deletions | additions       diff --git a/section_Discussion_textit_F_tularensis__.tex b/section_Discussion_textit_F_tularensis__.tex  index 8d0ec56..e9be284 100644  --- a/section_Discussion_textit_F_tularensis__.tex  +++ b/section_Discussion_textit_F_tularensis__.tex 
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Temperature is known to regulate expression of \textit{F. tularensis} virulence factors \cite{18842136}. One of the advantages of insect systems in comparison with mammalian hosts is the ability to experimentally manipulate the temperature at which the host-pathogen interaction occurs. When we varied the temperature of incubation, we saw that as temperature decreased, so did \textit{F. tularensis} LVS virulence (\textbf{Figure 2, Table 1}). This observation raises the intriguing possibility to collect gene expression data from \textit{F. tularensis} LVS growing at different temperatures \textit{in vivo} and comparing the resulting patterns with cells grown at different temperatures \textit{in vitro}.   Host immune function is not static, rather it often varies dramatically across developmental stages \cite{25730277}. We therefor sought to determine if OS cockroach susceptibility to \textit{F. tularensis} LVS varied by developmental stage. We determined the killing kinetics and LD_{50}s of \textit{F. tularensis} LVS against late-stage juvenile, adult female, and adult male OS cockroaches. The susceptibility pattern of juveniles (which we used for all other experiments reported here) and adult females were highly similar. But interestingly, adult males showed enhanced susceptibility in comparison, with a shorter median time to death (\textbf{Figure 4}) and a lower LD_{50} (\textbf{Table 1}). The reason for this difference is currently unknown and future experiments aimed at uncovering the mechanistic differences in immune responses between these groups could identify important anti-\textit{F. tularensis} host pathways. In order to begin to define the genetic requirements for virulence in this model, we examined the virulence of a small panel of \textit{F. tularensis} LVS mutants that are attenuated in other model systems (\textbf{Table 1}). All four of the mutants examined showed significant attenuation in OS cockroaches, which correlates with previous findings in mice and chick embryos. This finding supports the idea that \textit{F. tularensis}} tularensis}  uses a similar virulence program to evade immunity and cause disease across extremely diverse host organisms. Since \textit{F. tularensis} is considered a facultative intracellular pathogen, we sought to determine the proportion of bacteria that were located in intracellular and extracellular compartments throughout infection of OS cockroaches. As seen in \textbf{Figure 5}, intracellular bacteria can be recovered as early as six hours post injection. The intracellular population continues to grow throughout the infection process, as does the total bacterial population. Initially, we were surprised that the majority of the bacterial population at each time point is located in an extracelllular environment. However, this is similar to what others observed for \textit{F. novicida} in \textit{D. melanogaster} \cite{20865166}. Since gentamicin rescued OS cockraoches from lethality when injected into the hemocoel (\textbf{Figure 7}), the extracellular population is likely critical to the outcome of infection as has been recently suggested \cite{22795971}. While the intracellular phase of \textit{F. tularensis} pathogenesis is well appreciated, our findings suggest that the OS cockroach may be a useful model for elucidating the mechanisms by which \textit{F. tularensis} survives, grows, and moves within the extracellular environment.