Instituto Regional de Investigación y Desarrollo Agroalimentario y Forestal de Castilla-La Mancha

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Selection for heat tolerant (HT) animals in dairy production has been so far linked to estimation of declines in production using milk recording and meteorological information on the day of control using reaction norms. Results from these models show that there is a reasonable amount of genetic variability in the individual response to high heat loads, which makes feasible selection of HT animals at low costs. However, the antagonistic relationship between level of production and response to heat stress (HS) implies that selection for HT animals under this approach must be done with caution so that productivity is not damaged. Decomposition of the genetic variability in principal components (PC) can provide selection criteria independent of milk production level although biological interpretation of PC is difficult. Moreover, given that response to heat stress for each animal is estimated with very sparse information collected under different physiological and management circumstances, biased (normally underestimation) and lack of accuracy may be expected. Alternative phenotypic characterization of HT can come from the use of physiological traits, which have also shown moderate heritability. However, costs of a large scale implementation based on physiological characteristics has precluded its use. Another alternative is the use of biomarkers that define heat tolerance. A review of biomarkers of HS from more recent studies is provided. Of particular interest are milk biomarkers, which together with infrared spectra prediction equations can provide useful tools for genetic selection. In the 'omics' era, genomics, transcriptomics, proteomics and metabolomics have been already used to detect genes affecting HT. A review of findings in these areas is also provided. Except for the slick hair gene, there are no other genes for which variants have been clearly associated with HT. However, integration of omics information could help in pointing at knots of the HS control network and, in the end, to a panel of markers to be used in the selection of HT animals. Overall, HT is a complex phenomenon that requires integration of fine phenotypes and omics information to provide accurate tools for selection without damaging productivity. Technological developments to make on-farm implementation feasible and with greater insight into the key biomarkers and genes involved in HT are needed.

Abstract Beekeepers have various options to control the parasitic mite Varroa destructor in honey bee colonies, but no empirical data are available on the methods they apply in practice. We surveyed 28,409 beekeepers maintaining 507,641 colonies in 30 European countries concerning Varroa control methods. The set of 19 different Varroa diagnosis and control measures was taken from the annual COLOSS questionnaire on honey bee colony losses. The most frequent activities were monitoring of Varroa infestations, drone brood removal, various oxalic acid applications and formic acid applications. Correspondence analysis and hierarchical clustering on principal components showed that six Varroa control options (not necessarily the most used ones) significantly contribute to defining three distinctive clusters of countries in terms of Varroa control in Europe. Cluster I (eight Western European countries) is characterized by use of amitraz strips. Cluster II comprises 15 countries from Scandinavia, the Baltics, and Central-Southern Europe. This cluster is characterized by long-term formic acid treatments. Cluster III is characterized by dominant usage of amitraz fumigation and formed by seven Eastern European countries. The median number of different treatments applied per beekeeper was lowest in cluster III. Based on estimation of colony numbers in included countries, we extrapolated the proportions of colonies treated with different methods in Europe. This suggests that circa 62% of colonies in Europe are treated with amitraz, followed by oxalic acid for the next largest percentage of colonies. We discuss possible factors determining the choice of Varroa control measures in the different clusters.

The microsporidia Nosema apis and Nosema ceranae are major honey bee pathogens that possess different characteristics in terms of the signs they produce, as well as disease development and transmission. Although the ventricular epithelium is generally considered the target tissue, indirect observations led to speculation that N. ceranae may also target other structures, possibly explaining at least some of the differences between these 2 species. To investigate the tropism of Nosema for honey bee tissues, we performed controlled laboratory infections by orally administering doses of 50 000 or 100 000 fresh mature spores of either species. The fat body was isolated from the infected bees, as well as organs from the digestive (esophagus, ventriculus, ileum, rectum), excretory (Malpighian tubules), circulatory (aorta, heart), respiratory (thoracic tracheas), exocrine (hypopharyngeal, mandibular and labial, cephalic, thoracic salivary glands), and sensory/nervous (brain, eyes and associated nerve structures, thoracic nerve ganglia) systems. Tissues were examined by light and electron microscopy at 7, 10, and 15 days postinfection. Both Nosema species were found to infect epithelial cells and clusters of regenerative cells in the ventriculus, and while the ileum and rectum contained spores of the microsporidia in the lumen, these structures did not show overt lesions. No stages of the parasites or cellular lesions were detected in the other organs tested, confirming the high tropism of both species for the ventricular epithelium cells. Thus, these direct histopathological observations indicate that neither of these 2 Nosema species exhibit tropism for honey bee organs other than the ventriculus.

Pollination services to increase crop production are becoming more and more important, as we are facing both climate change and a growing world population. Both are predicted to impact food security worldwide. High-density, commercial beekeeping has become a key link in the food supply chain, and diseases have become a central issue in hive losses around the world. American Foulbrood (AFB) disease is a highly contagious bacterial brood disease in honey bees ( Apis mellifera ), leading to hive losses worldwide. The causative agent is the Gram+ bacterium Paenibacillus larvae , which is able to infect honey bee larvae during the first 3 days of their lives. It can be found in hives around the world with viable spores for decades. Antibiotics are largely ineffective in treating the disease as they are only efficient against the vegetative state. Once a hive shows the clinical manifestation of the disease, the only effective way to eradicate it and prevent the spread of the disease is by burning the hive, the equipment, and the colony. Because of its virulent nature and detrimental effects on honey bee colonies, AFB is classified as a notifiable disease worldwide. Effective, safe, and sustainable methods are needed to ensure the wellbeing of honey bee colonies. Even though insects lack antibodies, which are the main requisites for trans-generational immune priming (TGIP), they can prime their offspring against persisting pathogens. Here, we demonstrate an increased survival of infected honey bee larvae after their queen was vaccinated, compared to offspring of control queens (placebo vaccinated). These results indicate that TGIP in insects can be used to majorly enhance colony health, protect commercial pollinators from deadly diseases, and reduce high financial and material losses to beekeepers. Classification biological sciences, applied biological sciences

The present study aimed to examine the effects of exposure to adverse weather conditions on milk production to assess the thermotolerance capability of the Manchega breed, a dairy sheep reared in the Mediterranean area, and the extent of decline in production outside the thermal comfort zone. To achieve this purpose, we merged data from the official milk recording of the breed with weather information and used to describe the cold and heat stress response for production traits. Production data consisted of 1,094,804 test-day records from the first 3 lactations of 177,605 ewes gathered between years 2000 to 2010. For each production trait and climate variable, the thermal load production response was characterized by the estimation of cold and heat stress thresholds that define a thermoneutral zone and the slopes of production decay outside this thermoneutral zone. Overall, we observed a comfort region between 10 and 22°C for daily average temperature, 18 and 30°C for daily maximum temperature, and from 9 to 18 units for a temperature-humidity index (THI) for all traits. Decline in production due to cold stress effects was of a greater magnitude than heat stress effects, especially for milk yield. Production losses ranged between 7 and 16 and from 0.2 to 0.6g/d per °C (or THI unit) for milk and for fat and protein yields, respectively. For heat stress, the observed decline in production was of 1 to 5 and 0.1 to 0.3g/d per °C (or THI unit) above the threshold for milk yield and for fat and protein yields, respectively. Highly productive animals showed a narrower comfort zone and higher slopes of decay. The study of lagged effects of thermal load showed how consequences of cold and heat stress are already visible in the first hours after exposure. Thus, production losses were due mainly to climate conditions on the day of control and the day before, with conditions on the previous days having a smaller effect. Annual economic losses due to thermal (cold and heat) stress ranged from 0.1 up to 4% of total profit depending on which climate variable was considered. Although of small magnitude, the effect of adverse climatic conditions on total annual farm profit is not negligible; thus, we consider the implementation of strategies aimed at reducing these losses to be important. These strategies could be target improving the mitigation strategies as well as obtaining more thermotolerant animals through selection.

This review attempts to reflect the importance of different factors that affect the environmental quality of dairy farms and must, therefore, be taken into account when considering the importance of environmental microbiology as a tool in the improvement of the quality of milk and dairy products. The effect of a factor such as temperature is vital for the dairy farm environment, especially when the temperatures are extreme, because a proper choice of temperature range improves the quality of the air and, thus, animal welfare. Similarly, the appropriate level of relative humidity in the environment should be taken into consideration to avoid the proliferation of microorganisms on the farm. Air quality, well-designed livestock housing, proper hygienic practices on the farm, stocking density, and the materials used in the livestock houses are all important factors in the concentration of microorganisms in the environment, promoting better welfare for the animals. In addition, a ventilation system is required to prevent the pollution of the farm environment. It is demonstrated that proper ventilation reduces the microbial load of the environment of dairy farms, enhancing the quality of the air and, therefore, the wellbeing of the animals. All this information is very useful to establish certain standards on dairy farms to improve the quality of the environment and, thereby, achieve better quality milk and dairy products.

Bumblebees are constantly exposed to a wide range of biotic and abiotic stresses which they must defend themselves against to survive. Pathogens and pesticides represent important stressors that influence bumblebee health, both when acting alone or in combination. To better understand bumblebee health, we need to investigate how these factors interact, yet experimental studies to date generally focus on only one or two stressors. The aim of this study is to evaluate how combined effects of four important stressors (the gut parasite Nosema ceranae, the neonicotinoid insecticide thiamethoxam, the pyrethroid insecticide cypermethrin and the EBI fungicide tebuconazole) interact to affect bumblebees at the individual and colony levels. We established seven treatment groups of colonies that we pulse exposed to different combinations of these stressors for 2 weeks under laboratory conditions. Colonies were subsequently placed in the field for 7 weeks to evaluate the effect of treatments on the prevalence of N. ceranae in inoculated bumblebees, expression levels of immunity and detoxification-related genes, food collection, weight gain, worker and male numbers, and production of worker brood and reproductives. Exposure to pesticide mixtures reduced food collection by bumblebees. All immunity-related genes were upregulated in the bumblebees inoculated with N. ceranae when they had not been exposed to pesticide mixtures, and bumblebees exposed to the fungicide and the pyrethroid were less likely to have N. ceranae. Combined exposure to the three-pesticide mixture and N. ceranae reduced bumblebee colony growth, and all treatments had detrimental effects on brood production. The groups exposed to the neonicotinoid insecticide produced 40%-76% fewer queens than control colonies. Our findings show that exposure to combinations of stressors that bumblebees frequently come into contact with have detrimental effects on colony health and performance and could therefore have an impact at the population level. These results also have significant implications for current practices and policies for pesticide risk assessment and use as the combinations tested here are frequently applied simultaneously in the field. Understanding the interactions between different stressors will be crucial for improving our ability to manage bee populations and for ensuring pollination services into the future.

Phenotypic variability within forest species populations is considered of special relevance for local adaptation under new environments, albeit it has been analyzed to a lesser extent than inter-population phenotypic variability. A common garden study was carried out to assess phenotypic variability in response to water stress in half-sibling families from a marginal population of Fagus sylvatica L. at its south-western range edge distribution in Europe. Two irrigation regimes were applied, well-watered (WW) seedlings and those submitted to weekly cycles of drying-rewatering of growth media. Seedling growth and their leaf functional traits were recorded during the last cycle of water stress. Most of the phenotypic changes were explained by phenotypic plasticity in response to water stress, but there was also a significant effect of family in the expression of some of the studied traits. The relationship of carbon isotope fractioning with gas exchange traits across families under WW conditions did not follow the same pattern as the phenotypic trends. The leaf net photosynthesis across families was modified by the nitrogen content on a leaf mass basis that was in turn correlated positively with leaf nitrogen isotope fractionation. The results point to an important role of leaf nitrogen in determining the intrinsic water-use efficiency (WUE) across families. Variation in WUE was ruled mainly by control of stomatal conductance to water vapor under water stress, but by leaf net photosynthesis under wet conditions. Relatively high inter-family phenotypic variability in growth and functional traits were observed. Within-population phenotypic variability, and the plasticity of some of the studied traits, is of fundamental importance to cope with the harsher environments beech will have to endure in the future at different points in its distribution range.

Non-ferrous metal mining is considered one of the largest sources of toxic metal released to the environment and may threaten ecosystems, notably biota. We explored how birds that inhabit non-ferrous metal mining sites are exposed to mercury, lead, and other trace elements by analyzing their feathers and verifying which factors may influence element concentrations in feathers. We sampled a total of 168 birds, representing 26 species, with different feeding habits and migration patterns in a non-polluted reference site and two historical metal mining areas: Almadén, which is considered one of the most heavily mercury-contaminated sites worldwide, and the Sierra Madrona mountains where lead has been mined since ancient times. The quantification of aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se), thorium (Th), thallium (Tl), uranium (U), vanadium (V) and zinc (Zn) was performed by inductively coupled plasma mass spectrometry (ICP-MS). Feather analysis revealed contamination by Hg and Pb, in Almadén and Sierra Madrona, respectively. We found that granivorous birds had the lowest feather Hg levels compared to those found in omnivorous, insectivorous, and piscivorous species, whereas feather Pb was about twice as high in granivores and omnivores, than in insectivorous and piscivorous birds. We also found differences among study sites in 13 elements and confirmed the influence of feather age, migratory patterns of the birds, and external deposition of elements, on metal concentrations in the feathers. Our results highlight that despite the cessation of metal mining in the study areas, local avifauna are being exposed to Hg and Pb from abandoned mines and old tailings sites, indicating that appropriate measures are needed to protect biota from overexposure to these toxic metals.

Nosemosis is a serious microsporidian disease of adult European honey bees caused by the spore-forming unicellular fungi Nosema apis and Nosema ceranae. In this paper we describe the currently known techniques for nosemosis prevention and control including Good Beekeeping Practices (GBPs) and biosecurity measures (BMBs). Topics such as queen renewal, nosema-resistant bees and hygienic and control methods are described. Strong efforts are currently provided to find more a sustainable solution than the use of antibiotics. So far, it seems that the best way to approach nosemosis is given by an “integrated pest management strategy”, which foresees the contemporary application of different, specific GBPs and BMBs.

Migratory beekeeping is a widely extended practice aimed at increasing the yield of products and pollination services of honey bee colonies. However, it represents a stress factor, as it facilitates the dissemination of diseases and may compromise the genetic identity of the colonies involved. To analyze the extent of these effects, pathogens infestation rate and genetic composition were monitored in a field experiment comparing stationary and migratory colonies sharing the same environmental conditions but differing in management (stationary vs. migratory) and genetic background. We studied the pathogens infestation rate (Varroa destructor, Nosema spp., and Deformed Wing Virus (DWV)) at four different times: before migratory operation, two weeks later, at the end of the migratory period, and two weeks after the return of the migratory hives. An increased incidence of V. destructor and Nosema ceranae and a lower DWV viral load were found in migratory colonies. Temporary changes in genetic diversity were detected regardless of colony type, suggesting that stressors other than management affect the genetic diversity of the colonies. Our study demonstrates that migratory practices have variable effects on the health and genetic diversity of honey bee colonies, which should be taken into account for the development of sustainable beekeeping.

Water stress is considered to be the most influential type of abiotic stress to which plants may be exposed. In grapevines (Vitis vinifera L.), it is a common practice to keep plants under water stress at different stages of the season with the aim of reducing yield and improving the composition of the fruit. The objective of this study was to evaluate foliar development and yield of ‘Merlot’ grapevines grown in the field when they are subjected to different levels of water stress in a semi-arid Mediterranean climate. Four treatments with different levels of water stress were applied during two phenological intervals (flowering-veraison and veraison-maturity) to 128 grapevines for a period of two consecutive years. The levels of water stress were none-light, light-moderate, moderate-intense, and intense-intense for the flowering-veraison and veraison-maturity intervals, respectively. The results revealed that the total leaf area, the exposed leaf area, and the yield all decreased as the degree of water stress increased. The weight of the berry was a decisive factor in determining yield. The least restrictive water regime treatment gave the heaviest berries and bunches and, as a result, the highest yields.

The ability to obtain different wines with a singular organoleptic profile is one of the main factors for the wine industry’s growth, in order to appeal to a broad cross section of consumers. Due to this, white wines made from the novel grape genotypes Albillo Dorado and Montonera del Casar (Vitis vinifera L.) were studied and compared to the well-known Airén at two consecutive years. Wines were evaluated by physicochemical, spectrophotometric, high-performance liquid chromatography–diode array detection–mass spectrometry, gas chromatography–mass spectrometry and sensory analyses. The chromatic characteristics of the new wines were defined by more color purity than Airén, with greenish highlights. In general, the phenolic profile of the Albillo Dorado wines showed a higher flavonol and hydroxycinnamic acid derivative content. Several volatile compounds were determined, and their odor activity values were calculated to determine their impact on wine aroma. A fruity series dominated the wine aromatic composition, but spicier and greener notes characterized the aroma profile of Airén wines. Albillo Dorado and Montonera del Casar were sensory evaluated as wines with a less fresh taste compared to Airén. Unique chemical and sensory profiles were determined for wines made from these novel grape genotypes, providing alternative monovarietal wines to encourage the wine market growth and extend the offer to consumers.

BACKGROUND: Due to the current dominance of a few grape varieties in the wine market, the aim of this work was to study the detailed phenolic and volatile composition, chromatic characteristics, and sensorial properties of red wines elaborated with new Vitis vinifera grapes (Moribel and Tinto Fragoso) identified using the High Perfomance Liquid Chromatography-Diode Array Detector-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-DAD-ESI-MS/MS), Gas Chromatography- Mass Spectrometry (GC-MS), CIELab color space, and Napping ® techniques. RESULTS: Tinto Fragoso wine showed higher phenolic content than Moribel, with more anthocyanins, flavonols, and stilbenes. These wines also contained anthocyanin diglucosides not reported for Vitis vinifera wines. The odor activity values of free volatile compounds were calculated to indicate their influence on wine aroma, the fruity aromas of Moribel standing out particularly. The wines studied were positively evaluated by the tasters. Moribel's sensory profile was characterized by red fruit aromas and Tinto Fragoso showed more aromatic intensity and persistence in the mouth. CONCLUSION: The results obtained from the first characterization of red wines made from novel Vitis vinifera grapes suggest that Moribel and Tinto Fragoso could be appropriate raw materials for the elaboration of quality young red wines. Tinto Fragoso provided wines with sensorial properties different from those of Tempranillo, and could be an alternative to the well known red wines on the market. © 2018 Society of Chemical Industry.

To evaluate the influence that parasites have on the losses of Apis mellifera it is essential to monitor their presence in the colonies over time. Here we analysed the occurrence of nosematids, trypanosomatids and neogregarines in five homogeneous colonies for up to 21 months until they collapsed. The study, which combined the use of several molecular markers with the application of a massive parallel sequencing technology, provided valuable insights into the epidemiology of these parasites: (I) it enabled the detection of parasite species rarely reported in honeybees (Nosema thomsoni, Crithidia bombi, Crithidia acanthocephali) and the identification of two novel taxa; (II) it revealed the existence of a high rate of co-infections (80% of the samples harboured more than one parasite species); (III) it uncovered an identical pattern of seasonal variation for nosematids and trypanosomatids, that was different from that of neogregarines; (IV) it showed that there were no significant differences in the fraction of positive samples, nor in the levels of species diversity, between interior and exterior bees; and (V) it unveiled that the variation in the number of parasite species was not directly linked with the failure of the colonies.

European Apis mellifera and Asian Apis cerana honeybees are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of the 16S rRNA from bacteria and the internally transcribed spacer (ITS) regions from fungi and plants allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollen collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees collected at various time points from anthropogenic landscapes such as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial content of honeybee intestine as well as fungi and pollens. Furthermore, isolated DNA was used as the template for screening pathogens: Nosema apis, N. ceranae, N. bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi). We conclude that differences between samples were mainly influenced by the bacteria, plant pollen and fungi, respectively. Moreover, honeybees feeding on a sugar based diet were more prone to fungal pathogens (Nosema ceranae) and neogregarines. In most samples Nosema sp. and neogregarines parasitized the host bee at the same time. A higher load of fungi, and bacteria groups such as Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and other unidentified bacteria was observed for Nosema ceranae and neogregarine infected honeybees. Healthy honeybees had a higher load of plant pollen, and bacteria groups such as: Orbales, Gilliamella, Snodgrassella, and Enterobacteriaceae. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations, and hence pathogen attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.

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.

Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy–Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.

Nosema ceranae is an intracellular parasite that infects honeybees' gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or β-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees' age had a significant impact in shaping the bacteriome structure and composition of the bees' gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees' microbiome.