deletions | additions       diff --git a/section_Discussion_textit_F_tularensis__.tex b/section_Discussion_textit_F_tularensis__.tex  index 771ceb4..2abc2f5 100644  --- a/section_Discussion_textit_F_tularensis__.tex  +++ b/section_Discussion_textit_F_tularensis__.tex 
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Other insect species such as \textit{D. melanogaster} and \textit{G. mellonella} have been used extensively as experimental hosts for mammalian pathogens. However, there are significant trade-offs associated with each of those insects that we wished to avoid. Therefore, we sought to identify an experimental host that is commercially-available, simple to rear in the laboratory, thrives at mammalian temperatures for long periods of time, is large enough to deliver consistent bacterial inoculations using standard needle-syringe combinations, and is vigorous enough to withstand multiple injections of bacteria and/or antibiotics. We found that the \textit{B. dubia} OS cockroach satisfied all of these requirements.   Like wax moth larvae, OS cockroaches are commercially-available from a large number of suppliers that produce them for the pet industry (primarily used as food for captive reptiles). However, whereas wax moths are relatively difficult to rear in captivity, maintenance of an OS cockroach breeding colony in the laboratory is simple and straightforward. Compared to other species, OS cockroaches are relatively docile and easy to handle. Given adequate access to water, OS cockroaches can be maintained at temperatures above 37°C (up to 40°C in our studies) indefinitely. OS cockroaches undergo incomplete metamorphosis, with each developmental stage (or instar) lasting between 20 and 45 days. In total, it takes approximately 6 months for OS cockroaches to reach adulthood. The juvenile cockroaches used in this study were infected during the 6th instar (next to last), leaving between 30 and 60 days of experimental observation before they would have molted into the adult stage. In contrast, we often observed that significant percentages (>25\%, data not shown) of wax moth larvae pupated during a 7 day experiment. Importantly, deaths of OS cockroaches were are  extremely rare in our PBS-only or no-injection control groups (none occurred during the experiments presented here)  and the LD_{50} of a non-pathogenic bacteria, bacterium,  \textit{Escherichia coli} DH5$\alpha$, was determined to be nearly 10^{7} CFU (\textbf{Table 1}), indicating that OS cockroaches are capable of mounting protective immune responses to non-pathogenic microorganisms. However, intrahemoceol injection of \textit{F. tularensis} LVS resulted in dose-dependent lethality (\textbf{Figure 1}) with an estimated LD_{50} of 1.7 to 3.5 x 10^4 CFU (with overlapping confidence intervals) for two different laboratory stocks (\textbf{Table 1}). Thus \textit{F. tularensis} LVS is able to evade OS immunity and cause disease in this experimental host. Temperature is known to regulate expression of \textit{F. tularensis} virulence factors \cite{18842136}. One of the advantages of insect models, in comparison with mammalian models, mammals,  is the ability to experimentally manipulate the temperature at which the host-pathogen interactions occur. When we varied the temperature of incubation following cockroach infection, we observed that as the temperature decreased, so did \textit{F. tularensis} LVS lethality (\textbf{Figure 2, Table 1}). Since \textit{F. tularensis} can be spread by environmental arthropods \cite{24057273,21529386,20482589,18950590,22530023,21612530,20885922, 20810833, 21845949}, temperature may provide an important environmental cue that allows \textit{F. tularensis} to dampen virulence pathways that would otherwise kill these vectors before they have an opportunity to transmit the bacterium to another host mammal. The OS cockroach system may provide investigators a new tool for analysis of the important but understudied environmental stage of the \textit{F. tularensis} lifecycle. Host immune function is not static, rather it often varies dramatically across developmental stages \cite{25730277}. We therefore 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 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. In comparison, adult males showed enhanced susceptibility, 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.