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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.

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.

Many biological processes are partitioned among organs and tissues, necessitating tissue-specific or organ-specific analysis (particularly for comparative -omics studies). Standardised techniques for tissue identification and dissection are therefore imperative for comparing among studies. Here we describe dissection protocols for isolating six key tissues/organs from larvae of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae): the supraoesophageal ganglion, posterior midgut, hindgut, Malpighian tubules, fat body, and thoracic muscle. We also describe how to extract haemolymph and preserve whole larvae for measurements such as protein, lipid, and carbohydrate content. We include dissection protocols for both fresh-killed and previously frozen specimens. Although this protocol is developed for A. glabripennis, it should allow standardised tissue collection from larvae of other cerambycids and be readily transferrable to other beetle taxa with similar larval morphology.

In North America Amylostereum areolatum (Chaillet ex Fr.) Boidin is a fungal symbiont associated with both the non-native Sirex noctilio Fabricius (Hymenoptera: Siricidae) and less commonly the native Sirex nigricornis Fabricius (Hymenoptera: Siricidae) woodwasps. The relationship between S. noctilio and A. areolatum constitutes a serious threat to pine plantation in the southern hemisphere. Studies have shown evidence of exchange of symbionts between non-native and native Sirex species. Our objectives were 1) to identify and assemble a panel of rDNA intergenic spacer–single nucleotide polymorphisms (IGS-SNPs) for genotyping strains of A. areolatum symbionts associated with Sirex species in North America, and 2) to develop genetic markers for monitoring the spread of specific A. areolatum haplotypes associated with S. noctilio across regions. The IGS-SNPs panel analyzed included haplotypes B1, B2, D1, D2 (from known IGS type B and D), E, and F. Genetic markers and haplotype-specific primers were designed to detect the IGS haplotypes D and E of A. areolatum. We found that haplotype D was absent in A. areolatum from S. nigricornis in Louisiana, while haplotype E was detected in all A. areolatum from S. nigricornis in Canada and Louisiana. Both haplotype D and E were co-detected in approximately 5% of samples from Canada. The IGS-SNP markers detected specific haplotypes accurately. Observing haplotype D in any A. areolatum from the native S. nigricornis likely indicates the presence of the potentially harmful S. noctilo-A. areolatum complex. The work highlights how IGS-SNPs can help in early detection without direct occurrence/observations of the non-native species of concern.

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.

Invasive species pose significant threats to forest ecosystems. Early intervention strategies are the most cost-effective means to control biological invasions, but are reliant on robust biosurveillance. State-of-the-art genomic approaches can provide an unprecedented opportunity to access detailed information on the invasion process and adaptive potential of invasive insects that pose an immediate threat to forests environments. Genomics can improve diagnostics of the invader and identify its route of invasion by determining the source population(s), assess its probability of establishment and patterns of spread, as well as provide evidence of adaptation. Applied biosurveillance efforts by plant health regulatory agencies will benefit substantially from the detailed insights that genomic data bring to our understanding of biological invasions.

Early intervention, effective management, and regulations are essential to mitigate the potential negative impacts of invasive forest insects. Biosurveillance provides the necessary knowledge to inform management, and regulatory practices. Genomic approaches can contribute valuable information to this process. Unfortunately, adoption and incorporation of genomic tools into biosurveillance frameworks is not straightforward. To realize the full potential of genomic knowledge, researchers must work together with end users to ensure full adoption, standardization, validation, and interpretation of genomic results.

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.

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 soil arthropod biodiversity assessments. Processing individually collected field samples recovered significantly higher richness (539-596 ESVs) than pooling the same number of field samples (126-154 ESVs), and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Variations in the number of individual or composite samples or DNA extractions resulted in similar sample clustering based on community dissimilarities. 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 of 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.

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.

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

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.

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.

The emerald ash borer (Agrilus planipennis) is an important invasive insect pest of Fraxinus spp. that feeds on host tissues containing high levels of sucrose. However, little is known about how it digests sucrose. Here, using larval midgut transcriptome data and preliminary genome sequence efforts, two β-fructofuranosidase-encoding ScrB genes, AplaScrB-1 and AplaScrB-2, were identified, and proved to reside within the A. planipennis genome. Homology and phylogenetic analysis revealed that they were acquired by A. planipennis via horizontal gene transfer (HGT) from bacteria, possibly an event independent from that reported in bark beetles (eg ScrB genes). Microsynteny between A. planipennis DNA scaffold #2042940, which hosts AplaScrB-1, and a region in the Tribolium castaneum chromosome LG4 suggested that A. planipennis gained this gene after the separation of Buprestidae and Tenebrionidae. Although both of the putative AplaScrB proteins have conserved β-fructofuranosidase motifs, only AplaScrB-2 was predicted to be a secretory protein. Expression of AplaScrB-1 seemed constitutive during development and in all tissues examined, whereas AplaScrB-2 showed a peak expression in adults and in the midgut. We propose that acquisition of these genes by A. planipennis from bacteria is adaptive, and specifically AplaScrB-2 is involved in breaking down dietary sucrose to obtain energy for development.

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.

The insect exoskeleton is present as a rigid structure above a monolayer of epidermal cells and together both form the integument. The cuticle is composed of an outer, multilayered epicuticle and an inner procuticle containing the chitinous exo- and endo- cuticle. The non-living cuticle is replaced during each instar to accommodate growth and development by sequentially degrading the old cuticle and replacing it with a new one. The entire molting cycle and the shedding of the old cuticle are precisely regulated by a series of endocrine and neuroendocrine cues. This chapter addresses the various cuticular components, cuticular metabolism and cuticular biogenesis regulatory systems that have been targeted for pest management. Major classes of synthetic compounds, including benzoylphenyl ureas, benzoyl hydrazines and etoxazole that target chitin synthesis and the molting cycle are presented. In addition, we focus on recent developments toward the targeting of additional components of the cuticle or processes of cuticle biogenesis that may find future applications in pest management.

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.

Current influenza vaccines containing primarily hypervariable haemagglutinin and neuraminidase proteins must be prepared against frequent new antigenic variants. Therefore, there is an ongoing effort to develop influenza vaccines that also elicit strong and sustained cytotoxic responses against highly conserved determinants such as the matrix (M1) protein and nucleoprotein (NP). However, their antigenic presentation properties in humans are less defined. Accordingly, we analysed MHC class I and class II presentation of endogenously processed M1 and NP in human antigen presenting cells and observed expansion of both CD8+- and CD4+-specific effector T lymphocytes secreting gamma interferon and tumour necrosis factor. Further enhancement of basal MHC-II antigenic presentation did not improve CD4+ or CD8+ T-cell quality based on cytokine production upon challenge, suggesting that endogenous M1 and NP MHC-II presentation is sufficient. These new insights about T-lymphocyte expansion following endogenous M1 and NP MHC-I and -II presentation will be important to design complementary heterosubtypic vaccination strategies.

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.

Proteins that are capable of binding chitin play essential roles in the synthesis and structural integrity of the insect cuticle and peritrophic matrix. In the course of developing expressed sequence tag (EST) libraries for the eastern spruce budworm, Choristoneura fumiferana, we identified an abundant cDNA encoding a homolog of the Drosophila “gasp” gene (Gene Analogous to Small Peritrophins). For the present work, we undertook the characterization of this new homolog, CfGasp, in an effort to identify its role during larval development. As shown for DmGasp, the C. fumiferana homolog was found to contain three type-2 chitin-binding domains (CBDs), which were also found in Gasp orthologs retrieved from GenBank. In a phylogenetic analysis, these Gasp proteins formed a tight cluster, distinct from the midgut-specific peritrophins with which they share the cysteine-containing CBDs so far considered absent from cuticular proteins. However, unlike what has been shown for peritrophins, CfGasp transcript levels were low in larval midguts and most abundant in epidermis, while they were low in trachea and ovaries. Transcript levels increased during larval molts in a pattern similar to that observed for exocuticular proteins in other insects. In addition, the recombinant protein was shown to be capable of binding chitin. Altogether, these results suggest a structural role for CfGasp in exocuticle formation.

A trypsin-like molting-related serine protease cDNA (CfMRSP) was cloned from the spruce budworm, Choristoneura fumiferana. The full-length CfMRSP comple- mentary DNA (cDNA) encoded a 43 kDa protein that contained a trypsin-like serine protease catalytic domain, but no clip domain. The C-terminal extension contained five cystein residues, which may allow the protein to form a homodimer through interchain disulfide bonds and regulate the activity of CfMRSP. Phylogenetic tree analysis showed that CfMRSP clusters with lepidopteran homologues such as serine protease 1 of Lonomia obliqua, hemolymph proteinase 20 (HP20), pattern recognition serine proteinase precursor (ProHP14) and a trypsin-like protein of Manduca sexta. Northern blot analysis of devel- opmental expression of CfMRSP indicated that its transcripts were found primarily in the epidermis and were produced during all of the tested stadia, from 4th instar larvae to pupae, but increased levels of CfMRSP transcripts were always found after each molt. A high level of the protein was found in the epidermis by immunohistochemistry analysis. Altogether these data suggest that CfMRSP plays a role in the epidermis during molting and metamorphosis.

Four cDNAs (Cfserpin-1a, Cfserpin-1b, Cfserpin-1c and Cfserpin-1d) of the Choristoneura fumiferana serpin-1 gene were cloned from an epidermis cDNA library. Analysis of the deduced amino acid sequences indicated that the cloned cDNAs encode four different proteins displaying identical N- but distinct C-termini, the latter region containing the inhibitory loop. The entire CfSerpin-1 gene is transcribed while the variants are generated. Antibodies generated against the purified recombinant serpins cross-reacted with the other three. Each of the four Cfserpin-1 cDNA variants was transcribed throughout larval development, from the 4th to the 6th instar, but transcript levels during the intermolt phases were generally higher than during the molting phase. The epidermis and fat body had higher levels of Cfserpin-1 transcripts than the midgut. Cfserpin-1 proteins, detected with the Cfserpin-1a antibody, were found in the epidermis, midgut, fat body, plasma and molting fluid of 6th instar larvae and pre-pupae. Prepupal and pupal insects had higher levels of the proteins than the 6th instar feeding larvae, despite a drop in transcript levels. Cfserpin-1a could bind with the serine proteinase elastase and form a complex in vitro. We hypothesize that the cloned serpins could be involved in the regulation of cuticle degradation during the insect molting cycle.

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 ( Cf PKC1) 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 Cf PKC1 was distributed ubiquitously in various tissues and at different developmental stages. Activation of Cf PKC1 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 Cf PKC1 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 Cf PKC1 is involved in the 20E-response gene expression in C. fumiferana .

Immune challenge in arthropods is frequently accompanied by melanization of the hemolymph, a reaction triggered by the activa- tion 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 ~3.3 kb (for Cf PPO1) and 3.0 kb (for Cf PPO2) and possess structural features typical of other insect PPOs. While there appears to be a single Cf PPO2 gene in the C. fumiferana genome, we detected three Cf PPO1 mRNA variants displaying insertions/deletions in the 3' untranslated region, suggesting that there may be more than one Cf PPO1 gene copy. Both Cf PPO1 and Cf PPO2 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 Cf PPO1 and Cf PPO2 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. Arch. Insect Biochem. Physiol. 67:188-201, 2008. Published 2008 Wiley-Liss, Inc. †

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

Organisms have to constantly respond to environmental conditions to maintain a status of dynamic homeostasis for survival. As single celled organisms evolved into multicellular animals, intercellular and inter-organ communications became indispensable not only to orchestrate homeostasis but also to control the many events punctuating metazoan development. To accomplish this need, a signal transduction system was evolved, consisting of an arborized network of nerves as well as a collection of oligopeptide neurotransmitters (neuropeptides) along with their cognate receptors. We review here the current state of our understanding of the physiology of neuropeptides in the most species-rich group of animals, the crustaceans and insects. The vast majority of neuropeptides signal through cell-surface guanosine-protein coupled receptors (GPCRs). These neuropeptide-receptor systems control a variety of physiological functions, for instance the numerous involuntary movements of internal ducts that are precisely timed for transporting reproductive, digestive and secretory materials. The rigid exoskeleton in Crustaceans and Insects require periodic molts to accomplish growth and metamorphosis and this is harmoniously regulated by a cascade of neuropeptides. Neuropeptides also regulate various events in the life of organisms including biological clocks and behavior. Technological advances in peptidomics, through the routine use MALDI-TOF mass spectrometry and in genomics, with in silico identification of neuropeptide receptor genes have revealed a staggering diversity of extracellular peptide-based signaling systems. This diversity underscores that communication between the afferent (nervous) system and the appropriate organs for an efferent response must be done in an unambiguous manner without any short circuits or reversal of directions. While we have a broad understanding of how neuropeptides and their receptors play a vital role in signal transduction, much of the details remain to be unraveled.

Not surprisingly, in the spruce budworm, Choristoneura fumiferana, antifreeze protein (AFP) gene expression is most abundant in the second instar, overwintering stage. However, low level RNA and protein expression was also found in the sixth instar larvae, a summer stage. In situ hybridization further confirmed the presence of AFP mRNA in sixth instar midgut tissues. Sequencing of cDNAs corresponding to ‘‘summer-expressed’’ transcripts revealed an isoform that was not apparent in a cDNA library made to second instar larvae. Although similar to AFP cDNAs obtained from overwintering larvae, this AFP-like isoform (CfAFP6) has two Cys substitutions. Since AFPs from this species fold into a b-helix that is stabilized by disulfide bonds, it was of interest to determine if this summerexpressed isoform had AFP activity. No thermal hysteresis activity was found when CfAFP6 was cloned and expressed in E. coli, even after in vitro denaturation and refolding. As well, there was no activity detected when the sequence of a known, active isoform was changed to mimic the Cys substitutions in CfAFP6. Since CfAFP6 does not appear to contribute to freeze resistance, its apparent absence in the overwintering second instar should not in itself be considered curious. r 2006 Elsevier Ltd. All rights reserved.

Spruce budworm (Choristoneura) species survive sub-zero winter temperatures by producing antifreeze proteins (AFPs) encoded by a multigene family of short and long isoforms. We report in this study the first analysis of antifreeze proteins from related Choristoneura sister species. The additional thirty amino acid insert found in the longer AFP isoforms maintains the proteins beta-helix and original fifteen amino acid (Thr-X-Thr) repeat motif. Analysis of the beta-helix region shows more divergent residues surround the conserved Thr residues. Maintaining the beta-helix structure and conserved Thr residues appear to be paramount for AFP function and surviving sub-zero winter temperatures. Two other species within the same lepidopteran clade, Ditrysia, do not appear to contain any AFP-like sequences.

Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, we raised antibodies against a tumorous blood cell line and identified an antigenic determinant that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, we induced a dramatic increase in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis.

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face.

The freeze-intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp-Lu1 encodes a approximately 9-kDa AFP isoform, and Afp-Iu1 encodes a approximately 12-kDa AFP isoform. Both CfAFP genes have similar structures with a single 3- to 3.6-kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new approximately 9-kDa isoform, was found 3.7 kb upstream of Afp-Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68-76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved 'Thr button' (Thr-Xaa-Thr) ice-binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are approximately 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp-Lu1 and Afp-Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

Tranosema rostrale rostrale (Brishke) (subfamily Campopleginae) is a parasitoid of the eastern spruce budworm, Choristoneura fumiferana (Clemens), in Canada and the United States. T. r. rostrale has often been confused with the closely related Enytus montanus (Ashmead), another parasitoid of C. fumiferana. These 2 species are defined and characters distinguishing them are compared and illustrated. A survey across southern Quebec indicates that >99% of all T. r. rostrale/E. montanus-like parasitoids recovered from spruce budworm larvae implanted in fir stands over a period of 1 wk belong to the former species. T. r. rostrale oviposits in all postdiapause instars of C. fumiferana, as well as in several alternative hosts, starting in late May in the Quebec City region. At 20°C, development inside the host takes 14 d and the parasitoid goes through 3 stadia. T. r. rostrale is believed to complete 2 or 3 additional generations on alternate hosts. Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, P.O. Box 3800, Sainte-Foy, QC, Canada G1V 4C7. Eastern Cereal and Oil Seed Research Centre, Agriculture and Agri-Food Canada, Research Branch, Ottawa, ON, Canada K1A 0C6. Departement de biologie, Universite Laval, Sainte-Foy, QC, Canada G1K 7P4.

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.

The identification of specific viral and tumor antigen epitopes recognized by CD4(+) or CD8(+) T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8(+) or CD4(+) T lymphocytes.This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3'end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA's sensitivity to degradation, we also insert a control define epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and capacity to be translated.

We studied the effect of parasitism by Tranosema rostrale (Brischke)* (Hymenoptera: Ichneumonidae) on the developmental rate and growth of its host, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae). In the laboratory, T. rostrale completed its larval development in 13–14 days and its prepupae always emerged from 6th instar hosts, irrespective of host age (day 2 of the 4th, 5th or 6th instar) at the time of parasitization. Parasitism by T. rostrale did not affect the developmental rate of C. fumiferana parasitized as 4th instar larvae, but significantly lengthened the developmental period of hosts parasitized as 5th or 6th instars, with a doubling of duration in the latter case. In addition, T. rostrale significantly reduced the growth of its host: the maximum fresh mass reached by larvae parasitized as 4th, 5th or 6th instars was about half that of unparasitized larvae and their dry mass gain varied between 7 and 20% of that measured for their unparasitized counterparts. Parasitism also significantly lowered the production of faeces by host larvae. We examined the possibility that accessory fluids from the female wasp were responsible for the observed parasitism-induced alterations. Calyx fluid (CxF) injected into 6th instar larvae lengthened the time to pupation whereas venom had no effect on development. Although total faeces production was not affected by injection of either fluid, the rate of faeces production was significantly reduced in larvae injected with CxF at 0.5 female-equivalent. Injection of CxF treated with psoralen and UV did not delay pupation of C. fumiferana. These results indirectly suggest that polydnavirus transcription is required for this developmental alteration to be effected.

Oviposition by the endoparasitoid Tranosema rostrale1 (Hymenoptera: Ichneumonidae) in larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae) was accompanied by significant changes in total hemocyte counts (THCs) and phenoloxidase (PO) activity of host hemolymph. Three days after parasitization, THCs were half those measured in control larvae and PO activity had undergone a considerable reduction. However, while the parasitoid was able to evade the host's defense reaction, parasitism did not prevent encapsulation of glass rods implanted in 6th instar hosts 3 days following oviposition, although it inhibited melanization of capsules and whole hemolymph. Differential hemocyte counts (DHCs) were also affected by parasitism but the differences were transient. Injection of calyx fluid (CxF), at a concentration of 0.1 female equivalent (FE), mimicked the effect of parasitism on THCs and PO activity, but DHCs were not affected. Injected alone, venom (0.1 FE) had no significant effect on the above variables and, injected together with CxF, it did not modulate the effect of the latter fluid.

The identification of specific viral and tumor antigen epitopes recognized by CD4(+) or CD8(+) T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8(+) or CD4(+) T lymphocytes.This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3'end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA's sensitivity to degradation, we also insert a control define epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and capacity to be translated.

Chitin is a polymer of N-acetyl glucosamine that forms the protective exoskeleton of all arthropods and is replaced periodically during growth and development. Chitin biosynthesis starts with the disaccharide trehalose, culminating in the polymerization of the N-acetyl glucosamine subunits by chitin synthase to produce chitin microfibrils. Chitin in the old exoskeleton is degraded by chitinases, deacetylases and hexosaminidases and recycled. Chitin synthesis has been used as a target for developing biorational insecticides such as benzoylphenyl ureas, diflubenzuron being the original such compound. Several benzoylphenyl ureas with diverse activity spectra have since been synthesized and widely used for pest control. Newer pesticides targeting not only chitinase and chitin synthase but also other novel sites are being developed. Understanding the various nuances of chitin metabolism and regulation with all the genomic resources on hand will undoubtedly pave the way for developing more target-oriented softer control agents that have minimal impact on the environment.

The identification of specific viral and tumor antigen T-cell epitopes remains a challenge. Indeed, epitope mapping methods are generally costly and time-consuming. Thus, few techniques allow for efficient CD4 + T-lymphocyte epitope identification. Here, we introduce a novel polymerase chain reaction-based mRNA epitope identification method, called mPEC, to rapidly and precisely identify relevant T-cell epitopes recognized by CD8 + or CD4 + T lymphocytes. This method is based on the use of mRNA fragments synthesized from polymerase chain reaction-amplified cDNA with a choice of 3'end deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to deduce an epitope's localization in a given protein antigen. Considering mRNA's sensitivity to degradation, we also inserted a defined epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and its capacity to be translated. Using this method, we rapidly and successfully identified the specific epitope of 2 CD8 + and 1 CD4 + T-lymphocyte clones derived from influenza model antigens. Hence, mPEC could be used to identify new, in vivo-relevant T-cell epitopes for cancer immunotherapy and vaccination in general.

The Bousquet 2 mine is a deformed pyritic polymetallic Archean gold deposit located within a zone of intense ductile-brittle deformation composed of highly sheared and altered metamorphic tectonites of contrasting rheological properties. The ore occurs in a metamorphosed zone of aluminous alteration and is composed of foliation-parallel massive and semimassive pyritic lenses overprinted by late en echelon extensional veinlets containing Au-Ag-Cu mineralization.Structural and textural analyses show that the gold-bearing sulfide bodies and enclosing host rocks contain discrete tectono-metamorphic features resulting from two principal episodes of metamorphism, M 1 and M 2 . The earlier prograde event, M 1 , and coeval regional deformation, D 1 , were accompanied by overall ductile deformation of sulfides and by recrystallization and remobilization of the mineralization parallel to foliation. M 1 caused development of a pervasive east-west-trending metamorphic layering of the pyritic mineralization, transposition and elongation of the pyritic bodies parallel to mineral stretching L 1 lineations, meter-scale folding of sulfides, sulfide brecciation, coarsening and annealing recrystallization of pyrite, and ductile remobilization of incompetent sulfides and gold into interstices and triple-point junctions of juxtaposed pyrite grains. Extensive differential remobilization of ductile sulfides (mainly sphalerite, pyrrhotite, galena, chalcopyrite, chalcocite, and bornite) and gold took place during M 1 and gave rise to planar zones enriched in Au-Ag-Cu mineralization along the interfaces of pyritic lenses and their adjacent silicate rocks.M 2 metamorphism is chronologically related to a late brittle incremental stage of the progressive deformation and is characterized by high fluid influx, pervasive retrogression of peak metamorphic minerals, and remobilization of sulfides and gold into transgressive structures. The retrograde mineralization displays a wide range of small-scale structures resulting from fluid-assisted remobilization of ductile sulfides, including piercement and injection structures, as well as foliation-oblique extensional veinlets. Metamorphic remobilization of ore constituents took place mainly at ore-wall rock interfaces. Fe-, Pb-Zn-, and Au-Ag-Cu- bearing extensional veinlets generally occur in foliation-parallel protores of similar mineralogical composition and are interpreted as having resulted from in situ metamorphic remobilization.Gold and copper show a corelationship throughout the deposit; the highest gold and copper values occur within extensional veins overprinting previously Au-Cu-enriched planar zones. Mineralogical zoning in the sulfide masses is structurally controlled and can be related directly to abundant Au-Cu-bearing veinlets that represent mineralized segments of a systematic joint system.Structural and textural evidence suggests that gold mineralization with its present textural relations formed during M 1 and M 2 , but that a large proportion of the structurally controlled vein mineralization resulted from in situ remobilization rather than from additions from an external source.

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.

The widespread distribution of insects over many ecological niches is a testimony to their evolutionary success. The colonization of environments at high latitudes or altitudes required the evolution of biochemical strategies that reduced the impact of cold or freezing stress. This review focuses on our current interests in some of the genes and proteins involved in low temperature survival in insects. Although the most widespread form of protection is the synthesis of low molecular weight polyol cryoprotectants, proteins with intrinsic protective properties, such as the thermal hysteresis or antifreeze proteins are also important. These have been cloned and characterized in certain moths and beetles. Molecular techniques allowing the isolation of genes differentially regulated by low temperatures have revealed that heat shock proteins, cold stress proteins, membrane protectants, as well as ice nucleators and other less well characterized proteins likely also play a role in cold hardiness.