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Purpose: Several reports have revealed a difference in various populations regarding the variability of intraocular pressure (IOP) and corneal hysteresis (CH). This retrospective study was created to determine a profile for U.S. Veterans in a large outpatient clinical setting. OVERVIEW AND METHODOLOGY: The objective was to evaluate a random cohort of patient records in this clinical setting to determine the mean and standard deviation (SD) of the following parameters: Goldmann-correlated IOP (IOPg), CH, waveform score, and central corneal thickness from the Reichert Ocular Response Analyzer.Results: All patient records were reviewed by Veteran Health Affairs Eye clinical staff which resulted in data collected on 538 eyes. The mean IOP of this cohort for IOPg was 17.39 (SD 10.08). The mean for CH was calculated at 9.61 (SD 3.04) with a waveform score mean of 5.66 (SD 2.18) and central corneal thickness for 207 eyes was a mean of 545.09 (SD 51.09) nm.Conclusion: From the results, IOPg appears to be significantly higher and CH significantly lower than other populations reported in the literature. This information provides an accurate baseline for the evaluation of the U.S. veteran population for future. [ABSTRACT FROM AUTHOR]

Major evolutionary transitions, such as the shift of cetaceans from terrestrial to marine life, can put pressure on sensory systems to adapt to a new set of relevant stimuli. Relatively little is known about the role of smell in the evolution of mysticetes (baleen whales). While their toothed cousins, the odontocetes, lack the anatomical features to smell, it is less clear whether baleen whales have retained this sense, and if so, when the pressure on olfaction diverged in the cetacean evolutionary lineage. We examined eight genes encoding olfactory signal transduction pathway components and key chaperones for signs of inactivating mutations and selective pressures. All of the genes we examined were intact in all eight mysticete genomes examined, despite inactivating mutations in odontocete homologs in multiple genes. We also tested several models representing various hypotheses regarding the evolutionary history of olfaction in cetaceans. Our results support a model where olfactory ability is specifically reduced in the odontocete lineage following their split from stem cetaceans and serve to clarify the evolutionary history of olfaction in cetaceans., Competing Interests: Declarations. Competing Interests: The authors declare they have no financial interests. Ethical approval: The Byrde’s whale tissue sample used in this study was collected from a dead beach-cast whale on 29 March 2010. The sample is curated at the University of Auckland in the New Zealand Cetacean Tissue Archive, an archive of national significance on behalf of the New Zealand Department of Conservation in partnership with the Indigenous people—Maori., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This article is the third of 3 articles in a series about managing the care of physicians as patients. In part 1, the authors reviewed unique characteristics of physicians as patients with some general guidance for how to approach their care. Part 2 highlighted role clarity for the treating physician with discussion of the physical and cognitive issues that commonly arise when treating physician-patients along with licensure issues and reporting requirements. This final installment will focus on physician mental health and work-related stress., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This second installment in a 3-part series about physicians as patients explores challenges in communication and role definition while managing their care and safe return to work. In the first article of the series, authors reviewed unique characteristics that make physicians different as patients, with some general guidance about how to approach their care. Although most treating physicians receive little occupational training, health issues commonly have an impact on work with imperative to address work issues promptly for best outcome. This paper demystifies the challenge of managing work status and discusses navigating common physical and cognitive issues while maintaining role clarity. The treating clinician reading this paper will learn to avoid common pitfalls and be better equipped to provide initial assessments and interventions to keep physicians working safely, keeping in mind licensure issues and reporting requirements. Part Three of the series will focus on the most common mental health issues seen in physicians., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This is the first article of a 3-part series about physician health. In this installment, we outline the unique characteristics of physicians as patients, challenges and opportunities presented by physician-patients, and recommendations for treating physicians. Future articles will delve into role clarity, occupational considerations, mental health, and interactions with third parties such as the physician's employer or licensing board. Ultimately, this series will help treating clinicians provide the best care to their physician-patients and successfully navigate the unique challenges that may arise, especially when the diagnosis may have an impact on their ability to practice medicine., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The light brown apple moth, Epiphyas postvittana is an invasive, polyphagous pest of horticultural systems around the world. With origins in Australia, the pest has subsequently spread to New Zealand, Hawaii, California and Europe, where it has been found on over 500 plants, including many horticultural crops. We have produced a genomic resource, to understand the biological basis of the polyphagous and invasive nature of this and other lepidopteran pests. The assembled genome sequence encompassed 598 Mb and has an N50 of 301.17 kb, with a BUSCO completion rate of 97.9%. Epiphyas postvittana has 34% of its assembled genome represented as repetitive sequences, with the majority of the known elements made up of longer DNA transposable elements (14.07 Mb) and retrotransposons (LINE 17.83 Mb). Of the 31,389 predicted genes, 28,714 (91.5%) were assigned to 11,438 orthogroups across the Lepidoptera, of which 945 were specific to E. postvittana . Twenty gene families showed significant expansions in E. postvittana , including some likely to have a role in its pest status, such as cytochrome p450s, glutathione-S-transferases and UDP-glucuronosyltransferases. Finally, using a RAD-tag approach, we investigated the population genomics of this pest, looking at its likely patterns of invasion.

Sex pheromones facilitate species-specific sex communication within the Lepidoptera. They are detected by specialised pheromone receptors (PRs), most of which to date fall into a single monophyletic receptor lineage (frequently referred to as "the PR clade") within the odorant receptor (OR) family. Here we investigated PRs of the invasive horticultural pest, Epiphyas postvittana, commonly known as the light brown apple moth. Ten candidate PRs were selected, based on their male-biased expression in antennae or their relationship to the PR clade, for functional assessment in both HEK293 cells and Xenopus oocytes. Of these, six ORs responded to compounds that include components of the E. postvittana ('Epos') sex pheromone blend or compounds that antagonise sex pheromone attraction. In phylogenies, four of the characterised receptors (EposOR1, 6, 7 and 45) fall within the PR clade and two other male-biased receptors (EposOR30 and 34) group together well outside the PR clade. This new clade of pheromone receptors includes the receptor for (E)-11-tetradecenyl acetate (EposOR30), which is the main component of the sex pheromone blend for this species. Interestingly, receptors of the two clades do not segregate by preference for compounds associated with behavioural response (agonist or antagonist), isomer type (E or Z) or functional group (alcohol or acetate), with examples of each scattered across both clades. Phylogenetic comparison with PRs from other species supports the existence of a second major clade of lepidopteran ORs including, EposOR30 and 34, that has been co-opted into sex pheromone detection in the Lepidoptera. This second clade of sex pheromone receptors has an origin that likely predates the split between the major lepidopteran families., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

With the ever-increasing number of publicly available eukaryotic genome assemblies and user-friendly bioinformatics tools, there are increasing opportunities for researchers to use genomic resources in their research. While there are multiple dimensions to genome quality, it is often reduced to a single score that may not be correlated with other metrics, or appropriate for all applications of an assembly. To assess whether the commonly reported N50 value could reliably predict a separate dimension of genome quality, gene space completeness, we performed a meta-analysis of 611 published articles on eukaryotic genomes that used BUSCO scores, in addition to the typical N50 score. We found that although assemblies with relatively high contig and scaffold N50 values consistently had high BUSCO scores, a high BUSCO score could also be obtained from assemblies with a low N50. This reinforces that despite its ubiquity, N50 is not a perfect proxy for all measures of genome accuracy. Our data also suggests that variations in BUSCO scores among assemblies with poor N50 scores may be related to the number of introns in conserved eukaryotic genes. We stress the importance of screening and evaluating assembly quality based on the appropriate tools and urge increased reporting of additional genome assessment metrics in addition to N50. We also discuss the potential limitations of BUSCO and suggest improvements for assessing gene space within genome assemblies., (© 2021 John Wiley & Sons Ltd.)

Some animal groups, such as stick insects (Phasmatodea), have repeatedly evolved alternative reproductive strategies, including parthenogenesis. Genomic studies have found modification of the genes underlying meiosis exists in some of these animals. Here we examine the evolution of copy number, evolutionary rate, and gene expression in candidate meiotic genes of the New Zealand geographic parthenogenetic stick insect Clitarchus hookeri. We characterized 101 genes from a de novo transcriptome assembly from female and male gonads that have homology with meiotic genes from other arthropods. For each gene we determined copy number, the pattern of gene duplication relative to other arthropod orthologs, and the potential for meiosis-specific expression. There are five genes duplicated in C. hookeri, including one also duplicated in the stick insect Timema cristinae, that are not or are uncommonly duplicated in other arthropods. These included two sister chromatid cohesion associated genes (SA2 and SCC2), a recombination gene (HOP1), an RNA-silencing gene (AGO2) and a cell-cycle regulation gene (WEE1). Interestingly, WEE1 and SA2 are also duplicated in the cyclical parthenogenetic aphid Acyrthosiphon pisum and Daphnia duplex, respectively, indicating possible roles in the evolution of reproductive mode. Three of these genes (SA2, SCC2, and WEE1) have one copy displaying gonad-specific expression. All genes, with the exception of WEE1, have significantly different nonsynonymous/synonymous ratios between the gene duplicates, indicative of a shift in evolutionary constraints following duplication. These results suggest that stick insects may have evolved genes with novel functions in gamete production by gene duplication., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana 1,2 -is an iconic species that is endemic to New Zealand 2,3 . A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes 2,4 . Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Background: US public health strategy for eliminating tuberculosis (TB) prioritizes treatment of latent TB infection (LTBI). Healthcare personnel (HCP) are less willing to accept treatment than other populations. Little is known about factors associated with HCP LTBI therapy acceptance and completion., Methods: We conducted a retrospective chart review to identify all employees with LTBI at time of hire at a large academic medical center during a 10-year period. Personal demographics, occupational factors, and clinic visit variables were correlated with LTBI treatment acceptance and completion rates using multivariate logistic regression., Results: Of 470 HCP with LTBI for whom treatment was recommended, 193 (41.1%) accepted treatment, while 137 (29.1%) completed treatment. Treatment adherence was better with 4 months of rifampin than 9 months of isoniazid (95% vs 68%, P < .005). Increased age of the healthcare worker was independently associated with lower rates of treatment acceptance (odds ratio [95% confidence interval]: 0.97 [0.94-0.99] per year), as was having an occupation of clinician (0.47 [0.26-0.85]) or researcher (0.34 [0.19-0.64]). Male gender was associated with higher treatment acceptance (1.90 [1.21-2.99]). Treatment completion was associated with being from a low- (9.49 [2.06-43.73]) or medium- (8.51 [3.93-18.44]) TB-burden country., Conclusions: Geographic and occupational factors affect acceptance and completion of LTBI therapy. Short-course regimens may improve adherence. Physicians, researchers, and HCP from high-TB-burden countries have lower treatment rates than other HCP. Improving LTBI treatment in HCP will require attending to cultural and occupational differences., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2 N242I and DAD2 F135A , having substitutions in the core α/β hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2 D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2 N242I and DAD2 D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2 N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators., (© 2020 Lee et al.)

Invertebrates are a major component of terrestrial ecosystems, however, estimating their biodiversity is challenging. We compiled an inventory of invertebrate biodiversity along an elevation gradient on the temperate forested island of Hauturu, New Zealand, by DNA barcoding of specimens obtained from leaf litter samples and pitfall traps. We compared the barcodes and biodiversity estimates from this data set with those from a parallel DNA metabarcoding analysis of soil from the same locations, and with pre-existing sequences in reference databases, before exploring the use of combined data sets as a basis for estimating total invertebrate biodiversity. We obtained 1,282 28S and 1,610 COI barcodes from a total of 1,947 invertebrate specimens, which were clustered into 247 (28S) and 366 (COI) OTUs, of which ≤ 10% were represented in GenBank. Coleoptera were most abundant (730 sequenced specimens), followed by Hymenoptera, Diptera, Lepidoptera, and Amphipoda. The most abundant OTU from both the 28S (153 sequences) and COI (140 sequences) data sets was an undescribed beetle from the family Salpingidae. Based on the occurrences of COI OTUs along the elevation gradient, we estimated there are ~1,000 arthropod species (excluding mites) on Hauturu, including 770 insects, of which 344 are beetles. A DNA metabarcoding analysis of soil DNA from the same sites resulted in the identification of similar numbers of OTUs in most invertebrate groups compared with the DNA barcoding, but less than 10% of the DNA barcoding COI OTUs were also detected by the metabarcoding analysis of soil DNA. A mark-recapture analysis based on the overlap between these data sets estimated the presence of approximately 6,800 arthropod species (excluding mites) on the island, including ~3,900 insects. Estimates of New Zealand-wide biodiversity for selected arthropod groups based on matching of the COI DNA barcodes with pre-existing reference sequences suggested over 13,200 insect species are present, including 4,000 Coleoptera, 2,200 Diptera, and 2,700 Hymenoptera species, and 1,000 arachnid species (excluding mites). These results confirm that metabarcoding analyses of soil DNA tends to recover different components of terrestrial invertebrate biodiversity compared to traditional invertebrate sampling, but the combined methods provide a novel basis for estimating invertebrate biodiversity., (© 2019 by the Ecological Society of America.)

Exposure to low temperatures requires an organism to overcome physiological challenges. New Zealand wētā belonging to the genera Hemideina and Deinacrida are found across a wide range of thermal environments and therefore subject to varying selective pressures. Here we assess the selection pressures across the wētā phylogeny, with a particular emphasis on identifying genes under positive or diversifying selection. We used RNA-seq to generate transcriptomes for all 18 Deinacrida and Hemideina species. A total of 755 orthologous genes were identified using a bidirectional best-hit approach, with the resulting gene set encompassing a diverse range of functional classes. Analysis of ortholog ratios of synonymous to nonsynonymous amino acid changes found 83 genes that are under positive selection for at least one codon. A wide variety of Gene Ontology terms, enzymes, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways are represented among these genes. In particular, enzymes involved in oxidative phosphorylation, melanin synthesis, and free-radical scavenging are represented, consistent with physiological and metabolic changes that are associated with adaptation to alpine environments. Structural alignment of the transcripts with the most codons under positive selection revealed that the majority of sites are surface residues, and therefore have the potential to influence the thermostability of the enzyme, with the exception of prophenoloxidase where two residues near the active site are under selection. These proteins provide interesting candidates for further analysis of protein evolution., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objective: To assess current medical surveillance monitoring practices for health care workers who prepare, handle, or administer hazardous medications., Methods: A cross-sectional survey was distributed to members of the American College of Occupational and Environmental Medicine and the National Comprehensive Cancer Network., Results: Forty-six of the 91 survey respondents indicated that their institution had a hazardous medication surveillance program. We identified the most frequent laboratory (complete blood count) and physical (skin) examination components. A health history was frequently used. Statistical analysis did not suggest an association between institutions with greater resources and presence of a surveillance program., Conclusions: A consensus standard for medical monitoring was not reported by the respondents. We recommend using a standardized surveillance questionnaire and applying uniform laboratory testing across institutions, in addition to establishing a national repository for surveillance data.

To understand the ecosystem dynamics that underpin the year-round presence of a large generalist consumer, the Bryde's whale (Balaenoptera edeni brydei), we use a DNA metabarcoding approach and systematic zooplankton surveys to investigate seasonal and regional changes in zooplankton communities and if whale diet reflects such changes. Twenty-four zooplankton community samples were collected from three regions throughout the Hauraki Gulf, New Zealand, over two temperature regimes (warm and cool seasons), as well as 20 samples of opportunistically collected Bryde's whale scat. Multi-locus DNA barcode libraries were constructed from 18S and COI gene fragments, representing a trade-off between identification and resolution of metazoan taxa. Zooplankton community OTU occurrence and relative read abundance showed regional and seasonal differences based on permutational analyses of variance in both DNA barcodes, with significant changes in biodiversity indices linked to season in COI only. In contrast, we did not find evidence that Bryde's whale diet shows seasonal or regional trends, but instead indicated clear prey preferences for krill-like crustaceans, copepods, salps and ray-finned fishes independent of prey availability. The year-round presence of Bryde's whales in the Hauraki Gulf is likely associated with the patterns of distribution and abundance of these key prey items.

Objective: The National Institute for Occupational Safety and Health recommends that institutions establish a medical surveillance program for workers who handle hazardous drugs. Our aim was to investigate current practices with occupational medicine practice (OMP) national leaders., Methods: A series of qualitative telephone interviews were conducted with 11 OMP national leaders from medical centers in 10 states. Interviews were recorded, transcribed, and coded using a directed content analysis. Codes were organized into themes., Results: All respondents were board-certified physicians in medical center OMP. Interviews up to 45 minutes found three themes: policy interpretation, benefits and barriers to surveillance, and potential respondent-generated solutions. Three of 10 medical centers provided medical surveillance., Conclusions: Medical surveillance for hazardous drugs is infrequent, and consensus is lacking regarding standard practices. Further work is needed to minimize risk to health care workers.

Background: Sex pheromone communication in moths has attracted the attention of evolutionary biologists due to the vast array of pheromone compounds used, addressing questions of how this diversity arose and how male reception has evolved in step with the female signal. Here we examine the role of changing gene expression in the evolution of mate recognition systems in leafroller moths, particularly focusing on genes involved in the biosynthetic pathways of sex pheromones in female pheromone glands and the peripheral reception repertoire in the antennae of males. From tissue-specific transcriptomes we mined and compared a database of genes expressed in the pheromone glands and antennae of males and females of four closely related species of leafroller moths endemic to New Zealand, Ctenopseutis herana and C. obliquana, and Planotortrix excessana and P. octo. The peculiarity of this group, compared to other Lepidoptera, is the use of (Z)-5-tetradecenyl acetate, (Z)-7-tetradecenyl acetate, and (Z)-8-tetradecenyl acetate as sex pheromone components., Results: We identify orthologues of candidate genes from the pheromone biosynthesis pathway, degradation and transport, as well as genes of the periphery olfactory repertoire, including large families of binding proteins, receptors and odorant degrading enzymes. The production of distinct pheromone blends in the sibling species is associated with the differential expression of two desaturase genes, deast5 and desat7, in the pheromone glands. In male antennae, three odorant receptors, OR74, OR76a and OR30 are over-expressed, but their expression could not be clearly associated with the detection of species-specific pheromones components. In addition these species contain duplications of all three pheromone binding proteins (PBPs) that are also differentially expressed among species., Conclusions: While in females differences in the expression of desaturases may be sufficient to explain pheromone blend differences among these New Zealand leafroller species, in males differential expression of several genes, including pheromone binding proteins, may underpin differences in the response by males to changing pheromone components among the species.

Objective: The impact of "real world" collaborative care on depression and absenteeism for depressed employees seen in primary care practices using objective employer absence data., Methods: A retrospective cohort study comparing depressed employees seen in primary care practices who enrolled for a "real world" collaborative care program to practice as usual (PAU) on objective absence days and depression response and remission at 6, and 12-month time periods., Results: Absence days were more in the collaborative care group compared with the PAU group at 3 and 6 months but at 12 months the difference was no longer statistically significant. Collaborative care led to better response and remission depression scores compared with PAU at 12 months., Conclusions: Collaborative care led to faster improvement in depression symptoms but did not translate to less time away from work.

Background: Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects., Results: The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified., Conclusions: The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects.

Animal reproductive proteins, especially those in the seminal fluid, have been shown to have higher levels of divergence than non-reproductive proteins and are often evolving adaptively. Seminal fluid proteins have been implicated in the formation of reproductive barriers between diverging lineages, and hence represent interesting candidates underlying speciation. RNA-seq was used to generate the first male reproductive transcriptome for the New Zealand tree weta species Hemideina thoracica and H. crassidens. We identified 865 putative reproductive associated proteins across both species, encompassing a diverse range of functional classes. Candidate gene sequencing of nine genes across three Hemideina, and two Deinacrida species suggests that H. thoracica has the highest levels of intraspecific genetic diversity. Non-monophyly was observed in the majority of sequenced genes indicating that either gene flow may be occurring between the species, or that reciprocal monophyly at these loci has yet to be attained. Evidence for positive selection was found for one lectin-related reproductive protein, with an overall omega of 7.65 and one site in particular being under strong positive selection. This candidate gene represents the first step in the identification of proteins underlying the evolutionary basis of weta reproduction and speciation.

Pheromone receptors (PRs) are essential in moths to detect sex pheromones for mate finding. However, it remains unknown from which ancestral proteins these specialized receptors arose. The oldest lineages of moths, so-called non-ditrysian moths, use short-chain pheromone components, secondary alcohols, or ketones, so called Type 0 pheromones that are similar to many common plant volatiles. It is, therefore, possible that receptors for these ancestral pheromones evolved from receptors detecting plant volatiles. Hence, we identified the odorant receptors (ORs) from a non-ditrysian moth, Eriocrania semipurpurella (Eriocraniidae, Lepidoptera), and performed functional characterization of ORs using HEK293 cells. We report the first receptors that respond to Type 0 pheromone compounds; EsemOR3 displayed highest sensitivity toward (2S, 6Z)-6-nonen-2-ol, whereas EsemOR5 was most sensitive to the behavioral antagonist (Z)-6-nonen-2-one. These receptors also respond to plant volatiles of similar chemical structures, but with lower sensitivity. Phylogenetically, EsemOR3 and EsemOR5 group with a plant volatile-responding receptor from the tortricid moth Epiphyas postvittana (EposOR3), which together reside outside the previously defined lepidopteran PR clade that contains the PRs from more derived lepidopteran families. In addition, one receptor (EsemOR1) that falls at the base of the lepidopteran PR clade, responded specifically to β-caryophyllene and not to any other additional plant or pheromone compounds. Our results suggest that PRs for Type 0 pheromones have evolved from ORs that detect structurally-related plant volatiles. They are unrelated to PRs detecting pheromones in more derived Lepidoptera, which, in turn, also independently may have evolved a novel function from ORs detecting plant volatiles., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: The New Zealand collembolan genus Holacanthella contains the largest species of springtails (Collembola) in the world. Using Illumina technology we have sequenced and assembled a draft genome and transcriptome from Holacanthella duospinosa (Salmon). We have used this annotated assembly to investigate the genetic basis of a range of traits critical to the evolution of the Hexapoda, the phylogenetic position of H. duospinosa and potential horizontal gene transfer events., Results: Our genome assembly was ~375 Mbp in size with a scaffold N50 of ~230 Kbp and sequencing coverage of ~180×. DNA elements, LTRs and simple repeats and LINEs formed the largest components and SINEs were very rare. Phylogenomics (370,877 amino acids) placed H. duospinosa within the Neanuridae. We recovered orthologs of the conserved sex determination genes thought to play a role in sex determination. Analysis of CpG content suggested the absence of DNA methylation, and consistent with this we were unable to detect orthologs of the DNA methyltransferase enzymes. The small subunit rRNA gene contained a possible retrotransposon. The Hox gene complex was broken over two scaffolds. For chemosensory ability, at least 15 and 18 ionotropic glutamate and gustatory receptors were identified, respectively. However, we were unable to identify any odorant receptors or their obligate co-receptor Orco. Twenty-three chitinase-like genes were identified from the assembly. Members of this multigene family may play roles in the digestion of fungal cell walls, a common food source for these saproxylic organisms. We also detected 59 and 96 genes that blasted to bacteria and fungi, respectively, but were located on scaffolds that otherwise contained arthropod genes., Conclusions: The genome of H. duospinosa contains some unusual features including a Hox complex broken over two scaffolds, in a different manner to other arthropod species, a lack of odorant receptor genes and an apparent lack of environmentally responsive DNA methylation, unlike many other arthropods. Our detection of candidate horizontal gene transfer candidates confirms that this phenomenon is occurring across Collembola. These findings allow us to narrow down the regions of the arthropod phylogeny where key innovations have occurred that have facilitated the evolutionary success of Hexapoda.

Background: Available information is insufficient to guide determination of whether tuberculin skin test (TST) conversions of health care workers (HCWs) within 2 years of two-step testing are related to occupational exposures or to other causes, including late boosting., Aims: To describe the epidemiologic factors of TST conversion in HCWs, comparing early TST conversion (≤2 years after two-step testing) with late conversion to possibly distinguish late boosting phenomenon from occupational TST conversion., Methods: Retrospective analysis of a database of TSTs of HCWs from 1 January 1998, through 31 May 2014, in the United States Midwest., Results: In total, 40142 HCWs had 197932 tests over the 16 years, with 123 conversions (conversion rate: 0.3%; 95% CI 0.3-0.4%). Among 61 HCWs with a negative two-step TST, 30 (49%) were found to have early TST conversion within 2 years; 31 (51%) had late conversion, with likely occupational exposure but no identifiable community risks. Persons with early conversion were more likely to be born outside the USA (89% versus 57%; P < 0.05), had a higher rate of prior bacille Calmette-Guérin (BCG) vaccination (89% versus 52%; P < 0.05) and had no identifiable risk factors for conversion (63% versus 58%; P < 0.05)., Conclusions: Early conversions among HCWs after negative two-step TST are associated with various nonoccupational factors, including international birth and BCG vaccination history. Therefore, conversion is not a reliable indicator of recent tuberculosis contact in this population, and two-step TST is insufficient to discount a delayed boosting response for HCWs., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

The Hessian fly, Mayetiola destructor Say (Diptera, Cecidomyiidae), is a pest of wheat and belongs to a group of gall-inducing herbivores. This species has a unique life history and several ecological features that differentiate it from other Diptera such as Drosophila melanogaster and blood-feeding mosquitoes. These features include a short, non-feeding adult life stage (1-2 days) and the use of a long-range sex pheromone produced and released by adult females. Sex pheromones are detected by members of the odorant receptor (OR) family within the Lepidoptera, but no receptors for similar long-range sex pheromones have been characterized from the Diptera. Previously, 122 OR genes have been annotated from the Hessian fly genome, with many of them showing sex-biased expression in the antennae. Here we have expressed, in HEK293 cells, five MdesORs that display male-biased expression in antennae, and we have identified MdesOR115 as a Hessian fly sex pheromone receptor. MdesOR115 responds primarily to the sex pheromone component (2S,8E,10E)-8,10-tridecadien-2-yl acetate, and secondarily to the corresponding Z,E-isomer. Certain sensory neuron membrane proteins (i.e., SNMP1) are important for responses of pheromone receptors in flies and moths. The Hessian fly genome is unusual in that it encodes six SNMP1 paralogs, of which five are expressed in antennae. We co-expressed each of the five antennal SNMP1 paralogs together with each of the five candidate sex pheromone receptors from the Hessian fly and found that they do not influence the response of MdesOR115, nor do they confer responsiveness in any of the non-responsive ORs to any of the sex pheromone components identified to date in the Hessian fly. Using Western blots, we detected protein expression of MdesOrco, all MdesSNMPs, and all MdesORs except for MdesOR113, potentially explaining the lack of response from this OR. In conclusion, we report the first functional characterization of an OR from the Cecidomyiidae, extending the role of ORs as long-range sex pheromone detectors from the Lepidoptera into the Diptera.

Background: Communication skills and professionalism are two competencies in graduate medical education that are challenging to evaluate. We aimed to develop, test and validate a de novo instrument to evaluate these two competencies., Methods: Using an Objective Standardized Clinical Examination (OSCE) based on a medication error scenario, we developed an assessment instrument that focuses on distinctive domains [context of discussion, communication and detection of error, management of error, empathy, use of electronic medical record (EMR) and electronic medical information resources (EMIR), and global rating]. The aim was to test feasibility, acceptability, and reliability of the method., Results: Faculty and standardized patients (SPs) evaluated 56 trainees using the instrument. The inter-rater reliability of agreement between faculty was substantial (Fleiss k = 0.71) and intraclass correlation efficient was excellent (ICC = 0.80). The measured agreement between faculty and SPs evaluation of resident was lower (Fleiss k = 0.36). The instrument showed good conformity (ICC = 0.74). The majority of the trainees (75 %) had satisfactory or higher performance in all six assessed domains and 86 % found the OSCE to be realistic. Sixty percent reported not receiving feedback on EMR use and asked for subsequent training., Conclusion: An OSCE-based instrument using a medical error scenario can be used to assess competency in professionalism, communication, using EMRs and managing medical errors.

In this work, we examine the possibility of improving the prediction performance of an olfactory biosensor through the use of temporal spiking data. We present an Artificial Neural Network (ANN), in the form of an optimal hybrid Multi-Layer Perceptron (MLP) system for the classification of chemical odorants from olfactory receptor neuron spike responses of the Drosophila melanogaster fruit fly (DmOrs). The data used in this study contains the responses to 34 odorants from 6 individual DmOrs, of which we exploit the temporal spiking responses of a 500ms odorant stimulus window. We report, for the first time, the difference between the classification performance of the temporal spiking data to an equivalent spontaneous scalar dataset that we have reported previously. We demonstrate that a higher prediction (%) was obtained when using the temporal data, in which a greater number of validation odorants are identified to their correct chemical class. This work presents a novel technique to improve the classification performance of an olfactory biosensor, whilst maintaining a limited sensory array of 6 DmOr receptors.

Background: The cost of workplace absenteeism and presenteeism due to depression in the USA is substantial., Aims: To assess the frequency of depression and its impact at the point of care in an occupational health (OH) practice., Methods: Patients presenting to an OH practice completed a standardized depression screening tool and were compared to an unscreened group in the same clinic. Respondents with a nine-item Patient Health Questionnaire (PHQ-9) score >15 and untreated for depression were referred for further evaluation per usual practice. A comparison group of unscreened patients were selected from the same clinic from 1 year prior and records were reviewed for evidence of prior depression, treatment and outcomes. After 1 year, frequency of depression, PHQ-9 scoring for screened patients, days absent from work, days on restricted duties and permanent restrictions were recorded for both groups., Results: Two hundred and five patients were screened for depression. Screening was associated with increased frequency of a diagnosis of current depression (30 versus 4%; P < 0.05). Screening was associated with similar rates of absenteeism but lower number of days on restricted duties (97 versus 159 days; P < 0.001). After adjusting for age, sex, history of and treatment for depression, screening was associated with lower odds of being on work restrictions [odds ratio (OR) 0.55; 95% confidence interval (CI) 0.38-0.78] or permanent restrictions (OR 0.35; 95% CI 0.23-0.52)., Conclusions: Depression was common in this OH practice. Screening for depression, with appropriate recognition and referral, may reduce time for employed patients on restricted duties and permanent restrictions., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Phasmatodea, more commonly known as stick insects, have been poorly studied at the molecular level for several key traits, such as components of the sensory system and regulators of reproduction and development, impeding a deeper understanding of their functional biology. Here, we employ de novo transcriptome analysis to identify genes with primary functions related to female odour reception, digestion, and male sexual traits in the New Zealand common stick insect Clitarchus hookeri (White). The female olfactory gene repertoire revealed ten odorant binding proteins with three recently duplicated, 12 chemosensory proteins, 16 odorant receptors, and 17 ionotropic receptors. The majority of these olfactory genes were over-expressed in female antennae and have the inferred function of odorant reception. Others that were predominantly expressed in male terminalia (n = 3) and female midgut (n = 1) suggest they have a role in sexual reproduction and digestion, respectively. Over-represented transcripts in the midgut were enriched with digestive enzyme gene families. Clitarchus hookeri is likely to harbour nine members of an endogenous cellulase family (glycoside hydrolase family 9), two of which appear to be specific to the C. hookeri lineage. All of these cellulase sequences fall into four main phasmid clades and show gene duplication events occurred early in the diversification of Phasmatodea. In addition, C. hookeri genome is likely to express γ-proteobacteria pectinase transcripts that have recently been shown to be the result of horizontal transfer. We also predicted 711 male terminalia-enriched transcripts that are candidate accessory gland proteins, 28 of which were annotated to have molecular functions of peptidase activity and peptidase inhibitor activity, two groups being widely reported to regulate female reproduction through proteolytic cascades. Our study has yielded new insights into the genetic basis of odour detection, nutrient digestion, and male sexual traits in stick insects. The C. hookeri reference transcriptome, together with identified gene families, provides a comprehensive resource for studying the evolution of sensory perception, digestive systems, and reproductive success in phasmids.

Moths use their sense of smell to find food sources, mating partners and oviposition sites. For this they possess a family of odorant receptors (ORs). Some ORs are used by both sexes whereas others have sex-specific roles. For example, male moths possess ORs specifically tuned to sex pheromones produced by conspecific females. Here we identify sets of ORs from the antennae of New Zealand endemic leafroller moths Planotortrix octo (48 ORs) and P. excessana (47 ORs) using an RNA-Seq approach. Two orthologous ORs show male-biased expression in the adult antennae of both species (OR7 and OR30) and one other OR in each species was female-biased in its expression (PoctOR25, PexcOR14) by qPCR. PAML analysis conducted on male-biased ORs indicated positive selection acting on the male-biased OR7. The fact that OR7 is likely under positive selection, that it is male-biased in its expression and that its orthologue in C. obliquana, CoblOR7, responds to sex pheromone components also utilised by Planotortrix species, suggests that this receptor may also be important in sex pheromone reception in Planotortrix species.

Insects have co-opted a unique family of seven transmembrane proteins for odour sensing. Odorant receptors are believed to have evolved from gustatory receptors somewhere at the base of the Hexapoda and have expanded substantially to become the dominant class of odour recognition elements within the Insecta. These odorant receptors comprise an obligate co-receptor, Orco, and one of a family of highly divergent odorant "tuning" receptors. The two subunits are thought to come together at some as-yet unknown stoichiometry to form a functional complex that is capable of both ionotropic and metabotropic signalling. While there are still no 3D structures for these proteins, site-directed mutagenesis, resonance energy transfer, and structural modelling efforts, all mainly on Drosophila odorant receptors, are beginning to inform hypotheses of their structures and how such complexes function in odour detection. Some of the loops, especially the second extracellular loop that has been suggested to form a lid over the binding pocket, and the extracellular regions of some transmembrane helices, especially the third and to a less extent the sixth and seventh, have been implicated in ligand recognition in tuning receptors. The possible interaction between Orco and tuning receptor subunits through the final intracellular loop and the adjacent transmembrane helices is thought to be important for transducing ligand binding into receptor activation. Potential phosphorylation sites and a calmodulin binding site in the second intracellular loop of Orco are also thought to be involved in regulating channel gating. A number of new methods have recently been developed to express and purify insect odorant receptor subunits in recombinant expression systems. These approaches are enabling high throughput screening of receptors for agonists and antagonists in cell-based formats, as well as producing protein for the application of biophysical methods to resolve the 3D structure of the subunits and their complexes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: There is an increasing demand for rapid biodiversity assessment tools that have a broad taxonomic coverage. Here we evaluate a suite of environmental DNA (eDNA) markers coupled with next generation sequencing (NGS) that span the tree of life, comparing them with traditional biodiversity monitoring tools within ten 20×20 meter plots along a 700 meter elevational gradient., Results: From six eDNA datasets (one from each of 16S, 18S, ITS, trnL and two from COI) we identified sequences from 109 NCBI taxonomy-defined phyla or equivalent, ranging from 31 to 60 for a given eDNA marker. Estimates of alpha and gamma diversity were sensitive to the number of sequence reads, whereas beta diversity estimates were less sensitive. The average within-plot beta diversity was lower than between plots for all markers. The soil beta diversity of COI and 18S markers showed the strongest response to the elevational variation of the eDNA markers (COI: r=0.49, p<0.001; 18S: r=0.48, p<0.001). Furthermore pairwise beta diversities for these two markers were strongly correlated with those calculated from traditional vegetation and invertebrate biodiversity measures., Conclusions: Using a soil-based eDNA approach, we demonstrate that standard phylogenetic markers are capable of recovering sequences from a broad diversity of eukaryotes, in addition to prokaryotes by 16S. The COI and 18S eDNA markers are the best proxies for aboveground biodiversity based on the high correlation between the pairwise beta diversities of these markers and those obtained using traditional methods.

Recent work suggests that Drosophila and Saccharomyces yeasts may establish a mutualistic association, and that this is driven by chemical communication. While individual volatiles have been implicated in the attraction of D. melanogaster, the semiochemicals affecting the behavior of the sibling species D. simulans are less well characterized. Here, we scrutinized a broad range of volatiles produced by attractive and repulsive yeasts to experimentally evaluate the chemical nature of communication between these species. When grown in liquid or on agar-solidified grape juice, attraction to S. cerevisiae was driven primarily by 3-methylbutyl acetate (isoamyl acetate) and repulsion by acetic acid, a known attractant to D. melanogaster (also known as vinegar fly). By using T-maze choice tests and synthetic compounds, we showed that these responses are strongly influenced by compound concentration. Moreover, the behavioral response is impacted further by the chemical context of the environment. Thus, chemical communication between yeasts and flies is complex, and is not driven simply by the presence of single volatiles, but modulated by compound interactions. The ecological context of chemical communication needs to be taken into consideration when testing for ecologically realistic responses.

The lightbrown apple moth, Epiphyas postvittana is an increasingly global pest of horticultural crops. Like other moths, E. postvittana relies on olfactory cues to locate mates and oviposition sites. To detect these cues, moths have evolved families of genes encoding elements of the peripheral olfactory reception system, including odor carriers, receptors and degrading enzymes. Here we undertake a transcriptomic approach to identify members of these families expressed in the adult antennae of E. postvittana, describing open reading frames encoding 34 odorant binding proteins, 13 chemosensory proteins, 70 odorant receptors, 19 ionotropic receptors, nine gustatory receptors, two sensory neuron membrane proteins, 27 carboxylesterases, 20 glutathione-S-transferases, 49 cytochrome p450s and 18 takeout proteins. For the odorant receptors, quantitative RT-PCR corroborated RNAseq count data on steady state transcript levels. Of the eight odorant receptors that group phylogenetically with pheromone receptors from other moths, two displayed significant male-biased expression patterns, one displayed significant female-biased expression pattern and five were expressed equally in the antennae of both sexes. In addition, we found two male-biased odorant receptors that did not group with previously described pheromone receptors. This suite of olfaction-related genes provides a substantial resource for the functional characterization of this signal transduction system and the development of odor-mediated control strategies for horticultural pests.

Background: Physicians may face unique challenges in accessing health care and managing their own health., Aims: To evaluate physicians' perceptions of their health care needs and desired services., Methods: A written survey, distributed and collected anonymously among attendees at a large primary care continuing medical education conference., Results: The survey was given to 346 physicians and 141 (41%) responded. The majority of physicians (53%) reported having difficulty accessing health care and reverting to self-diagnosis and treatment (63%). Over 83% reported having or knowing a colleague who had a career-threatening illness and 42% had experienced concern about a colleague's ability to practise safely., Conclusions: Physicians as an occupational group have challenges in accessing health care, very commonly suffer career-limiting illnesses and revert to self-diagnosis and treatment. Programmes tailored to providing health care to physicians are needed., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The model organism, Drosophila melanogaster, and the mosquito Anopheles gambiae use 60 and 79 odorant receptors, respectively, to sense their olfactory world. However, a commercial "electronic nose" in the form of an insect olfactory biosensor demands very low numbers of receptors at its front end of detection due to the difficulties of receptor/sensor integration and functionalization. In this letter, we demonstrate how computation via artificial neural networks (ANNs), in the form of multilayer perceptrons (MLPs), can be successfully incorporated as the signal processing back end of the biosensor to drastically reduce the number of receptors to three while still retaining 100% performance of odorant detection to that of a full complement of receptors. In addition, we provide a detailed performance comparison between D. melanogaster and A. gambiae odorant receptors and demonstrate that A. gambiae receptors provide superior olfaction detection performance over D. melanogaster for very low receptor numbers. The results from this study present the possibility of using the computation of MLPs to discover ideal biological olfactory receptors for an olfactory biosensor device to provide maximum classification performance of unknown odorants.

How new mate recognition systems evolve when changes are required in both the male and female components remains a conundrum. Here, we investigated the molecular basis of pheromone reception in two species of tortricid (leafroller) moth, Ctenopseustis obliquana and C. herana. Male C. obliquana are attracted to a 90:10 blend of (Z)-8-tetradecenyl acetate (Z8-14:OAc) and (Z)-5-tetradecenyl acetate (Z5-14:OAc), whereas C. herana males are attracted to Z5-14:OAc alone. We used a transcriptome sequencing approach from adult male and female antennae to identify 47 olfactory receptors (ORs) from each species and assessed their expression levels in male and female antennae using RNA-Seq counting and quantitative RT-PCR. Three male-biased and one female-biased OR were identified in C. obliquana by quantitative RT-PCR, and four male-biased and one female-biased receptor in C. herana. The male-biased receptors, CoblOR7, CoblOR30, CherOR7, CherOR30, CherOR1a and CherOR1b were tested for their ability to respond to sex pheromone components in a HEK293 cell calcium assay. CoblOR7 and CherOR7 responded to Z8-14:OAc, however, no receptor for Z5-14:OAc was identified. In addition to Z8-14:OAc, CherOR7 also responded to Z7-14:OAc, indicating that this receptor may be under relaxed constraint. Of the 29 amino acid differences between CoblOR7 and CherOR7, significantly more are located in the third and the sixth transmembrane domain regions. Overall, these findings are consistent with studies revealing the presence of neurons tuned to both Z8-14:OAc and Z5-14:OAc in both species, but that for C. herana males, the ability to detect Z8-14:OAc is currently not required.

Olfactory receptors (ORs) are the key elements of the molecular machinery responsible for the detection of odors in insects. Since their initial discovery in Drosophila melanogaster at the beginning of the twenty-first century, insect ORs have been the focus of intense research, both for fundamental knowledge of sensory systems and for their potential as novel targets for the development of products that could impact harmful behaviors of crop pests and disease vectors. In recent years, studies on insect ORs have entered the genomic era, with an ever-increasing number of OR genes being characterized every year through the sequencing of genomes and transcriptomes. With the upcoming release of genomic sequences from hundreds of insect species, the insect OR family could very well become the largest multigene family known. This extremely rapid identification of ORs in many insects is driving the necessity for the development of high-throughput technologies that will allow the identification of ligands for this unprecedented number of receptors. Moreover, such technologies will also be important for the development of agonists or antagonists that could be used in the fight against pest insects., (© 2015 Elsevier Inc. All rights reserved.)

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca(2+) levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Niche construction theory explains how organisms' niche modifications may feed back to affect their evolutionary trajectories. In theory, the evolution of other species accessing the same modified niche may also be affected. We propose that this niche construction may be a general mechanism driving the evolution of mutualisms. Drosophilid flies benefit from accessing yeast-infested fruits, but the consequences of this interaction for yeasts are unknown. We reveal high levels of variation among strains of Saccharomyces cerevisiae in their ability to modify fruits and attract Drosophila simulans. More attractive yeasts are dispersed more frequently, both in the lab and in the field, and flies associated with more attractive yeasts have higher fecundity. Although there may be multiple natural yeast and fly species interactions, our controlled assays in the lab and field provide evidence of a mutualistic interaction, facilitated by the yeast's niche modification., (© 2014 John Wiley & Sons Ltd/CNRS.)

Identifying tobacco use status is essential to address use and provide resources to help patients quit. Being able to collect this information in an electronic format will become increasingly important, as the Centers for Medicare and Medicaid Services has included the assessment of tobacco use as part of its Stage 1 Meaningful Use criteria. The objective was to compare the accuracy of online vs. paper assessment methods to ascertain cigarette smoking status using a face-to-face structured interview as the gold standard. This was a retrospective analysis of a stratified opportunity sample of consecutive patients, reporting in 2010 for a periodic health evaluation, who completed either a scannable paper-based form or an online questionnaire and underwent a standardized rooming interview. Compared with face-to-face structured interview, the overall observed agreement and kappa coefficient for both methods combined (paper and online) were 97.7% and 0.69 (95% confidence interval (CI) 0.51-0.86) . For the online form they were 97.4% and 0.61 (95% CI 0.33-0.90), and for the paper form they were 97.9% and 0.75 (95% CI 0.54-0.96). There was no statistically significant difference in agreement between the online and paper-based methods (P=0.76) compared with a face-to-face structured interview. Online assessment of tobacco use status is as accurate as a paper questionnaire, and both methods have greater than 97% observed agreement with a face-to-face structured interview. The use of online assessment of tobacco use status has several advantages and more widespread use should be explored.

In widespread and genetically structured populations, temperature variation may lead to among-population differentiation of thermal biology. The New Zealand stick insect genus Micrarchus contains four species that inhabit different thermal environments, two of which are geographically widespread. RNA-Seq and quantitative PCR were used to investigate the transcriptional responses to cold shock among lowland and alpine species to identify cold-responsive transcripts that differ between the species and to determine whether there is intraspecific geographical variation in gene expression. We also used mitochondrial DNA, nuclear 28S ribosomal DNA and transcriptome-wide SNPs to determine phylogeographic structure and the potential for differences in genetic backgrounds to contribute to variation in gene expression. RNA-Seq identified 2160 unigenes that were differentially expressed as a result of low-temperature exposure across three populations from two species (M. hystriculeus and M. nov. sp. 2), with a majority (68% ± 20%) being population specific. This extensive geographical variation is consistent across years and is likely a result of background genetic differences among populations caused by genetic drift and possibly local adaptation. Responses to cold shock shared among alpine M. nov. sp. 2 populations included the enrichment of cuticular structure-associated transcripts, suggesting that cuticle modification may have accompanied colonization of low-temperature alpine environments and the development of a more cold-hardy phenotype., (© 2014 John Wiley & Sons Ltd.)

Odor learning induces structural and functional modifications throughout the olfactory system, but it is currently unknown whether this plasticity extends to the olfactory receptors (Or) in the sensory periphery. Here, we demonstrate that odor learning induces plasticity in olfactory receptor expression in the honeybee, Apis mellifera. Using quantitative RT-PCR analysis, we show that six putative floral scent receptors were differentially expressed in the bee antennae depending on the scent environment that the bees experienced. Or151, which we characterized using an in vitro cell expression system as a broadly tuned receptor binding floral odorants such as linalool, and Or11, the specific receptor for the queen pheromone 9-oxo-decenoic acid, were significantly down-regulated after honeybees were conditioned with the respective odorants in an olfactory learning paradigm. Electroantennogram recordings showed that the neural response of the antenna was similarly reduced after odor learning. Long-term odor memory was essential for inducing these changes, suggesting that the molecular mechanisms involved in olfactory memory also regulate olfactory receptor expression. Our study demonstrates for the first time that olfactory receptor expression is experience-dependent and modulated by scent conditioning, providing novel insight into how molecular regulation at the periphery contributes to plasticity in the olfactory system., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Sexual communication in the Lepidoptera typically involves a female-produced sex pheromone that attracts males of the same species. The most common type of moth sex pheromone comprises individual or blends of fatty acyl derivatives that are synthesized by a specific enzymatic pathway in the female's pheromone gland, often including a desaturation step. This reaction is catalyzed by fatty acyl desaturases that introduce double bonds at specific locations in the fatty acid precursor backbone. The two tortricid moths, Ctenopseustis obliquana and C. herana (brown-headed leafrollers), which are endemic in New Zealand, both use (Z)-5-tetradecenyl acetate as part of their sex pheromone. In C. herana, (Z)-5-tetradecenyl acetate is the sole component of the pheromone. Labeling experiments have revealed that this compound is produced via an unusual Δ5-desaturation of myristic acid. Previously six desaturases were identified from the pheromone glands of Ctenopseustis and its sibling genus Planotortrix, with one differentially regulated to produce the distinct blends used by individual species. However, none were able to conduct the Δ5-desaturation observed in C. herana, and presumably C. obliquana. We have now identified an additional desaturase gene, desat7, expressed in the pheromone glands of both Ctenopseustis species, which is not closely related to any previously described moth pheromone desaturase. The encoded enzyme displays Δ5-desaturase activity on myristic acid when heterologously expressed in yeast, but is not able to desaturate any other fatty acid (C8-C16). We conclude that desat7 represents a new group of desaturases that has evolved a role in the biosynthesis of sex pheromones in moths.

Current electronic noses, or e-noses, that employ insect odorant receptors (Ors) as their sensory front end are potentially limited by the fact that the Ors come from a single species. In addition, a realistic e-nose also demands low numbers of Ors at its sensory front end due to the difficulties of receptor/sensor integration and functionalisation. In this work, we report the first investigations of a `Super E-Nose' that incorporates Ors from both the model organism Drosophila melanogaster fruit fly (DmOr) and the mosquito, Anopheles gambiae (AgOr). Furthermore, we report how an Artificial Neural Network (ANN), in the form of a hybrid double hidden layer Multi-Layer Perceptron (MLP), can be used to determine the optimal Ors that provide the best prediction performance in the classification of unknown odorants into their respective chemical class. Our findings demonstrate how 3-Or arrays consisting of DmOr only, AgOr only, or cross-species DmOr-AgOr combinations correctly classified all unknown odorants of the validation set. In addition, we report that all 3-Or combinations perform equally well as the complete 74 DmOr-AgOr array. Thus, the results of this work support further investigation into cross-species `Super E-noses' coupled with hybrid MLPs for the classification of unknown odorants.

In our previous work, we have investigated the classification of odorants based on their chemical classes only, e.g. Alcohol, Terpene or Ester, using Artificial Neural Networks (ANN) as the signal processing backend of an insect olfactory electronic nose, or e-nose. However, potential applications of e-noses in the food and beverage industry which include the assessment of a fruit's ripeness, quality of wines or identifying bacterial contamination in products, demand the ability to predict beyond chemical class and to identify exact chemicals, known as specific Volatile Organic Compounds (VOCs) and blends of chemical that present themselves as aromas, known as blended VOCs (BVOCs). In this work, we demonstrate for the first time how it is possible to predict such VOCs and also BVOCs at varying concentration levels. We achieve this goal by using ANNs in the form of hybrid Multi-Layer Perceptrons (MLPs), to analyze the firing rate responses of the model organism Drosophila melanogaster's odorant receptors (DmOrs), in order to predict the specific VOCs and BVOCs. We report for the raw and noise injected data how the highest MLP prediction for specific VOCs occurred at a 10(-4)mol.dm(-3) concentration in which all the VOC validation vectors were identified and at a concentration of 10(-2)mol.dm(-3) for BVOCs in which 8/9 or 88.9% were identified. The results demonstrate for the first time the power of using MLPs and insect odorant receptors (Ors) to predict specific VOCs and BVOCs.

Background: The glycolytic pathway is central to cellular energy production. Selection on individual enzymes within glycolysis, particularly phosphoglucose isomerase (Pgi), has been associated with metabolic performance in numerous organisms. Nonetheless, how whole energy-producing pathways evolve to allow organisms to thrive in different environments and adopt new lifestyles remains little explored. The Lanceocercata radiation of Australasian stick insects includes transitions from tropical to temperate climates, lowland to alpine habitats, and winged to wingless forms. This permits a broad investigation to determine which steps within glycolysis and what sites within enzymes are the targets of positive selection. To address these questions we obtained transcript sequences from seven core glycolysis enzymes, including two Pgi paralogues, from 29 Lanceocercata species., Results: Using maximum likelihood methods a signature of positive selection was inferred in two core glycolysis enzymes. Pgi and Glyceraldehyde 3-phosphate dehydrogenase (Gaphd) genes both encode enzymes linking glycolysis to the pentose phosphate pathway. Positive selection among Pgi paralogues and orthologues predominately targets amino acids with residues exposed to the protein's surface, where changes in physical properties may alter enzyme performance., Conclusion: Our results suggest that, for Lancerocercata stick insects, adaptation to new stressful lifestyles requires a balance between maintaining cellular energy production, efficiently exploiting different energy storage pools and compensating for stress-induced oxidative damage.