Beeheal: standardization of laboratory methods for sample processing, nucleic acids extraction and PCR for microsporidia and viruses analysis

BEEHEAL is a project designed to determine the phenology and interaction of Nosema ceranae and viruses in four Mediterranean countries: Spain, France, Portugal and Israel, including some territories where Varroa destructor is not present (Azores and Ouessant islands). This will allow us to study and compare the interactions between pathogens in a wide range of hosts, beekeeping and climatic conditions. The honey bee samples collected along the year in the different countries will be analysed for pathogens in three laboratories. This requires a standardization of methods to compare the results in order to assign the effect of every variable in a reliable way. To that end, the participating laboratories have been working together to establish the sampling methodology, the conservation of the samples, the nucleic acids extraction and the PCR analysis. We analyzed the sample processing for nucleic acid extraction on TE buffer (with or without Proteinase K), CTAB buffer or commercial kits (Qiagen). The maceration of bees (either individually or in composite samples) in TE buffer and posterior incubation at 96ºC for 20 minutes showed a good sensibility level and good value for N. ceranae DNA extraction. This method also allowed the conservation of RNA at -80ºC for a month in the TE solution for later RNA extraction. A joint protocol for sample processing, DNA and RNA extraction and PCR analysis has been developed but adjusted to the particular conditions and equipment of each laboratory. The standardization of methods to be implemented by each participating laboratory will avoid the biases on conclusions based on the diverse methods applied.This work has been developed under the BEEHEAL project. BEEHEAL is funded through the ARIMNet2 2016 Call by the following funding agencies: INIA (Spain), MOARD (Israel), ANR (France), and FCT (Portugal). ARIMNet2 (ERA-NET) has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127.info:eu-repo/semantics/publishedVersio

A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.)

With a growing number of parasites and pathogens experiencing large-scale range expansions, monitoring diversity in immune genes of host populations has never been so important because it can inform on the adaptive potential to resist the invaders. Population surveys of immune genes are becoming common in many organisms, yet they are missing in the honey bee (Apis mellifera L.), a key managed pollinator species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in a wide range of honey bee immune genes and developed a medium-density assay targeting a subset of these genes. Using a discovery panel of 123 whole-genomes, representing seven A. mellifera subspecies and three evolutionary lineages, 180 immune genes were scanned for SNPs in exons, introns (< 4 bp from exons), 3’ and 5´UTR, and < 1 kb upstream of the transcription start site. After application of multiple filtering criteria and validation, the final medium-density assay combines 91 quality-proved functional SNPs marking 89 innate immune genes and these can be readily typed using the high-sample-throughput iPLEX MassARRAY system. This medium-density-SNP assay was applied to 156 samples from four countries and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that honey bee ancestry can be delineated from functional variation. In addition to allowing analysis of immunogenetic variation, this newly-developed SNP assay can be used for inferring genetic structure and admixture in the honey bee.We are deeply indebted to Frank Aguiar, Luís Silva, Edgardo Melo, João Martins, João Melo, Manuel Moura, Manuel Viveiros, and Ricardo Sousa from "Direção Regional da Agricultura e Desenvolvimento Rural dos Açores" (Portugal), and to Laura Garreau, Laurent Maugis, Pascale Sauvage and Jacques Kermagoret, from “Association Conservatoire de l’Abeille Noir Bretonne” (France), for sampling the apiaries in São Miguel, Santa Maria, and Ouessant islands. Genotyping was outsourced to the Epigenetics and Genotyping laboratory, Central Unit for Research in Medicine (UCIM), University of Valencia, Spain. Data analyses were performed using computational resources at the Research Centre in Digitalization and Intelligent Robotics (CeDRI), Instituto Politécnico de Bragança. Ana Rita Lopes is supported by a PhD scholarship (SFRH/BD/143627/2019) from the Foundation for Science and Technology (FCT), Portugal. FCT provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020).This research was funded through the projects BEEHAPPY (POCI-01-0145- FEDER-029871, FCT and COMPETE/QREN/EU) and BEEHEAL. BEEHEAL was funded by the ARIMNet2 2016 Call by the following agencies: INIA (Spain), MOARD (Israel), ANR (France) and FCT (Portugal). ARIMNet2 (ERA-NET) received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127.info:eu-repo/semantics/publishedVersio

Projeto BEEHEAL: promover a saúde da abelha para uma agricultura sustentável

O BEEHEAL, com o título original “Promoting bee health for sustainable agriculture”, é um projeto internacional colaborativo aprovado no âmbito da Ação ERA-Net ARIMNet2 (Coordination of Agricultural Research in the Mediterranean). O projeto é coordenado por Raquel Martín- Hernández, investigadora do “Centro de Investigación Apícola y Agroambiental de Marchamalo” (CAR), Espanha. Para além deste centro de investigação, representado por Raquel Martín-Hernández e Mariano Higes, o consórcio inclui mais três instituições, nomeadamente: o Centro de Investigação de Montanha (CIMO) do Instituto Politécnico de Bragança, representado por M. Alice Pinto e Ana Rita Lopes, o “Centre de Recherche Provence-Alpes-Côte d’Azur Unité: Abeilles et Environnement do “Institut National de la Recherche Agronomique” (INRA), França, representado por Yves Le conte, Anne Dalmon e Maritza Maritza Reyes-Carreno, e o “Volcani Center” da “Agricultural Research Organization” (ARO), Israel, representado por Nor Chevjanovsky e Victoria Soroker. As populações de abelha melífera (Apis mellifera L.) têm vindo a sofrer perdas acentuadas em todo o mundo. Estas perdas estão relacionado com vários factores, que podem atuar sozinhos ou em combinação, incluindo (i) propagação de parasitas e agentes patogénicos exóticos , como por exemplo o ácaro ectoparasita Varroa destructor, o qual serve de vetor de transmissão de vários vírus, e o fungo microsporídeo Nosema ceranae, (ii) exposição das colónias a agro-químicos, (iii) má nutrição, (iv) alterações climáticas, entre outros (vanEngelsdorp & Meixner, 2010; Potts et al., 2010).Ao Paulo Ventura pelo acompanhamento técnico feito ao apário no primeiro ano do projeto. O BEEHEAL é financiado por ARIMNet2 (2016) com os financiadores nacionais Instituto Nacional de Investigación y Teccnologia Agraria y alimentaria (INIA – Espanha), Agence Nationale de la recherche (ARN – France), Ministry off Agriculture & Rural Development, (MOARD – Israel) e Fundação para a Ciência e a Tecnologia (FCT – Portugal)info:eu-repo/semantics/publishedVersio

Epidemiology of the Microsporidium Nosema ceranae in Four Mediterranean Countries

Nosema ceranae is a highly prevalent intracellular parasite of honey bees’ midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control

Susceptibilidad a insecticidas y actividad enzimática de Cydia pomonella L. (Lepidoptera: Tortricidae) Proveniente de Tres Huertos de la Región del Maule, Chile

Cydia pomonella L., the major pest of pome fruits and walnuts in Chile, has been controlled almost exclusively with applications of organophosphates insecticides. However, during the last few seasons, increasing levels of fruit damage at harvest have been observed. Given that this insect has evolved insecticide resistance in several countries, the susceptibility to diagnostic dosages of azinphos methyl and tebufenozide were evaluated on diapausing larvae in populations from three apple orchards in Maule Region and one susceptible laboratory strain (S); as well as the activity of detoxifying enzymes on adults emerging from the aforementioned. Both mixed function oxidases (OFM) and glutathione-S-tranferases (GST) activities were evaluated by fluorometry, while esterase activity (EST) was determined by absorption. Larval mortality for azinphosmethyl was significantly lower in the populations from Molina and Teno (30 and 85.4%, respectively) than in the susceptible laboratory strain (95.3%). For tebufenozide larval mortality was significantly lower in the Molina strain (35.1%) than the S strain (88.6%). GST activity was statistically higher in two of the three analyzed orchards (13,679 fluorescence insect-1 units in Teno, and 13,096 fluorescence insect-1 units in Molina). Similarly, OFM activity was significantly higher in the same orchards, with values of 25.08 and 17.95 picograms (pg) of 7OH insect-1 min1 in Molina and Teno, respectively. The S strain had significantly higher EST activity in relation to the other populations, which seems to be unrelated with lower susceptibility to the insecticides evaluate

Respuesta de cultivares de Musa spp. al estrés hídrico in vitro inducido con polietilenglicol 6000

Bananas and plantains are crops very sensitive to water deficit. Increasingly prolonged drought condition suggests the need for tolerant plants to this factor. The early selection of these plants in in vitro conditions save time and allow working with large volumes of individuals. This requires having genotypes with favorable response to in vitro water deficit. The aim of this work was to determine the response of Musa spp. cultivars, with different genotype contribution, to in vitro water stress induced by polyethylene glycol 6000 (PEG-6000). Cultivars ‘Pelipita’ (ABB), ‘Manzano’ (AAB) and ‘Grande naine’ (AAA) were cultured in semisolid multiplication medium supplemented with PEG-6000 30 g/L. Different stress indicator traits were evaluated after 30 days (morphological: height and number of shoots per explant; physiological: fresh and dry weight; and biochemical: proline, hydrogen peroxide and malondialdehyde content). As results, osmotic stress affected only plant height in ‘Pelipita’ cultivar. However, in the other cultivars all variables were affected, except dry weight in ‘Manzano’. Moreover cv. ‘Grande naine’ and ‘Manzano’ increased proline, hydrogen peroxide and malondialdehyde content. These results indicate that osmotic pressure could induce oxidative stress and cell membrane damages in these cultivars. The obtained results suggested that these genotypes could be used as a tolerant (‘Pelipita’) and sensitive (‘Grande naine’ and ‘Manzano’) controls in in vitro selection of drought-tolerant plants in future breeding programs.Los plátanos y bananos son cultivos sensibles al déficit hídrico. Las sequías cada vez más prolongadas sugieren la necesidad de obtener plantas tolerantes a este factor; la selección temprana de estas plantas, comparada con la selección en campo, permite ahorrar tiempo y trabajar con mayores volúmenes de individuos. Para ello es conveniente contar con cultivares patrones cuya respuesta al déficit hídrico in vitro sea favorable. El objetivo del presente trabajo fue determinar la respuesta de cultivares de Musa spp. con diferente composición genómica al estrés hídrico inducido in vitro con polietilenglicol 6000 (PEG-6000). Se estudiaron los cultivares ‘Pelipita’ (ABB), ‘Manzano’ (AAB) y ‘Grande naine’ (AAA). El estrés se indujo con 30 g/L de PEG-6000 en medio de cultivo semisólido de multiplicación. A los 30 días se evaluaron variables indicadoras de estrés morfológicas (altura y número de brotes por explante), fisiológicas (masa fresca y masa seca) y bioquímicas (contenido prolina, peróxido de hidrógeno y malondialdehido). En el cultivar ‘Pelipita’ se afectó solamente la altura de las plantas, mientras que en los demás se afectaron todas las variables excepto la masa seca en el ‘Manzano’. En este último y en el ‘Grande naine’ se incrementó la prolina, el peróxido de hidrógeno y el malondialdehido, lo que evidenció un mayor estrés oxidativo y daño en las membranas celulares. Los cultivares estudiados, pudieran emplearse como controles de tolerancia (‘Pelipita’) y sensibilidad (‘Grande naine’ y ‘Manzano’) en la selección in vitro de plantas tolerantes a la sequía, en futuros programas de mejoramiento genético

IN PRESS - Mitochondrial and nuclear diversity of colonies of varying origins: contrasting patterns inferred from the intergenic tRNAleu-cox2 region and immune SNPs

In this study, we gathered sequence data from the tRNAleu-cox2 intergenic mitochondrial (mtDNA) region concurrently with single nucleotide polymorphism (SNP) data from 91 loci of nuclear DNA (ncDNA). The data was obtained from 156 colonies sampled in six apiaries from four countries. The full dataset was analysed and discussed for genetic patterns with a focus on cytonuclear diversity and admixture levels.This research was funded through the projects BEEHAPPY (POCI-01-0145-FEDER-029871, FCT and COMPETE/QREN/EU) and BEEHEAL. BEEHEAL was funded by the ARIMNet2 2016 Call by the following agencies: INIA (Spain), MOARD (Israel), ANR (France) and FCT (Portugal). ARIMNet2 (ERANET) received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. Ana Rita Lopes is supported by a PhD scholarship (SFRH/BD/143627/2019) from the Foundation for Science and Technology (FCT), Portugal. FCT provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020).info:eu-repo/semantics/publishedVersio

Memorias VIII Coloquio Perspectivas Metodológicas y Pedagógicas de la Investigación en Lengua Materna

La investigación como campo de actuación profesional requiere que las dimensiones del ser y del hacer del profesor se expliciten mediante actitudes y aptitudes desde las cuales se incida en procesos de innovación y transformación educativa y cultural, coherentes con el contexto histórico-social del momento. "Perspectivas de la Investigación en Pedagogía de la Lengua Materna y la Literatura" presenta el trabajo de discusión académica desarrollado en el VIII Coloquio en Pedagogía de la Lengua Materna, a propósito de los estatutos metódicos, teóricos y pedagógicos involucrados en el devenir de las investigaciones sobre la enseñanza -aprendizaje de la lengua materna y de la literatura, como también sobre el proceso de formación del docente - investigador en este campo.Research as a field of professional action requires that the dimensions of the being and of the teacher be made explicit through attitudes and skills from which the processes of innovation and educational and cultural transformation are based, coherent with the historical-social context of the moment. "Perspectives of Research in Pedagogy of the Mother Tongue and Literature" presents the academic discussion work developed in the VIII Colloquium in Pedagogy of the Mother Tongue, with regard to the methodical, theoretical and pedagogical statutes involved in the evolution of research on teaching - learning the mother tongue and literature, as well as the process of teacher training - researcher in this field.Bogotá-Colombi