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Background: A comprehensive examination of the sport-specific activities and circumstances being performed at the time of injury is important to hypothesise mechanisms, develop prevention strategies and inform future investigations. Results reported in the literature are inconsistent because inciting activities are reported using different classifications. Hence the aim was to develop a standardised system for the reporting of inciting circumstances. Methods: The system was developed using a modified Nominal Group Technique. The initial panel included 12 sports practitioners and researchers from four continents with respectively ≥ 5 years of experience working in professional football and/or conducting injury research. The process consisted of six phases: idea generation, two surveys, one online meeting and two confirmations. For answers to the closed questions, consensus was deemed achieved if ≥ 70% of respondents agreed. Open-ended answers were qualitatively analysed and then introduced in subsequent phases. Results: Ten panellists completed the study. The risk of attrition bias was low. The developed system includes a comprehensive range of inciting circumstances across five domains: contact type, ball situation, physical activity, session details, contextual information. The system also distinguishes between a core set (essential reporting) and an optional set. The panel deemed all the domains to be important and easy to use both in football and in research environments. Conclusion: A system to classify inciting circumstances in football was developed. Given the extent of reporting inconsistency of inciting circumstances in the available literature, this can be used while further studies evaluate its reliability. [ABSTRACT FROM AUTHOR]

Background: A comprehensive examination of the sport-specific activities performed around the time of injury is important to hypothesise injury mechanisms, develop prevention strategies, improve management, and inform future investigations. The aim of this systematic review is to summarise the current literature describing the activities performed around the time of injury in football (soccer). Methods: A systematic search was carried out in PubMed, Web of Science, SPORTDiscus, and OpenGrey. Studies were included if participants were football players aged > 13 years old and the activities performed at the time of injury were reported together with the total number of injuries. Risk of bias was assessed using an adapted version of checklists developed for prevalence studies. The activities reported by the studies were grouped to account for inconsistent reporting, and the proportion of each injury activity was calculated. Data were not meta-analysed due to high heterogeneity of methods and classification criteria. Results: We included 64 studies reporting on 56,740 injuries in total. ACL injures were analysed by 12 studies, ankle/foot and knee injuries were analysed by five studies, thigh injuries were analysed by four studies, hip/groin injuries were analysed by three studies, and hamstring injuries were analysed by two studies. Five studies analysed more than one type of injury and 38 studies did not specify the type of injuries analysed. Running and kicking were the predominant activities leading to thigh and hamstring injuries. Changing direction and kicking were the predominant activities leading to hip and groin injuries and duels were the predominant activities leading to ankle injuries. Duels and pressing seem the predominant activities leading to ACL injuries, while results for other knee and general injuries were inconsistent. Conclusions: A qualitative summary of the activities performed at the time of injury has been reported. The results need to be interpreted carefully due to the risk of bias observed in the included studies. If we are to meaningfully progress our knowledge in this area, it is paramount that future research uses consistent methods to record and classify injuries and activities leading up to and performed at the time of injury. Registration: The protocol of this systematic review was registered at the Open Science Framework (https://doi.org/10.17605/OSF.IO/U96KV). [ABSTRACT FROM AUTHOR]

Abstract: In Drosophila, primary pair-rule genes establish the parasegmental boundaries and indirectly control the periodic expression of the segment polarity genes engrailed (en) and wingless (wg) via regulation of secondary pair-rule genes. Although orthologs of some Drosophila pair-rule genes are not required for proper segmentation in Tribolium, segmental expression of Tc-en and Tc-wg is conserved. To understand how these segment polarity genes are regulated, we examined the results of expressing one or two pair-rule genes in the absence of the other known pair-rule genes. Expression of one or both of the secondary pair-rule genes, Tc-sloppy-paired (Tc-slp) and Tc-paired (Tc-prd), activated Tc-wg in the absence of the primary pair-rule genes, Tc-even-skipped (Tc-eve), Tc-runt (Tc-run) and Tc-odd-skipped (Tc-odd). Tc-eve alone failed to activate Tc-wg or Tc-en, but in combination with Tc-run or Tc-prd activated Tc-en. These results, interpreted within the pair-rule gene expression patterns, suggest separate models for the genetic regulation of the juxtaposed expression of Tc-wg and Tc-en at odd- and even-numbered parasegmental boundaries, respectively. Conserved interactions between eve and prd at the anterior boundary of odd-numbered parasegments may reflect an ancestral segmentation mechanism that functioned in every segment prior to the evolution of pair-rule segmentation. [Copyright &y& Elsevier]

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi. [ABSTRACT FROM AUTHOR]

In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway ( Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these small spherical embryos. On the other hand, RNAi analysis of a negative regulator ( Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en–wg–hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect. [ABSTRACT FROM AUTHOR]

Abstract: In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. [Copyright &y& Elsevier]

Protein–DNA interaction plays a pivotal role in transcriptional regulation, DNA metabolism and chromatin formation. Although structural data are available for a few hundreds of protein–DNA complexes, the molecular recognition pattern is still poorly understood. With the rapid accumulation of sequence data from many genomes, it is important to develop predictive methods for identification of potential DNA-binding residues in proteins. In this study, neural networks have been trained using five sequence-derived features for prediction of DNA-binding residues. These features include the molecular mass, hydrophobicity index, side chain pKa value, solvent accessible surface area and conservation score of an amino acid. Interestingly, the side chain pKa value appears to be the best feature for prediction, suggesting that the ionization state of amino acid side chains is important for DNA-binding. The predictive performance is enhanced by using multiple features for classifier construction. The classifier that has been constructed using all the five features predicts at 72.71% sensitivity and 67.73% specificity. This is by far the most accurate classifier reported for prediction of DNA-binding residues from sequence data. The classifier has also been evaluated by using the Receiver Operating Characteristic curve and by examining the predictions made for different classes of DNA-binding proteins. Supplementary materials including the datasets are available at . [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to ascertain patients' and health professionals' requirements from a rheumatology helpline and how such a service should be delivered. Methods: Self-completed questionnaires were administered to both users of rheumatology helplines, patients and health professionals (HPs), and providers of rheumatology services at five UK NHS trusts. Additional data were sought from experts in the field. Information was elicited on access to the service, preferred waiting time for response to a call, acceptability of an answerphone and choice of HP responding. Quantitative data were analysed using percentages and Chi-squared tests. Open questions were analysed as free text responses, and organized into clusters of themes. Results: A total of 607 questionnaires were returned, 523 from users (411 patients, 112 health professionals) and 84 from providers. There were no significant differences across the five Trusts. The top six reasons for patients contacting the helpline were: advice on changes in condition, drug information, understanding symptoms, blood results, information on diagnosis, and appointment queries. All groups recommended a return call on the same day and were happy to leave an answerphone message. HP users requested additional access via e-mail and fax. Rheumatology practitioners were deemed the most appropriate personnel to staff a helpline. Conclusions: The findings of this study have contributed a valuable insight into the essential components of a rheumatology helpline service from a user and provider perspective. These data will add to existing recommendations for the management of a rheumatology helpline service and ultimately contribute to a proposal for national guidelines. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to investigate the impact of the pilot education programmes, entitled `Focus on Lupus' and `Focus on Scleroderma', upon participants' management of their disease, Methods: Five people with lupus and five with scleroderma were invited to take part in individual semi-structured interviews. Qualitative analysis of transcripts was performed using a framework approach. Views relating to changes in knowledge and behaviour, increased empowerment, meeting others and the format and delivery of the programmes were explored. Results: Analysis of the interviews revealed overall satisfaction with both programmes, however people with lupus had more positive feelings about their attendance. Both disease groups considered it valuable to meet others with the same disease and welcomed the involvement of a patient educator within the programme planning team. The main difference between the two groups concerned behaviour change. The lupus group revealed more definite life changes. Participants unanimously regarded the format of the programme highly and most were satisfied with the content. [ABSTRACT FROM AUTHOR]

Evidence from Drosophila suggests that Hox genes not only specify regional identity, but have the additional function of repressing antennal development within their normal domains. This is dramatically demonstrated by a series of Hox mutants in the red flour beetle, Tribolium castaneum, and is likely an ancient function of Hox genes in insetcs. [Copyright &y& Elsevier]

Examines the sequence of Tribolium casteneum homeotic complex. Transcription factors in regulating hox gene encoding; Polymorphism of Tribolium DNA; Comparison of Tribolium and Drosophila genes.

Studies the cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase. Wild-type strains; Degenerate polymerase chain reaction; Isolation of cDNA and genomic clones; DNA sequencing and analysis.

SUMMARY Null mutations in the Drosophila melanogaster homeotic gene proboscipedia (pb) cause transformation of the adult labial palps to legs. The similar phenotype produced by mutations in the Tribolium castaneum homeotic complex (HOMC) gene maxillopedia (mxp) has led to suggestions that the two genes may be orthologous. We have cloned the Tribolium ortholog of pb, which predicts a protein with a homeodomain identical to that of Drosophila Pb. The two proteins also share several additional regions of identity, including an N-box, a motif unique to Pb orthologs. We have identified a frameshift mutation within Tribolium pb associated with an mxp null mutation, demonstrating that Tribolium pb corresponds to the mxp genetic locus. Thus, we will refer to the cloned gene as mxp. In addition, we have begun to construct a molecular map of the Tribolium HOMC. Two overlapping BAC clones which span the mxp locus also include the Tribolium labial ortholog (Tclabial ) and part of Tczerknüllt, indicating that the order of these genes in the HOMC is conserved between Drosophila and Tribolium. [ABSTRACT FROM AUTHOR]

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies. [ABSTRACT FROM AUTHOR]

Examines a genetic map based on randomly amplified polymorphic DNA (RAPD) of Tribolium castaneum. Efficiency of RAPD mapping; Conversion of RAPD to sequence-tagged site markers; Identification of single-strand conformational polymorphisms; RAPD map as tool for genomics; Correlation between sex chromosomes and linkage group.

Life table data and intrinsic rates of increase were obtained for Myzus persicae, Brachycaudus helichrysi and Macrosiphoniella sanborni on chrysanthemum and for Aphis gossypii on cucumber, at 18 and 24° C, with daylengths of 8 and 16 h and light intensities of 800, 4000 and 8000 lux, in all combinations, using a modified Munger cell technique. In General, Aphis gossypii matured earliest and produced most young, followed by Myzus persicae. Pre-reproductive time, mean generation time and longevity were prolonged by low temperature, but were little affected by other factors. The number of progeny was greatly reduced by poor light. Daylength had no general effect but produced strong and complex interactions with other factors. [ABSTRACT FROM AUTHOR]

For more than a decade the artificial sweeteners acesulfame (ACE) and sucralose (SUC) have been applied as tracers of the input of wastewater to environmental waters. Recently concerns have been raised that degradation of ACE during treatment may hinder or restrict its use as a wastewater tracer. In this study the value of ACE and SUC as tracers was reassessed based on samples of wastewater at 12 municipal wastewater treatment (MWWT) plants and from 7 septic systems and associated septic plumes in groundwater. The results indicated stability of SUC during MWWT at most plants, and variable removal of both sweeteners during some MWWT and in the septic wastewater systems. However, the residual concentrations of ACE and SUC in municipal effluent and in septic plumes indicate that both sweeteners remain valuable wastewater tracers. The mass ratio SUC/ACE was found to be a useful parameter for examining the relative persistence of these sweeteners. Image 1 • Synthetic sweeteners acesulfame and sucralose persist during wastewater treatment. • They remain useful as tracers of wastewater in environmental waters. • Analysis of these chemicals as co-tracers is advantageous. [ABSTRACT FROM AUTHOR]

This study presents the first reported detections and concentrations of alkyl phenoxy-benzenesulfonate surfactants (APBS) in municipal wastewater. A semi quantitative direct injection LC/MS/MS method was developed. Samples of raw influent and final effluent were obtained from fourteen municipal wastewater treatment plants (WWTPs) at various locations in Canada and were analyzed for APBS, including five homologues of monoalkyldiphenylether disulfonates (MADS) and one monoalkyldiphenylether sulfonate (MAMS) homologue. APBS were detected in all 42 of the wastewater raw influent samples and in 37 of the 42 wastewater final effluent samples; the other 5 final effluent samples had trace levels below the minimum detection limit. In the samples of raw influent from the fourteen municipal treatment plants, the dissolved concentrations of APBS (total) ranged from 0.9 to 13.6 μg/L. In samples of final effluent from the same plants the total APBS ranged from below detection to 4 μg/L. The APBS were more resistant to loss during wastewater treatment compared to previous studies of linear alkylbenzene sulfonates in wastewaters. The most effective wastewater treatments for removal of APBS were those that involved either secondary treatment with aeration or advanced treatment including biological nutrient removal. Available information on ecotoxicity is lacking for evaluating the impacts of APBS surfactants when released to the environment. • First detections of alkyl phenoxy-benzenesulfonate surfactants in wastewater. • Detections in all raw influent samples and in most final effluent samples. • Seem more resistant to wastewater treatment than linear alkylbenzene sulfonates. [ABSTRACT FROM AUTHOR]

Motion capture systems are used in the analysis and interpretation of athlete movement patterns for a variety of reasons, but data integrity remains critical regardless. The extent to which marker location or constraining degrees of freedom (DOF) in the biomechanical model impacts on this integrity lacks consensus. Ten elite academy footballers performed bilateral overhead squats using a marker-based motion capture system. Kinematic data were calculated using four different marker sets with 3DOF and 6DOF configurations for the three joint rotations of the right knee. Root mean squared error differences between marker sets ranged in the sagittal plane between 1.02 and 4.19 degrees to larger values in the frontal (1.30–6.39 degrees) and transverse planes (1.33 and 7.97 degrees). The cross-correlation function of the knee kinematic time series for all eight marker-sets ranged from excellent for sagittal plane motion (>0.99) but reduced for both coronal and transverse planes (<0.9). Two-way ANOVA repeated measures calculated at peak knee flexion revealed significant differences between marker sets for frontal and transverse planes (p < 0.05). Pairwise comparisons showed significant differences between some marker sets. Marker location and constraining DOF while measuring relatively large ranges of motion in this population are important considerations for data integrity. [ABSTRACT FROM AUTHOR]

PROBLEM STATEMENT: To define the Oncology Nursing Society Research Agenda for 2019-2022. DESIGN: Multimethod, consensus-building approach by members of the Research Agenda Project Team. DATA SOURCES: Expert opinion, literature review, surveys, interviews, focus groups, town hall, and review of research priorities from other cancer care organizations and funding agencies. ANALYSIS: Content analysis and descriptive statistics were used to synthesize research priority themes that emerged. FINDINGS: Three priority areas for scientific development were identified: symptom science, health disparities, and palliative and psychosocial care in oncology. In addition, cross-cutting themes that provide context and elaboration for these priorities emerged. IMPLICATIONS FOR NURSING: The Research Agenda can be used to focus oncology nurses' research, scholarship, leadership, and health policy efforts to advance quality cancer care, inform research funding priorities, and align initiatives and resources across the ONS enterprise. [ABSTRACT FROM AUTHOR]

While it is recognized that groundwater contaminant plumes can impact surface waters, there remains little information on the magnitude, spatial extent, and especially temporal variability of the resulting exposure to the variety of aquatic organisms, particularly for stagnant surface waters (e.g., ponds). The present study of a historic landfill plume discharging to a pond investigated contaminant exposure to multiple aquatic zones (endobenthic, epibenthic, pelagic) over approximately 1 year within a temperate climate. Landfill tracers included the artificial sweetener saccharin, ammonium, chloride, and specific conductance. Sampling of pond sediment porewater (upwelling groundwater) and continuous geophysical imaging of the subsurface showed a relatively stable plume footprint covering approximately 26% of the pond, although with spatially varying leachate composition, revealing year‐round exposure to endobenthic (within sediments) organisms. Substantial and variable contaminant exposure to epibenthic organisms within the plume footprint was shown by elevated specific conductance measured directly above the sediment interface. Exposure varied daily at times and increased through winter to values representing undiluted plume groundwater. Exposure to pelagic organisms (overlying water) covered a larger area (~50%) due to in‐pond circulation. The stream outlet concentrations were stable at approximately 10 times dilution for chloride and saccharin, but were substantially less in summer for ammonium due to in‐pond processes. Whereas groundwater contaminants are typically assumed elevated at base flows, the outlet stream contaminant mass discharges to downstream receptors were notably higher in winter than summer, following stream flow patterns. Insights from the present study into the timings and locations of contaminant plume exposure to multiple ecological zones of a pond can provide guidance to contaminated site and aquatic ecosystem managers on improved monitoring, assessment, and remediation protocols. Environ Toxicol Chem 2023;42:1667–1684. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada. [ABSTRACT FROM AUTHOR]

Monitoring of a seasonal‐use, on‐site wastewater disposal system (septic system) in Canada, over a 33‐year period from 1988 to 2021, showed that during recent sampling the groundwater plume had TIN (total inorganic nitrogen) averaging 12.2 mg/L that was not significantly different than early values, representing 80% removal, whereas SRP (soluble reactive phosphate), although higher than early values averaging 0.08 mg/L, was still 99% lower than the effluent concentration. Evidence suggests that the anammox reaction and possibly also denitrification contribute to TIN removal, whereas SRP removal is primarily the result of mineral precipitation. Most of the removal occurs in close proximity to the drainfield infiltration pipes (within about 1 m) demonstrating that reaction rates are relatively fast in the context of typical groundwater plume residence times. This long‐term consistency demonstrates that sustainable nutrient treatment can be achieved with conventional on‐site wastewater disposal systems that have low capital costs and require minimal energy input and maintenance. [ABSTRACT FROM AUTHOR]

Monitoring of a well-defined septic system groundwater plume and groundwater discharging to two urban streams located in southern Ontario, Canada, provided evidence of natural attenuation of background low level (ng/L) perchlorate ( ClO4−) under denitrifying conditions in the field. The septic system site at Long Point contains ClO4− from a mix of waste water, atmospheric deposition, and periodic use of fireworks, while the nitrate plume indicates active denitrification. Plume nitrate ( NO3−-N) concentrations of up to 103 mg/L declined with depth and downgradient of the tile bed due to denitrification and anammox activity, and the plume was almost completely denitrified beyond 35 m from the tile bed. The ClO4− natural attenuation occurs at the site only when NO3−-N concentrations are <0.3 mg/L, after which ClO4− concentrations decline abruptly from 187 ± 202 to 11 ± 15 ng/L. A similar pattern between NO3−-N and ClO4− was found in groundwater discharging to the two urban streams. These findings suggest that natural attenuation (i.e., biodegradation) of ClO4− may be commonplace in denitrified aquifers with appropriate electron donors present, and thus, should be considered as a remediation option for ClO4− contaminated groundwater. [ABSTRACT FROM AUTHOR]

Artificial sweeteners have been widely incorporated in human food products for aid in weight loss regimes, dental health protection and dietary control of diabetes. Some of these widely used compounds can pass non-degraded through wastewater treatment systems and are subsequently discharged to groundwater and surface waters. Measurements of artificial sweeteners in rivers used for drinking water production are scarce. In order to determine the riverine concentrations of artificial sweeteners and their usefulness as a tracer of wastewater at the scale of an entire watershed, we analyzed samples from 23 sites along the entire length of the Grand River, a large river in Southern Ontario, Canada, that is impacted by agricultural activities and urban centres. Municipal water from household taps was also sampled from several cities within the Grand River Watershed. Cyclamate, saccharin, sucralose, and acesulfame were found in elevated concentrations despite high rates of biological activity, large daily cycles in dissolved oxygen and shallow river depth. The maximum concentrations that we measured for sucralose (21 µg/L), cyclamate (0.88 µg/L), and saccharin (7.2 µg/L) are the highest reported concentrations of these compounds in surface waters to date anywhere in the world. Acesulfame persists at concentrations that are up to several orders of magnitude above the detection limit over a distance of 300 km and it behaves conservatively in the river, recording the wastewater contribution from the cumulative population in the basin. Acesulfame is a reliable wastewater effluent tracer in rivers. Furthermore, it can be used to assess rates of nutrient assimilation, track wastewater plume dilution, separate human and animal waste contributions and determine the relative persistence of emerging contaminants in impacted watersheds where multiple sources confound the usefulness of other tracers. The effects of artificial sweeteners on aquatic biota in rivers and in the downstream Great Lakes are largely unknown. [ABSTRACT FROM AUTHOR]

In Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other arthropods, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. Recently, the cyclic nature of the pair-rule gene Tc-odd-skipped was demonstrated in the growth zone of Tribolium, indicating that a vertebrate-like segmentation clock is employed in the germband stage of its development. This suggests that two mechanisms might function in the same organism: a Drosophila-like mechanism in the blastoderm, and a vertebrate-like mechanism in the germband. Here, we show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, we show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clockbased segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Moreover, by tracking cells in live embryos and by analyzing mitotic profiles, we found that neither cell movement nor oriented cell division could explain the observed wave dynamics of Tc-eve. These results pose intriguing evolutionary questions, as Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms. [ABSTRACT FROM AUTHOR]

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects. [ABSTRACT FROM AUTHOR]

Summary: There is little information available on the prevalence of artificial sweeteners in groundwater, though these compounds may prove to be useful tracers of human wastewater, especially in urban settings with complex hydrology. In this study, the artificial sweetener acesulfame was detected in groundwater at all eight urban sites investigated (from five different urban areas in Canada), often at high concentrations (i.e., μg/L-scale). In a municipal wastewater plume at Jasper, Alberta, acesulfame was strongly correlated with chloride and was positively correlated with other wastewater-related contaminants indicating that this sweetener has potential to be a good tracer of young wastewater (<20years residence time) in Canada. Three other artificial sweeteners were detected in urban groundwater: saccharin at six of the sites, sucralose at three sites, and cyclamate at five of seven sites where it was analyzed. The occurrence of sucralose may have been affected by its detection limit, which was much higher than for the other sweeteners. These results, and those of a parallel study, are the first reported detections of saccharin and cyclamate in groundwater, and suggest that these sweeteners may be more common than previously anticipated. In general, fewer samples from each site contained these other three sweeteners compared to acesulfame. At Barrie, Ontario, adjacent to an old landfill, the concentration of saccharin was higher than acesulfame in many samples. These results suggest that analyses of multiple sweeteners, rather than just acesulfame, may provide useful information on contaminant sources and groundwater conditions in urban settings. Further work is needed to address this potential use. [ABSTRACT FROM AUTHOR]

Early stages of lineage divergence in insect herbivores are often related to shifts in host plant use and divergence in mating capabilities, which may lead to sexual isolation of populations of herbivorous insects. We examined host preferences, degree of differentiation in mate choice, and divergence in cuticular morphology using near-infrared spectroscopy in the grasshopper Hesperotettix viridis aiming to understand lineage divergence. In Kansas (USA), H. viridis is an oligophagous species feeding on Gutierrezia and Solidago host species. To identify incipient mechanisms of lineage divergence and isolation, we compared host choice, mate choice, and phenotypic divergence among natural grasshopper populations in zones of contact with populations encountering only one of the host species. A significant host-based preference from the two host groups was detected in host-paired feeding preference studies. No-choice mate selection experiments revealed a preference for individuals collected from the same host species independent of geographic location, and little mating was observed between individuals collected from different host species. Female mate choice tests between males from the two host species resulted in 100% fidelity with respect to host use. Significant differentiation in colour and cuticular composition of individuals from different host plants was observed, which correlated positively with host choice and mate choice. No evidence for reinforcement in the zone of contact was detected, suggesting that divergent selection for host plant use promotes sexual isolation in this species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 866–878. [ABSTRACT FROM AUTHOR]

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624–1629, ; Angelini and Kaufman Dev Biol 283:409–423, ; Miyawaki et al. Mech Dev 121:119–130, ). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine ( Tc-porc) or Tc-pangolin ( Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624–1629, ). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560–6564, ), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt ( Tc-run) and Tc-even-skipped ( Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone . Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone. [ABSTRACT FROM AUTHOR]

Abstract: The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum α-Tubulin1 (TcαTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcαTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages. [Copyright &y& Elsevier]

In Drosophila, a hierarchy of maternal, gap, pair-rule, and segment polarity gene interactions regulates virtually simultaneous blastoderm segmentation. For the last decade, studies have focused on revealing the extent to which Drosophila segmentation mechanisms are conserved in other arthropods where segments are added sequentially from anterior to posterior in a cellular environment. Despite our increased knowledge of individual segmentation genes, details of their interactions in non-Drosophilid insects are not well understood. We analyzed the Tribolium orthologs of Drosophila pair-rule genes, which display pair-rule expression patterns. Tribolium castaneum paired (Tc-prd) and sloppy-paired (Tc-slp) genes produced pair-rule phenotypes when their transcripts were severely reduced by RNA interference. In contrast, similar analysis of T. castaneum even-skipped (Tc-eve), runt (Tc-run), or odd-skipped (Tc-odd) genes produced severely truncated, almost completely asegmental phenotypes. Analysis of interactions between pair-rule components revealed that Tc-eve, Tc-run, and Tc-odd form a three-gene circuit to regulate one another as well as their downstream targets, Tc-prd and Tc-slp. The complement of primary pair-rule genes in Tribolium differs from Drosophila in that it includes Tc-odd but not Tc-hairy. This gene circuit defines segments sequentially in double segment periodicity. Furthermore, this single mechanism functions in the early blastoderm stage and subsequently during germ-band elongation. The periodicity of the Tribolium pair-rule gene interactions reveals components of the genetic hierarchy that are regulated in a repetitive circuit or clock-like mechanism. This pair-rule gene circuit provides insight into short-germ segmentation in Tribolium that may be more generally applicable to segmentation in other arthropods. [ABSTRACT FROM AUTHOR]

Anterior morphogens have long been postulated to function in early pattern formation in several insect taxa. Genetic studies inDrosophilarevealed that the Bicoid protein performs as such in the fly. Maternally providedbicoid(bcd) mRNA is localized at the anterior pole of the oocyte and, upon fertilization, acts as the source of a morphogenetic gradient of Bicoid protein. Despite its central role inDrosophila, it is believed thatbicoidarose rather recently during dipteran evolution through duplication of theHox3orthologzen.Here we show that in the red flour beetleTribolium castaneum, RNAs of the homologs of theDrosophilatranscription factorseagle(Tc′ eagle) andpangolin(Tc′ pan) are maternally localized at the anterior pole of the egg. It is possible that underlying mechanisms for the anterior localization of maternal RNAs evolved independently in the two clades. However, considering the complexity of the process inDrosophila, it is more tempting to speculate that the molecular machinery evolved only once during insect evolution. Under this assumption, it was present beforebicoidevolved as an anterior morphogen. To become concentrated at the anterior pole of the eggbicoidmay only have had to acquire a signal sequence that was recognized by the preexisting localization machinery. Although we were not able to demonstrate a function in early embryogenesis forTc′ eagleorTc′ pan, our findings suggest that other factors likely function as anterior determinants inTribolium, and other nondipteran insects. [ABSTRACT FROM AUTHOR]

Examines the role Hox gene expression in the morphological evolution of insects. Determination of embryonic labial development; Dependence of labium identity on homeotic genes; Establishment on gnathal segment identities.

Background: The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results: We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions: We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting. [ABSTRACT FROM AUTHOR]

Quantitative information about fluxes of perchlorate in the environment is lacking. This study reports analyses of perchlorate in various environmental waters sampled from rural headwater catchments in the Thames River basin in southern Ontario (Canada) that provide evidence about the fluxes and fate of perchlorate in the environment. Concentrations in streams (16 to 1047 ng/L) were used to estimate exports from these rural catchments (228–1843 mg/(ha·year)), atmospheric deposition (1480 ± 230 mg/(ha·year)), as well as variable rates of microbial degradation of perchlorate, which appeared to be enhanced in catchments with higher percentages of wetlands. Groundwater data supported earlier evidence that degradation of perchlorate occurs in the subsurface under oxygen-depleted conditions. The stream data suggest that the rate of degradation varies strongly between catchments and ranges up to >1000 mg/(ha·year). Unlabelled Image • Perchlorate concentrations in streams used to estimate exports from rural catchments. • Atmospheric deposition of perchlorate inferred to be higher than earlier estimates. • Evidence for enhanced microbial degradation of perchlorate in wetlands and groundwater [ABSTRACT FROM AUTHOR]

High quality gene models are necessary to expand the molecular and genetic tools available for a target organism, but these are available for only a handful of model organisms that have undergone extensive curation and experimental validation over the course of many years. The majority of gene models present in biological databases today have been identified in draft genome assemblies using automated annotation pipelines that are frequently based on orthologs from distantly related model organisms and usually have minor or major errors. Manual curation is time consuming and often requires substantial expertise, but is instrumental in improving gene model structure and identification. Manual annotation may seem to be a daunting and cost-prohibitive task for small research communities but involving undergraduates in community genome annotation consortiums can be mutually beneficial for both education and improved genomic resources. We outline a workflow for efficient manual annotation driven by a team of primarily undergraduate annotators. This model can be scaled to large teams and includes quality control processes through incremental evaluation. Moreover, it gives students an opportunity to increase their understanding of genome biology and to participate in scientific research in collaboration with peers and senior researchers at multiple institutions. [ABSTRACT FROM AUTHOR]

Abstract The pair-rule gene teneurin-m/odd Oz (ten-m/odz) is required for the patterning of alternate segment boundaries in the early Drosophila embryo. Mutant phenotypes of ten-m/odz display a typical pair-rule phenotype in which odd-numbered segments are eliminated. Consistent with its pair-rule function, Ten-m/Odz protein is expressed in a seven-stripe pattern before the onset of gastrulation. While expression of ten-m/odz orthologues have been characterized in several vertebrate species, their expression patterns in non- Drosophila arthropods during embryonic segmentation have yet to be reported. Here, we have identified a Tribolium orthologue of ten-m/odz (Tc-ten-m/odz) and analyzed its expression patterns during embryonic segmentation. Tc-ten-m/odz expression was observed in a region of the growth zone, which appeared to be a potential mesodermal region, during germband elongation. Later, segmental expression appeared in the trunk after segments had already formed. In contrast to Drosophila , apparently Tc-ten-m/odz was neither expressed in the ectoderm of the growth zone where segmentation occurs, nor the ectoderm of trunk regions where segmentation is maintained. Our findings suggest that Tc-ten-m/odz may not function as a pair rule gene in Tribolium segmentation. Highlights • The first report of ten-m/odz expression during segmentation in a non- Drosophila arthropod. • During segmentation, ten-m/odz is expressed in the growth zone of Tribolium germbands. • ten-m/odz is expressed in stripes in the trunk of germbands during segmentation. [ABSTRACT FROM AUTHOR]

Abstract In Hemiptera, presumptive stylets for each consecutive postembryonic instar are manufactured prior to ecdysis to replace the ecdysial stylets discarded with the exuviae. With the discovery that the bacterium “ Candidatus ” Liberibacter solanacearum accesses the tissues involved in the stylet replacement process of the potato psyllid, a hypothesis was formed that the bacterium could adhere to the stylets of freshly emerged instars and hence gain access to the host plant when feeding is resumed. Although unproven, it was imperative that a model for stylet replacement be built. Stylet morphology and the stylet replacement process of the Asian citrus psyllid (ACP), vector of “ C. ” L. asiaticus, causal pathogen of citrus greening disease, are comparable to the potato psyllid model system. Morphology consists of a basal terminus with its tab-shaped auricle, a base, shaft, and an apical terminus. Each of the four auricles act as a platform for the replacement apparatus, which is compacted into a tight aggregate of cells, the ‘end-cap’. As modeled, on apolysis of larval instar hypodermis, the aggregate ‘deconstructs’ and expands into a snail shell-shaped tube, the ‘atrium’, that houses the presumptive stylet as it is synthesized. Completed stylets then despool from the atrium and are fitted into their functional positions as the next instar emerges from its exuviae. Graphical abstract Image 1 Highlights • Asian citrus psyllid stylets and potato psyllid stylets are comparable in structure. • Stylet end-caps of the adult and atria of the pharate adult are comparable also. • Apparent presence of epicuticle cells at the stylet base is not proven. • The Asian citrus psyllid stylet base has numerous, unique pore-like structures. • Liberibacter may possibly use the stylet replacement apparatus for transmission. [ABSTRACT FROM AUTHOR]

Background: The de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly. Methods: We used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome. Results: We improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies. Conclusions: We describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes. [ABSTRACT FROM AUTHOR]

During the anterior-posterior fate specification of insects, anterior fates arise in a nonelongating tissue (called the "blastoderm"), and posterior fates arise in an elongating tissue (called the "germband"). However, insects differ widely in the extent to which anterior-posterior fates are specified in the blastoderm versus the germband. Here we present a model in which patterning in both the blastoderm and germband of the beetle Tribolium castaneum is based on the same flexible mechanism: a gradient that modulates the speed of a genetic cascade of gap genes, resulting in the induction of sequential kinematic waves of gap gene expression. The mechanism is flexible and capable of patterning both elongating and nonelongating tissues, and hence converting blastodermal to germband fates and vice versa. Using RNAi perturbations, we found that blastodermal fates could be shifted to the germband, and germband fates could be generated in a blastoderm-like morphology. We also suggest a molecular mechanism underlying our model, in which gradient levels regulate the switch between two enhancers: One enhancer is responsible for sequential gene activation, and the other is responsible for freezing temporal rhythms into spatial patterns. This model is consistent with findings in Drosophila melanogaster, where gap genes were found to be regulated by two nonredundant "shadow" enhancers. [ABSTRACT FROM AUTHOR]

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of meth-ylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior. [ABSTRACT FROM AUTHOR]

Background: Genome assembly remains an unsolved problem. Assembly projects face a range of hurdles that confound assembly. Thus a variety of tools and approaches are needed to improve draft genomes. Results: We used a custom assembly workflow to optimize consensus genome map assembly, resulting in an assembly equal to the estimated length of the Tribolium castaneum genome and with an N50 of more than 1 Mb. We used this map for super scaffolding the T. castaneum sequence assembly, more than tripling its N50 with the program Stitch. Conclusions: In this article we present software that leverages consensus genome maps assembled from extremely long single molecule maps to increase the contiguity of sequence assemblies. We report the results of applying these tools to validate and improve a 7x Sanger draft of the T. castaneum genome. [ABSTRACT FROM AUTHOR]

In the short-germ beetle Tribolium castaneum, waves of pair-rule gene expression propagate from the posterior end of the embryo towards the anterior and eventually freeze into stable stripes, partitioning the anterior-posterior axis into segments. Similar waves in vertebrates are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. However, neither a molecular candidate nor a functional role has been identified to date for such a frequency gradient, either in vertebrates or elsewhere. Here we provide evidence that the posterior gradient of Tc-caudal expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. We show this by analyzing the spatiotemporal dynamics of Tc-even-skipped expression in strong and mild knockdown of Tc-caudal, and by correlating the extension, level and slope of the Tc-caudal expression gradient to the spatiotemporal dynamics of Tc-even-skipped expression in wild type as well as in different RNAi knockdowns of Tc-caudal regulators. Further, we show that besides its absolute importance for stripe generation in the static phase of the Tribolium blastoderm, a frequency gradient might serve as a buffer against noise during axis elongation phase in Tribolium as well as vertebrates. Our results highlight the role of frequency gradients in pattern formation. [ABSTRACT FROM AUTHOR]

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes. [ABSTRACT FROM AUTHOR]

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, we analyzed the expression and function of axin, which encodes a highly conserved negative regulator of the pathway. We found Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development. [ABSTRACT FROM AUTHOR]

Abstract: Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin®) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level. [Copyright &y& Elsevier]

Background: The Wnt genes encode secreted glycoprotein ligands that regulate a wide range of developmental processes, including axis elongation and segmentation. There are thirteen subfamilies of Wnt genes in metazoans and this gene diversity appeared early in animal evolution. The loss of Wnt subfamilies appears to be common in insects, but little is known about the Wnt repertoire in other arthropods, and moreover the expression and function of these genes have only been investigated in a few protostomes outside the relatively Wnt-poor model species Drosophila melanogaster and Caenorhabditis elegans. To investigate the evolution of this important gene family more broadly in protostomes, we surveyed the Wnt gene diversity in the crustacean Daphnia pulex, the chelicerates Ixodes scapularis and Achaearanea tepidariorum, the myriapod Glomeris marginata and the annelid Platynereis dumerilii. We also characterised Wnt gene expression in the latter three species, and further investigated expression of these genes in the beetle Tribolium castaneum. Results: We found that Daphnia and Platynereis both contain twelve Wnt subfamilies demonstrating that the common ancestors of arthropods, ecdysozoans and protostomes possessed all members of all Wnt subfamilies except Wnt3. Furthermore, although there is striking loss of Wnt genes in insects, other arthropods have maintained greater Wnt gene diversity. The expression of many Wnt genes overlap in segmentally reiterated patterns and in the segment addition zone, and while these patterns can be relatively conserved among arthropods and the annelid, there have also been changes in the expression of some Wnt genes in the course of protostome evolution. Nevertheless, our results strongly support the parasegment as the primary segmental unit in arthropods, and suggest further similarities between segmental and parasegmental regulation by Wnt genes in annelids and arthropods respectively. Conclusions: Despite frequent losses of Wnt gene subfamilies in lineages such as insects, nematodes and leeches, most protostomes have probably maintained much of their ancestral repertoire of twelve Wnt genes. The maintenance of a large set of these ligands could be in part due to their combinatorial activity in various tissues. [ABSTRACT FROM AUTHOR]

Summary: wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved . Wnt signaling is also implicated in posterior patterning and germband elongation , but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions . Tribolium contains additional Wnt family genes that are also expressed in the growth zone . After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods and vertebrates . [Copyright &y& Elsevier]

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell–cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control. [ABSTRACT FROM AUTHOR]