Genetic Transformation And Molecular Study Of Transformed Ascosphaera Apis Mutants And Their Pathogenicity In Apis Mellifera Honeybees

Honeybees are one of the most well-known economically beneficial social insects which can be kept almost anywhere in the globe as long as there are flowering plants that produce nectar and pollen. These insects are accomplishing about80%of all crop insect pollinations. However, this ecologically important social insect is suffering from various diseases caused by various pathogens, like fungal diseases. Of course, fungi are economically important organisms whose pathogenicity continues to be the focus of extensive research. Among the fungal diseases, chalkbrood is the one known to be an invasive mycosis in honeybees, caused by Ascosphaera apis, that exclusively affects honeybee brood. The disease has been reported to cause about5%-37%reduction in honey production and80%brood death. Consequently, the severity of the disease, these days, has increased the use of pesticides and frequent long-distance transportation of colonies which is believed to provide an additional opportunity for A. apis infection globally. Furthermore, the fact that A. apis is so widespread in the globe and its viable spores can be found in stored honey, pollen, pollen capsules/tablets, used hive components, used beekeeping tools and equipments, and possibly in soil around infected apiaries for more than15years, makes the possibility of its eradication most unlikely. Here, even though a broad range of experimental works have been conducted safe control of the disease has been noticed to be difficult. Thus, genetic engineering works for better manipulation of the fungus, molecular investigation of pathogenesis and possible identification of strategies to control the disease shall take current issue of research. With this, this work was conducted with the objectives of understanding and developing an efficient and reproducible technique for the ultimate purpose of making this fungal species amenable to genetic studies and transformation. Furthermore, this work was aimed to study the pathogenicity of restricted enzyme mediated integration engineered A. apis mutants in honeybee(Apis melifera L.) larvae. Consequently, as restricted enzyme-mediated integration enables the investigation of the infectivity of transformed pathogens to host organisms, this work has utilized protoplast isolation and transformation techniques as a tool to understand this fungus further to meet set objectives. According to the results obtained, the fungus showed varying responses in terms of yield, size, and regeneration rates on different mediums, and osmotic stabilizer combinations. In this way, it was confirmed that younger incubation time has yielded better mycelium suitable for highest protoplast isolation while use of driselase was the best enzyme treatment, yielded about98.36×105mL-1of protoplasts. Young aged and exponentially growing mycelial culture provided the highest protoplast formation with higher protoplast viability. Use of citric acid monohydrate with NaCl as an osmotic stabilizer supported a51.06%viable protoplasts to regenerate and able to form mycelia colonies, while a combination of4hrs enzymolysis time with this osmotic stabilizer have supported53.06%protoplast regeneration. Following the successful protoplast isolation and restricted enzyme-mediated integration, PCR, ClustalW multiple sequence alignment, and Southern blot analyses confirmed the successful integration of a foreign DNA, as an insert, to the host chromosome targeting to cause mutation. An in-vitro bioassay experiment included in this work has confirmed that all engineered mutants had notable differences in pathogenicity among themselves and with the wild strain to the honeybee larvae (p<0.01). Disease infectivity data confirmed that the original (wild type) fungus caused the maximum (19.9%) amount of larval death starting from the5th day post inoculation, and its disease index was recorded to be33.5. Even though the mortality of engineered mutants was not significantly augmented compared to that of the wild strain, mutants4and8had16.8%and14.3%larval death with28.9and23.7of disease index, respectively. More specifically, transformed A. apis mutants were relatively less pathogenic to in vitro-reared honeybee larvae than those of the wild-type. In addition, higher larval death and faster mummification was detected in the wild-type too. Thus, the pathogenicity differences among strains, even in the natural conditions, have magnified the significance of further investigations on the reasons for variations among transformants and their wild types. Even if we have studied this fungus for a while, control of this fungus is still a mystery. Thus, we, generally, recommend that further understanding on this fungus and different investigations shall be carried out as a part of future important activities.