Coordination of the Agricultural Research in the Mediterranean

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Organizational Unit

Publications

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

Publication . Martín-Hernández, Raquel; Benito, M.; Chejanovsky, Nor; Le Conte, Yves; Dalmon, Anne; Higes, Mariano; Pinto, M. Alice; Reyes-Carreño, Maritza; Soroker, Victoria

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.

2018Conference object

Open access

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

Publication . Henriques, Dora; Lopes, Ana; Chejanovsky, Nor; Dalmon, Anne; Higes, Mariano; Jabal-Uriel, Clara; Le Conte, Yves; Reyes-Carreño, Maritza; Soroker, Victoria; Martín-Hernández, Raquel; Pinto, M. Alice

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.

2021Journal article

Open access

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

Publication . Henriques, Dora; Lopes, Ana; Chejanovsky, Nor; Dalmon, Anne; Higes, Mariano; Jabal-Uriel, Clara; Le Conte, Yves; Reyes-Carreño, Maritza; Soroker, Victoria; Martín-Hernández, Raquel; Pinto, M. Alice

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.

2021Journal article

Open access