Expression of the gonad-specific small heat shock protein, CfHSP20.2, in the spruce budworm, Choristoneura fumiferana (Clem.).

Small heat shock proteins (sHSPs) play important roles in insect development and stress resistance. However, the in vivo functions and mechanisms of action remain largely unknown or unclear for most members of the sHSPs in insects. This study investigated the expression of CfHSP20.2 in the spruce budworm, Choristoneura fumiferana (Clem.) under normal and heat-stress conditions. Under normal conditions, CfHSP20.2 transcript and protein were highly and constantly expressed in the testes of male larvae, pupae and young adults and in the ovaries of female late-stage pupae and adults. After adult eclosion, CfHSP20.2 remained highly and almost constantly expressed in the ovaries, but in contrast, was downregulated in the testes. Upon heat stress, CfHSP20.2 was upregulated in the gonads and non-gonadal tissues in both sexes. These results indicate that CfHSP20.2 expression is gonad-specific and heat-inducible. This provides evidence that the CfHSP20.2 protein plays important roles during reproductive development under normal environmental conditions, while under heat-stress conditions, it may also enhance the thermal tolerance of the gonads and non-gonadal tissues.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.

Development of a Loop-Mediated Isothermal Amplification Assay as an Early-Warning Tool for Detecting Emerald Ash Borer (Coleoptera: Buprestidae) Incursions.

The emerald ash borer (EAB), Agrilus planipennis (Fairmaire), is the most destructive invasive insect species of ash (Fraxinus spp.) in North America. An accurate method for early detection of this noxious insect pest is indispensable to providing adequate warning of A. planipennis infestation. A loop-mediated isothermal amplification (LAMP) assay (EAB-LAMP) was developed based on mitochondrial cytochrome c oxidase subunit I (COI) gene. The EAB-LAMP required only 30 min at 65°C to amplify A. planipennis DNA from specimens collected from geographically distinct locations. There was no cross-reactivity with other Agrilus and insect species. The developed EAB-LAMP differentially detected traces of A. planipennis genome (COI) within frass from various Fraxinus species. EAB-LAMP was also able to distinguish among A. planipennis DNA and other Agrilus species and nontarget insect species in trap captures. By detecting A. planipennis DNA in two additional trap captures (in situ), the EAB-LAMP was more sensitive and reliable than visual inspection. We tested the quantitative nature of the assay by evaluating pooled trap samples and demonstrated that the EAB-LAMP was capable of functioning optimally using a pool size of at least five individual trap samples. This potentially circumvents the need to perform large-scale individual analysis for processing trap samples. Considering its performance, specificity, sensitivity, and repeatability, the developed EAB-LAMP could be a valuable tool to support strategy and operation of large-scale surveillance for A. planipennis and could profitably be used in routine monitoring programs for effective management of A. planipennis.

Molecular characterization of eight ATP-dependent heat shock protein transcripts and their expression profiles in response to stresses in the spruce budworm, Choristoneura fumiferana (L.).

Heat shock proteins (HSPs) greatly contribute to insect stress tolerance and enhance survival and adaptation in severe environmental conditions. To investigate the potential roles of HSPs in the spruce budworm, Choristoneura fumiferana (L.), an important native pest of forests in North America, we found eight ATP-dependent HSP transcripts (CfHSPs). Based on molecular characteristics, the identified HSP genes were classified into HSP70 and HSP90 families, and phylogenetic results showed that they had orthologues in other insects. The transcript levels of these HSPs were measured using RT-qPCR under normal and stressful conditions in the laboratory. Under normal conditions, three HSP genes were consistently expressed in all life stages, whereas expression of the other five genes was dependent on the developmental stage. In the larvae, most CfHSP transcripts displayed similar expression levels among different tissues. Under heat shock conditions, one HSP70 gene and one HSP90 gene were upregulated in all life stages. One HSP70 gene was upregulated after cold injury in the larval stage. With starvation, HSP gene expression exhibited complex expression patterns; most of them were downregulated. These results suggest that the ATP-dependent HSPs have multiple roles during normal development as well as under stressful conditions including heat, cold injury and starvation.

Expression profiles of 14 small heat shock protein (sHSP) transcripts during larval diapause and under thermal stress in the spruce budworm, Choristoneura fumiferana (L.).

Diapause is an important strategy for certain insect species to survive unfavorable environmental conditions, including low temperatures experienced when they overwinter in cold climate. Many studies have indicated that the increased expression of heat shock proteins during diapause improves the thermal tolerance of insects. However, the relationship between small heat shock proteins (sHSPs) and diapause is not clear or well-researched. In this study, we investigated the transcript levels of 14 sHSP genes in the spruce budworm, Choristoneura fumiferana, a major pest of spruce and fir in Canada, during pre-diapause, diapause, and post-diapause under normal rearing conditions and in response to a heat shock treatment. We found that sHSP expression profiles could be classified into five patterns under normal laboratory conditions: pattern I was upregulated only during pre-diapause, pattern II was upregulated only during diapause, pattern III was constantly expressed throughout diapause, pattern IV was upregulated in both pre-diapause and diapause, and pattern V was upregulated only during post-diapause. After heat shock, five different expression patterns were observed: pattern I responded weakly or not at all throughout diapause, pattern II responded weakly during the diapause stage but strongly at the onset of diapause and in the post-diapause period, pattern III was upregulated only during post-diapause, pattern IV was strongest during diapause, and pattern V was strongest only in early diapause. These complex expression profiles lead us to suggest that most of the sHSP genes are involved in the diapause process and that they may have multiple and important roles in different phases of this process.

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.

The complete mitogenome of the Emerald Ash Borer (EAB), Agrilus planipennis (Insecta: Coleoptera: Buprestidae).

The complete mitogenome of the Emerald Ash Borer (EAB, Agrilus planipennis) was obtained by gleaning mitochondrial sequences from whole-genome Illumina sequencing data. The circular genome has 15,942 base pairs and contains 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and an A-T-rich region. All PCGs begin with ATN codons. The nucleotide composition is highly asymmetric (31.65% A, 40.25% T, 17.39% G, 10.71% C), with an overall A-T content of 71.9%. Phylogenetic analysis based on insect mitogenomes indicated that EAB is closely related to other Buprestoidea species, clustering most closely with Chrysochroa fulgidissima.

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.

A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3(oe) mutant.

The w-3(oe) silkworm mutant has white eyes and eggs due to the absence of ommochrome pigments in the eye pigment cells and serosa cells. The mutant is also characterized by translucent larval skin resulting from a deficiency in the transportation of uric acid, which acts as a white pigment in larval epidermal cells. A silkworm homolog of the fruitfly white gene, Bmwh3, a member of ATP-binding cassette transporter superfamily, was mapped on the w-3 locus. The w-3(oe) mutant has a single-base deletion in exon 2 and a premature stop codon at the 5' end of exon 3. These results show that w-3 is equivalent to Bmwh3 and is responsible for the transportation of ommochrome precursors and uric acid into pigment granules and urate granules, respectively.

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.

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.

Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.

Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus.

An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN.