Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the network of National Reference Laboratories: Determination of aflatoxin B1 in defatted peanut powder

The Joint Research Centre (JRC), a Directorate-General of the European Commission, operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise proficiency tests (PTs) among appointed National Reference Laboratories (NRLs). This report presents the results of the PT on the determination of aflatoxins in defatted peanut powder. The test items for this PT were two contaminated defatted peanut powder samples. The materials were produced by the JRC and dispatched to the participants mid October 2016. Each participant received one bottle per test material containing approximately 55 g each. Fifty-six participants from thirty countries (among them 40 NRLs and 16 official food control laboratories) registered for the exercise and 54 sets (Sample A and B) of results were reported. The assigned values, established by an exact-matching double isotope dilution mass spectrometric technique, were 2.80 µg/kg (± 0.19 µg/kg) aflatoxin B1 in sample A and 3.20 µg/kg (± 0.20 µg/kg) in sample B. Participants' results were rated with z-scores and zeta-scores for aflatoxin B1 in accordance with ISO 13528:2015. The z-score compares the participant's deviation from the reference value with the target standard deviation accepted for the PT, whereas the zeta-score provides an indication of whether the participant's estimate of uncertainty is consistent with the observed deviation from the assigned value. Only z-scores were used for the evaluation whether an individual laboratory underperformed. In total, 96 % of the attributed z scores were below an absolute value of two for sample A and and 92 % for sample B. This indicates that most of the participants performed satisfactorily. The few participants that had z-scores above an absolute value of 2 will have to investigate the reasons for the deviation (root-cause analysis) and report the planned corrective actions to the EURL.JRC.F.5-Food and Feed Complianc

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in tea and herbal infusions

Tropane alkaloids (TAs) are toxins found in a wide variety of plant species growing in mild climates. The most well-known are Datura, Atropa and Hyoscyamus sp., belonging to the Solanaceae family. The TAs family comprises more than 200 compounds, of which atropine and scopolamine are the most active producing anticholinergic symptoms (e.g. blurred vision, dry mouth, muscle spasms, tachycardia and death) if ingested in toxic quantities. The presence of botanical impurities (e.g. seeds, leaves and roots) has been reported in a variety of tea and herbal blends, stressing the need to control the quality of these products in the EU market. The European Union Reference Laboratory (EURL) for Mycotoxins organised a proficiency test (PT) on the determination of TAs (atropine and scopolamine) in tea and herbal infusions upon request from DG SANTE. The measurand levels were targeted to provide insight on the measurement capabilities of EU Member States' laboratories at concentrations close to the recommended limit of quantification (LOQ) established by the Commission Recommendation 2015/976 (preferably below 5 μg/kg and not higher 10 μg/kg). Additionally, the ratio of atropine to scopolamine was kept as native in the plant materials in some samples. Three matrices appropriately processed were provided to the participants: black tea, peppermint leaves and fennel seeds. The concentrations of atropine varied from 8.3 to 42.2 μg/kg while those of scopolamine ranged from 1.5 to 20.8 μg/kg. The participants were asked to determine atropine and scopolamine in 6 contaminated samples (2 per matrix) and 3 blank materials spiked by them with a TAs solution of unknown concentration. This setup was also aimed to allow a preliminary assessment of the robustness of the EURL-developed method. Thirty-three laboratories from 11 Member States joined the PT, with a very significant participation from Germany. The performance of the laboratories was assessed using z-scores with regard to the assigned values obtained by exact matching double isotope dilution mass spectrometry (EMD-IDMS), in line with the ISO 13528:2015. In all cases, the consensus values derived from the participants' data were within the range of the assigned values, considering the respective confidence intervals. On average, eighty-seven percent of the z-scores for atropine and 84 % for scopolamine fell in the acceptable range (|z| ≤ 2). The success rate varied from 83 to 94 % for atropine and from 67 to 94 % for scopolamine, across the distributed matrices and concentration levels. The robust standard deviations of the reported results for both TAs were in good agreement with the target standard deviation (22 %). The results of this PT indicate that EU Member States’ laboratories can determine atropine and scopolamine reliably in tea and herbal infusions at levels relevant to the current legislation (Commission Recommendation 2015/976).JRC.F.5-Food and Feed Complianc

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in cereal products for infants and young children

Tropane alkaloids (TAs) are plant toxins that occur mainly in Datura, Atropa and Hyoscyamus sp, belonging to the Solanaceae family, besides a variety of other families such as Erythroxylaceae, Brassicaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. The TAs occur in all parts of the plants and botanical impurities have been found in a range of crops due to accidental contamination during harvesting. The intoxication via the food leads to anticholinergic effects (e.g. blurred vision, pupil dilation, dry mouth, vomiting, muscle spasms, tachycardia, etc.), culminating in severe intoxications and death. The EFSA CONTAM Panel established a group Acute Reference Dose (ARfD) of 0.016 μg/kg body weight (b.w.) expressed as the sum of (-)-hyoscyamine and ( ) scopolamine, assuming equivalent potency. Infants and young children are the most exposed age classes as they consume a higher amount of cereal-based products per body weight. EFSA estimated that the dietary exposure of toddlers could be up to seven times the group ARfD. Recently, two European legislation acts were published in this field: Commission Recommendation (EU) 2015/976, recommending the monitoring of tropane alkaloids in certain food categories, and Commission Regulation (EU) 2016/239, enforcing maximum levels of tropane alkaloids in certain cereal-based foods for infants and young children. The EURL-Mycotoxins organised a proficiency test (PT) concerning the determination of atropine and scopolamine in cereal-based baby food, aiming to underpin and assess the measurement capability of Member States' (MS) laboratories. Particular focus was given to levels relevant for enforcement of legislation. Two samples were distributed to the participants: one sample labelled “C” – biscuits for infants containing approx. 1.2 µg/kg of atropine and 0.2 µg/kg of scopolamine, and one sample labelled “E” – cereals for porridge containing approx. 7.4 µg/kg of atropine and 1.0 µg/kg of scopolamine. Forty-eight datasets from 18 EU MS laboratories were received. Overall, 81 % of the z-scores were in the range of -2 to 2 and 90 % were in the range of -3 to 3. For the lowest TA level (sample C) still 75 % of z-scores fell into an acceptable range (|z| ≤ 2), while the mass fraction of scopolamine was far below the target level of 1 µg/kg. In line with this observation, the vast majority of reported LOQs were below 1.0 µg/kg. The methodologies used by the participants can be clustered into three groups: the method supplied by the EURL; the RIKILT SOP A1070 and methods based on QuEChERS. The instrumental determination was by LC-MS/MS, with one exception (GC-MS). The recoveries reported by the participants were close to 95 %. No statistically significant dependence of the z scores on the analytical methodology was observed. These results support the assumption that atropine and scopolamine can be reliably determined at the maximum levels proposed by the EU to ensure the protection of infants and young children's health using state-of-the-art analytical instrumentation.  JRC.F.5-Food and Feed Complianc

Intersex related gene expression profiles in clams Scrobicularia plana : molecular markers and environmental application

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction

Report on the 2017 Proficiency Test of the European Union Reference Laboratory for Mycotoxins: Determination of deoxynivalenol in wheat

The Joint Research Centre (JRC), a Directorate-General of the European Commission operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise proficiency tests (PTs) among designated National Reference Laboratories (NRLs). This report presents the results of the PT on the determination of deoxynivalenol in wheat. The test materials for this PT were four naturally contaminated wheat materials. The test items were dispatched to the participants at the end of April 2017. Each participant received one test item per material containing approximately 55 g each. Fifty-nine participants from 32 countries (among them 41 NRLs and 18 official food control laboratories-OCLs) registered for the exercise and 59 sets of results for test items A, B, C and D) were reported. The assigned values, established by an exact-matching double isotope dilution mass spectrometric technique (EMD-IDMS), were 551 µg/kg (± 37 µg/kg) deoxynivalenol for material A, 1556 µg/kg (± 83 µg/kg) for material B, 4405 µg/kg (± 265 µg/kg) for material C and 1160 µg/kg (± 60 µg/kg) for material D. Participants' results were rated with z-scores and zeta-scores in accordance with ISO 13528:2015. The z-score compares the participant's deviation from the reference value with the target standard deviation accepted for the PT, whereas the zeta-score indicates whether the participant's estimate of uncertainty is consistent with the observed deviation from the assigned value. Only z-scores were used for the evaluation of whether an individual laboratory underperformed. In total, 93 % of the attributed z-scores were below an absolute value of two for test items A and C, 95 % for test item B and 92 % for test item D. This indicates that most of the participants performed satisfactorily. One NRL had a z-score above an absolute value of 3 and will have to investigate the reasons for the deviation (root-cause analysis) and report the planned corrective actions to the EURL. Participants were requested to assess the compliance of the sample against legislative limits. Eighty-five percent to 100 % of the participants assessed the compliance/non-compliance of the test materials A, C and D correctly. Only 36 % of the laboratories assessed correctly the non-compliance of Material B, and 56 % classified the test material as compliant providing a proper justification (taking into account their measurement result and reported uncertainty).  JRC.F.5 - Food and Feed Complianc

Assignment of a Reference Value of Total Cow’s Milk Protein Content in Baked Cookies Used in an Interlaboratory Comparison

Interlaboratory comparisons (ILC) in the food allergens field mainly rely on the use of consensus values per applied methodology or even per type of an ELISA test kit. Results suggest good reproducibility; however, possible biases may not be recognized since metrological traceability to an independent reference is lacking. The work presented here utilizes isotope dilution mass spectrometry (IDMS) to assign a reference value of the total cow’s milk protein (TCMP) content in a baked cookie and its associated uncertainty. TCMP consists of several individual proteins, of which five (representing 92%) served us as markers for TCMP. Per marker, one to four proteotypic peptides were selected for the quantification. These were synthesized, and the mass fractions of respective reference solutions were determined with peptide-impurity-corrected amino acid analysis to establish traceability to SI units. Stable isotope labelled (“heavy”) analogues of the proteotypic peptides were also synthesized and blended with extracts of the test material or the reference solutions for IDMS. Through careful measurement design minimizing biases, well-defined model equations were developed, allowing appropriate estimation of the associated uncertainty. The determined reference value of 11.8 ± 1.1 mg TCMP/kg cookie was used for scoring of a novel ILC

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins: Determination of regulated mycotoxins and enniatins and beauvericin in cereals

The number of known mycotoxins, their precursors and metabolites has been steadily increasing over the past years. The European Commission puts special emphasis on the need to monitor the co-occurrence of mycotoxins of various families at levels that allow for a sound risk assessment, taking into account possible additive or synergistic effects. Prior any regulatory action is taken for mycotoxins for which a health concern has been expressed (e.g. enniatins and beauvericin) valid data on their prevalence in food is required. LC-MS-based multi-mycotoxin methods have the potential of streamlining and widening the monitoring work carried out by the official control laboratories. Although many practical advantages have been recognised, such methodologies have not been adopted by all routine laboratories and only a handful of such methods are just on the verge to become standardised. It is of great interest to assess how well laboratories using diverse sample preparation methodologies and determination techniques perform. Therefore, a proficiency test was organised by the European Union Reference Laboratory (EURL) for Mycotoxins for this purpose. The focus was the assessment of the measurement performance of EU Member States laboratories regarding the determination of aflatoxin B1, deoxynivalenol, zearalenone, fumonisins B1 & B2,T-2 & HT-2 toxins, enniatins B, B1, A, A1 and beauvericin in two test materials (corn and oat) using single- or multi-mycotoxin methodologies. Fifty-three laboratories, among them thirty-six National Reference Laboratories for mycotoxins in food and feed from the 28 EU Member States and 17 Official Control Laboratories, participated in the PT. For the regulated mycotoxins, 83.7 % of the results were rated with satisfactory z-scores. The performance of the laboratories was best for AFB1 (94 %), followed by DON (91 %), ZON (89 %), FB1 (87 %), FB2 (78 %), T-2 (75 %) and HT-2 (64 %). Additionally, 11 laboratories submitted results for all enniatins and beauvericin. LC-MS/MS is gaining much preference as it allowed for the determination of all the proposed analytes (12) in the test materials. Nevertheless, the esults provided by multi-mycotoxin methodologies did not differ statistically from those produced by singleanalyte procedures. Many participants uphold the will to implement a methodology to analyse enniatins and beauvericin in the near future, while other laboratories' methods require improvements in the extraction efficiency and sensitivity.JRC.F.5 - Food and Feed Complianc

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain

[EN] Liberibacteris a bacterial group causing different diseases and disorders in plants. Among liberibacters,CandidatusLiberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future worksThis research was funded by Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), grant numbers AT2016-007 and RTA2014-00008-C04-03-E, co-financed by FEDER.Ruiz-Padilla, A.; Redondo, C.; Asensio, A.; Garita-Cambronero, J.; Martinez, C.; Perez-Padilla, V.; Marquinez, R.... (2020). Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain. Microorganisms. 8(9):1-19. https://doi.org/10.3390/microorganisms8091446S11989Haapalainen, M. (2014). Biology and epidemics ofCandidatusLiberibacter species, psyllid-transmitted plant-pathogenic bacteria. Annals of Applied Biology, 165(2), 172-198. doi:10.1111/aab.12149Raddadi, N., Gonella, E., Camerota, C., Pizzinat, A., Tedeschi, R., Crotti, E., … Alma, A. (2010). ‘Candidatus Liberibacter europaeus’ sp. nov. that is associated with and transmitted by the psyllid Cacopsylla pyri apparently behaves as an endophyte rather than a pathogen. Environmental Microbiology, 13(2), 414-426. doi:10.1111/j.1462-2920.2010.02347.xWang, N., Pierson, E. A., Setubal, J. C., Xu, J., Levy, J. G., Zhang, Y., … Martins, J. (2017). The Candidatus Liberibacter–Host Interface: Insights into Pathogenesis Mechanisms and Disease Control. Annual Review of Phytopathology, 55(1), 451-482. doi:10.1146/annurev-phyto-080516-035513Morris, J., Shiller, J., Mann, R., Smith, G., Yen, A., & Rodoni, B. (2017). Novel ‘Candidatus Liberibacter’ species identified in the Australian eggplant psyllid, Acizzia solanicola. Microbial Biotechnology, 10(4), 833-844. doi:10.1111/1751-7915.12707Alfaro-Fernández, A., Hernández-Llopis, D., & Font, M. I. (2017). Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain. European Journal of Plant Pathology, 149(1), 127-131. doi:10.1007/s10658-017-1172-2Haapalainen, M., Wang, J., Latvala, S., Lehtonen, M. T., Pirhonen, M., & Nissinen, A. I. (2018). Genetic Variation of ‘Candidatus Liberibacter solanacearum’ Haplotype C and Identification of a Novel Haplotype from Trioza urticae and Stinging Nettle. Phytopathology®, 108(8), 925-934. doi:10.1094/phyto-12-17-0410-rHaapalainen, M., Latvala, S., Wickström, A., Wang, J., Pirhonen, M., & Nissinen, A. I. (2019). A novel haplotype of ‘Candidatus Liberibacter solanacearum’ found in Apiaceae and Polygonaceae family plants. European Journal of Plant Pathology, 156(2), 413-423. doi:10.1007/s10658-019-01890-0Mauck, K. E., Sun, P., Meduri, V. R., & Hansen, A. K. (2019). New Ca. 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Genome Announcements, 3(4). doi:10.1128/genomea.00733-15Wulff, N. A., Zhang, S., Setubal, J. C., Almeida, N. F., Martins, E. C., Harakava, R., … Gabriel, D. W. (2014). The Complete Genome Sequence of ‘Candidatus Liberibacter americanus’, Associated with Citrus Huanglongbing. Molecular Plant-Microbe Interactions®, 27(2), 163-176. doi:10.1094/mpmi-09-13-0292-rDuan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., … Gottwald, T. (2009). Complete Genome Sequence of Citrus Huanglongbing Bacterium, ‘CandidatusLiberibacter asiaticus’ Obtained Through Metagenomics. Molecular Plant-Microbe Interactions®, 22(8), 1011-1020. doi:10.1094/mpmi-22-8-1011Katoh, H., Miyata, S., Inoue, H., & Iwanami, T. (2014). Unique Features of a Japanese ‘Candidatus Liberibacter asiaticus’ Strain Revealed by Whole Genome Sequencing. PLoS ONE, 9(9), e106109. doi:10.1371/journal.pone.0106109Leonard, M. T., Fagen, J. R., Davis-Richardson, A. G., Davis, M. J., & Triplett, E. W. (2012). Complete genome sequence of Liberibacter crescens BT-1. Standards in Genomic Sciences, 7(2), 271-283. doi:10.4056/sigs.3326772Teresani, G. R., Bertolini, E., Alfaro-Fernández, A., Martínez, C., Tanaka, F. A. O., Kitajima, E. W., … Font, M. I. (2014). Association of ‘Candidatus Liberibacter solanacearum’ with a Vegetative Disorder of Celery in Spain and Development of a Real-Time PCR Method for Its Detection. Phytopathology®, 104(8), 804-811. doi:10.1094/phyto-07-13-0182-rLi, W., Hartung, J. S., & Levy, L. (2006). Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods, 66(1), 104-115. doi:10.1016/j.mimet.2005.10.018Munyaneza, J. E., Sengoda, V. G., Crosslin, J. M., De la Rosa-Lozano, G., & Sanchez, A. (2009). First Report of ‘Candidatus Liberibacter psyllaurous’ in Potato Tubers with Zebra Chip Disease in Mexico. Plant Disease, 93(5), 552-552. doi:10.1094/pdis-93-5-0552aPhillips, J. L., & Gnanakaran, S. (2014). A data-driven approach to modeling the tripartite structure of multidrug resistance efflux pumps. Proteins: Structure, Function, and Bioinformatics, 83(1), 46-65. doi:10.1002/prot.24632Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35(6), 1547-1549. doi:10.1093/molbev/msy096Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. (1993). Molecular Biology and Evolution. doi:10.1093/oxfordjournals.molbev.a040023Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J. C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S. E., & Sánchez-Gracia, A. (2017). DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution, 34(12), 3299-3302. doi:10.1093/molbev/msx248Liao, J., Wiedmann, M., & Kovac, J. (2017). Genetic Stability and Evolution of the sigB Allele, Used for Listeria Sensu Stricto Subtyping and Phylogenetic Inference. Applied and Environmental Microbiology, 83(12). doi:10.1128/aem.00306-17Tamura, K., Battistuzzi, F. U., Billing-Ross, P., Murillo, O., Filipski, A., & Kumar, S. (2012). Estimating divergence times in large molecular phylogenies. Proceedings of the National Academy of Sciences, 109(47), 19333-19338. doi:10.1073/pnas.1213199109Tamura, K., Tao, Q., & Kumar, S. (2018). Theoretical Foundation of the RelTime Method for Estimating Divergence Times from Variable Evolutionary Rates. Molecular Biology and Evolution, 35(7), 1770-1782. doi:10.1093/molbev/msy044López-Hermoso, C., de la Haba, R. R., Sánchez-Porro, C., Papke, R. T., & Ventosa, A. (2017). Assessment of MultiLocus Sequence Analysis As a Valuable Tool for the Classification of the Genus Salinivibrio. Frontiers in Microbiology, 8. doi:10.3389/fmicb.2017.01107Hajri, A., Loiseau, M., Cousseau-Suhard, P., Renaudin, I., & Gentit, P. (2017). Genetic Characterization of ‘Candidatus Liberibacter solanacearum’ Haplotypes Associated with Apiaceous Crops in France. Plant Disease, 101(8), 1383-1390. doi:10.1094/pdis-11-16-1686-reFang, Y., Wang, Y., Liu, Z., Dai, H., Cai, H., Li, Z., … Wang, D. (2019). Multilocus Sequence Analysis, a Rapid and Accurate Tool for Taxonomic Classification, Evolutionary Relationship Determination, and Population Biology Studies of the Genus Shewanella. Applied and Environmental Microbiology, 85(11). doi:10.1128/aem.03126-18Konstantinidis, K. T., Ramette, A., & Tiedje, J. M. (2006). Toward a More Robust Assessment of IntraspeciesDiversity, Using Fewer GeneticMarkers. Applied and Environmental Microbiology, 72(11), 7286-7293. doi:10.1128/aem.01398-06Ajene, I. J., Khamis, F., Ballo, S., Pietersen, G., van Asch, B., Seid, N., … Mohamed, S. (2020). Detection of Asian Citrus Psyllid (Hemiptera: Psyllidae) in Ethiopia: A New Haplotype and its Implication to the Proliferation of Huanglongbing. Journal of Economic Entomology, 113(4), 1640-1647. doi:10.1093/jee/toaa113Thapa, S. P., De Francesco, A., Trinh, J., Gurung, F. B., Pang, Z., Vidalakis, G., … Coaker, G. (2020). Genome‐wide analyses of Liberibacter species provides insights into evolution, phylogenetic relationships, and virulence factors. Molecular Plant Pathology, 21(5), 716-731. doi:10.1111/mpp.12925Antolinez, C. A., Fereres, A., & Moreno, A. (2017). Risk assessment of ‘Candidatus Liberibacter solanacearum’ transmission by the psyllids Bactericera trigonica and B. tremblayi from Apiaceae crops to potato. Scientific Reports, 7(1). doi:10.1038/srep45534Antolínez, Moreno, Ontiveros, Pla, Plaza, Sanjuan, … Fereres. (2019). Seasonal Abundance of Psyllid Species on Carrots and Potato Crops in Spain. Insects, 10(9), 287. doi:10.3390/insects10090287Wang, J., Haapalainen, M., Schott, T., Thompson, S. M., Smith, G. R., Nissinen, A. I., & Pirhonen, M. (2017). Genomic sequence of «Candidatus Liberibacter solanacearum» haplotype C and its comparison with haplotype A and B genomes. PLOS ONE, 12(2), e0171531. doi:10.1371/journal.pone.0171531Katsir, L., Zhepu, R., Santos Garcia, D., Piasezky, A., Jiang, J., Sela, N., … Bahar, O. (2018). Genome Analysis of Haplotype D of Candidatus Liberibacter Solanacearum. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.02933Quintana-González de Chaves, M., Teresani, G. R., Hernández-Suárez, E., Bertolini, E., Moreno, A., Fereres, A., … Siverio, F. (2020). ‘Candidatus Liberibacter Solanacearum’ Is Unlikely to Be Transmitted Spontaneously from Infected Carrot Plants to Citrus Plants by Trioza Erytreae. Insects, 11(8), 514. doi:10.3390/insects1108051

Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve Mytilus edulis

Background: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. Methodology/Principal Findings: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. Conclusions: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure