SUMMARYThe pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

For many organisms, there is agreement on the specific genomic region used for developing barcode markers. With nematodes, however, it has been found that the COI region designated for most animals lacks the taxonomic coverage (ability to amplify a diverse group of taxa) required of a metabarcoding marker. For that reason, studies on metabarcoding of nematodes thus far have utilized primarily regions within the highly conserved 18S ribosomal DNA. Two popular markers within this region are the ones flanked by the primer pairs NF1-18Sr2b and SSUF04-SSUR22. The NF1-18Sr2b primer pair, especially, has been critiqued as not being specific enough for nematodes leading to suggestions for other candidate markers while the SSUF04-SSUR22 region has hardly been tested on soil nematodes. The current study aimed to evaluate these two markers against other alternative ones within the 28S rDNA and the COI region for their suitability for nematode metabarcoding. The results showed that the NF1-18Sr2b marker could offer wide coverage and good resolution for characterizing soil nematodes. Sufficient availability of reference sequences for this region was found to be a significant factor that resulted in this marker outperforming the other markers, particularly the 18S-based SSUFO4-SSUR22 marker. None of the other tested regions compared with this marker in terms of the proportion of the taxa recovered. The COI-based marker had the lowest number of taxa recovered, and this was due to the poor performance of its primers and the insufficient number of reference sequences in public databases. In summary, this study highlights how dependent the success of metabarcoding is on the availability of a good reference sequence collection for the marker of choice as well as its taxonomic coverage.

Since its conception, DNA barcoding has seen a rapid uptake within the research community. Nevertheless, as with many new scientific tools, progression towards the point of routine deployment within diagnostic laboratories has been slow. In this paper, we discuss the application of DNA barcoding in the Defra plant health diagnostic laboratories, where DNA barcoding is used primarily for the identification of invertebrate pests. We present a series of case studies that demonstrate the successful application of DNA barcoding but also reveal some potential limitations to expanded use. The regulated plant pest, Bursephalenchus xylophilus, and one of its vectors, Monochamus alternatus, were found in dining chairs. Some traded wood products are potentially high risk, allowing the movement of longhorn beetles; Trichoferus campestris, Leptura quadrifasciata, and Trichoferus holosericeus were found in a wooden cutlery tray, a railway sleeper, and a dining chair, respectively. An outbreak of Meloidogyne fallax was identified in Allium ampeloprasum and in three weed species. Reference sequences for UK native psyllids were generated to enable the development of rapid diagnostics to be used for monitoring following the release of Aphalara itadori as a biological control agent for Fallopia japonica.Key words: plant health, regulated quarantine pest, DNA barcoding, diagnostics, invertebrate.Résumé : Depuis son développement, le codage à barres de l'ADN a connu une adoption rapide au sein de la communauté scientifique. Néanmoins, comme pour plusieurs nouveaux outils scientifiques, les avancées en vue d'un usage routinier au sein de laboratoires de diagnostic ont été lentes. Dans ce travail, les auteurs discutent de l'emploi du codage à barres au sein des laboratoires de diagnostique phytosanitaire de DEFRA, où le codage à barres est principalement employé pour l'identification des ravageurs invertébrés. Les auteurs présentent une série d'études de cas le codage à barres a été mis en oeuvre avec succès tout en révélant des limitations potentielles à son usage accru. Le parasite réglementé, Bursephalenchus xylophilus, et l'un de ses vecteurs, Monochamus alternatus, ont été retrouvés dans des chaises de salle à manger. Certains produits du bois sont potentiellement à haut risque en facilitant le mouvement des longicornes; Trichoferus campestris, Leptura quadrifasciata et Trichoferus holosericeus ont été trouvés, respectivement, dans un range-couvert en bois, une traverse ferroviaire, et une chaise de salle à manger. Une épidémie du Meloidogyne fallax a été identifiée chez l'Allium ampeloprasum et chez trois adventices. Des séquences de référence pour des psylles indigènes du Royaume-Uni ont été générées pour permettre le développe-ment d'outils diagnostiques rapides en vue de surveiller l'Aphalara itadori suite à son introduction en tant qu'agent de lutte biologique contre le Fallopia japonica. [Traduit par la Rédaction] Mots-clés : santé des végétaux, organisme nuisible réglementé, codage à barres de l'ADN, diagnostique...

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BackgroundPancreatic β-cells require a constant supply of zinc to maintain normal insulin secretory function. Following co-exocytosis with insulin, zinc is replenished via the Zrt- and Irt-like (ZIP; SLC39A) family of transporters. However the ZIP paralogues of particular importance for zinc uptake, and associations with β-cell function and Type 2 Diabetes remain largely unexplored. We retrieved and statistically analysed publically available microarray and RNA-seq datasets to perform a systematic review on the expression of β-cell SLC39A paralogues. We complemented results with experimental data on expression profiling of human islets and mouse β-cell derived MIN6 cells, and compared transcriptomic and proteomic sequence conservation between human, mouse and rat.ResultsThe 14 ZIP paralogues have 73–98% amino sequence conservation between human and rodents. We identified 18 datasets for β-cell SLC39A analysis, which compared relative expression to non-β-cells, and expression in response to PDX-1 activity, cytokines, glucose and type 2 diabetic status. Published expression data demonstrate enrichment of transcripts for ZIP7 and ZIP9 transporters within rodent β-cells and of ZIP6, ZIP7 and ZIP14 within human β-cells, with ZIP1 most differentially expressed in response to cytokines and PDX-1 within rodent, and ZIP6 in response to diabetic status in human and glucose in rat. Our qPCR expression profiling data indicate that SLC39A6, −9, −13, and − 14 are the highest expressed paralogues in human β-cells and Slc39a6 and −7 in MIN6 cells.ConclusionsOur systematic review, expression profiling and sequence alignment reveal similarities and potentially important differences in ZIP complements between human and rodent β-cells. We identify ZIP6, ZIP7, ZIP9, ZIP13 and ZIP14 in human and rodent and ZIP1 in rodent as potentially biologically important for β-cell zinc trafficking. We propose ZIP6 and ZIP7 are key functional orthologues in human and rodent β-cells and highlight these zinc importers as important targets for exploring associations between zinc status and normal physiology of β-cells and their decline in Type 2 Diabetes.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4119-2) contains supplementary material, which is available to authorized users.