Fotónica integrada

From the lifespan and developmental assays, there is little evidence of this stock having a severe longevity problem with genomic loss of the miR-137 gene. The mean average ages are not significantly different (p-value > 0.05), and correlate with documented lifespan from various studies. When cultivated on a sugar yeast diet, Linford et al. (2013) found that wild types had a mean age of 50 days, and a Sun et al. (2002) reported 46-52 days being within acceptable parameters of control D. melanogaster lifespan. While investigating differences in diet effect on lifespan, Bass et al. (2007) reported that mixed gender vials of canton-S

(another widely used control strain) produced an average individual lifespan of around 49 days when on standard laboratory food mix including maize, yeast, and sugar. In a screen for miRNA mutants, no significant change in lifespan was found for males or females with miR-

137 knockout (Chen et al. 2014).

0 20 40 60 80 100 Egg 1st 2nd 3rd Adult % sur vi val Developmental stage w1118 miR137KO

104 As the flies age, there is a noticeable difference in the survival graph following ~41days old (figure 4.2). The miR-137KO strain survival rate drops much faster at this stage than the controls, though this does not appear to influence the mean age (figure 4.1, which has a t-test p-value > 0.05). The rate of survival decrease looks to be more severe in the controls at a later stage, which could then be masking the average life span across the population. This drop ~41 days could infer a maintenance role for miR-137 that helps delay or manage the onset of age-related mechanisms.

Developmentally, there appears to be a small change in the percentage of individuals which survive from imago hatching to post-eclosion adult (figure 4.5). The survival rate graph shows the expected overall trend for both, with less surviving individuals at each developmental stage. There is an overall lower yield of adults in the miR-137KO strain, with 46% survival compared to 56% in the w1118 controls where the biggest decrease difference was during the pupal stage between 3rd _{instar and adulthood. The knockout strain had a 14%}

decrease in survival compared to 4% in the controls, potentially implying a role for miR-137 through the metamorphosis period. During this stage it is known that the organism undergoes huge changes in terms of biological anatomy and neural development (Technau and

Heisenberg 1982: Singh and Singh 1999: Pauls et al. 2010), so genes responsible for CNS maintenance or proliferation would be important. This developmental lethality during pupation is contrasting to results by Chen et al. (2014) which did not find any significant difference in fly survival to adulthood.

The rate at which miR-137KO flies reach development stage is has a slight but

significant difference when compared w1118. This trend was reversed when looking at time to reach adulthood, where the controls were faster. This switch is because the knockout strain spends an increased amount of time in the 3rd instar and pupal stages, further inferring miR-

105 the t-test p-values, the significance cut-off value is changed to 0.0167. This new threshold means that the difference in the time taken for the flies to develop from egg to 3rd instar larva is no longer significant (non-corrected p-value of 0.0468). This however does not change the significance of time spent in pupation or overall time to adult fly.

The knockout strain has had the miR-137 locus missing for a long period of time and with each generation it is more likely to have background mutations, which allow the stock to cope with the miRNA LOF. These modifiers could have a masking effect causing the flies to display a subdued phenotype. As longevity is widely research in D. melanogaster, there are many genes which are linked to a change in lifespan. For example, when overexpressing antioxidative enzymes, explicitly mitochondrial superoxide dismutase mnSOD, there is an increase in mean lifespan likely because of the increased enzyme expression reducing the accumulated effects of oxidative stress in an aging individual (Sun et al.2002). TOR

signalling pathways are also widely believed to result in an increase in lifespan if reduced in adults, an effect linked to insulin signalling and dietary restriction (Stefana et al. 2017).

4.4

Conclusion

There is a noticeable change within some of the characteristics contributed to measuring drosophila health in the miR-137 deficient flies. Most apparent is a significant change within developmental survival and overall longevity particularly in the old aged flies. The severity of the phenotype resultant of losing a ~100bp non-coding gene demonstrates the biological importance of miR-137, further backed up with a high evolutionary conservation of the gene across a multitude of species. The lack of significant difference in average lifespan means that objective A hypothesis 1 must reject the alternate hypothesis and accept H0

106 Developmentally, the miR-137KO flies exhibit a developmental delay and an increased mortality rate within the 3rd instar and pupal stages when compared to controls, which means that null hypothesis from objective A hypotheses 2 and 3 can be rejected in favour of the alternate hypotheses (Chapter 1.8). The pupal stage is a natural phenomenon which puts excessive stress on the organism as it undergoes metamorphosis. It is likely vital for the correct expression of genes responsible for maintenance and proliferation of the CNS at this stage, and the loss of miR-137 could be affecting successful CNS development.

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