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Beneficial Microorganisms for Honey Bees Health

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Beneficial Microorganisms for Honey Bees Health Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN Scienze e tecnologie agrarie, ambientali e alimentari Ciclo XXX Settore Concorsuale: 07/I1 Settore Scientifico disciplinare: AGR/16 (Microbiologia Agraria) Beneficial microorganisms for honey bees health Presentata da: Daniele Alberoni Coordinatore Dottorato Supervisore Prof. Giovanni Dinelli Prof. Diana Di Gioia Co- Supervisori Dr. Loredana Baffoni Dr. Francesca Gaggìa Esame finale anno 2018 1 2 Thesis Abstract Honeybees (Apis mellifera and other species) are considered as the most economically important insect species for humans and the ecosystems, not only as honey producers but also and especially as pollinators of agricultural, horticultural crops and wild plants (approximately 90 different farm-grown foods, including many fruits and nuts, depend on honeybees), contributing at the pollination of 35% of the global food production. Unfortunately, honeybee decline started about 30 years ago, with the arrival from Asia of the bee mite Varroa destructor. Since then, honeybees have been damaged by different kinds of biotic and abiotic stressor factors, cumulating any kind of damages, and posing a serious threat to the agricultural field. Many scientists agree that bee decline is a multifactorial process in which a mechanism seems to be more important in a given period of the year than in another, and different mechanisms may predominate in another period or in other environments. Of those multifactorial processes, leading factors are the new emergent pathogens, such as Nosema ceranae a gut pathogen causing serious threat to bees and the consequent death of the colony; Viruses such as “deformed wing virus”, “Black queen cell virus”, “chronic paralysis virus” and many others that are often over transmitted by the mite parasite Varroa destructor. Pesticides and other environmental stress factors are furthering enhancing the high pathogenicity on bees, weakening more and more the delicate beehive superorganism balance. The major science concern about the bees usually regards the study of the bee pathogens and their interaction with an increasingly anthropized environment (e.g.: pollution and sub lethal poisonings). Only few research projects (of high scientific importance) have been carried out using an approach aimed to fix the problems linked with it. Even less are the researches investigating probiotic microorganisms as growth promoter, in order to obtain a better wealth and wellbeing of the bees. In the light of these possibilities the aim of my research is the development of -environmental friendly- microbial technologies aimed to increase the health of the bees. Keywords: Hymenoptera, Apoidea, Apis mellifera, gut microbiota, beneficial bacteria, Nosema ceranae, EFB, Lactobacillus, Bifidobacterium, Bacteriocins, antimicrobial activity, DGGE, next generation sequencing (NGS), climate change. 3 PhD Thesis Conceptual Flow: C State of Art: Environment Paper 1: Diseases and Paper 2: Beneficial h apter apter 1 and honey bees: environment, an obligate Microorganisms for Correlation of climate co-evolution potentially honey bees, problems change to honey bee leading to lethal synergies and progresses: : I health. and symptomatology State of the art in the n troduction One of the major modifications: research field, and anthropogenic pressure Like the bees, also possible implementation influencing decision pathogenic bacteria, viruses for biotic and abiotic honey making in the beekeeping and parasites are bee stressors mitigation business coevolving. Isolation: List of microorganisms isolated from Apoideae and Vespulae, and habitat. Chapter 2: Isolation of gut microorganisms from Hymenopetra: Paper 3: Bifidobacterium xylocopum sp. Identification of bacterial species inhabiting nov. and Bifidobacterium aemilianum sp. the gut microbiota, and their potential nov., from the carpenter bee (Xylocopa violacea) digestive tract. antimicrobial activity Antimicrobial Activity: Screening of antimicrobial activity of isolated strains. Paper 4: Lactobacillus spp. from Apis mellifera gut are a source of Helveticin like bacteriolysins. Chapter 3: Honey bee gut microbiota exploited with NGS studies: Paper 5: Gut microbiota of the Maltese How environmental factors and honey bee Apis mellifera ruttneri SHEP. anthropogenic pressure are affecting the gut Paper 6: Impact of antibiotics and natural microbial community population? medicaments on the microbial community of honey bee Apis mellifera subsp. ligustica SPIN. Paper 7: Effect of dietary supplementation of Bifidobacterium and Lactobacillus Chapter 4: Counteracting diseases for a strains in Apis mellifera L. against Nosema better honey bee wealth and health: ceranae. Beneficial microorganisms against the gut parasite Nosema ceranae. Paper 8: Impact of beneficial bacteria supplementation on the gut microbiota, colony growth and productivity of Apis mellifera L. Chapter 5: Conclusions Future perspectives for beneficial microorganism-based strategy implementation. 4 THESIS INDEX Beneficial microorganisms for honey bees (Apis mellifera spp.) health ................................................. 1 Thesis Abstract .............................................................................................................................................. 3 PhD Thesis Conceptual Flow:......................................................................................................................... 4 CHAPTER 1 INTRODUCTION .......................................................................................................................... 1 Environment and Honey bees: An Insights on the Emilia-Romagna region .............................................. 2 Microbial investigation on honey bee larvae showing atypical symptoms of European Foulbrood ........ 8 Beneficial Microorganisms for Honey bees, problems and progresses .................................................. 18 CHAPTER 2 Isolation of gut microorganisms from Hymenopetra .............................................................. 34 Isolation of gut bacteria from Apoideae and Vespulae, and related habitat. ......................................... 35 Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract .......................................................................................................... 48 Antimicronial Activity .............................................................................................................................. 75 Apis mellifera gut Lactobacillus are a source of Helveticin like bacteriolysins. ...................................... 79 CHAPTER 3 Honeybee gut microbiota exploited with NGS studies ............................................................ 90 Gut microbiota of the Maltese honeybee Apis mellifera ruttneri SHEP. ................................................. 91 Impact of antibiotics and natural medicaments on the microbial community of honeybee Apis mellifera subsp. ligustica SPIN. ............................................................................................................................... 99 CHAPTER 4 Counteracting diseases for a honeybee wealth and health .................................................. 106 Effect of dietary supplementation of Bifidobacterium and Lactobacillus strains in Apis mellifera L. against Nosema ceranae. ...................................................................................................................... 107 Impact of beneficial bacteria supplementation on the gut microbiota, colony growth and productivity of Apis mellifera L. ................................................................................................................................. 117 CHAPTER 5 CONCLUSIONS ........................................................................................................................ 129 ACKNOWLEDGMENTS ............................................................................................................................... 132 REFERENCES............................................................................................................................................... 133 5 CHAPTER 1 INTRODUCTION 1 Environment and Honey bees: An Insights on the Emilia-Romagna region Climate change, a look to a case study: the year 2017. Extreme and unusual hot temperatures and draught were the ingredients of the past summer 2017. An endless summer that represents a further step towards the overall climate warming of the Emilia-Romagna region, i.e. the Italian region where University of Bologna is located. A problem joined by most of regions facing the Mediterranean Sea. Having a look at the registered records of temperatures, Summer 2017 is the 3rd hottest summer since 1961, preceded only by Summer 2003 and 2012. Taking into account then average temperature from the year 1961 to the year 1991, temperatures have been of +4.5°C degrees above average in year 2003, +3.4°C in year 2012, whereas in 2017 temperatures were “only” +3°C above average (See Bar Chart 1 and Figure 1). Summer 2017 had the prerogative to celebrate its own first place in terms of absolute temperature: +42.2°C represents the highest temperature registered in the region in the last 150 years. This peak was registered the first week of August 2017, and, with the humidity typical of the region (continental climate), the perception by the human population (and consequently
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