Drosophila melanogaster as a perspective organism for plastic waste management: preliminary investigation integrating transgenic techniques, with gut microbiota modification to develop a plastic-degrader fly model

Plastics pollution management is one of the biggest challenges of our time; among plastics, polyolefins, such as polyethylene (PE), are the most recalcitrant for biodegradation, due to their chemical structure. Nevertheless, several microorganisms can grow using PE as sole carbon source, lowering its molecular weight, or introducing new functional groups on its surface. Some insects’ species have also shown the ability to consume PE, such as Galleria mellonella. G. mellonella saliva has been described as capable of PE oxidation and depolymerization. A novel enzyme, Demetra, has been purified from the greater wax worm saliva and shown to produce similar effects on PE. Currently, the low efficiency of the studied organisms is the main drawback to PE biodegradation. Merging enzymatic action with microorganisms’ metabolism hold potential to overcome this issue. This work laid the foundations to weaponize the model organism D. melanogaster against PE by combining transgenic techniques and gut microbiota modification. A chimeric gene was constructed that allows expression of a secreted form of Demetra. A Demetra transgenic line was created and cross-mated with transgenic enhancer trap lines expressing the GAL4 transcription factor in salivary glands. Husbandry flies were grown on food supplemented with PE microplastics and adult flies’ fitness was studied. Fertility, locomotory and life span assays suggest that while microplastics in the diet affect D. melanogaster fitness, the expression of Demetra could help recovering it. At the same time, this work aimed to deepen our knowledge about Demetra enzymatic mechanism and its role in PE metabolism in G. mellonella. Different approaches for Demetra enzyme purification were attempted but were not successful. Demetra gene expression profile in G. mellonella fed on different substrates was studied and a higher expression rate of Demetra gene correlates with PE enrichment of standard diet or with a honey wax-based diet but not with a PE - only one. Proof of concept experiment was realized to verify the possibility to implant external bacteria strains that usually can grow using chemically pre-treated PE (PE wax) as sole carbon source, in D. melanogaster larvae gut microbiota. Three strains isolated from PE – wax degrading consortium were able to survive on fly food supplemented with PE-wax. Larvae fed on it, retrieved all the three strains in their gut until pupation. Finally, to advance the field of transgenic technology and genome manipulation in non-model organisms with plastic-degrading capability, such as G. mellonella, we have characterized new promoters that can be implemented in these applications.