Molecular analysis of the genes expressed during feeding site establishment by first instar Lucilia cuprina

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The sheep blowfly L. cuprina is a major concern of the Australian sheep industries. First instars of L. cuprina are capable of initiating primary fly strike via initiation of a feeding site on the host‘s skin at around 6 hours post-hatching. The first instars do not feed prior to the establishment of the feeding site, giving rise to the hypothesis that gene expression profiles pre- and post-feeding site establishment may differ. Therefore, the primary aim of this thesis was to investigate and develop antigen candidates against L. cuprina larvae in sheep. - Chapter two constructed subtractive hybridisation cDNA libraries from instars at 4-6 hours development (pre-feeding) and 6-8 hours development (post-feeding). The results of this chapter showed that 40 genes from stage 6-8 hour larvae and 14 genes from stage 4-6 hour larvae were found. - Chapter three described the further validation of the expression changes of selected genes that were up-regulated in L. cuprina larvae in the stage of 6-8 hours of development post-hatch. Using quantitative RT-PCR, the zinc carboxypeptidase showed a reproducible 1.5 fold upregulation between 4-6 hours and 6-8 hours development and was chosen for recombinant protein production in an E. coli expression system. - Chapter four was to take a zinc carboxypeptidase that had first been identified as being differentially regulated by SSH and validated using qPCR, and to generate a recombinant protein with which antibodies could be raised and functionally tested. Specific anti-recombinant zinc carboxypeptidase polyclonal antibodies were used to identify the native zinc carboxypeptidase protein as a zymogen and active enzyme. There were apparent differences in zinc carboxypeptidase protein expression over the 4 to 8 hour period of first instar development and between unfed larvae and fed larvae. In artificial feeding experiments, a decrease in larval weight was observed between controls and anti-recombinant zinc carboxypeptidase groups after 20 (16% difference) and 48 hours (12% difference) of growth in vitro, but differences were not statistically significant. The findings in this thesis have therefore provided new avenues and insight to improve the potential of using a vaccination control against blowfly strike in sheep.