A glimmer of hope - ash genotypes with increased resistance to ash dieback pathogen show cross-resistance to emerald ash borer.

Plants rely on cross-resistance traits to defend against multiple, phylogenetically distinct enemies. These traits are often the result of long co-evolutionary histories. Biological invasions can force naïve plants to cope with novel, coincident pests, and pathogens. For example, European ash (Fraxinus excelsior) is substantially threatened by the emerald ash borer (EAB), Agrilus planipennis, a wood-boring beetle, and the ash dieback (ADB) pathogen, Hymenoscyphus fraxineus. Yet, plant cross-resistance traits against novel enemies are poorly explored and it is unknown whether naïve ash trees can defend against novel enemy complexes via cross-resistance mechanisms. To gain mechanistic insights, we quantified EAB performance on grafted replicates of ash genotypes varying in ADB resistance and characterized ash phloem chemistry with targeted and untargeted metabolomics. Emerald ash borer performed better on ADB-susceptible than on ADB-resistant genotypes. Moreover, changes in EAB performance aligned with differences in phloem chemical profiles between ADB-susceptible and ADB-resistant genotypes. We show that intraspecific variation in phloem chemistry in European ash can confer increased cross-resistance to invasive antagonists from different taxonomic kingdoms. Our study suggests that promotion of ADB-resistant ash genotypes may simultaneously help to control the ADB disease and reduce EAB-caused ash losses, which may be critical for the long-term stability of this keystone tree species.

Transcriptome profiling of Fraxinus excelsior genotypes infested by emerald ash borer.

European ash, Fraxinus excelsior is facing the double threat of ongoing devastation by the invasive fungal pathogen, Hymenoscyphus fraxineus and the imminent arrival of the non-native emerald ash borer (EAB), Agrilus planipennis. The spread of EAB which is currently moving westwards from European Russia and Ukraine into central Europe, poses an additional substantial threat to European ash, F. excelsior. While the molecular basis for resistance or variation in resistance among European ash genotypes is heavily investigated, comparatively little is known about the molecular ash traits involved in resistance against EAB. In this study we have gathered transcriptomic data from EAB inoculated genotypes of F. excelsior that have previously shown different levels of susceptibility to EAB. Resultant datasets show differential gene expression in susceptible and resistant genotypes in response to EAB infestation. This data will provide important information on the molecular basis of resistance to the EAB and allow the development of management plans to combat a pending threat of a culturally and ecologically important European tree species.

An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.

Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect.

Cloning, characterization and expression of two glutathione S-transferase cDNAs in the spruce budworm, Choristoneura fumiferana.

Two Choristoneura fumiferana glutathione S-transferase cDNAs (CfGSTs4 and CfGSTd5) were cloned from a cDNA library constructed using mRNA from the midgut cell line, CF-203. These cDNAs encoded two structurally different proteins with a predicted molecular mass of 23 and 24 kDa, respectively. Amino acid sequence analysis indicates that CfGSTs4 and CfGSTd5 contained Sigma and Delta GST domain, respectively. CfGSTs4 cDNA was expressed as a recombinant protein with the same molecular mass as predicted. Semi-quantitative reverse-transcription PCR analyses indicated that both of these genes were expressed in the epidermis, fat body, and midgut of the 6th instar larvae, as well as CF-203 cells. CfGSTs4 was highly and almost constantly expressed in all tissues during the 6th instar stage. There were higher levels of CfGSTs4 protein in the midgut and fat body than in the epidermis. CfGSTd5 was expressed in the fat body when the insects underwent pupal molting and was constantly expressed in the epidermis and midgut during 6th instar development. CfGSTs4 expression was not affected by ecdysone agonist tebufenozide (RH5992), whereas CfGSTd5 expression was slightly suppressed by the compound.

Cloning and characterization of a protein kinase C gene from the spruce budworm, Choristoneura fumiferana.

Recent studies have implicated protein kinase C (PKC) in the control of 20-hydroxyecdysone (20E)-dependent gene expression during molting and metamorphosis in insects. To further understand the role of this kinase in 20E signal transduction, we cloned a homolog of mammalian PKC by RT-PCR and 5'/3'-RACE from adult of the moth Choristoneura fumiferana. The full-length cDNA of the C. fumiferana PKC (CfPKC1) is 2.3 kb with an open reading frame encoding a protein of 669 amino acids. The deduced amino acid sequence contains all the characteristic features of the classical protein kinase C subfamily. Northern and Western blot analysis showed that CfPKC1 was distributed ubiquitously in various tissues and at different developmental stages. Activation of CfPKC1 with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in a rapid redistribution of the protein from the cytosol to the plasma membrane. Knock-down of the CfPKC1 gene by double-stranded RNA interference or treatment of the CF-203 cells with PKC-specific inhibitors reduces the expression of the 20E-responsive genes CHR3 and E75. This data suggests that CfPKC1 is involved in the 20E-response gene expression in C. fumiferana.

Identification and expression analysis of multiple small heat shock protein genes in spruce budworm, Choristoneura fumiferana (L.).

Fifteen small heat shock protein (sHSP) genes were identified from spruce budworm, Choristoneura fumiferana (L.), an important native forest pest in North America. The transcript levels of each CfHSP were measured under non-stress conditions in all life stages from egg to adult and in five different larval tissues. CfHSP transcript levels showed variation during development, with highest levels in adults and lowest in eggs. Most CfHSP transcripts are highly expressed in larval fat body and Malpighian tubules; two CfHSPs display extremely high expression in the head and epidermis. Upon heat stress, nine CfHSP genes are significantly upregulated, increasing by 50- to 2500-fold depending on developmental stage and tissue type. Upon starvation, eight CfHSPs are upregulated or downregulated, whereas six others retain constant expression. These results suggest that CfHSPs have important and multiple roles in spruce budworm development and in response to heat stress and starvation.

Transcriptome Analysis of the Emerald Ash Borer (EAB), Agrilus planipennis: De Novo Assembly, Functional Annotation and Comparative Analysis.

BACKGROUND: The Emerald ash borer (EAB), Agrilus planipennis, is an invasive phloem-feeding insect pest of ash trees. Since its initial discovery near the Detroit, US- Windsor, Canada area in 2002, the spread of EAB has had strong negative economic, social and environmental impacts in both countries. Several transcriptomes from specific tissues including midgut, fat body and antenna have recently been generated. However, the relatively low sequence depth, gene coverage and completeness limited the usefulness of these EAB databases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep RNA-Sequencing (RNA-Seq) was used to obtain 473.9 million pairs of 100 bp length paired-end reads from various life stages and tissues. These reads were assembled into 88,907 contigs using the Trinity strategy and integrated into 38,160 unigenes after redundant sequences were removed. We annotated 11,229 unigenes by searching against the public nr, Swiss-Prot and COG. The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific. Differential gene expression revealed that 290 unigenes are expressed during larval molting and 3,911 unigenes during metamorphosis from larvae to pupae, respectively (FDR< 0.01 and log2 FC>2). In addition, 1,167 differentially expressed unigenes were identified from larval and adult midguts, 435 unigenes were up-regulated in larval midgut and 732 unigenes were up-regulated in adult midgut. Most of the genes involved in RNA interference (RNAi) pathways were identified, which implies the existence of a system RNAi in EAB. CONCLUSIONS AND SIGNIFICANCE: This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date. Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

Characterization of a spruce budworm chitin deacetylase gene: stage- and tissue-specific expression, and inhibition using RNA interference.

Chitin deacetylase (CDA) catalyzes the conversion of chitin into chitosan, thereby modifying the physical properties of insect cuticles and peritrophic matrices. A lepidopteran chitin deacetylase gene (CfCDA2) was cloned from the spruce budworm, Choristoneura fumiferana, and found to generate two alternatively spliced transcripts, CfCDA2a and CfCDA2b. Transcriptional analysis using isoform-specific RT-PCR primers indicated that both isoforms were upregulated during the molt. Interestingly, CfCDA2b transcripts were most abundant in the head during the molting stage while those of CfCDA2a were predominant in the epidermis during the feeding period. Injection of CfCDA2-specific dsRNA into C. fumiferana larvae or pre-pupae induced both abnormal phenotypes and high mortality, which resulted from an inability to shed the old cuticle. These results suggest that CfCDA2 plays an important role in the molting process, and that the two alternatively spliced transcripts have different functions during insect development. This is the first detailed characterization of lepidopteran chitin deacetylase gene.

Cloning and characterization of two glutathione S-transferase cDNAs in the spruce budworm, Choristoneura fumiferana.

Two Choristoneura fumiferana glutathione S-transferase cDNAs were cloned from a cDNA library constructed using mRNA from the midgut cell line, CF-203. These cDNAs (CfGST2, CfGST3) encoded two structurally different proteins with a predicted molecular mass of 21 and 24 kDa, respectively, which was confirmed through protein expression in a bacterial system. Quantitative reverse-transcription PCR analyses revealed that the transcripts of these two genes were present in the epidermis, fat body, and midgut of the 6th instar larvae. CfGST2 was expressed in the fat body when the insects were close to pupal molting, while it was constantly expressed in the other two tissues during the 6th instar stage. CfGST3 gene was expressed highly and constantly in all of the tissues throughout the 6th instar stage. Immunohistochemistry analysis demonstrated that CfGST2 and CfGST3 proteins were present mainly in the fat body and epidermis and no protein was detected in the midgut. CfGST2 and CfGST3 were different from CfGST reported before (Feng et al., 1999: Insect Biochem Mol Biol 29:779-793) in amino acid sequence, expression pattern, and responsiveness to tebufenozide.

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae).

We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.

Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle, Anoplophora glabripennis (Motschulsky).

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.