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

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

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

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

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

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

It's Not Just Me. Parents' Perceptions of a Multi-Disciplinary Therapy Group for Infants with Disabilities.

Aim: To explore parent perceptions of Baby Club, a therapeutic program involving physiotherapy, occupational therapy, and speech and language therapy for infants with physical disabilities under 2 years and to better understand how the group addresses child and family needs. Methods: A qualitative descriptive design using semi-structured interviews and a brief demographics questionnaire. Eight parents who attended a Baby Club program between 2017 and 2018 were recruited from ErinoakKids Centre for Treatment and Development. Interviews were audio-recorded, transcribed, and de-identified. A content analysis approach was used with an inductive process to generate a coding scheme. Investigators developed overarching themes. Results: Three themes representing parents' perceptions of Baby Club were identified: (1) Learning Together through Play and Fun; (2) I'm Not the Only One - Sharing and Support; (3) The Value of Therapist Time and Expertise. Conclusions: Baby Club provides parents and infants with opportunities to learn new skills through play, providing enriching social experiences and networking in a supportive therapeutic group environment.

Every team needs a coach: Training for interprofessional clinical placements.

Despite growing awareness of the benefits of interprofessional education and interprofessional collaboration (IPC), understanding how teams successfully transition to IPC is limited. Student exposure to interprofessional teams fosters the learners' integration and application of classroom-based interprofessional theory to practice. A further benefit might be reinforcing the value of IPC to members of the mentoring team and strengthening their IPC. The research question for this study was: Does training in IPC and clinical team facilitation and mentorship of pre-licensure learners during interprofessional clinical placements improve the mentoring teams' collaborative working relationships compared to control teams? Statistical analyses included repeated time analysis multivariate analysis of variance (MANOVA). Teams on four clinical units participated in the project. Impact on intervention teams pre- versus post-interprofessional clinical placement was modest with only the Cost of Team score of the Attitudes Towards Healthcare Team Scale improving relative to controls (p = 0.059) although reflective evaluations by intervention team members noted many perceived benefits of interprofessional clinical placements. The significantly higher group scores for control teams (geriatric and palliative care) on three of four subscales of the Assessment of Interprofessional Team Collaboration Scale underscore our need to better understand the unique features within geriatric and palliative care settings that foster superior IPC and to recognise that the transition to IPC likely requires a more diverse intervention than the interprofessional clinical placement experience implemented in this study. More recently, it is encouraging to see the development of innovative tools that use an evidence-based, multi-dimensional approach to support teams in their transition to IPC.

Advancing Interprofessional Collaborative Teams in the Winnipeg Health Region.

This project developed an evaluation platform aimed at diagnosing team functioning using evidence-informed, measurable indicators to provide an actionable roadmap to guide teams in improving their interprofessional collaborative team performance. A scoping literature review, stakeholder consultation, survey and focus groups were conducted to inform both the final selection of eight indicators of effective, high-performing teams and the process to assess and evaluate teams against these indicators. The program was piloted with two interprofessional teams in the Winnipeg Health Region. Focus groups and questionnaires were used to evaluate the program.

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

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

Evaluation of the cyclooxygenase selectivity of robenacoxib and its effect on recovery of ischemia-injured jejunal mucosa in horses.

OBJECTIVE: To determine the cyclooxygenase (COX) selectivity of robenacoxib and its effect on recovery of jejunal mucosa following ischemic injury in horses. ANIMALS: 12 healthy horses. PROCEDURES: Half the maximal inhibition (EC50) of robenacoxib for COX-1 and COX-2 activity was established in bloods samples from 6 horses via measurement of thromboxane B2 (TXB2) and prostaglandin E2 concentrations, respectively; COX selectivity was subsequently calculated. Six other horses were anesthetized, and ischemia was induced in the jejunum for 2 hours. Control and ischemia-injured mucosa were collected and incubated with Ringer's solution (control treatment), flunixin meglumine (2.7 × 10⁻5M), or robenacoxib (2.7 × 10⁻5M). Transepithelial electrical resistance and mannitol flux were measured over a 4-hour recovery period. Bathing solution TXB2 and prostaglandin E metabolite concentrations were measured to assess COX-1 and COX-2 function, respectively. RESULTS: The mean ± SD EC50 value of robenacoxib for COX-1 and COX-2 was 11.46 ± 4.46 µM and 0.19 ± 0.07 µM, respectively, resulting in a COX selectivity ratio of 61.01. The transepithelial electrical resistance of ischemia-injured jejunum treated with flunixin meglumine was significantly lower than that of control and robenacoxib-treated tissues. A significant increase in concentrations of prostaglandin E metabolites and TXB2 was detected in control and robenacoxib-treated tissues but not flunixin meglumine-treated tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Robenacoxib selectively inhibited COX-2 and allowed recovery of barrier function in ischemia-injured equine jejunal tissue in vitro.

Demographic characteristics of horses donated to the North Carolina State University Equine Health Center, 1996-2008.

OBJECTIVE: To determine demographic characteristics of horses donated to the North Carolina State University Equine Health Center (EHC) between 1996 and 2008. DESIGN: Retrospective cohort study. ANIMALS: 122 horses donated to the EHC between January 1996 and December 2008, and 246 horses offered for donation to the EHC between January 2007 and December 2008. PROCEDURES: Telephone and medical records were examined. Data were collected in 5 categories: age, sex, breed, reason for donation, and use prior to donation. RESULTS: From January 1996 through December 2008, 122 horses were donated to the EHC (median, 3 horses/y; range, 0 to 39 horses/y). There were 131 and 115 horses offered for donation during 2007 and 2008, respectively, of which 38 and 23 were accepted. Mean +/- SD age of horses offered for donation during 2007 and 2008 was 12.7 +/- 6.7 years, with 75 of the 246 (30.5%) horses between 6 and 10 years old. Musculoskeletal disease was the most commonly listed reason horses were offered for donation (115/240 [47.9%]). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that unwanted horses donated to the EHC between 1996 and 2008 spanned a wide range of ages and breeds and included both males and females. The most common reason given for unwanted horses offered for donation during 2007 and 2008 was musculoskeletal disease, with degenerative joint disease, lameness of undetermined cause, laminitis, and navicular disease being the most common musculoskeletal conditions.

Neuropeptide-like protein diversity in phylum Nematoda.

This study reports the identification of nematode neuropeptide-like protein (nlp) sequelogs from the GenBank expressed sequence tag (EST) database, using BLAST (Basic Local Alignment Search Tool) search methodology. Search strings derived from peptides encoded by the 45 known Caenorhabditis elegans nlp genes were used to identify more than 1000 ESTs encoding a total of 26 multi-species nlp sequelogs. The remaining 18 nlps (nlp-4, -16, -24 through -36, -39, -41 and -45) were identified only in C. elegans, while the sole EST representative of nlp-23 was from Caenorhabditis remanei. Several ESTs encoding putative antibacterial peptides similar to those encoded by the C. elegans genes nlp-24-33 were observed in several parasite species. A novel gene (nlp-46) was identified, encoding a single, amidated dodecapeptide (NIA[I/T]GR[G/A]DG[F/L]RPG) in eight species. Secretory signal peptides were identified in at least one species representing each nlp sequelog, confirming that all 46 nematode nlp genes encode secretory peptides. A random sub-set of C. elegans NLPs was tested physiologically in Ascaris suum ovijector and body wall muscle bioassays. None of the peptides tested were able to modulate ovijector activity, while only three displayed measurable myoactivity on somatic body wall muscle. AFAAGWNRamide (from nlp-23) and AVNPFLDSIamide (nlp-3) both produced a relaxation of body wall muscle, while AIPFNGGMYamide (nlp-10) induced a transient contraction. Numerical analyses of nlp-encoding ESTs demonstrate that nlp-3, -13, -14, -15 and -18 are amongst the most highly represented transcripts in the dataset. Using available bioinformatics resources, this study delineates the nlp complement of phylum Nematoda, providing a rich source of neuropeptide ligands for deorphanisation of nematode neuropeptide receptors.

Molecular phylogeny of the pinworms of mice, rats and rabbits, and its use to develop molecular beacon assays for the detection of pinworms in mice.

Though pinworm infestation has been prevalent since the early years of laboratory animal medicine, the genomes of these parasites have not yet been sequenced. The authors used high-fidelity polymerase chain reaction to amplify a large portion of the ribosomal gene complex of four pinworm species commonly found in lab rodents and rabbits (Aspiculuris tetraptera, Passalurus ambiguus, Syphacia muris and Syphacia obvelata). They determined DNA sequences for these complexes and carried out phylogenetic analysis. Using this information, the authors developed real-time molecular beacon assays for pinworm detection, comparing the new diagnostic approach with traditional methods such as perianal tape testing, fecal flotation and direct examination of intestinal content.

Structure-activity relationships of an inhibitory nematode FMRFamide-related peptide, SDPNFLRFamide (PF1), on Ascaris suum muscle.

FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure-function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K+ channels in the same tissue. The use of K+ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals.

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.

Marcfortine and paraherquamide class of anthelmintics: discovery of PNU-141962.

Three distinct chemical classes for the control of gastrointestinal nematodes are available: benzimidazoles, imidazothiazoles, and macrocyclic lactones. The relentless development of drug resistance has severely limited the usefulness of such drugs and the search for a new class of compounds preferably with a different mode of action is an important endeavor. Marcfortine A (1), a metabolite of Penicillium roqueforti, is structurally related to paraherquamide A (2), originally isolated from Penicillium paraherquei. Chemically the two compounds differ only in one ring; in marcfortine A, ring G is six-membered and carries no substituents, while in paraherquamide A, ring G is five-membered with methyl and hydroxyl substituents at C14. Paraherquamide A (2) is superior to marcfortine A as a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has excellent nematocidal activity, a superior safely profile, and is the first semi-synthetic member of this totally new class of nematocides that is a legitimate candidate for development. This review describes the chemistry, efficacy and mode of action of PNU-141962.