Honeybees Apis mellifera L. in the Azores: developing innovative molecular tools and uncovering host-pathogen interactions in a pre- and post-Varroa destructor invasion

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In vitro antimicrobial activity of extracts and essential oils of Cinnamomum, Salvia, and Menthaspp. against foodborne pathogens: a meta‐analysis study

Publication . Ezzaky, Youssef; Elmoslih, Abdelkhaleq; Silva, Beatriz Nunes; Bonilla‐Luque, Olga M.; Possas, Arícia; Valero, Antonio; Cadavez, Vasco; Gonzales-Barron, Ursula; Achemchem, Fouad

Genetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.

2023Journal article

Open access

Frequent parasitism of Apis mellifera by Trypanosomatids in geographically isolated areas with restricted beekeeping movements

Publication . Aguado-López, Daniel; Bartolomé, Carolina; Lopes, Ana; Henriques, Dora; Segura, Sara K.; Maside, Xulio; Pinto, M. Alice; Higes, Mariano; Martín-Hernández, Raquel

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

2023Journal article

Open access

Colonization patterns of Nosema ceranae in the Azores archipelago

Publication . Lopes, Ana; Segura, Sara K.; Martín-Hernández, Raquel; Henriques, Dora; Pinto, M. Alice

Nosema ceranae and Nosema apis are pathogens of honey bees that cause nosemosis, a disease implicated in colony losses worldwide. They are obligate intracellular pathogens infecting the midgut epithelial cells of adult honey bees. Although N. ceranae was originally a pathogen specific of Apis cerana, currently, it is found in Apis mellifera throughout most of the world. Due to their confined environments, which limited pathogen transmission and dissemination, islands are unique places for epidemiological studies. There are only a few Varroa destructor-free and possibly even fewer N. ceranae-free honey bee sanctuaries in the world, with the Azores being one. Even though with the exception of Santa Maria and Flores, nosemosis has been present in the Azores since 2008, the causal species has yet to be identified. Hence, this study aimed to determine the prevalence and infection levels of Nosema spp. in the Azorean honey bees. In 2014/2015, 474 colonies were sampled on Faial, Flores, Pico, Graciosa, São Jorge, São Miguel, Santa Maria, and Terceira. Additionally, São Jorge, Santa Maria, Faial, and Terceira were re-sampled in 2020 with a total of 91 colonies. DNA was extracted, and the diagnosis and Nosema spp. loads were obtained by multiplex PCR and RT-qPCR. The findings indicate that N. ceranae appears to be the dominant species in the Azores. N. apis was only detected in 2014/2015 with a very low prevalence (5.1%). N. ceranae prevalence varied between 2.7%, on São Jorge, and 50.7%, on Pico. In 2020, N. ceranae positive colonies increased significantly on Terceira (57.1%) and São Jorge (50.0%). N. ceranae was not detected on Santa Maria in both periods. Average infection levels in positive N. ceranae colonies were usually medium to high (>10-7 ng/μl), with São Jorge colonies displaying the greatest infection intensity (>10-5 ng/ μl). This study highlights the Azores archipelago as a unique place for beekeeping, with islands free of N. ceranae and V. destructor, which are two important stressors that afflict honey bees in the world.

2022Conference object

Open access

Colonisation patterns of Nosema ceranae in the Azores Archipelago

Publication . Lopes, Ana Rita; Martín-Hernández, Raquel; Higes, Mariano; Segura, Sara K.; Henriques, Dora; Pinto, M. Alice

Nosema ceranae is a highly prevalent pathogen of Apis mellifera, which is distributed worldwide. However, there may still exist isolated areas that remain free of N. ceranae. Herein, we used molecular tools to survey the Azores to detect N. ceranae and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that N. ceranae was not only present but also the dominant species in the Azores. In 2014/2015, N. apis was rare and N. ceranae prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, N. ceranae prevalence increased significantly (p < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that N. ceranae colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the N. ceranae invasion, and it is remarkable that Santa Maria is also free of Varroa destructor, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.

2022Journal 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