Percorrer por autor "Miranda, Joachim"
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- Abelha melífera dos Açores: estudo epidemiológicaPublication . Lopes, Ana; Miranda, Joachim; Martín-Hernández, Raquel; Henriques, Dora; Pinto, M. AliceOs Açores são um local único para estudos epidemiológicos da abelha devido à distribuição heterogénea de um dos principais responsáveis pelo seu declínio mundial: Varroa destructor. Ademais, o fungo Nosema ceranae e vários vírus têm sido apontados como um problema sanitário. Assim, este é o primeiro estudo epidemiológico nos Açores para avaliar o estatuto da N. ceranae e os mais importantes vírus das abelhas: BQCV1, SBV2, CBPV3, LSV4, DWV5, AKI6 e BeeMLV7. Analisaram-se 474 amostras de oito das nove ilhas em 2014/2015 e 92 de quatro ilhas em 2020. O ADN e ARN foram extraídos e o diagnóstico e carga viral foram obtidos por RT-qPCR. Apenas Flores e Sta Maria não têm Nc, nas restantes ilhas a carga é variável, ocorrendo um aumento significativo em S. Jorge e Terceira em 2020. Os vírus BQCV e LSV estão em todas as ilhas amostradas sendo as cargas virais significativamente diferentes entre elas. Relativamente ao LSV, Flores apresentou a carga mais baixa e Pico a mais elevada. SBV existe apenas no Faial e Pico, sem diferenças nas cargas. CBPV tem prevalências baixas, tendo sido detetado no Pico, S. Miguel, Graciosa, Terceira, Faial, com cargas elevadas, à exceção de Graciosa e Pico. S. Jorge e Terceira não têm DWV, sendo que as restantes têm prevalências variáveis e cargas virais diferentes em ambos os anos. AKI e BeeMLV não foram detetados. Este estudo mostra que os Açores são um local privilegiado para a apicultura, com várias ilhas livres dos principais patógenos que afligem a abelha melífera no mundo.
- Cell-free layer measurements ofin vitroblood flow in a microfluidic network: an automatic and manual approachPublication . Bento, David; Pereira, Ana I.; Lima, José; Miranda, Joachim; Lima, Rui A.In microcirculation, the cell-free layer (CFL) is a well-known physiological phenomenon that plays an important role in reducing the flow resistance and in balancing nitric oxide (NO) production by endothelial cells and NO scavenging by red blood cells. To better understand this phenomenon, several blood flow studies have been performed in simple geometries at both in vivo and in vitro environments. However, to date little information is available regarding the effects imposed by a complex branching network on the CFL. The present study shows the CFL layer variation at a microchannel network. The images were captured using a high-speed video microscopy system and the thickness of the CFL was measured using both manual and automatic image analysis techniques. Using this methodology, it was possible to visualise the in vitro blood flowing through the network and to identify several flow phenomena that happen in microcirculation. Overall, the results have shown that the concentration of cells and the geometrical configuration of the network have a major impact on the CFL thickness. In particular, the thickness of the CFL decreases as the fluid flows through a microchannel network composed with successive smaller channels. It was also clear that, for the full length of the network, the CFL thickness tends to decrease with the increase of the concentration of cells. The automatic method developed becomes inaccurate for high haematocrit and needs be calibrated by manual methods for Hcts bigger than 10%. The results obtained from this study could help the development and validation of multiscale numerical models able to take into account the CFL for simulating microvascular blood flow.
- The honeybee (Apis mellifera) developmental state shapes the genetic composition of the deformed wing virus-A quasispecies during serial transmissionPublication . Yañez, Orlando; Chávez-Galarza, Julio; Tellgren-Roth, Christian; Pinto, M. Alice; Neumann, Peter; Miranda, JoachimThe main biological threat to the western honeybee (Apis mellifera) is the parasitic mite Varroa destructor, largely because it vectors lethal epidemics of honeybee viruses that, in the absence of this mite, are relatively innocuous. The severe pathology is a direct consequence of excessive virus titres caused by this novel transmission route. However, little is known about how the virus adapts genetically during transmission and whether this influences the pathology. Here, we show that upon injection into honeybee pupae, the deformed wing virus type-A (DWV-A) quasispecies undergoes a rapid, extensive expansion of its sequence space, followed by strong negative selection towards a uniform, common shape by the time the pupae have completed their development, with no difference between symptomatic and asymptomatic adults in either DWV titre or genetic composition. This suggests that the physiological and molecular environment during pupal development has a strong, conservative influence on shaping the DWV-A quasispecies in emerging adults. There was furthermore no evidence of any progressive adaptation of the DWV-A quasispecies to serial intra-abdominal injection, simulating mite transmission, despite the generation of ample variation immediately following each transmission, suggesting that the virus either had already adapted to transmission by injection, or was unaffected by it.
- 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.
- Origins, diversity, and adaptive evolution of DWV in the honey bees of the Azores: the impact of the invasive mite Varroa destructorPublication . Lopes, Ana Rita; Low, Matthew; Martín-Hernández, Raquel; Pinto, M. Alice; Miranda, JoachimDeformed wing virus (DWV) is a honey bee virus, whose emergence from relative obscurity is driven by the recent host-switch, adaptation, and global dispersal of the ectoparasitic mite Varroa destructor (a highly efficient vector of DWV) to reproduction on honey bees (Apis mellifera). Our study examines how varroa affects the continuing evolution of DWV, using the Azores archipelago, where varroa is present on only three out of the eight Islands, as a natural experimental system for comparing different evolutionary conditions and trajectories. We combined qPCR of 494 honey bee colonies sampled across the archipelago with amplicon deep sequencing to reveal how the DWV genetic landscape is altered by varroa. Two of the varroa-free Islands were also free of DWV, while a further two Islands were intriguingly dominated by the rare DWV-C major variant. The other four Islands, including the three varroa-infested Islands, were dominated by the common DWV-A and DWV-B variants. The varroa-infested Islands had, as expected, an elevated DWV prevalence relative to the uninfested Islands, but not elevated DWV loads, due the relatively high prevalence and loads of DWV-C on the varroa-free Islands. This establishes the Azores as a stable refuge for DWV-C and provides the most convincing evidence to date that at least some major strains of DWV may be capable of not just surviving, but actually thriving in honey bees in the absence of varroa-mediated transmission. We did not detect any change in DWV genetic diversity associated with island varroa status but did find a positive association of DWV diversity with virus load, irrespective of island varroa status.
- Patógenos detetados em colónias de Apis mellifera nos AçoresPublication . Lopes, Ana; Miranda, Joachim; Martín-Hernández, Raquel; Henriques, Dora; Pinto, M. AliceOs Açores são um local único para estudos epidemiológicos das doenças da abelha devido à distribuição heterogénea dos principais agentes invasores responsáveis pelo seu declínio a nível mundial: Varroa destructor e Nosema ceranae. Além disso, existem diversos vírus que têm sido apontados como um problema sanitário frequentemente associado à Vd e Nc. Enquanto a chegada da Vd ao Pico, Flores e Faial foi imediatamente reconhecida pela DGAV, o estatuto da Nc era desconhecido até este estudo. Este é o primeiro estudo epidemiológico nos Açores que teve como objetivo avaliar o estatuto da Nc e dos mais importantes vírus das abelhas: BQCV,SBV,CBPV,LSV,DWV,AKI, e BeeMLV. Analisaram-se 495 amostras do Faial, Flores, Pico, Graciosa, São-Jorge, São-Miguel, Santa-Maria e Terceira. O ADN e ARN foram extraídos e o diagnóstico e carga viral foram obtidos por RT-qPCR. Apenas Flores, Santa-Maria e Faial não têm Nc, nas restantes ilhas a carga é variável, nomeadamente entre 1.02E+06 e 2.55E+01 cópias/μl. Os vírus BQCV e LSV estão em todas as ilhas sendo as cargas virais significativamente diferentes entre elas. A carga mais baixa é em São-Miguel (3.18E+02 cópias/μl) e a mais elevada em Santa- Maria (1.00E+04 cópias/μl). Relativamente ao LSV, Flores apresentou a carga mais baixa (1.61E+02 cópias/μl) e Pico a mais elevada (4.77E+05 cópias/μl). SBV existe em duas ilhas, Faial e Pico, com prevalências de 50% e 18.3%, respetivamente, sem diferenças nas cargas. CBPV tem prevalências baixas (2.8-23%) tendo sido detetado em cinco ilhas (Pico,São- Miguel,Graciosa,Terceira,Faial) mas com cargas elevadas, à exceção de Graciosa e Pico. São-Jorge e Terceira não têm DWV, sendo que as restantes têm prevalências entre 11.1-82% mas sem diferenças significativas nas cargas. AKI e BeeMLV não foram detetados. Este estudo mostra que os Açores são um local privilegiado para a apicultura, com várias ilhas livres dos principais patógenos que afligem a abelha melífera no mundo.
- Projeto BEEHAPPY: sanidade da abelha melífera (Apis mellifera L.) dos Açores: comparação dos padrões epidemiológicos num laboratório natural únicoPublication . Pinto, M. Alice; Henriques, Dora; Rodrigues, Pedro João; Miranda, Joachim; Martín-Hernández, Raquel; Amaral, Joana S.O BEEHAPPY é um projeto financiado pela Fundação para a Ciência e a Tecnologia (FCT), é coordenado pelo CIMO e envolve uma equipa de 16 especialistas multidisciplinares provenientes de várias instituições nacionais (CIMO-Centro de Investigação de Montanha, REQUIMTE-Rede de Química e Tecnologia, CEDRI -Centro de Investigação em Digitalização e Robótica Inteligente) e estrangeiras (SLU-Swedish University of Agricultural Sciences, CIAPA-IRIAF- Centro de Investigación Apícola y Agroambiental- Instituto Regional de Investigación y Desarrollo Agralimentario y Forestal, INIA-Peru- Instituto Nacional de Innovación Agraria). O BEEHAPPY centra-se nas populações de abelha dos Açores e tem como principais objectivos (i) o estudo da interação entre a abelha e as suas principais doenças (viroses e Nosemose) e (ii) o desenvolvimento de ferramentas com vista a identificar as populações de abelha (ferramenta informática) e avaliar a autenticidade do mel dos Açores (ferramenta molecular), o qual apresenta um grande potencial comercial. Para se atingirem tais objetivos, irão ser executadas 10 tarefas dedicadas à abelha (Tarefas 1, 4, 5, 6, 7, 8, 9), patogénios (Tarefas 1, 2, 3) e mel (Tarefa 10; Figura 1).
- Standard methods for molecular research in Apis melliferaPublication . Evans, Jay D.; Schwarz, Ryan S.; Chen, Yan Ping; Budge, Giles; Cornman, Robert S.; De la Rúa, Pilar; Miranda, Joachim; Foret, Sylvain; Foster, Leonard; Gauthier, Laurent; Genersch, Elke; Gisder, Sebastian; Jarosch, Antje; Kucharski, Robert; Lopez, Dawn; Lun, Cheng Man; Moritz, Robin F.A.; Maleszka, Ryszard; Muñoz, Irene; Pinto, M. AliceFrom studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.
- 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.
- Varroa destructor shapes the unique viral landscapes of the honey bee populations of the Azores archipelagoPublication . Lopes, Ana Rita; Low, Matthew; Martín-Hernández, Raquel; Miranda, Joachim; Pinto, M. AliceThe worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands na & iuml;ve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.