Landesanstalt für Bienenkunde

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Auslöser und Ausprägung des Varroa Sensitiven Hygiene (VSH) Verhaltens im Zusammenhang mit der Reproduktion der Varroamilbe Varroa destructor
(2024) Fölsch, Lina; Hasselmann, Martin
The ectoparasite Varroa destructor causes massive economic damages to the Western honey bee (Apis mellifera) and thus threatens the health of honey bee colonies worldwide. When left untreated, varroa populations can lead to high winter colony losses (Genersch et al. 2010; Traynor et al. 2020; Le Conte et al. 2010). Beekeepers must thus routinely treat against varroa to keep their colonies healthy. A sustainable solution to overcome the varroa problem is selection for varroa resistant honey bees. In addition to studies on natural selection against varroa, there is much interest in breeding varroa resistant honey bees. During varroa resistant selection the focus is often on different selection criteria, such as mite non-reproduction (MNR) and varroa sensitive hygiene (VSH). The selection criteria MNR describes the proportion of non-reproductive mites in a colony (Virag et al. 2022). VSH encompasses the specialized removal behavior of mite infested brood by worker bees (Dietemann et al. 2013; Harbo und Harris 2005). This brood removal interrupts varroa reproduction, which leads to reduced varroa infestation in honey bee colonies. The experiments in thesis were conducted as part of the SETBie project. The SETBie (Selection and Establishment of Varroa-tolerant Bee Colonies) project was a four-year selection project whose objectives included breeding a varroa-resistant honey bee in Baden-Württemberg, Germany. Several institutions and beekeeper organizations cooperated in this project. This dissertation focuses on the factors that trigger VSH behaviour, in addition to documenting the reproductive success of the mite (MNR) in selected colonies. Initial experiments investigated the relationship between VSH and the reproductive success of female Varroa (MNR). A widely held hypothesis was that the reproduction of the mite and the associated nymphal stages in the brood cells triggered the removal behaviour of the workers. However, this hypothesis was disproved by placing mites in capped brood cells and comparing the removal behaviour of reproducing mites with that of mites whose reproduction had been blocked by a special procedure (publication 1). Both groups of mites were removed at a rate of about 40% and did not differ significantly from each other. Therefore, reproduction of the mite could be excluded as a trigger for removal behavior of the workers. This result was supported by simultaneous analysis of VSH behaviour and the proportion of non-reproductive mites (MNR) in a large number of pre-selected colonies. It was also assumed that the increased number of non-reproductive mites (MNR) within a colony is directly connected to VSH behaviour, because the workers would primarily remove the reproductive mites, leaving only non-reproductive mites behind. In addition to providing evidence that mite reproduction is not the trigger of removal behaviour, our analysis of the data collected during the SETBie project showed no correlation between MNR and VSH values (publication 3). The MNR value of a colony therefore does not allow any conclusions to be drawn about the VSH value, which should be taken into account in recommendations for breeding practices. Furthermore, an experiment was conducted to rule out the possibility that removal behaviour effects the normal reproduction of the mite in the next cycle (publication 3). The need for other triggering factors for removal behaviour to occur was clearly demonstrated in this first set of experiments. Varroa mites manipulated to remove specific factors and inanimate objects were placed in capped brood cells to determine what influences removal behaviour. We found that an object (bead) alone does not induce removal behaviour (publication 2). Dead and odour-reduced mites were removed more frequently than the bead and control, but not anywhere near as frequently as live mites. We can thus assume that, in addition to the odour of the mite, the movement of the mite in particular has a signalling effect that triggers this removal behaviour. Other as yet unknown factors such as the reaction of the larvae to the parasitism are likely to play an important role in the removal behaviour of workers. The SETBie project took place over a period of four years, which allowed us to analyse colonies over several generations. Although MNR and VSH are in principle selectable traits, our analysis has shown that they are difficult to improve over multiple generations (publication 4). Colonies with low MNR values could often be improved by crossing them with a drone (artificial insemination) from a colony with a high MNR value. However, it also became clear that it is difficult to maintain high MNR values. Methodological difficulties in targeted selection of MNR/VSH also became noticeable during the collaborative project, which involved the cooperation of numerous breeders. Despite prior infestation of the selected colonies with 180 mites, it was often not possible to collect enough mites to make a valid statement about the reproductive capability of the mite. Even after opening many cells, the minimum number of 10 single infested cells could not be found, which means that MNR values could not be determined for about 63 % of the evaluated colonies due to the presence of too few mites. The selection and breeding work involved in this project is extremely labour-intensive and therefore expensive and requires a high level of expertise among the breeders. It was shown that MNR and VSH are in principle selectable and thus theoretically a Varroa-resistant honey bee line can be bred. However, for future projects, methodological problems need to be resolved or the evaluation methods of the MNR and VSH traits need to be simplified. An optimal solution would be the identification of molecular markers to support selection via genetic heritability. However, this requires further fundamental knowledge about the Varroa mite, the mechanisms that trigger the behaviour of the currently used selection traits, and more precise phenotypic definition of the resistant behaviours.
Charakterisierung der Qualität von Blütenpollen in unterschiedlichen Regionen Baden-Württembergs
(2022) Friedle, Carolin Gertrud Maria; Hasselmann, Martin
Honey bees (Apis mellifera) collect nectar and pollen from plants to feed their brood. Pollen provides a wide range of nutrients, such as proteins and lipids, but also carbohydrates, vitamins and enzymes. Because of these ingredients, pollen is also attractive to humans and is used as a dietary supplement. However, honey bees collect pollen not only from wild plants, but also from flowering crops grown in agriculture. Accordingly, contamination from plant protection products can be found in bee pollen and bee bread. In order to get a deeper insight into the occurrence and distribution of pesticide residues during an entire season, a total of 102 daily pollen samples were collected from April to July 2018 using pollen traps in an orchard in southern Germany. Almost 90% of the pollen samples showed detectable levels of pesticide residues. A total of 29 pesticides were detected in the samples, with more than half being fungicides, followed by insecticides and herbicides. Maximum concentrations of up to 4500 ng/g could be measured at the end of April. Samples collected in early May and late June also showed high levels of pesticides. A general risk management was performed to assess the risk of the detected pesticide concentrations for honey bees. The microbial quality of bee pollen is highly dependent on its botanical and geographic origin, as well as climatic conditions and post-harvest processing steps by the beekeeper. If no processing steps such as freezing or drying follow after harvest, the growth of microorganisms can be promoted and the pollen quality can be influenced by negative side effects such as fermentation or the production of mycotoxins. Bacterial and fungal colonies can be determined both by culture-dependent methods such as colony counting on plates and by culture-independent methods such as 16-rRNA amplicon sequencing. Following the hypothesis that storage conditions influence the composition of microorganisms in bee pollen, freshly harvested bee pollen was stored for seven days in June 2018 and 2019 under defined conditions (cold, room temperature, warm) and analyzed by sequencing 16S and 18S PCR amplicons. The bacterial community varied slightly between the sites studied and showed no significant difference between the storage conditions. The fungal community showed significant differences both between the studied sites and between the different storage conditions. The dominant fungal genera in the pollen samples were Cladosporium, Aspergillus and Zygosaccharomyces. While Cladosporium was most dominant in freshly collected pollen and the percentage decreased during storage, Aspergillus and Zygosaccharomyces showed a significant increase especially under warm storage conditions. Other contaminants naturally produced by plants can also have negative impacts on human health. Pyrrolizidine alkaloids belong to a group of phytochemicals, of which more than 600 structures are known in around 3% of all flowering plants worldwide. PA are known to be able to cause both acute poisoning and chronic damage or cancer in animals and humans. In July 2019, pollen was collected at 57 locations in Baden-Württemberg and analyzed for 42 different PAs and their N-oxides in order to expand knowledge about PA contamination in pollen and to be able to estimate the risk of the concentrations. A total of 22 different PAs were detected in over 90% of all samples examined. Only 5% of the PA were obtained as PA from plants of Senecio sp. identified, while 95% of PAs with a botanical background are from Echium sp. and Eupatorium sp. could be identified. The maximum total concentration of PA per sample was determined to be 48,400 ng/g. According to the risk values calculated by the BfR, however, 42% of the samples represented an increased risk to human health.
The production of melezitose in honeydew and its impact on honey bees (Apis mellifera L.)
(2021) Seeburger, Victoria; Hasselmann, Martin
Honeydew honey is a honey type which is of high economic importance in Europe. Phloem sap feeding insects of the order Hemiptera (true bugs) excrete honeydew, the key component of honeydew honey. Beekeepers move their hives between forest regions so that their bees can process the honeydew into honey. In case of high osmolality in the phloem sap of the hemipterans’ host trees, they counteract osmotic pressure by osmoregulation and produce oligosaccharides such as melezitose. Melezitose-rich honeydew honey is a major issue for beekeepers; it crystallises and obstructs the combs, leading to an economical loss. Nevertheless, precise analyses of the conditions of the occurrence of melezitose have not been realised. Furthermore, it is not known which impacts the trisaccharide has on honey bee health and the honeydew flow disease documented in beekeepers’ journals can have one explanation in the nutrition on melezitose. In order to determine influence factors for the emergence of melezitose, more than 600 honeydew droplets from defined honeydew producer species were collected under different environmental conditions (hemipteran species (host tree specific), natural area, air temperature, relative humidity, altitude, time of the year and of the day) between 2016 and 2019. The sugar spectra were analysed by high performance anion exchange chromatography with pulsed amperometric detection. To obtain the impact of melezitose on honey bee health, additional feeding experiments with daily evaluation of food uptake, gut-body weight ratio and mortality have been realised between 2017 and 2019. Additionally, comprehensive 16S rRNA Illumina sequencing of the gut microbial community has been performed. Remarkable differences could be found in the amount of melezitose between honeydew samples collected from different honeydew producer species and according to different environmental conditions. Air temperature increases and decreases in relative humidity increased the melezitose production in honeydew by the observed seven hemipteran species. Both, scale insect species on Picea abies and aphid species on Abies alba produced significantly less honeydew containing melezitose than aphid species on Picea abies. Additionally, honeydew with increased melezitose content was significantly more frequent collected in natural areas with limited water reservoir capacities, at higher altitudes and years with low precipitation. All results lead to the conclusion that hemipteran species produce more melezitose when the host trees have less access to water, increasing the osmolality of the phloem sap and indirectly enhancing the osmoregulation with producing melezitose by hemipteran species. Bees fed with melezitose showed increased food uptake and higher gut-body weight ratio than the control groups. Furthermore, melezitose feeding caused disease symptoms such as swollen abdomen, abdomen tipping and impaired movement and a significantly higher mortality than in control groups. Gut microbiota analyses indicated a shift of the bacterial species Lactobacillus Firm-4 and Lactobacillus kunkeei in favour of Lactobacillus Firm-5 in melezitose fed bees. This PhD project provides the important knowledge about the indicators that point out an enhanced melezitose production. This is a valuable contribution to design a warning system for beekeepers that will help to prevent harmful nutrition for honey bees or crystallised honey in the future by timely removal of bee colonies from local regions at risk. Additionally, feeding experiments point out the high effort that is required for the degradation process of the large-molecule melezitose. This effort might lead to a higher uptake of food, heavier guts, shorter lifespan and a higher susceptibility to intestinal diseases. Finally, an evidence was presented that the lactic acid bacteria of the gut microbiota are significantly involved in the digestion of melezitose.

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