Molecular signatures of diapause in the Asian longhorned beetle: Gene expression.

Most previous studies on gene expression during insect diapause do not address among-tissue variation in physiological processes. We measured transcriptomic changes during larval diapause in the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae). We conducted RNA-seq on fat body, the supraesophageal ganglion, midgut, hindgut, and Malpighian tubules during pre-diapause, diapause maintenance, post-diapause quiescence, and post-diapause development. We observed a small, but consistent, proportion of genes within each gene expression profile that were shared among tissues, lending support for a core set of diapause-associated genes whose expression is tissue-independent. We evaluated the overarching hypotheses that diapause would be associated with cell cycle arrest, developmental arrest, and increased stress tolerance and found evidence of repressed TOR and insulin signaling, reduced cell cycle activity and increased capacity of stress response via heat shock protein expression and remodeling of the cytoskeleton. However, these processes varied among tissues, with the brain and fat body appearing to maintain higher levels of cellular activity during diapause than the midgut or Malpighian tubules. We also observed temperature-dependent changes in gene expression during diapause maintenance, particularly in genes related to the heat shock response and MAPK, insulin, and TOR signaling pathways. Additionally, we provide evidence for epigenetic reorganization during the diapause/post-diapause quiescence transition and expression of genes involved in post-translational modification, highlighting the need for investigations of the protein activity of these candidate genes and processes. We conclude that diapause development is coordinated via diverse tissue-specific gene expression profiles and that canonical diapause phenotypes vary among tissues.

Prediction of a conserved pheromone receptor lineage from antennal transcriptomes of the pine sawyer genus Monochamus (Coleoptera: Cerambycidae).

Longhorned beetles (Cerambycidae) are a diverse family of wood-boring insects, many species of which produce volatile pheromones to attract mates over long distances. The composition and structure of the pheromones remain constant across many cerambycid species, and comparative studies of those groups could, therefore, reveal the chemoreceptors responsible for pheromone detection. Here, we use comparative transcriptomics to identify a candidate pheromone receptor in the large and economically important cerambycid genus Monochamus, males of which produce the aggregation-sex pheromone 2-(undecyloxy)-ethanol ("monochamol"). Antennal transcriptomes of the North American species M. maculosus, M. notatus, and M. scutellatus revealed 60-70 odorant receptors (ORs) in each species, including four lineages of simple orthologs that were highly conserved, highly expressed in both sexes, and upregulated in the flagellomeres where olfactory sensilla are localized. Two of these orthologous lineages, OR29 and OR59, remained highly expressed and conserved when we included a re-annotation of an antennal transcriptome of the Eurasian congener M. alternatus. OR29 is also orthologous to a characterized pheromone receptor in the cerambycid Megacyllene caryae, suggesting it as the most likely candidate for a monochamol receptor and highlighting its potential as a conserved lineage of pheromone receptors within one of the largest families of beetles.

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.

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.

Cold tolerance of laboratory-reared Asian longhorned beetles.

Low winter temperatures in temperate climates can limit the success of non-native species. The Asian longhorned beetle, Anoplophora glabripennis, is an invasive wood-boring pest of hardwood trees in North America and Europe. Native A. glabripennis populations are spread across several climate zones in China and the Korean Peninsula and are likely to encounter low temperatures in at least some of this range. Understanding the lethal limits of the overwintering life stages of A. glabripennis is essential for accurately modeling the risk that invasive populations pose to non-native environments. In this study, we provide the first systematic characterization of the cold tolerance strategy and lower lethal limits of A. glabripennis eggs, larvae, and pupae. In diapausing larvae, the most common overwintering stage in this species, we measure hemolymph glycerol and osmolality and identify the effects of prolonged low temperature exposure. In developing pupae, we identify sublethal effects caused by low temperature exposure before freezing. Eggs and larvae were the most cold-tolerant life stages; eggs were freeze-avoidant with an average supercooling point of -25.8 °C and larvae were freeze tolerant with an LT90 of -25 °C. Hemolymph osmolality of freeze-tolerant larvae, on average, increased to 811 mOsm during chilling. This increase was primarily driven by a concurrent, average increase of 232 mM hemolymph glycerol. Pupae died upon exposure to freezing temperatures, but accumulate strong sublethal effects prior to freezing, indicating that they are chill susceptible. Taken together, these data will be useful to inform species distribution modeling in A. glabripennis.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis.

An insect's capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period - results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators.

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488-614 sequence variants) than pooling the same number of samples (165-191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Overexpression of TAT-PTD-diapause hormone fusion protein in tobacco and its effect on the larval development of Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUND: The diapause hormone (DH) has been shown either to induce or to terminate diapause, depending on the insect species. In a previous study we demonstrated that the DH from Clostera anastomosis (caDH) has biological activity in Helicoverpa armigera, which prompted us to examine the potential growth-inhibiting or antiherbivory effects of the TAT-caDH fusion protein when expressed in transgenic plants. RESULTS: In this study, we produced transgenic tobacco plants expressing either the TAT-caDH protein or a TAT-caDH-eGFP fusion version that allowed tracking of the fluorescent protein in plant tissues. Our results indicate that H. armigera larvae feeding on transgenic tobacco expressing TAT-caDH exhibited a significantly reduced survival rate and weight gain. However, larvae feeding on transgenic tobacco expressing TAT-caDH-eGFP were unaffected. While fusion of the eGFP gene influenced the bioactivity of caDH in larvae, TAT-caDH-eGFP can still penetrate the insect midgut cell membrane. CONCLUSION: TAT-caDH increases DH stability in oral delivery. Our results may help in targeting DH-dependent physiological processes in insects for improving herbivore tolerance in economically important crops. © 2016 Society of Chemical Industry.

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.

Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH.

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.

Establishment of a cell line from the ash and privet borer beetle Tylonotus bimaculatus Haldeman and assessment of its sensitivity to diacylhydrazine insecticides.

A novel cell line, NRCAN-Tb521, was developed from larvae of the longhorn beetle Tylonotus bimaculatus (Coleoptera: Cerambycidae), a pest of North American ash trees. The cell line has been successfully passaged more than 50 times and displayed very strong attachment to the substrate and a modal chromosomal count distribution of 19. Sequencing of a 649 bp fragment of the mitochondrial cytochrome oxidase I gene confirmed the identity of NRCAN-Tb521 as T. bimaculatus. The response of the cell line to 20-hydroxyecdysone and diacylhydrazine ecdysone agonist insecticides was also studied. At 10(-6) M, 20-hydroxyecdysone, tebufenozide, methoxyfenozide and halofenozide triggered the production of numerous filamentous cytoplasmic extensions, and the cells tended to form aggregates, indicative of a cell differentiation response. This response was followed by a strong decrease in viability after 4 d. Reverse transcription polymerase chain reaction (PCR) experiments and sequencing of PCR fragments showed that the 20E receptor gene EcR is expressed in the cells and that 20E, tebufenozide, methoxyfenozide and halofenozide also induce the expression of the nuclear hormone receptor gene HR3. This report establishes that NRCAN-Tb521 is a valuable in vitro model to study effects of ecdysone agonists in wood-boring cerambycids.

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.

New insights into the evolution of Entomopoxvirinae from the complete genome sequences of four entomopoxviruses infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata.

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.

Identification of odor-processing genes in the emerald ash borer, Agrilus planipennis.

BACKGROUND: Insects rely on olfaction to locate food, mates, and suitable oviposition sites for successful completion of their life cycle. Agrilus planipennis Fairmaire (emerald ash borer) is a serious invasive insect pest that has killed tens of millions of North American ash (Fraxinus spp) trees and threatens the very existence of the genus Fraxinus. Adult A. planipennis are attracted to host volatiles and conspecifics; however, to date no molecular knowledge exists on olfaction in A. planipennis. Hence, we undertook an antennae-specific transcriptomic study to identify the repertoire of odor processing genes involved in A. planipennis olfaction. METHODOLOGY AND PRINCIPAL FINDINGS: We acquired 139,085 Roche/454 GS FLX transcriptomic reads that were assembled into 30,615 high quality expressed sequence tags (ESTs), including 3,249 isotigs and 27,366 non-isotigs (contigs and singletons). Intriguingly, the majority of the A. planipennis antennal transcripts (59.72%) did not show similarity with sequences deposited in the non-redundant database of GenBank, potentially representing novel genes. Functional annotation and KEGG analysis revealed pathways associated with signaling and detoxification. Several odor processing genes (9 odorant binding proteins, 2 odorant receptors, 1 sensory neuron membrane protein and 134 odorant/xenobiotic degradation enzymes, including cytochrome P450s, glutathione-S-transferases; esterases, etc.) putatively involved in olfaction processes were identified. Quantitative PCR of candidate genes in male and female A. planipennis in different developmental stages revealed developmental- and sex-biased expression patterns. CONCLUSIONS AND SIGNIFICANCE: The antennal ESTs derived from A. planipennis constitute a rich molecular resource for the identification of genes potentially involved in the olfaction process of A. planipennis. These findings should help in understanding the processing of antennally-active compounds (e.g. 7-epi-sesquithujene) previously identified in this serious invasive pest.

Selection and characterization of Autographa californica multiple nucleopolyhedrovirus DNA polymerase mutations.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.

Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster.

Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1-Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila.

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 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.

Prophenoloxidases 1 and 2 from the spruce budworm, Choristoneura fumiferana: molecular cloning and assessment of transcriptional regulation by a polydnavirus.

Immune challenge in arthropods is frequently accompanied by melanization of the hemolymph, a reaction triggered by the activation of prophenoloxidase (PPO). Because their immature stages are spent inside the hemocoel of insect larvae, endoparasitoids have evolved strategies to escape or counter melanin formation. Very little molecular information is available on these endoparasitoid counterstrategies. We have sought to shed light on the inhibition of melanization in the spruce budworm, Choristoneura fumiferana, by the parasitic wasp Tranosema rostrale, by cloning two host PPO homologs and studying their transcriptional regulation after parasitization. The two polypeptides are encoded by transcripts of approximately 3.3 kb (for CfPPO1) and 3.0 kb (for CfPPO2) and possess structural features typical of other insect PPOs. While there appears to be a single CfPPO2 gene in the C. fumiferana genome, we detected three CfPPO1 mRNA variants displaying insertions/deletions in the 3' untranslated region, suggesting that there may be more than one CfPPO1 gene copy. Both CfPPO1 and CfPPO2 were expressed at high levels in C. fumiferana 6th instars, and parasitization by T. rostrale had no apparent impact on the level of their transcripts. Injection of a large dose (0.5 female-equivalent) of polydnavirus-laden calyx fluid extracted from T. rostrale, which is known to inhibit melanization in C. fumiferana, only caused a transient decrease in CfPPO1 and CfPPO2 transcript accumulation at 2-3 d post injection. It thus appears that transcriptional downregulation of C. fumiferana PPO by T. rostrale plays a minor role in the inhibition of hemolymph melanization in this host-parasitoid system.