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Nourishing PRObiotics to Bees to Mitigate Stressors

Periodic Reporting for period 1 - NO PROBleMS (Nourishing PRObiotics to Bees to Mitigate Stressors)

Reporting period: 2018-01-01 to 2019-12-31

The main issue addressed by the NO PROBLEMS project is the honeybee gut microsporidium N. ceranae, a pathogen and parasite showing great synergies with biotic and abiotic environmental conditions such as viruses, (e.g. in the case of co-infection of N. ceranae and Chronic Bee Paralysis Virus), gut microbes dysbiosis, pollen diet, varroa, pesticides and climate change, thus leading to colony collapse and honey productivity losses. A secondary but not less importan project aim is the reinforcment of honeybee immuno system.

In relation to the importance for society, to counteract the proliferation of this pathogen is crucial because its control guarantees stability in the beekeeping sector. This project addresses the issues with eco-friendly approaches based on beneficial bacteria and plants extracts, aiming at their final validation as feed additive.
A reduced mortality of honey bees will guarantee a number of benefits for society such as:
- A constant pollination service (both of natural environments and crops)
- Reduced honeybee colony losses
- Higher honey (and other honeybee hives products) production
- Economic stability for beekeeping farms
- Protection of biodiversity through ecofriendly approaches.

The overall objectives of the NO PROBLEMS project are:
1. Design of a product based on probiotics and plant resources acting against N. ceranae proliferation. while preserving gut symbionts.
2. Implement scientific outputs in the field of essential oils and beneficial microorganisms.
3. Scale-up production processes.
4. Perform in field tests on a large scale of the designed product.
5. Develop a business strategy for the final product.
6. Organize training activities.
The first two years of project dealt with three kind of cage tests aiming at the inhibition of the pathogen Nosema ceranae, with the aim of obtaining indicative information on the effectiveness of:
1. Selection of single strain beneficial bacterium or a mixture of different bacterial strains.
2. Test novel antimicrobial compounds produced as secondary metabolites from a set of isolated bacteria.
3. Test of a number of plant extracts.
The activities were carried out using positive control compounds already present on the market as an external reference standard, when possible.

Moreover, a number of field tests have been carried out after the cage tests to effectively validate the efficacy of beneficial bacteria and plant extracts.
The main findings confirmed that beneficial bacteria administered alone, or as a mixture of different strains, are effective in the control of N. ceranae, even if they were not as effective as the reference antibiotic Fumagillin when applied in cage tests. On the other hand, beneficial bacteria were particularly well performing in field tests, where a reduction of 2 Log of Nosema sp. spores load was detected. Beneficial bacteria were shown to increase of about 50% the production of honeybees antimicrobial peptides like defensin, abaecin and imenoptericin. Moreover, the vitellogenin content of hemolymph (an indicator of honeybee health) was significatively higher if compared with control honeybees when single strain beneficial bacteria were administered to honeybees. In conclusion beneficial bacteria are a promising feed additive capable to increase honeybee health and counteract N. ceranae.
About plant extracts, bot neem oil and the thymol based feed additive developed by the partner ADVSCI showed their effectiveness against N. ceranae. In particular, thymol is very effective against N. ceranae in cage and in field tests. Moreover, when both beneficial bacteria and thymol are administered in field (via a separate bus successive administration), additive effects are underlined.
To date, the assessment of the impact of beneficial bacteria on Nosema sp. was based on a wide set of application methods lacking standardization:
i) International literature reports the use of the sole fermented growing media deprived of bacteria or purified antimicrobials only or bacteria pelleted and mixed with sugar syrup at different concentrations, lacking a combined approach.
ii) On the N. ceranae side, different N. ceranae spore doses ranging from 10^4 to 10^6 were used for individual infections of honeybees, significatively changing the final results. Moreover, also different strains of N. ceranae, with different virulence were used worldwide to carry out the experiments.
iii) Different approaches influencing the environmental condition were used, ranging from cage tests with different RH and incubation temperature, or direct testing in field on developing or fully developed honeybee hives.
iv) The feed supplement often comprised just sugar or proteinaceous additives like monofloral or polyfloral pollen, sterile or unsterile.

Finally, data available on the use of beneficial bacteria and plants extracts were sparse, dated, carried out with old methodologies and often inconsistent.

The progresses beyond the state of the art consist in:
i) The final validation of beneficial bacteria and some plants extracts as effective control methods for N. ceranae, with standardized experimental conditions.

ii) The tests carried out in the framework of NO PROBLEMS, involved the most advanced molecular techniques applied to a large number of experimental theses (51 theses) composed of individually infected honeybees (over 13,000 bees). The reliability of obtained data therefore should be considered as a milestone in the scientific knowledge about Nosema and organic control methods.
By the end of the project the expected result is a prototype feed additive for honeybees, based on beneficial bacteria and validated in field for its efficacy against N. ceranae but also proven as booster of honeybee immune system.

A beneficial bacteria-based feed additive for honeybees, proven for its efficacy and presenting open access data of the experiments involving it, will definitely pave the way for a microbial resource management of the honeybee superorganisms. This fact is very important because an extensive literature, which shows how naturally occurring bacteria and fungi can significantly increase the nutritional power of honeybee natural feed (pollen and nectar) and drive honeybee health, already exists. What is missing is the knowledge on the management of these resources for a better performance of the beekeeping sector, and NO PROBLEMS project has strong implications on these aspects paving the way to new solutions and approaches
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