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.

Genomic evidence for the widespread presence of GH45 cellulases among soil invertebrates.

Lignocellulose is a major component of vascular plant biomass. Its decomposition is crucial for the terrestrial carbon cycle. Microorganisms are considered primary decomposers, but evidence increases that some invertebrates may also decompose lignocellulose. We investigated the taxonomic distribution and evolutionary origins of GH45 hydrolases, important enzymes for the decomposition of cellulose and hemicellulose, in a collection of soil invertebrate genomes. We found that these genes are common in springtails and oribatid mites. Phylogenetic analysis revealed that cellulase genes were acquired early in the evolutionary history of these groups. Domain architectures and predicted 3D enzyme structures indicate that these cellulases are functional. Patterns of presence and absence of these genes across different lineages prompt further investigation into their evolutionary and ecological benefits. The ubiquity of cellulase genes suggests that soil invertebrates may play a role in lignocellulose decomposition, independently or in synergy with microorganisms. Understanding the ecological and evolutionary implications might be crucial for understanding soil food webs and the carbon cycle.

Duplication of horizontally acquired GH5_2 enzymes played a central role in the evolution of longhorned beetles.

The rise of functional diversity through gene duplication contributed to the adaption of organisms to various environments. Here we investigate the evolution of putative cellulases of the subfamily 2 of glycoside hydrolase family 5 (GH5_2) in the Cerambycidae (longhorned beetles), a megadiverse assemblage of mostly xylophagous beetles. Cerambycidae originally acquired GH5_2 from a bacterial donor through horizontal gene transfer (HGT), and extant species harbor multiple copies that arose from gene duplication. We ask how these digestive enzymes contributed to the ability of these beetles to feed on wood. We analyzed 113 GH5_2, including the functional characterization of 52 of them, derived from 25 species covering most subfamilies of Cerambycidae. Ancestral gene duplications led to five well-defined groups with distinct substrate specificity, allowing these beetles to break down, in addition to cellulose, polysaccharides that are abundant in plant cell walls (PCWs), namely, xyloglucan, xylan, and mannans. Resurrecting the ancestral enzyme originally acquired by HGT, we show it was a cellulase that was able to break down glucomannan and xylan. Finally, recent gene duplications further expanded the catalytic repertoire of cerambycid GH5_2, giving rise to enzymes that favor transglycosylation over hydrolysis. We suggest that HGT and gene duplication, which shaped the evolution of GH5_2, played a central role in the ability of cerambycid beetles to use a PCW-rich diet and may have contributed to their successful radiation.

Genome sequencing provides insights into the evolution of gene families encoding plant cell wall-degrading enzymes in longhorned beetles.

With more than 36,000 species, the longhorned beetles (family Cerambycidae) are a mega-diverse lineage of mostly xylophagous insects, all of which are represented by the sole sequenced genome of the Asian longhorned beetle (Anoplophora glabripennis; Lamiinae). Their successful radiation has been linked to their ability to degrade plant cell wall components using a range of so-called plant cell wall-degrading enzymes (PCWDEs). Our previous analysis of larval gut transcriptomes demonstrated that cerambycid beetles horizontally acquired genes encoding PCWDEs from various microbial donors; these genes evolved through multiple duplication events to form gene families. To gain further insights into the evolution of these gene families during the Cerambycidae radiation, we assembled draft genomes for four beetle species belonging to three subfamilies using long-read nanopore sequencing. All the PCWDE-encoding genes we annotated from the corresponding larval gut transcriptomes were present in these draft genomes. We confirmed that the newly discovered horizontally acquired glycoside hydrolase family 7 (GH7), subfamily 26 of GH43 (GH43_26), and GH53 (all of which are absent from the A. glabripennis genome) were indeed encoded by these beetles' genome. Most of the PCWDE-encoding genes of bacterial origin gained introns after their transfer into the beetle genome. Altogether, we show that draft genome assemblies generated from nanopore long-reads offer meaningful information to the study of the evolution of gene families in insects. We anticipate that our data will support studies aiming to better understand the biology of the Cerambycidae and other beetles in general.

First evidence of a horizontally-acquired GH-7 cellobiohydrolase from a longhorned beetle genome.

Xylophagous larvae of longhorned beetles (Coleoptera; Cerambycidae) efficiently break down polysaccharides of the plant cell wall, which make the bulk of their food, using a range of carbohydrate-active enzymes (CAZymes). In this study, we investigated the function and evolutionary history of the first identified example of insect-encoded members of glycoside hydrolase family 7 (GH7) derived from the Lamiinae Exocentrus adspersus. The genome of this beetle contained two genes encoding GH7 proteins located in tandem and flanked by transposable elements. Phylogenetic analysis revealed that the GH7 sequences of E. adspersus were closely related to those of Ascomycete fungi, suggesting that they were acquired through horizontal gene transfer (HGT) from fungi. However, they were more distantly related to those encoded by genomes of Crustacea and of protist symbionts of termites and cockroaches, supporting that the same enzyme family was recruited several times independently in Metazoa during the course of their evolution. The recombinant E. adspersus GH7 was found to primarily break down cellulose polysaccharides into cellobiose, indicating that it is a cellobiohydrolase, and could also use smaller cellulose oligomers as substrates. Additionally, the cellobiohydrolase activity was boosted by the presence of calcium chloride. Our findings suggest that the combination of GH7 cellobiohydrolases with other previously characterized endo-ß-1,4-glucanases and ß-glucosidases allows longhorned beetles like E. adspersus to efficiently break down cellulose into monomeric glucose.

Direct evidence for a new mode of plant defense against insects via a novel polygalacturonase-inhibiting protein expression strategy.

Plant cell wall-associated polygalacturonase-inhibiting proteins (PGIPs) are widely distributed in the plant kingdom. They play a crucial role in plant defense against phytopathogens by inhibiting microbial polygalacturonases (PGs). PGs hydrolyze the cell wall polysaccharide pectin and are among the first enzymes to be secreted during plant infection. Recent studies demonstrated that herbivorous insects express their own PG multi-gene families, raising the question whether PGIPs also inhibit insect PGs and protect plants from herbivores. Preliminary evidence suggested that PGIPs may negatively influence larval growth of the leaf beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) and identified BrPGIP3 from Chinese cabbage (Brassica rapa ssp. pekinensis) as a candidate. PGIPs are predominantly studied in planta because their heterologous expression in microbial systems is problematic and instability and aggregation of recombinant PGIPs has complicated in vitro inhibition assays. To minimize aggregate formation, we heterologously expressed BrPGIP3 fused to a glycosylphosphatidylinositol (GPI) membrane anchor, immobilizing it on the extracellular surface of insect cells. We demonstrated that BrPGIP3_GPI inhibited several P. cochleariae PGs in vitro, providing the first direct evidence of an interaction between a plant PGIP and an animal PG. Thus, plant PGIPs not only confer resistance against phytopathogens, but may also aid in defense against herbivorous beetles.

Analyzing the Substrate Specificity of a Class of Long-Horned-Beetle-Derived Xylanases by Using Synthetic Arabinoxylan Oligo- and Polysaccharides.

Xylophagous long-horned beetles thrive in challenging environments. To access nutrients, they secrete plant-cell-wall-degrading enzymes in their gut fluid; among them are cellulases of the subfamily 2 of glycoside hydrolase family 5 (GH5_2). Recently, we discovered that several beetle-derived GH5_2s use xylan as a substrate instead of cellulose, which is unusual for this family of enzymes. Here, we analyze the substrate specificity of a GH5_2 xylanase from the beetle Apriona japonica (AJAGH5_2-1) using commercially available substrates and synthetic arabinoxylan oligo- and polysaccharides. We demonstrate that AJAGH5_2-1 processes arabinoxylan polysaccharides in a manner distinct from classical xylanase families such as GH10 and GH11. AJAGH5_2-1 is active on long oligosaccharides and cleaves at the non-reducing end of a substituted xylose residue (position +1) only if: 1) three xylose residues are present upstream and downstream of the cleavage site, and 2) xylose residues at positions -1, -2, +2 and +3 are not substituted.

Effects of class-specific, synthetic, and natural proteinase inhibitors on life-history traits of the cotton bollworm Helicoverpa armigera.

Herbivorous insects have more difficulty obtaining proteins from their food than do predators and parasites. The scarcity of proteins in their diet requires herbivores to feed voraciously, thus heavily damaging their host plants. Plants respond to herbivory by producing defense compounds, which reduce insect growth, retard development, and increase mortality. Herbivores use both pre- and postdigestive response mechanisms to detect and avoid plant defense compounds. Proteinase inhibitors (PIs) are one example of plant compounds produced as a direct defense against herbivory. Many insects can adapt to PIs when these are incorporated into artificial diets. However, little is known about the effect of PIs on diet choice and feeding behavior. We monitored the diet choice, life-history traits, and gut proteinase activity of Helicoverpa armigera larvae using diets supplemented with synthetic and natural PIs. In choice experiments, both neonates and fourth-instar larvae preferred the control diet over PI-supplemented diets, to varying degrees. Larvae that fed on PI-supplemented diets weighed less than those that fed on the control diet and produced smaller pupae. Trypsin-specific PIs had a stronger effect on mean larval weight than did other PIs. A reduction of trypsin activity but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets. Therefore, behavioral avoidance of feeding on plant parts high in PIs could be an adaptation to minimize the impact of this plant's defensive strategy.

Functional diversification of horizontally acquired glycoside hydrolase family 45 (GH45) proteins in Phytophaga beetles.

BACKGROUND: Cellulose, a major polysaccharide of the plant cell wall, consists of ß-1,4-linked glucose moieties forming a molecular network recalcitrant to enzymatic breakdown. Although cellulose is potentially a rich source of energy, the ability to degrade it is rare in animals and was believed to be present only in cellulolytic microbes. Recently, it has become clear that some animals encode endogenous cellulases belonging to several glycoside hydrolase families (GHs), including GH45. GH45s are distributed patchily among the Metazoa and, in insects, are encoded only by the genomes of Phytophaga beetles. This study aims to understand both the enzymatic functions and the evolutionary history of GH45s in these beetles. RESULTS: To this end, we biochemically assessed the enzymatic activities of 37 GH45s derived from five species of Phytophaga beetles and discovered that beetle-derived GH45s degrade three different substrates: amorphous cellulose, xyloglucan and glucomannan. Our phylogenetic and gene structure analyses indicate that at least one gene encoding a putative cellulolytic GH45 was present in the last common ancestor of the Phytophaga, and that GH45 xyloglucanases evolved several times independently in these beetles. The most closely related clade to Phytophaga GH45s was composed of fungal sequences, suggesting this GH family was acquired by horizontal gene transfer from fungi. Besides the insects, other arthropod GH45s do not share a common origin and appear to have emerged at least three times independently. CONCLUSION: The rise of functional innovation from gene duplication events has been a fundamental process in the evolution of GH45s in Phytophaga beetles. Both, enzymatic activity and ancestral origin suggest that GH45s were likely an essential prerequisite for the adaptation allowing Phytophaga beetles to feed on plants.

Phyllotreta striolata flea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system.

The ability of a specialized herbivore to overcome the chemical defense of a particular plant taxon not only makes it accessible as a food source but may also provide metabolites to be exploited for communication or chemical defense. Phyllotreta flea beetles are adapted to crucifer plants (Brassicales) that are defended by the glucosinolate-myrosinase system, the so-called "mustard-oil bomb." Tissue damage caused by insect feeding brings glucosinolates into contact with the plant enzyme myrosinase, which hydrolyzes them to form toxic compounds, such as isothiocyanates. However, we previously observed that Phyllotreta striolata beetles themselves produce volatile glucosinolate hydrolysis products. Here, we show that P. striolata adults selectively accumulate glucosinolates from their food plants to up to 1.75% of their body weight and express their own myrosinase. By combining proteomics and transcriptomics, a gene responsible for myrosinase activity in P. striolata was identified. The major substrates of the heterologously expressed myrosinase were aliphatic glucosinolates, which were hydrolyzed with at least fourfold higher efficiency than aromatic and indolic glucosinolates, and ß-O-glucosides. The identified beetle myrosinase belongs to the glycoside hydrolase family 1 and has up to 76% sequence similarity to other ß-glucosidases. Phylogenetic analyses suggest species-specific diversification of this gene family in insects and an independent evolution of the beetle myrosinase from other insect ß-glucosidases.

Intracellular symbiont Symbiodolus is vertically transmitted and widespread across insect orders.

Insects engage in manifold interactions with bacteria that can shift along the parasitism-mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species S. clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus' intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus' success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.

Coconut rhinoceros beetle digestive symbiosis with potential plant cell wall degrading microbes.

Coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is an invasive palm pest whose larvae eat wood, yet lack the necessary digestive enzymes. This study confirmed endogenous CRB cellulase is inactive, suggesting microbial fermentation. The inner lining of the CRB hindgut has tree-like structures covered with a conspicuous biofilm. To identify possible symbionts, 16 S rRNA amplicon sequencing was used on individuals from across Taiwan. Several taxa of Clostridia, an anaerobic class including many cellulolytic bacteria, were highly abundant in most individuals from all locations. Whole metagenome sequencing further confirmed many lignocellulose degrading enzymes are derived from these taxa. Analyses of eggs, larvae, adults, and soil found these cellulolytic microbes are not transmitted vertically or transstadially. The core microbiomes of the larval CRB are likely acquired and enriched from the environment with each molt, and enable efficient digestion of wood.

Paleocene origin of a streamlined digestive symbiosis in leaf beetles.

Timing the acquisition of a beneficial microbe relative to the evolutionary history of its host can shed light on the adaptive impact of a partnership. Here, we investigated the onset and molecular evolution of an obligate symbiosis between Cassidinae leaf beetles and Candidatus Stammera capleta, a γ-proteobacterium. Residing extracellularly within foregut symbiotic organs, Stammera upgrades the digestive physiology of its host by supplementing plant cell wall-degrading enzymes. We observe that Stammera is a shared symbiont across tortoise and hispine beetles that collectively comprise the Cassidinae subfamily, despite differences in their folivorous habits. In contrast to its transcriptional profile during vertical transmission, Stammera elevates the expression of genes encoding digestive enzymes while in the foregut symbiotic organs, matching the nutritional requirements of its host. Despite the widespread distribution of Stammera across Cassidinae beetles, symbiont acquisition during the Paleocene (∼62 mya) did not coincide with the origin of the subfamily. Early diverging lineages lack the symbiont and the specialized organs that house it. Reconstructing the ancestral state of host-beneficial factors revealed that Stammera encoded three digestive enzymes at the onset of symbiosis, including polygalacturonase-a pectinase that is universally shared. Although non-symbiotic cassidines encode polygalacturonase endogenously, their repertoire of plant cell wall-degrading enzymes is more limited compared with symbiotic beetles supplemented with digestive enzymes from Stammera. Highlighting the potential impact of a symbiotic condition and an upgraded metabolic potential, Stammera-harboring beetles exploit a greater variety of plants and are more speciose compared with non-symbiotic members of the Cassidinae.

The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer.

The primary plant cell wall comprises the most abundant polysaccharides on the Earth and represents a rich source of energy for organisms which have evolved the ability to digest them. Enzymes able to degrade plant cell wall polysaccharides are widely distributed in micro-organisms but are generally absent in animals, although their presence in insects, especially phytophagous beetles from the superfamilies Chrysomeloidea and Curculionoidea, has recently begun to be appreciated. The observed patchy distribution of endogenous genes encoding these enzymes in animals has raised questions about their evolutionary origins. Recent evidence suggests that endogenous plant cell wall degrading enzymes-encoding genes have been acquired by animals through a mechanism known as horizontal gene transfer (HGT). HGT describes how genetic material is moved by means other than vertical inheritance from a parent to an offspring. Here, we provide evidence that the mustard leaf beetle, Phaedon cochleariae, possesses in its genome genes encoding active xylanases from the glycoside hydrolase family 11 (GH11). We also provide evidence that these genes were originally acquired by P. cochleariae from a species of gammaproteobacteria through HGT. This represents the first example of the presence of genes from the GH11 family in animals.

An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin.

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt-expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.

An Assassin's Secret: Multifunctional Cytotoxic Compounds in the Predation Venom of the Assassin Bug Psytalla horrida (Reduviidae, Hemiptera).

Predatory assassin bugs produce venomous saliva that enables them to overwhelm, kill, and pre-digest large prey animals. Venom from the posterior main gland (PMG) of the African assassin bug Psytalla horrida has strong cytotoxic effects, but the responsible compounds are yet unknown. Using cation-exchange chromatography, we fractionated PMG extracts from P. horrida and screened the fractions for toxicity. Two venom fractions strongly affected insect cell viability, bacterial growth, erythrocyte integrity, and intracellular calcium levels in Drosophila melanogaster olfactory sensory neurons. LC-MS/MS analysis revealed that both fractions contained gelsolin, redulysins, S1 family peptidases, and proteins from the uncharacterized venom protein family 2. Synthetic peptides representing the putative lytic domain of redulysins had strong antimicrobial activity against Escherichia coli and/or Bacillus subtilis but only weak toxicity towards insect or mammalian cells, indicating a primary role in preventing the intake of microbial pathogens. In contrast, a recombinant venom protein family 2 protein significantly reduced insect cell viability but exhibited no antibacterial or hemolytic activity, suggesting that it plays a role in prey overwhelming and killing. The results of our study show that P. horrida secretes multiple cytotoxic compounds targeting different organisms to facilitate predation and antimicrobial defense.

Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle.

BACKGROUND: The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. RESULTS: Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (ß-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level. CONCLUSIONS: Combining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.

Comparative proteomic analysis of Helicoverpa armigera cells undergoing apoptosis.

Apoptosis is of crucial importance in the life of multicellular organisms. In holometabolous insects, particularly in Lepidoptera, apoptosis is essential in biological processes such as metamorphosis and defense against pathogens. Apoptosis is tightly regulated and involves many proteins, among them caspases, which play a central role. In mammals, almost 300 targets of caspases have been described, and the expression of more than a hundred proteins has been shown to be altered in apoptotic cells. To date, the molecular pathways controlling apoptosis are poorly understood in Lepidoptera. Here, we used a comparative approach aiming to identify candidate proteins potentially implicated in these pathways. We examined changes occurring, in the proteome of a Helicoverpa armigera-derived cell line, upon induction by actinomycin D. We identified 13 proteins for which the relative abundance was significantly altered. Among these, the abundance of procaspase-1 decreased in apoptotic cells, reflecting its processing into the active form. We characterized its properties by heterologous expression and correlated the observed substrate specificity with changes in caspase activity in HaAM1 cells after induction. We also identified three chaperones as well as several putative pro- and anti-apoptotic proteins. Altogether, these data suggest that apoptotic pathways in Lepidoptera share similarities with the ones described in mammals.

A comprehensive characterization of the caspase gene family in insects from the order Lepidoptera.

BACKGROUND: The cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species. RESULTS: In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their Drosophila counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species. CONCLUSIONS: The basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.

Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.

BACKGROUND: The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. RESULTS: Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication). BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. CONCLUSION: Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.