Longhorned beetles (Cerambycidae) are the most diverse group of predominantly wood‐feeding (xylophagous) insects on Earth. Larvae of most species feed within tissues of plants made up of large amounts of plant cell wall (PCW), which is notoriously difficult to digest. To efficiently access nutrients from their food source, cerambycid larvae have to deconstruct PCW polysaccharides – such as cellulose, hemicelluloses and pectin – requiring them to possess a diversity of plant cell wall degrading enzymes (PCWDEs) in their digestive tract. Genomic data for Cerambycidae are mostly limited to notorious forest pests and are lacking for most of the taxonomic groups. Consequently, our understanding of the distribution and evolution of cerambycid PCWDEs is quite limited. We addressed the numbers, kinds and evolution of cerambycid PCWDEs by surveying larval midgut transcriptomes from 23 species representing six of the eight recognized subfamilies of Cerambycidae and each with very diverse host types (i.e., gymnosperms, angiosperms, xylem, phloem, fresh or dead plant tissues). Using these data, we identified 340 new putative PCWDEs belonging to ten carbohydrate active enzyme families, including two gene families (GH7 and GH53) not previously reported from insects. The remarkably wide range of PCWDEs expressed by Cerambycidae should allow them to break down most PCW polysaccharides. Moreover, the observed distribution of PCWDEs encoded in cerambycid genomes agreed more with phylogenetic relationship of the species studied than with the taxonomic origin or quality of the host plant tissues.

Specialist herbivores have often evolved highly sophisticated mechanisms to counteract defenses mediated by major plant secondary-metabolites. Plant species of the herbivore host range often display high chemical diversity and it is not well understood how specialist herbivores respond to this chemical diversity. Pieris larvae overcome toxic products from glucosinolate hydrolysis, the major chemical defense of their Brassicaceae hosts, by expressing nitrile-specifier proteins (NSP) in their gut. Furthermore, Pieris butterflies possess so-called major allergen (MA) proteins, which are multi-domain variants of a single domain major allergen (SDMA) protein expressed in the guts of Lepidopteran larvae. Here we show that Pieris larvae fine-tune NSP and MA gene expression depending on the glucosinolate profiles of their Brassicaceae hosts. Although the role of MA is not yet fully understood, the expression levels of NSP and MA in larvae that fed on plants whose glucosinolate composition varied was dramatically changed, whereas levels of SDMA expression remained unchanged. In addition, we found a similar regulation pattern among these genes in larvae feeding on Arabidopsis mutants with different glucosinolate profiles. Our results demonstrate that Pieris larvae appear to use different host plant adaptive genes to overcome a wide range of glucosinolate profiles in their host plants.

Plants defend themselves against herbivores not only by a single trait but also by diversified multiple defense strategies. It remains unclear how these multiple defense mechanisms are effectively organized against herbivores. In this study, we focused on Brassicaceae plants, which have one of the most diversified secondary metabolites, glucosinolates (GSLs), as a defense against herbivores. By analyzing various defense traits including GSL profiles among 12 species (11 genera) of Brassicaceae plants, it is revealed that their defense strategies can be divided into three categories as multiple defenses. The GSL profiles differed between these three categories: (i) high nutritional level with long-chain aliphatic GSLs; (ii) low nutritional level and high physical defenses with short-chain aliphatic GSLs; and (iii) high nutritional level and low defense. The feeding experiment was conducted using two types of herbivores, Pieris rapae (Lepidoptera: Pieridae) as a specialist herbivore and the Eri silkmoth Samia cynthia ricini (Lepidoptera: Saturniidae) as a generalist, to assess the ability of each plant in multiple defense strategy. It was observed that the Eri silkmoth's performance differed according to which defense strategy it was exposed to. However, the growth rate of P. rapae did not vary among the three categories of defense strategy. These results suggest that the diversified defense strategies of Brassicaceae species have evolved to cope with diversified herbivores.

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. K E Y W O R D Sarms-race, host plant adaptation, insects, selection | 4959 OKAMURA et Al.

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.