Análisis de los patrones de diversidad genética de Nosema ceranae, un patógeno emergente de Apis mellifera

La abeja doméstica occidental Apis mellifera es parasitada por dos microsporidios, N. apis, históricamente su único microsporidio parásito, y N. ceranae, una especie inicialmente descrita en una abeja del Sureste Asiático, A. cerana, y encontrado en A. mellifera a comienzos del siglo XXI. Desde entonces, N. ceranae es el microsporidio más predominante en A. mellifera y fue señalado como posible responsable del despoblamiento de las colonias, que amenaza la población de A. mellífera en todo el planeta. Motivados por la escasez de datos genéticos de este parásito emergente, en esta tesis abordamos la descripción de los patrones de polimorfismo genético de las poblaciones naturales de N. ceranae mediante el estudio de varios genes de copia única, con objeto de reconstruir su historia demográfica reciente, investigar si existe alguna relación entre variantes genéticas y sus propiedades patogénicas, y profundizar en el conocimiento de su biología la existencia de procesos de recombinación. Asimismo, planteamos un estudio paralelo en N. apis con el fin de conocer su historia demográfica y compararla con la de su congénere. N. ceranae mostró niveles moderados de variabilidad en todas las colonias de A. mellifera independientemente del año y el lugar de procedencia. Los patrones de variación genética: i.e. exceso de variantes a baja frecuencia con respecto a las expectativas asumiendo equilibrio mutación-deriva, niveles moderados de diversidad, presencia de genotipos idénticos del parásito en distintos continentes y la ausencia de una clina geográfica, sugieren que la población de N. ceranae a A. mellifera tiene un origen reciente, posiblemente en el SE asiático, y que experimentó un crecimiento demográfico muy acentuado. La diferente patogenicidad de N. ceranae en las colonias de A. mellifera no parece estar asociada con una hipotética diferenciación genética, sino más bien con aspectos relativos a su hospedador o el manejo de las colonias en diferentes áreas geográficas. Además, el análisis de haplotipos reveló la participación de procesos de recombinación, aunque a tasa ligera, en la configuración de la variabilidad genética. En comparación, N. apis presentó una tasa de polimorfismo genético similar a la de N. ceranae y también mostró signos de recombinación genética. Sin embargo, su historia demográfica reciente es distinta de la de su congénere y sugiere que la parasitación de A. mellifera por N. apis es más antigua y que existe una incipiente diferenciación del parásito en las poblaciones de su hospedador

Population Genetics of Nosema apis and Nosema ceranae: One Host (Apis mellifera) and Two Different Histories

Two microsporidians are known to infect honey bees: Nosema apis and Nosema ceranae. Whereas population genetics data for the latter have been released in the last few years, such information is still missing for N. apis. Here we analyze the patterns of nucleotide polymorphism at three single-copy loci (PTP2, PTP3 and RPB1) in a collection of Apis mellifera isolates from all over the world, naturally infected either with N. apis (N = 22) or N. ceranae (N = 23), to provide new insights into the genetic diversity, demography and evolution of N. apis, as well as to compare them with evidence from N. ceranae. Neutral variation in N. apis and N. ceranae is of the order of 1%. This amount of diversity suggests that there is no substantial differentiation between the genetic content of the two nuclei present in these parasites, and evidence for genetic recombination provides a putative mechanism for the flow of genetic information between chromosomes. The analysis of the frequency spectrum of neutral variants reveals a significant surplus of low frequency variants, particularly in N. ceranae, and suggests that the populations of the two pathogens are not in mutation-drift equilibrium and that they have experienced a population expansion. Most of the variation in both species occurs within honey bee colonies (between 62%-90% of the total genetic variance), although in N. apis there is evidence for differentiation between parasites isolated from distinct A. mellifera lineages (20%-34% of the total variance), specifically between those collected from lineages A and C (or M). This scenario is consistent with a long-term host-parasite relationship and contrasts with the lack of differentiation observed among host-lineages in N. ceranae (< 4% of the variance), which suggests that the spread of this emergent pathogen throughout the A. mellifera worldwide population is a recent event.This study was supported by funds from the Instituto Nacional de Investigación y Tecnología Agraria (INIA; http://www.inia.es/; grant numbers RTA2013-00042-C10-05 and 06), the Regional Government of Murcia (Fundación Séneca; http://fseneca.es/; grant number 19908/GERM/2015) and the Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA; Plan Apícola Nacional 2014; http://www.magrama.gob.es). PDR is presently a member and receives support from COST Action FA1307, Sustainable pollination in Europe: joint research on bees and other pollinators, SUPER-B (http://www.cost.eu/COST_Actions/fa/Actions/FA1307)S

Survival of honey bees (Apis mellifera) infected with Crithidia mellificae spheroid forms (Langridge and McGhee: ATCC® 30254™) in the presence of Nosema ceranae

Crithidia mellificae, a trypanosomatid parasite of Apis mellifera, has been proposed to be one of the pathogens responsible for the serious honey bee colony losses produced worldwide in the last decade, either alone or in association with Nosema ceranae. Since this pathogenic effect contradicts the results of the experimental infections originally performed by Langridge and McGhee nearly 40 years ago, we investigated the potential linkage of this protozoan with colony decline under laboratory conditions. Nosema-free and trypanosomatid-free honey bees from three different colonies were experimentally infected with fresh C. mellificae spheroid forms (reference strain ATCC30254), with N. ceranae fresh spores and with both parasites at the same time. Replicate cages were kept at 27 °C and used to analyse survival. C. mellificae spheroid forms did not reduce significantly the survival of the worker bees (64.5% at 30 days post-infection vs. 77.8% for the uninfected bees used as controls; differences were non statistically significant) under these experimental conditions. In contrast, the cages infected with N. ceranae exhibited higher rates of mortality from the 20th day post-infection onwards, irrespective of the presence of C. mellificae, suggesting that the spheroid forms of the latter have no pathological effect on A. melliferaINIA-FEDER (RTA2013-00042-C10-06 and E-RTA2014-00003-C03)S

Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts

Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.Spanish Programme for Knowledge Generation and Scientific and Technological Strengthening of the R+D+I System, grant PID2021-126938OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF/EU"Grant PGC2018-098929-A-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe"Maria Zambrano postdoctoral fellowship program of the Spanish Ministry of Science, Innovation and Universities (RD289/2021)Doctoral Program in Fundamental and Systems Biology (University of Granada, UGR

Holistic screening of collapsing honey bee colonies in Spain: a case study

Background: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. Results: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. Conclusion: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified

Holistic screening of collapsing honey bee colonies in Spain: A case study

Background: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. Results: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. Conclusion: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified

Where, What and With Whom to Eat: Towards an integrative study of foraging behaviour

International audienc

The gut parasite Nosema ceranae impairs olfactory learning in bumblebees

Abstract Pollinators are exposed to numerous parasites and pathogens when foraging on flowers. These biological stressors may affect critical cognitive abilities required for foraging. Here, we tested whether exposure to Nosema ceranae , one of the most widespread parasite of honey bees also found in wild pollinators, impacts cognition in bumblebees. We investigated different forms of olfactory learning and memory using conditioning of the proboscis extension reflex. Seven days after feeding parasite spores, bumblebees showed lower performance in absolute and differential learning, and reversal learning than controls. Long-term memory was also slightly reduced. The consistent effect of N. ceranae exposure across different types of olfactory learning indicates that its action was not specific to particular brain areas or neural processes. We discuss the potential mechanisms by which N. ceranae impairs bumblebee cognition and the broader consequences for populations of pollinators

Supplementation in vitamin B3 counteracts the negative effects of tryptophan deficiencies in bumble bees

Increasing evidence highlights the importance of diet content in 9 essential amino acids for bee physiological and behavioral performance. However, the tenth essential amino acid, tryptophan, has been overlooked as its experimental measurement requires a specific hydrolysis. Tryptophan is the precursor of serotonin and vitamin B3, which together modulate cognitive and metabolic functions in most animals. Here, we investigated how tryptophan deficiencies influence the longevity and behavior of bumble bees. Tryptophan-deficient diets led to a moderate increase in food intake, aggressiveness and mortality compared to the control diet. Vitamin B3 supplementation in tryptophan-deficient diets tended to buffer these effects on food intake and survival, and significantly reduced workers aggressiveness. Considering that the pollens of major crops and common plants, such as corn and dandelion,