Draft whole-genome sequences of Bacillus strains of potential probiotic for poultry.

Bacillus species have been widely selected and used as probiotics for humans and animals. In this article, we reported draft whole-genome sequences of four Bacillus strains isolated from sourdough and chicken cecum and previously selected as potential probiotics for poultry. These genome sequences will provide a foundation for further characterization and understanding of their probiotic attributes.

Draft whole-genome sequences of competitive exclusion Ligilactobacillus salivarius strains for poultry.

Competitive exclusion (CE) bacteria have been used to control the colonization of chickens by major foodborne pathogens. In this article, we report draft whole-genome sequences of three Ligilactobacillus salivarius strains isolated from chicken gastrointestinal tracts and previously selected as CE for poultry. These genome sequences will provide a foundation for further characterization and understanding of their CE attributes.

Prevalence of Shiga Toxin-Producing Escherichia coli and Salmonella in Native Pecan Orchards as Influenced by Waiting Periods between Grazing and Harvest.

ABSTRACT: Animals (grazing, working, or intrusion) in produce production areas may present a potential contamination source of foodborne pathogens on produce. Cattle grazing on native pecan production orchards, a common practice in the southern United States, provides an opportunity to study the impact of grazing practice and waiting periods on contamination rates of foodborne pathogens of tree nuts. Therefore, the objective of this study was to determine the prevalence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in native pecan production orchards as influenced by waiting periods between grazing cattle and pecan harvest. Soil (10 g), cattle feces (10 g), and in-shell pecans (25 g) were sampled from five cattle-grazed orchards in areas with cattle removed 2 or 4 months before harvest and not removed. Five nongrazing orchards were sampled at harvest for comparison. Detection and isolation of the pathogens were performed by enrichment, selective isolation, and multiplex PCR. Statistical analyses were performed using contingency tables with Pearson's chi-square test. The prevalence of STEC (36%) and Salmonella (29%) in cattle-grazed orchards was significantly higher than in nongrazed orchards (13 and 7%, respectively). STEC prevalence in cattle-grazed orchards was higher (38%) in areas with cattle at harvest than in fenced areas where cattle were removed 2 (29%) and 4 (27%) months before harvest. Salmonella prevalence was similar in areas without fencing (31%) and areas with cattle removed at 2 (22%) and 4 (30%) months before harvest. However, there were no significant differences (P > 0.05) in contamination rates between waiting periods for either pathogen, suggesting a limited impact of waiting periods on reducing the risk of contamination.

Isolation and characterization of Bacillus spp. strains as potential probiotics for poultry.

Probiotics have become one of the potential solutions to global restriction on antibiotic use in food animal production. Bacillus species have been attractive probiotics partially due to their long-term stability during storage. In this study, 200 endospore-forming bacteria isolates were recovered from sourdough and the gastrointestinal tract (GIT) of young broiler chicks. Based on the production of a series of exoenzymes and survivability under stress conditions similar to those in the poultry GIT, 42 isolates were selected and identified by 16S rRNA gene sequencing. Seven strains with a profile of high enzymatic activities were further evaluated for sporulation efficiency, biofilm formation, compatibility among themselves (Bacillus spp.), and antagonistic effects against three bacteria pathogenic to poultry and humans: Enterococcus cecorum, Salmonella enterica, and Shiga-toxin-producing Escherichia coli. The strains from sourdough were identified as Bacillus amyloliquefaciens whereas the ones from the chicks' GIT were Bacillus subtilis. These strains demonstrated remarkable potential as probiotics for poultry.

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

As an emerging sterilization technology, cold atmospheric plasma offers a dry, non-thermal, rapid process that is minimally damaging to a majority of substrates. However, the mechanisms by which plasma interacts with living cells are poorly understood and the plasma generation apparatuses are complex and resource-intensive. In this study, the roles of reactive oxygen species (ROS), nitric oxide (NO), and charged particles (ions) produced by surface dielectric barrier discharge (SDBD) plasma on prokaryotic (Listeria monocytogenes (Gram-positive)) and eukaryotic (human umbilical vein endothelial cells (HUVEC)) cellular function were evaluated. HUVEC and bacterial oxidative stress responses, the accumulation of nitrite in aqueous media, air ion density, and bacterial inactivation at various distances from SDBD actuators were measured. SDBD actuator designs were also varied in terms of electrode number and length to evaluate the cellular effects of plasma volume and power distribution. NO and ions were found to contribute minimally to the observed cellular effects, whereas ROS were found to cause rapid bacterial inactivation, induce eukaryotic and prokaryotic oxidative stress, and result in rapid oxidation of bovine muscle tissue. The results of this study underscore the dominance of ROS as the major plasma generated species responsible for cellular effects, with ions and RNS having a secondary, complimentary role.

Importance of Frontal Horn Ratio and Optimal CSF Drainage in the Treatment of Very Low-Pressure Hydrocephalus.

Introduction Unlike low-pressure hydrocephalus, very low pressure hydrocephalus (VLPH) is a rarely reported clinical entity previously described to be associated with poor outcomes and to be possibly refractory to treatment with continued cerebrospinal fluid (CSF) drainage at subatmospheric pressures. 1, 2 We present four cases of VLPH following resection of suprasellar lesions and hypothesize that untreatable patients can be identified early, thereby avoiding futile prolonged external ventricular drainage in ICU. Methods We performed a retrospective chart review of four cases of VLPH encountered between 2007 and 2015 in two different institutions and practices and tried to identify factors contributing to successful treatment. We hypothesized that normalization of frontal horn ratio (FHR), optimization of volume of CSF drained, and avoidance of fluid shifts would contribute to improved Glasgow Coma Score (GCS). We examined fluid shifts by studying net fluids shifts and serum levels of sodium, urea, and creatinine. We used Pearson and Spearman correlations to identify measures that would correlate with improved GCS. Results Our study reveals that improving GCS is positively correlated with decreased FHR and increased CSF drainage within an optimal range. The most important determinant of good outcome is retention of brain viscoelasticity as evidenced by restoration and maintenance of good GCS score despite fluctuations in FHR. Conclusion Futile prolonged subatmospheric drainage can be avoided by declining to continue treatment in patients who have permanently altered brain compliance secondary to unsealed CSF leaks, irremediable ventriculitis, and who are therefore unable to sustain an improved neurologic examination.

Small power and lighting load time series data for 27 departments across 8 UK hospitals.

The electricity consumption of 27 departments was measured across 8 medium to large General Acute hospitals in England (largely by the authors, some data was donated and authorised for publication by the respective hospitals). The departments fall into 6 different categories which have been selected due to their prevalence in General Acute Hospitals (wards), their high energy intensities (theatres, laboratories, imaging and radiotherapy) or their distinct operating hours (day clinics). This data article provides floor areas and the time series of departmental power loads, mostly encompassing lighting and small power (but excluding central electricity use for ventilation, pumping and medical gas services). Comparative interpretations of the data are published in doi: 10.1016/j.enbuild.2016.02.052 [1].

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups-O26, O111, O103, O121, O45, and O145-and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks.

Quantitative metabolome, proteome and transcriptome analysis of midgut and fat body tissues in the mountain pine beetle, Dendroctonus ponderosae Hopkins, and insights into pheromone biosynthesis.

Bark beetles (Coleoptera: Scolytinae) are pests of many forests around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant pest of western North American pine forests. The MPB is able to overcome the defences of pine trees through pheromone-assisted aggregation that results in a mass attack of host trees. These pheromones, both male and female produced, are believed to be biosynthesized in the midgut and/or fat bodies of these insects. We used metabolite analysis, quantitative proteomics (iTRAQ) and transcriptomics (RNA-seq) to identify proteins and transcripts differentially expressed between sexes and between tissues when treated with juvenile hormone III. Juvenile hormone III induced frontalin biosynthesis in males and trans-verbenol biosynthesis in females, as well as affected the expression of many proteins and transcripts in sex- and tissue-specific ways. Based on these analyses, we identified candidate genes involved in the biosynthesis of frontalin, exo-brevicomin, and trans-verbenol pheromones.

Surface survival and internalization of salmonella through natural cracks on developing cantaloupe fruits, alone or in the presence of the melon wilt pathogen Erwinia tracheiphila.

Outbreaks of foodborne illness attributed to the consumption of Salmonella-tainted cantaloupe have occurred repeatedly, but understanding of the ecology of Salmonella on cantaloupe fruit surfaces is limited. We investigated the interactions between Salmonella enterica Poona, the plant pathogenic bacterium Erwinia tracheiphila, and cantaloupe fruit. Fruit surfaces were inoculated at the natural cracking stage by spreading S. enterica and E. tracheiphila, 20 µl at 107 cfu/ml, independently or together, over a 2×2 cm rind area containing a crack. Microbial and microscopic analyses were performed at 0, 9 and 24 days post inoculation (DPI). Even at 24 DPI (fruit maturity) S. enterica was detected on 14% and 40% of the fruit inoculated with S. enterica alone and the two-pathogen mixture, respectively. However, the population of S. enterica declined gradually after initial inoculation. E. tracheiphila, inoculated alone or together with Salmonella, caused watersoaked lesions on cantaloupe fruit; but we could not conclude in this study that S. enterica survival on the fruit surface was enhanced by the presence of those lesions. Of fruit inoculated with E. tracheiphila alone and sampled at 24 DPI, 61% had watersoaked lesions on the surface. In nearly half of those symptomatic fruits the watersoaking extended into the sub-rind mesocarp, and E. tracheiphila was recovered from that tissue in 50% of the symptomatic fruit. In this work, E. tracheiphila internalized through natural cracks on developing fruits. S. enterica was never detected in the fruit interior (ca. 2-3 mm below rind surface) under the limited conditions of our experiments, but the possibility that it, or other human pathogens that contaminate fresh produce, might also do so should be investigated under a wider range of conditions and produce types.

Survival and growth of foodborne pathogens in pesticide solutions routinely used in leafy green vegetables and tomato production.

BACKGROUND: The consumption of fresh produce has increased tremendously in the past few years as have outbreaks of foodborne illnesses associated with these commodities. Pesticides routinely used in crop production could influence the outcomes of foodborne pathogen contamination of fresh produce. Experiments were performed to determine the effects of pesticides on the survival and growth characteristics of Escherichia coli O157:H7 and Salmonella spp. Eight commercial fungicides and insecticides commonly used for disease and insect pest control on leafy green vegetables and tomatoes were evaluated. RESULTS: Among the pesticides tested, copper hydroxide, acetamiprid, cypermethrin and permethrin were found to be significantly (P < 0.05) inhibitory to pathogens while no effect was observed for chlorothalonil, flonicamid and methoxyfenozide. At the highest concentration tested (2.66%), azoxystrobin had a significant (P < 0.05) stimulatory effect on the growth of E. coli O157:H7 after 24 h incubation. The results indicated that some pesticides can stimulate the growth of human pathogens if contaminated water is used in their preparation, whereas others were likely to inhibit or reduce pathogen populations. CONCLUSION: This information is helpful in mitigating the risk of microbial contamination in fresh produce, which is critical to public health and safety.

The lion's mane sign: surgical results using the bilateral fronto-orbito-nasal approach in large and giant anterior skull base meningiomas.

OBJECT: Concerns about extreme peritumoral edema and its ensuing surgical and perioperative complications led the authors to use the bilateral fronto-orbito-nasal approach to remove midline anterior skull base meningiomas that were larger than 4 cm. The authors hypothesize that extreme vasogenic edema exemplified by finger-like hyperintensities extending into the bifrontal white matter and external capsule and/or the extreme capsule, coined the lion's mane sign (LMS), would help identify patients with a challenging postoperative course. They hypothesize that the LMS would better predict symptomatic postoperative cerebral edema than the edema index (EI). METHODS: This is an observational case series of 9 patients. The authors noted the grade, pathology, tumor volume, EI, and the presence or absence of the LMS in all tumors. They used the intensive unit care (ICU) length of stay as a nonspecific measure reflecting postoperative symptomatic cerebral edema. Comparisons of edema-related postoperative complications and the EI were made between patients with and without an LMS. RESULTS: Bifrontal hyperintensities, extending into at least three-eighths of the length of the external capsules on T2-weighted MRI, seen in 4 of 9 patients, portended a longer postoperative ICU stay. The presence of an LMS better predicted postoperative complications related to cerebral edema than tumor grade, pathology, volume, or EI. CONCLUSIONS: The LMS predicts an increased duration of stay in the ICU after a bilateral fronto-orbito-nasal approach for resection of large and giant anterior skull base meningiomas. Furthermore, the LMS better predicted increased length of stay in the ICU than did the EI.

CYP345E2, an antenna-specific cytochrome P450 from the mountain pine beetle, Dendroctonus ponderosae Hopkins, catalyses the oxidation of pine host monoterpene volatiles.

The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a significant pest of western North American pine forests. This beetle responds to pheromones and host volatiles in order to mass attack and thus overcome the terpenoid chemical defences of its host. The ability of MPB antennae to rapidly process odorants is necessary to avoid odorant receptor saturation and thus the enzymes responsible for odorant clearance are an important aspect of host colonization. An antenna-specific cytochrome P450, DponCYP345E2, is the most highly expressed transcript in adult MPB antenna. In in vitro assays with recombinant enzyme, DponCYP345E2 used several pine host monoterpenes as substrates, including (+)-(3)-carene, (+)-ß-pinene, (-)-ß-pinene, (+)-limonene, (-)-limonene, (-)-camphene, (+)-α-pinene, (-)-α-pinene, and terpinolene. The substrates were epoxidized or hydroxylated, depending upon the substrate. To complement DponCYP345E2, we also functionally characterized the NADPH-dependent cytochrome P450 reductase and the cytochrome b5 from MPB. DponCYP345E2 is the first cytochrome P450 to be functionally characterized in insect olfaction and in MPB.

Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

Transcriptome resources and functional characterization of monoterpene synthases for two host species of the mountain pine beetle, lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana).

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae) epidemic has affected lodgepole pine (Pinus contorta) across an area of more than 18 million hectares of pine forests in western Canada, and is a threat to the boreal jack pine (Pinus banksiana) forest. Defence of pines against MPB and associated fungal pathogens, as well as other pests, involves oleoresin monoterpenes, which are biosynthesized by families of terpene synthases (TPSs). Volatile monoterpenes also serve as host recognition cues for MPB and as precursors for MPB pheromones. The genes responsible for terpene biosynthesis in jack pine and lodgepole pine were previously unknown. RESULTS: We report the generation and quality assessment of assembled transcriptome resources for lodgepole pine and jack pine using Sanger, Roche 454, and Illumina sequencing technologies. Assemblies revealed transcripts for approximately 20,000 - 30,000 genes from each species and assembly analyses led to the identification of candidate full-length prenyl transferase, TPS, and P450 genes of oleoresin biosynthesis. We cloned and functionally characterized, via expression of recombinant proteins in E. coli, nine different jack pine and eight different lodgepole pine mono-TPSs. The newly identified lodgepole pine and jack pine mono-TPSs include (+)-α-pinene synthases, (-)-α-pinene synthases, (-)-ß-pinene synthases, (+)-3-carene synthases, and (-)-ß-phellandrene synthases from each of the two species. CONCLUSION: In the absence of genome sequences, transcriptome assemblies are important for defence gene discovery in lodgepole pine and jack pine, as demonstrated here for the terpenoid pathway genes. The product profiles of the functionally annotated mono-TPSs described here can account for the major monoterpene metabolites identified in lodgepole pine and jack pine.

Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest.

BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. RESULTS: We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. CONCLUSIONS: Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae).

BACKGROUND: The European spruce bark beetle, Ips typographus, and the North American mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae), are severe pests of coniferous forests. Both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host trees modulate the pheromone response. Thus, the bark beetle olfactory sense is of utmost importance for fitness. However, information on the genes underlying olfactory detection has been lacking in bark beetles and is limited in Coleoptera. We assembled antennal transcriptomes from next-generation sequencing of I. typographus and D. ponderosae to identify members of the major chemosensory multi-gene families. RESULTS: Gene ontology (GO) annotation indicated that the relative abundance of transcripts associated with specific GO terms was highly similar in the two species. Transcripts with terms related to olfactory function were found in both species. Focusing on the chemosensory gene families, we identified 15 putative odorant binding proteins (OBP), 6 chemosensory proteins (CSP), 3 sensory neuron membrane proteins (SNMP), 43 odorant receptors (OR), 6 gustatory receptors (GR), and 7 ionotropic receptors (IR) in I. typographus; and 31 putative OBPs, 11 CSPs, 3 SNMPs, 49 ORs, 2 GRs, and 15 IRs in D. ponderosae. Predicted protein sequences were compared with counterparts in the flour beetle, Tribolium castaneum, the cerambycid beetle, Megacyllene caryae, and the fruit fly, Drosophila melanogaster. The most notable result was found among the ORs, for which large bark beetle-specific expansions were found. However, some clades contained receptors from all four beetle species, indicating a degree of conservation among some coleopteran OR lineages. Putative GRs for carbon dioxide and orthologues for the conserved antennal IRs were included in the identified receptor sets. CONCLUSIONS: The protein families important for chemoreception have now been identified in three coleopteran species (four species for the ORs). Thus, this study allows for improved evolutionary analyses of coleopteran olfaction. Identification of these proteins in two of the most destructive forest pests, sharing many semiochemicals, is especially important as they might represent novel targets for population control.

Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera.