Expression patterns of immune associated genes in Euoniticellus intermedius and characterization of the embryonic cell line
Date
2013-02-01
Authors
Alaouna, Mohamed
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Abstract
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.