. Enzyme activity assays with crude beetle protein extracts revealed that glucosinolate sulfatase (GSS) activity is associated with the gut membrane and has narrow substrate specificity towards the benzenic glucosinolate sinalbin. In agreement with GSS activity localization in vivo, we identified six genes encoding arylsulfatase-like enzymes with a predicted C-terminal transmembrane domain, of which five showed GSS activity upon heterologous expression in insect cells. PcGSS1 and PcGSS2 used sinalbin and indol-3-ylmethyl glucosinolate as substrates, respectively, whereas PcGSS3, PcGSS4, and PcGSS5 showed weak activity in enzyme assays. RNAi-mediated knock-down of PcGSS1 and PcGSS2 expression in adult beetles confirmed their function in vivo. In a phylogenetic analysis of coleopteran and lepidopteran arylsulfatases, the P. chrysocephala GSSs formed a cluster within a coleopteran-specific sulfatase clade distant from the previously identified GSSs of the diamondback moth, Plutella xylostella, suggesting an independent evolution of GSS activity in ermine moths and flea beetles.

Adaptation of flea beetles to Brassicaceae: host plant associations and geographic distribution of Psylliodes Latreille and Phyllotreta Chevrolat (Coleoptera, Chrysomelidae).

The cosmopolitan flea beetle genera Phyllotreta and Psylliodes (Galerucinae, Alticini) are mainly associated with host plants in the family Brassicaceae and include economically important pests of crucifer crops. In this review, the host plant associations and geographical distributions of known species in these genera are summarised from the literature, and their proposed phylogenetic relationships to other Alticini analysed from published molecular phylogenetic studies of Galerucinae. Almost all Phyllotreta species are specialised on Brassicaceae and related plant families in the order Brassicales, whereas Psylliodes species are associated with host plants in approximately 24 different plant families, and 50% are specialised to feed on Brassicaceae. The current knowledge on how Phyllotreta and Psylliodes are adapted to the characteristic chemical defence in Brassicaceae is reviewed. Based on our findings we postulate that Phyllotreta and Psylliodes colonised Brassicaceae independently from each other.

Chemical convergence between plants and insects: biosynthetic origins and functions of common secondary metabolites.

Despite the phylogenetic distance between plants and insects, these two groups of organisms produce some secondary metabolites in common. Identical structures belonging to chemical classes such as the simple monoterpenes and sesquiterpenes, iridoid monoterpenes, cyanogenic glycosides, benzoic acid derivatives, benzoquinones and naphthoquinones are sometimes found in both plants and insects. In addition, very similar glucohydrolases involved in activating two-component defenses, such as glucosinolates and cyanogenic glycosides, occur in both plants and insects. Although this trend was first noted many years ago, researchers have long struggled to find convincing explanations for such co-occurrence. In some cases, identical compounds may be produced by plants to interfere with their function in insects. In others, plant and insect compounds may simply have parallel functions, probably in defense or attraction, and their co-occurrence is a coincidence. The biosynthetic origin of such co-occurring metabolites may be very different in insects as compared to plants. Plants and insects may have different pathways to the same metabolite, or similar sequences of intermediates, but different enzymes. Further knowledge of the ecological roles and biosynthetic pathways of secondary metabolites may shed more light on why plants and insects produce identical substances.

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the ß-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

Idesia polycarpa (Salicaceae) leaf constituents and their toxic effect on Cerura vinula and Lymantria dispar (Lepidoptera) larvae.

Phytochemical investigation of Idesia polycarpa (Salicaceae) resulted in the structure elucidation of nine previously undescribed phenolic natural products along with six known compounds. The compounds are structurally related to salicinoids that are known defense compounds from Salix and Populus species. The I. polycarpa diet was toxic, as shown in feeding experiments with larvae of Lymantria dispar, an herbivorous broadleaf tree generalist insect, and with larvae of Cerura vinula, a specialist adapted to poplar. The survival rate and mass gain of larvae was significantly lower when they fed on I. polycarpa leaves, compared to larvae fed on Populus nigra leaves. Potential reasons for the poor performance of both herbivores on I. polycarpa leaves are discussed.

Tissue-Specific Emission of (E)-α-Bergamotene Helps Resolve the Dilemma When Pollinators Are Also Herbivores.

More than 87% of flowering plant species are animal-pollinated [1] and produce floral scents and other signals to attract pollinators. These floral cues may however also attract antagonistic visitors, including herbivores [2]. The dilemma is exacerbated when adult insects pollinate the same plant that their larvae consume. It remains largely unclear how plants maximize their fitness under these circumstances. Here we show that in the night-flowering wild tobacco Nicotiana attenuata, the emission of a sesquiterpene, (E)-α-bergamotene, in flowers increases adult Manduca sexta moth-mediated pollination success, while the same compound in leaves is known to mediate indirect defense against M. sexta larvae [3, 4]. Forward and reverse genetic analyses demonstrated that both herbivory-induced and floral (E)-α-bergamotene are regulated by the expression of a monoterpene-synthase-derived sesquiterpene synthase (NaTPS38). The expression pattern of NaTPS38 also accounts for variation in (E)-α-bergamotene emission among natural accessions. These results highlight that differential expression of a single gene that results in tissue-specific emission of one compound contributes to resolving the dilemma for plants when their pollinators are also herbivores. Furthermore, this study provides genetic evidence that pollinators and herbivores interactively shape the evolution of floral signals and plant defense.

The Aggregation Pheromone of Phyllotreta striolata (Coleoptera: Chrysomelidae) Revisited.

Aggregations of the striped flea beetle Phyllotreta striolata on their crucifer host plants are mediated by volatiles emitted from feeding males. The male-specific sesquiterpene, (6R,7S)-himachala-9,11-diene (compound A), was shown previously to be physiologically and behaviorally active, but compound A was attractive only when combined with unnaturally high doses of the host plant volatile allyl isothiocyanate (AITC) in field trapping experiments. This indicated that our understanding of the chemical communication in this species is incomplete. Another male-specific sesquiterpenoid, (3S,9R,9aS)-3-hydroxy-3,5,5,9-tetramethyl-5,6,7,8,9,9a-hexahydro-1H-benzo[7]annulen-2(3H)-one (compound G), has been reported from an American P. striolata population. We confirmed the presence of compound G, and investigated its interaction with compound A and AITC in a P. striolata population in Taiwan. Compound G was attractive to Taiwanese P. striolata in laboratory bioassays, but significantly more beetles were attracted to a blend of compounds A and G. Under the same conditions, P. striolata showed no preference for the blend of A and G combined with a range of doses of AITC over the sesquiterpenoid blend alone. The sesquiterpenoid blend was tested further in field trapping experiments and attracted significantly more beetles than traps baited with compound A and ecologically relevant amounts of AITC. We conclude that A and G are components of the male-specific aggregation pheromone of P. striolata in Taiwan, and that the attractiveness of the pheromone is not reliant on the presence of AITC. Our results further indicate that the male-specific sesquiterpenoid blends differ qualitatively between the Taiwanese and American populations of P. striolata.

How does plant chemical diversity contribute to biodiversity at higher trophic levels?

Plants, perhaps Earth's most accomplished chemists, produce thousands of specialized metabolites having no direct role in cell division or growth. These phytochemicals vary by taxon, with many taxa producing characteristic substance classes; and within taxa, with individual variation in structural variety and production patterns. Observations of corresponding variation in herbivore metabolism, behavior, and diet breadth motivated the development of chemical ecology research. We discuss the importance of plant biodiversity in general and phytochemical diversity in particular for biodiversity and ecological interactions at higher trophic levels. We then provide an overview of the descriptive, molecular and analytical tools which allow modern biologists to investigate phytochemical diversity and its effects on higher trophic levels, from physiological mechanisms to ecological communities.