Percorrer por autor "Pinto, Maria Alice"
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- COI Metabarcoding as a Novel Approach for Assessing the Honey Bee Source of European HoneyPublication . Honrado, Mónica; Quaresma, Andreia; Henriques, Dora; Pinto, M. Alice; Amaral, Joana S.Honey is a widely consumed food product frequently subjected to adulteration, with the mislabeling of its botanical or geographical origin being one of the most common practices. Determining the entomological origin of honey is particularly challenging but of high relevance for ensuring its authenticity, especially for products with protected designation of origin (PDO) labels. This study presents a novel DNA metabarcoding approach targeting a highly informative 406 bp fragment of the cytochrome c oxidase I (COI) gene to differentiate among the three major mitochondrial lineages (A, M, and C) of honey bees (Apis mellifera L.) native to Europe. The target region was selected based on the calculated fixation index (FST), which is frequently used in Population Genetics as a measure of differentiation between populations. The approach was validated with 11 honey samples of known entomological origin and applied to 44 commercial honeys from 13 countries. The approach demonstrated high sensitivity, accurately identifying the entomological origin of honey, including samples produced by honey bees of varying ancestries, which could not be resolved by previous methods based on real-time PCR coupled with high-resolution melting (PCR-HRM) analysis. The results demonstrate the effectiveness of COI metabarcoding in verifying honey authenticity and highlight the predominance of C-lineage honey bees in the production of commercial honeys from northwestern Europe. This finding suggests a limited presence of the native M-lineage ancestry, underscoring the need for conservation efforts.
- Expression of Elongase‐ and Desaturase‐Encoding Genes Shapes the Cuticular Hydrocarbon Profiles of Honey BeesPublication . Rodríguez‐León, Daniel Sebastián; Schmitt, Thomas; Pinto, M. Alice; Thamm, Markus; Scheiner, RicardaMost terrestrial insects have a layer of cuticular hydrocarbons (CHCs) protecting them from desiccation and mediating chemical communication. The composition of these hydrocarbons is highly plastic and changes during their lifetime and with environmental conditions. How these changes in CHC composition are achieved is largely unknown. CHC profiles of Apis mellifera honey bees vary among castes, task groups and subspecies adapted to different climates. This makes A. melliferaan excellent model for studying the molecular mechanism underlying CHC biosynthesis. We correlated the expression of specific elongase‐ and desaturase‐encoding genes with the CHC composition in bees performing different social tasks in two highly divergent A. mellifera subspecies. Elongases are enzymes that lengthen the hydrocarbon chain, while desaturases introduce double bonds in it. We evaluated the hypothesis that the expression of the genes encoding these enzymes determines CHC profiles of the worker bees. Our results revealed that the specificity of desaturases and elongases shapes the CHC profiles of worker bees performing different social tasks. Expression of the desaturase‐encoding gene LOC100576797 and the elongase‐encoding gene LOC550828 seemed to be strongly associated with the abundance of compounds that were characteristic of the CHC profile of nurse bees. In contrast, the compounds that characterised the CHC profiles of the forager bees seemed to be associated with the desaturase‐encoding gene LOC551527 and the elongase‐encoding gene LOC409638. Our data shed light on the genetic basis for task‐specific CHC composition differences in social hymenopterans and paved the ground for unravelling the genetic underpinning of CHC biosynthesis.
- Incense honey (Pittosporum undulatum) in the Azores: Botanical authentication by using real-time PCR approachPublication . Lopes, Ana Rita; Moura, Monica B.M.V.; Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Pinto, M. Alice; Mafra, IsabelHoney is a widely consumed food and much appreciated for its nutritional, organoleptic, and health properties. The honey produced in the Azores archipelago can be categorised as incense honey or multifloral honey. Incense honey classification should account with >30% from Pittosporum undulatum pollen grains and it is generally perceived as a unique and high-quality product and, consequently, susceptible to be adulterated through incorrect labelling or admixing with low-cost and low-quality honeys. Therefore, assessing the authenticity of such highly appreciated honey is a key issue for its valorisation. In this work, a real-time PCR method targeting the ITS region was proposed for the first time to detect P. undulatum species. To that end, the DNA extracted from the leaves of P. undulatum from other endemic species (e.g. Eucaliptus spp., Acacia spp., Trifolium spp., Castanea sativa Mill., Hydrangea macrophylla, Rhododendro indicum, Hedychium gardnerianum, Pericallis malvifolia) of the Azores and mainland Portugal was used to test the cross-reactivity of the ITS primers by qualitative PCR, revealing full specificity for P. undulatum. Posteriorly, a realtime PCR approach was proposed, exhibiting high analytical performance (PCR efficiency= 97.4 ± 6.4%, R͖= 0.991 ± 0.003) and a limit of quantification of 0.01 pg of incense DNA. The method was successfully applied to the 22 honey samples, from which incense was detected in all 9 monofloral incense honeys and in 5 out of 10 multifloral samples from the Azores. Generally, the quantitative results for incense DNA were in good agreement with the melissopalynological data, showing that all samples, except two, were according to their labelled statements. Herein, a new, simple, cost-effective and reliable molecular approach was proposed to authenticate and valorise the Azores honey.
- Interactions between prevalent pathogens of honeybees (Apis mellifera)Publication . Jabal-Uriel, Clara; Bonjour-Dalmon, Anne; Chejanovsky, Nor; Higes, Mariano; Le Conte, Yves; Lopes, Ana Rita; Pinto, M. Alice; Reyes-Carreño, Maritza; Soroker, Victoria; Martín-Hernández, RaquelThe aim of the BEEHEAL project was to determine the phenology and interactions of the microsporidia Nosema ceranae and 5 highly prevalent and pathogenic honeybee viruses (DWV, BQCV, ABPV, IAPV and CBPV) in four Mediterranean countries: France, Israel, Portugal and Spain. The prevalence and phenology of these pathogens was monitored in four continental apiaries (one in each country) and three insular apiaries located on Ouessant Island (France) and on São Miguel and Santa Maria Islands (Portugal). Colonies were sampled during a 2-year study period, and honeybees were collected and analysed once every two months. Microsporidia detection was performed by conventional triplexPCR whereas viral loads were determined by RT-qPCR of the positive samples. Israel had the highest percentage of honeybees infected by Ǥ ” , followed by Spain, continental Portugal, and Ouessant, whereas the levels were the lowest in France mainland and it was not detected on the Portuguese Islands. The countries with the higher prevalence of viruses were France and Israel whereas the apiaries in Portugal and Spain had a lower percentage of colonies and individual honeybees infected by viruses. Moreover, CBPV was not detected in the latter countries. The viral load of colonies and honeybees coinfected with twoor more viruseswas positively correlated. Data analysedfrom all apiariestogether showed a negative correlation between the percentage of infected N. ceranae honeybees and the viral load. However, there was no correlation at the apiary level between N. ceranae and the viral load when analysed separately. Cross-tabulations showed a correlation in coinfection between N. ceranae with any of the three viruses: BQCV, IAPV and CBPV. In fact, honeybees coinfected by the microsporidia and one of these viruses had a higher viral load than honeybees infected only with the virus. This study showed that pathogen interactions differs greatly among countries and serves as a starting point for the development of epidemiological studies to unravel pathogen coinfections.
- Lake Sinai virus in Azorean Honey bees: Understanding the impact of Varroa destructor on prevalence, loads, and strain distributionPublication . Lopes, Ana Rita; Low, Matthew; Martín-Hernández, Raquel; Miranda, Joachim; Pinto, M. AliceLake Sinai virus (LSV) is a pathogen affecting honey bees worldwide. It was first discovered in 2009 in Lake Sinai, USA, and manifests as a multi-strain virus. The Azores archipelago comprises islands with and without Varroa destructor, offering a unique setting for studying viral epidemiology. Building upon insights from the heterogeneous distribution of Deformed wing virus (DWV) strains, which was modulated by varroa’s invasion, this study sought to evaluate prevalence, load, and diversity of LSV in the Azores, and explore potential changes in the viral landscape attributable to the mite’s presence. In July/August of 2014/15 and 2020, 494 colonies were sampled across the archipelago. These were screened for LSV using RT-qPCR with a primer pair that allowed detection of at least four strains (LSV-1,-2,-3, and -4). Positive samples were further examined by highthroughput sequencing (HTS). The impact of varroa on prevalence and loads was evaluated using general linear mixed models in the framework of Bayesian analysis. LSV was detected on all islands (prevalence range: 7.7%–89.9%), with varroa’s presence significantly increasing prevalence (mean increase: 19.5±9.5, Probability of increase=97.6%). HTS identified two known strains (LSV-2, LSV-3) and one novel strain (LSV-9). Phylogenetic reconstruction revealed a strong geographic structure in which LSV-2 was found dominating all varroa-infested islands (Flores, Faial, and Pico) and one varroa-free island (São Jorge). LSV-3 and LSV-9 dominated varroa-free islands, Graciosa and Terceira, and São Miguel and Santa Maria, respectively. Viral loads varied greatly among islands, from 4.77 log10 copies/bee to 8.71 log10 copies/bee. Varroa’s presence affected LSV-2 loads, with a mean increase of 2.5±0.7 log10 copies/bee (Pr=100%). Our Żndings highlight mitedriven evolutionary changes in LSV in the Azores and identify a novel strain dominating the easternmost islands, coinciding with the unique refuge of DWV-C existing in this part of the archipelago.
- Phenology of the Microsporidia Nosema ceranae in four mediterranean countriesPublication . Jabal-Uriel, Clara; Bonjour-Dalmon, A.; Chejanovsky, Nor; Higes, Mariano; Le Conte, Yves; Lopes, Ana Rita; Pinto, M. Alice; Reyes-Carreño, Maritza; Soroker, Victoria; Martín-Hernández, RaquelThe presence and prevalence of the Microsporidia Nosema ceranae was assessed as part of the BEEHEAL project during a 2-year study period (2018-2019). Monitoring took place in six apiaries located in four Mediterranean continental apiaries: France, Israel, Portugal and Spain, and two insular apiaries located on Ouessant (France) and on São Miguel (Azores archipelago, Portugal). Besides honeybee sample collection every two months, parameters on colony strength, honey production, beekeeping management and climate were recorded. The pathogen was not detected on São Miguel Island during the study. Infection at the apiary level showed that most of the colonies were positive to N. ceranae except in mainland France, where the percentage of infected colonies was the lowest and it fluctuated, with an increase in August in both years. The percentage of infected honeybees per colony varied among apiaries, with Israel having the highest mean levels (32.39%). Colonies in Spain and Portugal showed a similar pattern with the highest levels of infection in April 2018. Ouessant Island had a high percentage of infected honeybees in 2018 and it decreased on 2019. Temperature was not correlated with N. ceranae infection, although high temperatures combined with high relative humidity were associated to high infection levels in Israel. Intra-colony percentage of infection was positively correlated with honeybee population when all apiaries were analysed together. Honey production was not related with N. ceranae infection. Regarding queen replacement, infection levels in spring 2019 in mainland Portugal and Spain decreased in those colonies that had replaced the queen the previous summer (August 2018). Colony mortality varied amo ng apiaries, with mainland France and Spain showing the highest colony losses (47.6% and 39.1%, respectively). However, this parameter was not correlated with the levels of infection. These results allowed to compare the N. ceranae phenology among different countries showing that the infection is dependent on many factors. This information could assist in the development of beekeeping management strategies.
- Standard methods and good practices in Apis honey bee omics researchPublication . Techer, Maeva A.; Chakrabarti, Priyadarshini; Caesar, Lílian; Eynard, Sonia E.; Farrell, M. Catherine; Foster, Leonard J.; Gorrochategui-Ortega, June; Henriques, Dora; Li-Byarlay, Hongmei; Morré, Jeffrey T.; Newton, Irene L. G.; Parejo, Melanie; Pinto, M. Alice; Vignal, Alain; Zarraonaindia, Iratxe; McAfee, AlisonIn the past decades, COLOSS members have joined forces multiple times to develop and condense standard methods related to research on honey bees, their pests, pathogens, and colony products. This led to the publication of four open-access BEEBOOK volumes that have been utilized by researchers worldwide. Among the chapters, “Standard methods for molecular research in Apis mellifera,” written by Evans and collaborators in 2013, has been a cornerstone for the standardization of honey bee molecular studies. However, since sequencing technologies and analyzing algorithms have made tremendous progress, many described methods require updating. In parallel, other Apis species’ genomes have now been sequenced, thus opening new research avenues in a comparative framework. In this chapter, we add to the methods previously covered by Evans et al. in 2013 and provide updated methodology where necessary, including worked examples and bioinformatic analysis pipe-lines. We also cover topics which were not previously covered in depth, such as sequencing ancient samples, population genomics, proteomics, and sampling honey bee colony products for microbiome studies, among others. Our hope is for this to become a lasting resource for honey bee scientists as the field continues to advance.
- A survey of important honey bee viruses in the Azores archipelagoPublication . Lopes, Ana Rita; Miranda, Joachim; Martín-Hernández, Raquel; Pinto, M. AliceThe Azores archipelago is an interesting place to study honey bee viruses and their associations with Varroa destructor, given that there are islands with (Pico, Flores, and Faial) and without the mite. With the goal of establishing a baseline for future viruses studies, here we sampled 494 colonies distributed across eight islands, in the summers of 2014/2015 and 2020. These samples were screened for the most important honey bee viruses (BQCV, CBPV, SBV, BeeMLV, and the LSV-, DWV-, and AKI-complexes) and the viral loads were determined by RT-qPCR for the positive samples. Of the seven viruses, only BeeMLV and the AKI-complex were not detected. The BQCV was found on all the islands with a high prevalence (>79%) with a wide range of viral loads. São Miguel showed the lowest median BQCV loads (3.18x10͖copies/µL) and Terceira the highest (6.19x10͗copies/µL). In the 2020 sampling,the BQCV loads showed a significant increase on São Jorge and Santa Maria. The LSV was also found on all the islands. Notably, the LSV prevalence increased signiƤcantly in 2020, but not the viral loads. Only Faial and Pico (both with varroa) had SBV, but there was no statistical difference in viral loads between the two islands. CBPV was detected on Pico, São Miguel, Graciosa, Terceira, and Faial, but only in a few colonies (5.3%), although with high viral loads. DWV was never found on São Jorge and Terceira in either sampling period. With the exception of Faial, DWV prevalence was higher on the islands with varroa (Pico and Flores) than on the varroa-free islands. The viral loads were not statistically different between islands with and without varroa for any of the viruses, suggesting that varroa is not the only factor contributing to infection. This study suggests that, in addition to varroa- and varroa-free honey bee populations, some of the islands seem to harbor populations that are also free of some important viruses like DWV, CBPV, SBV, BeeMLV and AKI-complex.
- Viral genetic landscape in the honey bee populations of the Azores archipelagoPublication . Lopes, Ana Rita; Miranda, Joachim; Martín-Hernández, Raquel; Pinto, M. AliceHoney bee decline can be attributed to many stressors, including pathogens and parasites, such as varroa and its vectored viruses. The global honey bee trade poses an important risk of pathogen dissemination. The arrival of varroa in the Azores because of (illegal) queen importations is clear evidence of that. Varroa was Ƥrst detected on Pico in 2000, on Flores in 2001, and on Faial in 2008. The other six islands of the archipelago remain mite-free. Therefore, the Azores provide an interesting setting to study the impact of varroa in the viral landscapes, adding to previous studies. In July/August of 2014/2015 we sampled 402 colonies distributed across 8 islands. These were screened for DWV, BQCV and LSV by RT-qPCR and the positive samples were further examined by high-throughput sequencing (HTS) to uncover whether varroa has altered the viral genetic composition. Of the 8 islands, São Jorge and Terceira had no DWV-positive colonies and Pico and Flores showed the highest DWV prevalence. BQCV and LVS were detected on all the islands. The HST data indicated that DWV-A, -B, and -C variants were all present in the Azores, although with varying prevalences (DWV-A: 56%; DWV-C: 31.2%; DWV-B: 12.2%). The phylogenetic tree revealed a geographic pattern, in which most of the DWV variants from São Miguel and Santa Maria formed a clade with the DWV-C reference, whereas Pico, Faial, Flores, and Graciosa clustered with the DWV-A reference. Interestingly, the DWV-A sequences from Pico were more closely related with those from Faial than with those from Flores. If the DWV-A variant was introduced with varroa, then the illegal queen imports on Pico and Flores had independent origins, whereas the DWV-A variants on Faial might have originated from Pico. DWV-A and B have acquired a world-wide distribution in the wake of varroa, while variants DWV-C and DWV-D have become extremely rare, one plausible scenario is that DWV-C was one of the original genotypes in the Azores predating varroa and has been replaced by the variants A and B, due to their superior adaptation to varroamediated transmission. Also of interest is the distinct clade formed by the BQCV and LSV sequences of São Miguel and Santa Maria, as well as the independent cluster formed by Flores sequences supporting the previous scenario. All these Ƥndings will be discussed in this communication.
- Virus prevalence in egg samples collected from naturally selected and traditionally managed honey bee colonies across europePublication . Bouuaert, David Claeys; De Smet, Lina; Brunain, Marleen; Dahle, Bjørn; Blacquiere, Tjeerd; Dalmon, Anne; Dezmirean, Daniel; Elen, Dylan; Filipi, Janja; Giurgiu, Alexandru; Gregorc, Aleš; Kefuss, John; Locke, Barbara; Miranda, Joachim; Oddie, Melissa; Panziera, Delphine; Parejo, Melanie; Pinto, Maria Alice; Graaf, Dirk C. deMonitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the ‘suppressed in ovo virus infection’ trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time.