Expression patterns of immune associated genes in Euoniticellus intermedius and characterization of the embryonic cell line

• Main Author: Alaouna, Mohamed
• Format: Others
• Language: en
• Published: 2013
• Subjects: Dung beetles.; Embryos - Physiology.; Cell lines.; Genes.
• Online Access: http://hdl.handle.net/10539/12370

As bacteria are becoming resistant to conventional antibiotics, researchers are looking for new ways to combat microbial infection. We have begun to adopt genetic and functional genomic approaches to define the molecular determinants of pathogen resistance in the dung beetle, Euoniticellus intermedius. This dung beetle survives microbe-rich environments such as dung. This ability makes it a potential model for the study of infectious agents and ecological damage. To date, E. intermedius has not been studied at the molecular level. In this study, a range of complimentary analytical techniques were used to characterize the E. intermedius embryonic cell line established in our laboratory. These techniques characterize morphology, growth characteristics, karyotype, isoenzyme patterns and embryonic development. Complete characterization of the E. intermedius cell line is essential for the cell banks and for the regulatory requirements in biopharmaceutical production. This study followed gene sequences and their comparisons for both adult and cell line to confirm that the E. intermedius (EISA08) cell line is originated from the embryonic E. intermedius dung beetle. cDNA was synthesized from mRNA isolated from E. intermedius adult beetles and cell line (EISA08) was sequenced using GS (FLX) technology by a commercial facility, Inqaba Biotechnical Industries (Pty) Ltd, South Africa. In addition to characterization of the cell line, two genes, namely hopscotch and ribosomal protein S9 (RpS9), were selected from the Flylab genome data base. The E. intermedius database is a web-based system for the genome and transcriptome of the dung beetle to evaluate the immune system of the dung beetle (http://Flylab.wits.ac.za/). hopscotch was selected because it is believed to be involved in the JAK-STAT signalling pathway for anti-viral response, embryonic development and cell growth. Rsp9 was chosen as a loading control because it is expected to be a housekeeping gene. The conserved molecular signalling pathway JAK-STAT is used by E. intermedius (as in other insects and humans) for immune defence and early embryonic development. The project followed hopscotch and Rsp9 gene expression in all the E. intermedius life cycle developing stages; adult, pupae, larvae, embryo, and cell line cell growth, life cycle developing stages and embryonic development has was monitored. E. intermedius embryonic development is described as short germ-band. E. intermedius embryogenesis is regarded as basal and is observed in most arthropods. The study revealed that E. intermedius hopscotch is over expressed in the early developing stages, embryo, larvae, and pupae and in the newly established cell line EISA08. The results from this study lead to the suggestion that E. intermedius JAK-STAT pathway is activated early and has an important role in embryonic development, cell proliferation and immune defence. Studies of E. intermedius could provide more insight into the properties and evolution of innate immunity and embryonic development.