A TaqMan Assay for the Detection and Monitoring of Potentially Invasive Lasiocampids, With Particular Attention to the Siberian Silk Moth, Dendrolimus sibiricus (Lepidoptera: Lasiocampidae).

The Siberian silk moth, Dendrolimus sibiricus Tschetverikov, is a very serious pest of conifers in Russia and is an emerging threat in North America where an accidental introduction could have devastating impacts on native forest resources. Other Dendrolimus Germar species and related Eurasian lasiocampids in the genus Malacosoma (Hubner) could also present a risk to North America's forests. Foreign vessels entering Canadian and U.S. ports are regularly inspected for Lymantria dispar (Linnaeus) and for the presence of other potentially invasive insects, including suspicious lasiocampid eggs. However, eggs are difficult to identify based on morphological features alone. Here, we report on the development of two TaqMan (Roche Molecular Systems, Inc., Rotkreuz, Switzerland) assays designed to assist regulatory agencies in their identification of these insects. Developed using the barcode region of the cytochrome c oxidase I (COI) gene and run in triplex format, the first assay can detect Dendrolimus and Malacosoma DNA, and can distinguish North American from Eurasian Malacosoma species. The second assay is based on markers identified within the internal transcribed spacer 2 (ITS2) region and was designed to specifically identify D. sibiricus, while discriminating closely related Dendrolimus taxa. In addition to providing direct species identification in the context of its use in North America, the D. sibiricus assay should prove useful for monitoring the spread of this pest in Eurasia, where its range overlaps with those of the morphologically identical D. superans (Butler) and similar D. pini (Linnaeus). The assays described here can be performed either in the lab on a benchtop instrument, or on-site using a portable machine.

Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure.

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high-proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3',5'-hydroxylases and cytochrome b5 Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.

Genomic and Proteomic Analyses Indicate that Banchine and Campoplegine Polydnaviruses Have Similar, if Not Identical, Viral Ancestors.

UNLABELLED: Polydnaviruses form a group of unconventional double-stranded DNA (dsDNA) viruses transmitted by endoparasitic wasps during egg laying into caterpillar hosts, where viral gene expression is essential to immature wasp survival. A copy of the viral genome is present in wasp chromosomes, thus ensuring vertical transmission. Polydnaviruses comprise two taxa, Bracovirus and Ichnovirus, shown to have distinct viral ancestors whose genomes were "captured" by ancestral wasps. While evidence indicates that bracoviruses derive from a nudivirus ancestor, the identity of the ichnovirus progenitor remains unknown. In addition, ichnoviruses are found in two ichneumonid wasp subfamilies, Campopleginae and Banchinae, where they constitute morphologically and genomically different virus types. To address the question of whether these two ichnovirus subgroups have distinct ancestors, we used genomic, proteomic, and transcriptomic analyses to characterize particle proteins of the banchine Glypta fumiferanae ichnovirus and the genes encoding them. Several proteins were found to be homologous to those identified earlier for campoplegine ichnoviruses while the corresponding genes were located in clusters of the wasp genome similar to those observed previously in a campoplegine wasp. However, for the first time in a polydnavirus system, these clusters also revealed sequences encoding enzymes presumed to form the replicative machinery of the progenitor virus and observed to be overexpressed in the virogenic tissue. Homology searches pointed to nucleocytoplasmic large DNA viruses as the likely source of these genes. These data, along with an analysis of the chromosomal form of five viral genome segments, provide clear evidence for the relatedness of the banchine and campoplegine ichnovirus ancestors. IMPORTANCE: Recent work indicates that the two recognized polydnavirus taxa, Bracovirus and Ichnovirus, are derived from distinct viruses whose genomes integrated into the genomes of ancestral wasps. However, the identity of the ichnovirus ancestor is unknown, and questions remain regarding the possibility that the two described ichnovirus subgroups, banchine and campoplegine ichnoviruses, have distinct origins. Our study provides unequivocal evidence that these two ichnovirus types are derived from related viral progenitors. This suggests that morphological and genomic differences observed between the ichnovirus lineages, including features unique to banchine ichnovirus genome segments, result from evolutionary divergence either before or after their endogenization. Strikingly, analysis of selected wasp genomic regions revealed genes presumed to be part of the replicative machinery of the progenitor virus, shedding new light on the likely identity of this virus. Finally, these genes could well play a role in ichnovirus replication as they were overexpressed in the virogenic tissue.

Opposite action of R2R3-MYBs from different subgroups on key genes of the shikimate and monolignol pathways in spruce.

Redundancy and competition between R2R3-MYB activators and repressors on common target genes has been proposed as a fine-tuning mechanism for the regulation of plant secondary metabolism. This hypothesis was tested in white spruce [Picea glauca (Moench) Voss] by investigating the effects of R2R3-MYBs from different subgroups on common targets from distinct metabolic pathways. Comparative analysis of transcript profiling data in spruces overexpressing R2R3-MYBs from loblolly pine (Pinus taeda L.), PtMYB1, PtMYB8, and PtMYB14, defined a set of common genes that display opposite regulation effects. The relationship between the closest MYB homologues and 33 putative target genes was explored by quantitative PCR expression profiling in wild-type P. glauca plants during the diurnal cycle. Significant Spearman's correlation estimates were consistent with the proposed opposite effect of different R2R3-MYBs on several putative target genes in a time-related and tissue-preferential manner. Expression of sequences coding for 4CL, DHS2, COMT1, SHM4, and a lipase thio/esterase positively correlated with that of PgMYB1 and PgMYB8, but negatively with that of PgMYB14 and PgMYB15. Complementary electrophoretic mobility shift assay (EMSA) and transactivation assay provided experimental evidence that these different R2R3-MYBs are able to bind similar AC cis-elements in the promoter region of Pg4CL and PgDHS2 genes but have opposite effects on their expression. Competitive binding EMSA experiments showed that PgMYB8 competes more strongly than PgMYB15 for the AC-I MYB binding site in the Pg4CL promoter. Together, the results bring a new perspective to the action of R2R3-MYB proteins in the regulation of distinct but interconnecting metabolism pathways.

Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca).

BACKGROUND: Among the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. RESULTS: Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. CONCLUSIONS: While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction.

Development of a qPCR-based method for counting overwintering spruce budworm (Choristoneura fumiferana) larvae collected during fall surveys and for assessing their natural enemy load: a proof-of-concept study.

BACKGROUND: In eastern Canada, surveys of overwintering 2nd instar spruce budworm (Choristoneura fumiferana) larvae ('L2s') are carried out each fall to guide insecticide application decisions in the following spring. These surveys involve the collection of fir and spruce branches in selected stands, followed by the mechanical/chemical removal of larvae. The latter then are counted manually on filter papers, using a stereomicroscope. Considering the significant effort and difficulties which this manual counting entails, we developed a quantitative (q)PCR-based 'molecular counting' approach designed to make this step less tedious. RESULTS: Using the C. fumiferana mitochondrial cytochrome c oxidase 1 (COI) gene as a target for qPCR DNA quantification, we show that the amount of DNA in a larval extract is strongly correlated with the number of larvae used to generate that extract, and that molecular estimates of L2 counts are comparable to those generated using the manual approach. In addition, we used the same DNA extracts to monitor the microsporidian pathogen Nosema fumiferanae, and the hymenopteran parasitoids Glypta fumiferanae and Apanteles fumiferanae in overwintering L2s employing a subset of a TaqMan assay developed by Nisole et al. (2020) for the identification of budworm natural enemies. We show that the proportion of individuals affected by each natural enemy in samples containing a known number of larvae can be estimated from presence/absence data through the binomial probability distribution. CONCLUSION: The present proof-of-principle study shows that a molecular approach for counting L2s and assessing their natural enemy load is clearly possible and is expected to generate reliable results. © 2021 Her Majesty the Queen in Right of Canada. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Reproduced with the permission of the Minister of Natural Resources Canada.

Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process.

Adult conifers are notoriously recalcitrant in vegetative propagation and micropropagation that would result in the regeneration of juvenile propagules through somatic embryogenesis (SE) has not been demonstrated to date. Because SE-derived material is more amenable in subsequent tissue culture experiments compared with seed-derived material, a multi-year study was conducted to investigate induction of SE from primordial shoot (PS) explants that were excised from shoot buds of somatic embryo-derived white spruce. The SE induction experiments were carried out first with greenhouse-grown and later with field-grown trees each year from 2002 (2-year-old) to 2010 (10-year-old). Of the four genotypes tested, 893-2 and 893-12 never responded, 893-1 responded up to year 4 and 893-6 consistently responded every year. In 2010, for the first time, three of the 17 893-6 clonal trees produced male strobili as well as SE from cultured PS explants. SE induction was associated with formation of a nodule on the surface of an elongated needle primordium or in callus. Early somatic embryos were detectable after about 3 weeks of culture. Of 11 genes whose expression profiles were followed during the PS cultures, CHAP3A, VP1, WOX2 and SAP2C were expressed exclusively in the early stages of SE, and could potentially be used as markers of embryogenecity. Mature somatic embryos and plants were produced from the explants of responding genotype. Implication of these results for future research on adult conifer recalcitrance in micropropagation is discussed.

Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 (CHAP3A) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth.

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-beta-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed.

In Situ Processing and Efficient Environmental Detection (iSPEED) of tree pests and pathogens using point-of-use real-time PCR.

Global trade and climate change are responsible for a surge in foreign invasive species and emerging pests and pathogens across the world. Early detection and surveillance activities are essential to monitor the environment and prevent or mitigate future ecosystem impacts. Molecular diagnostics by DNA testing has become an integral part of this process. However, for environmental applications, there is a need for cost-effective and efficient point-of-use DNA testing to obtain accurate results from remote sites in real-time. This requires the development of simple and fast sample processing and DNA extraction, room-temperature stable reagents and a portable instrument. We developed a point-of-use real-time Polymerase Chain Reaction system using a crude buffer-based DNA extraction protocol and lyophilized, pre-made, reactions for on-site applications. We demonstrate the use of this approach with pathogens and pests covering a broad spectrum of known undesirable forest enemies: the fungi Sphaerulina musiva, Cronartium ribicola and Cronartium comandrae, the oomycete Phytophthora ramorum and the insect Lymantria dispar. We obtained positive DNA identification from a variety of different tissues, including infected leaves, pathogen spores, or insect legs and antenna. The assays were accurate and yielded no false positive nor negative. The shelf-life of the lyophilized reactions was confirmed after one year at room temperature. Finally, successful tests conducted with portable thermocyclers and disposable instruments demonstrate the suitability of the method, named in Situ Processing and Efficient Environmental Detection (iSPEED), for field testing. This kit fits in a backpack and can be carried to remote locations for accurate and rapid detection of pests and pathogens.

Extra-mitochondrial localization and likely reproductive function of a female-transmitted cytochrome c oxidase subunit II protein.

Our previous study documented a reproductive function for the male-transmitted mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit II (MCOX2) protein in a unionoid bivalve. Here, immunoblotting, immunohistochemistry and immunoelectron microscopy analyses demonstrate that the female-transmitted protein (FCOX2) is: (i) expressed in both male and female gonads; (ii) maximally expressed in ovaries just prior to the time of the annual fertilization event; (iii) displayed in the cytoplasm and more strongly in the plasma membrane (microvilli), vitelline matrix and vitelline envelope of mature ovarian eggs; and (iv) strongly localized to the vitelline matrix of some eggs just prior to fertilization. These findings represent evidence for the extra-mitochondrial localization of an mtDNA-encoded gene product and are consistent with multifunctionality for FCOX2 in eggs.

Genome-enhanced detection and identification of fungal pathogens responsible for pine and poplar rust diseases.

Biosurveillance is a proactive approach that may help to limit the spread of invasive fungal pathogens of trees, such as rust fungi which have caused some of the world's most damaging diseases of pines and poplars. Most of these fungi have a complex life cycle, with up to five spore stages, which is completed on two different hosts. They have a biotrophic lifestyle and may be propagated by asymptomatic plant material, complicating their detection and identification. A bioinformatics approach, based on whole genome comparison, was used to identify genome regions that are unique to the white pine blister rust fungus, Cronartium ribicola, the poplar leaf rust fungi Melampsora medusae and Melampsora larici-populina or to members of either the Cronartium and Melampsora genera. Species- and genus-specific real-time PCR assays, targeting these unique regions, were designed with the aim of detecting each of these five taxonomic groups. In total, twelve assays were developed and tested over a wide range of samples, including different spore types, different infected plant parts on the pycnio-aecial or uredinio-telial host, and captured insect vectors. One hundred percent detection accuracy was achieved for the three targeted species and two genera with either a single assay or a combination of two assays. This proof of concept experiment on pine and poplar leaf rust fungi demonstrates that the genome-enhanced detection and identification approach can be translated into effective real-time PCR assays to monitor tree fungal pathogens.

Functional Analysis of the PgCesA3 White Spruce Cellulose Synthase Gene Promoter in Secondary Xylem.

Cellulose is an essential structural component of the plant cell wall. Its biosynthesis involves genes encoding cellulose synthase enzymes and a complex transcriptional regulatory network. Three cellulose synthases have been identified in conifers as being potentially involved in secondary cell wall biosynthesis because of their preferential expression in xylem tissues; however, no direct functional association has been made to date. In the present work, we characterized the white spruce [Picea glauca (Moench) Voss] cellulose synthase PgCesA3 gene and 5' regulatory elements. Phylogenetic analysis showed that PgCesA1-3 genes grouped with secondary cell wall-associated Arabidopsis cellulose synthase genes, such as AtCesA8, AtCesA4, and AtCesA7. We produced transgenic spruce expressing the GUS reporter gene driven by the PgCesA3 promoter. We observed blue staining in differentiating xylem cells from stem and roots, and in foliar guard cells indicating that PgCesA3 is clearly involved in secondary cell wall biosynthesis. The promoter region sequence of PgCesA3 contained several putative MYB cis-regulatory elements including AC-I like motifs and secondary wall MYB-responsive element (SMRE); however, it lacked SMRE4, 7 and 8 that correspond to the sequences of AC-I, II, and III. Based on these findings and results of previous transient trans-activation assays that identified interactions between the PgCesA3 promoter and different MYB transcription factors, we performed electrophoretic mobility shift assays with MYB recombinant proteins and cis-regulatory elements present in the PgCesA3 promoter. We found that PgMYB12 bound to a canonical AC-I element identified in the Pinus taeda PAL promoter and two AC-I like elements. We hypothesized that the PgMYB12 could regulate PgCesA3 in roots based on previous expression results. This functional study of PgCesA3 sequences and promoter opens the door for future studies on the interaction between PgMYBs and the PgCesA3 regulatory elements.

Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR.

BACKGROUND: The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amplification efficiency determination. However, in addition to being highly resource intensive, the efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency as predicted by a standard curve. These limitations have driven efforts to develop methods for determining amplification efficiency by analyzing the fluorescence readings from individual amplification reactions. The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region. Nevertheless, a recently developed sigmoidal model has provided new insights that challenge such historically held views, dictating that amplification efficiency is not only dynamic, but is linearly coupled to amplicon DNA quantity. Called "linear regression of efficiency" or LRE, this kinetic-based approach redefines amplification efficiency as the maximal efficiency (Emax) generated at the onset of thermocycling. RESULTS: This study presents a critical evaluation of amplification efficiency determination, which reveals that potentially large underestimations occur when exponential mathematics is applied to the log-linear region. This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate. In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics. This paradoxical result implies that the quantitative efficacy of positional-based analysis relies not upon the exponential character of real-time PCR, but instead on the ability to precisely define the relative position of an amplification profile. CONCLUSION: In addition to presenting insights into the sigmoidal character of the polymerase chain reaction, LRE analysis provides a viable alternative to standard curves for amplification efficiency determination, based on analysis of high-quality fluorescence readings within the central region of SYBR Green I generated amplification profiles.

A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR.

BACKGROUND: Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach. RESULTS: Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of +/- 25% can be routinely achieved. Comparison with the LinReg and Miner automated qPCR data processing packages further demonstrated the superior performance of this kinetic-based methodology. CONCLUSION: Called "linear regression of efficiency" or LRE, this novel kinetic approach confers the ability to conduct high-capacity absolute quantification with unprecedented quality control capabilities. The computational simplicity and recursive nature of LRE quantification also makes it amenable to software implementation, as demonstrated by a prototypic Java program that automates data analysis. This in turn introduces the prospect of conducting absolute quantification with little additional effort beyond that required for the preparation of the amplification reactions.

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents.

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.

Executive management and IT innovation in health: identifying the barriers to adoption.

This study aims to understand IT investment decisions from the perspective of senior health system executives. A two-stage study was used to investigate this highly influential, very specialized and small population of interest. The first stage involved qualitative interviews with top health executives and analysed their opinions and beliefs using an innovation diffusion theory framework. The second stage involved quantitative surveys of senior health executives to gain an understanding of their opinions regarding the organizational and technological drivers (the independent variables) and the level of IT adoption (the dependent variable). It was found that the majority of drivers identified as being significant to organizational and technological innovation are degraded in respect to IT and health. It was concluded that health executives hold a range of views that potentially inhibit the increased adoption of IT in health. In particular, beliefs about the technology itself have been identified as the most influential deterrents.

High temperature induces downregulation of polydnavirus gene transcription in lepidopteran host and enhances accumulation of host immunity gene transcripts.

Endoparasitoids face the challenge of overcoming the immune reaction of their hosts, which typically consists of encapsulation and melanisation of parasitoid eggs or larvae. Some endoparasitic wasps such as the solitary Tranosema rostrale (Hymenoptera: Ichneumonidae) that lay their eggs in larvae of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), have evolved a symbiotic relationship with a polydnavirus (PDV), which in turn helps them suppress the host's immune response. We observed an increase in mortality of immature T. rostrale with increasing temperature, and we tested two hypotheses about the mechanisms involved: high temperatures (1) hamper the expression of T. rostrale PDV genes and (2) enhance the expression of spruce budworm immunity-related genes. Dissections of parasitized spruce budworm larvae reared at 30°C revealed that most parasitoid eggs or larvae had died as a result of encapsulation and melanisation by the host. A qPCR analysis of T. rostrale PDV (TrIV) gene expression showed that the transcription of several TrIV genes in host larvae was downregulated at high temperature. On the other hand, encapsulation, but not melanisation, of foreign bodies in spruce budworm larvae was enhanced at high temperatures, as shown by the injection of Sephadex™ beads into larvae. However, at the molecular level, the transcription of genes related to spruce budworm's melanisation process (prophenoloxidase 1 and 2) was upregulated. Our results support the hypothesis that a temperature-dependent increase of encapsulation response is due to the combined effects of reduced expression of TrIV genes and enhanced expression of host immune genes.

Comparative analysis of mitochondrial genomes of geographic variants of the gypsy moth, Lymantria dispar, reveals a previously undescribed genotypic entity.

The gypsy moth, Lymantria dispar L., is one of the most destructive forest pests in the world. While the subspecies established in North America is the European gypsy moth (L. dispar dispar), whose females are flightless, the two Asian subspecies, L. dispar asiatica and L. dispar japonica, have flight-capable females, enhancing their invasiveness and warranting precautionary measures to prevent their permanent establishment in North America. Various molecular tools have been developed to help distinguish European from Asian subspecies, several of which are based on the mitochondrial barcode region. In an effort to identify additional informative markers, we undertook the sequencing and analysis of the mitogenomes of 10 geographic variants of L. dispar, including two or more variants of each subspecies, plus the closely related L. umbrosa as outgroup. Several regions of the gypsy moth mitogenomes displayed nucleotide substitutions with potential usefulness for the identification of subspecies and/or geographic origins. Interestingly, the mitogenome of one geographic variant displayed significant divergence relative to the remaining variants, raising questions about its taxonomic status. Phylogenetic analyses placed this population from northern Iran as basal to the L. dispar clades. The present findings will help improve diagnostic tests aimed at limiting risks of AGM invasions.

Characterization and tissue-specific expression of two lepidopteran farnesyl diphosphate synthase homologs: implications for the biosynthesis of ethyl-substituted juvenile hormones.

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl-substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl-branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type-I FPPSs display unique active site substitutions, including several in and near the first aspartate-rich motif, whereas type-II proteins have a more "conventional" catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type-I enzymes are better suited than type-II for generating ethyl-substituted products, a quantitative real-time PCR assessment of their relative abundance in insect tissues indicated that type-I expression is ubiquitous whereas that of type-II is essentially confined to the JH-producing glands, where its transcripts are approximately 20 times more abundant than those of type-I. These results suggest that type-II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity.

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR.

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.