Nathan edited section_Discussion_textit_F_tularensis__.tex  over 8 years ago

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Other insect species such as the fruit fly, \textit{D. melanogaster}, and larvae of the greater wax moth, \textit{G. mellonella}, have been used extensively as experimental hosts for mammalian pathogens. However, there are significant trade-offs associated with each that we wanted to avoid. Therefore, we sought out to identify an experimental host that is available commercially but is also simple to rear in the laboratory, that thrives at mammalian temperatures for long periods of time, that is large enough for quantitative inoculations using standard needle-syringe combinations, and that is vigorous enough to withstand multiple injections, either of bacteria or antibiotics. We found that the OS cockroach, \textit{B. dubia}, 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 as food for captive reptiles). But unlike wax moths that 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. Most of 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 portions (>25\%, not shown) of wax moth larvae would pupate during a 7 day experiment. Importantly, death in our PBS-only or no-injection control groups are extremely rare and the LD_{50} of \textit{Escherichia coli} DH5$\alpha$ is greater than 10^{6} CFU (Figure 1 (\textbf{Figure 1}  and Table 1), \textbf{Table 1}),  indicating that OS cockroaches mount an effective immune response to non-pathogenic microorganisms. Intrahemoceol injection of \textit{F. tularensis} LVS resulted in dose-dependent lethality (Figure 1) with an estimated LD_50 LD_{50}  of 1.7 and 3.5 x 10^4 CFU (with overlapping confidence intervals) for two different laboratory stocks (Table 1). (\textbf{Table 1}).  Interestingly, this LD_50 LD_{50}  is similar to that observed for \textit{F. philomiragia}, an environmental isolate that can cause opportunistic infection \cite{26400786}, but that is generally considered avirulent \cite{22628499}. This similarity in virulence is not surprising or concerning given that \textit{F. tularensis} LVS is a laboratory derived strain known to be less virulent than wild-type \textit{F. tularensis} isolates \cite{19506014}. In the future, it will be useful and interesting to evaluate the pathogenesis of fully virulent isolates in this model. 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 (Figure (\textbf{Figure  2, Table 1). 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_50s 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 (Figure 4) (\textbf{Figure 4})  and a  lower LD_50 (Table 1). 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. Mutants. 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}} uses a similar virulence program to evade immunity and cause disease across extremely diverse host organisms.  In vivo growth. 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.  Antibiotic rescue.