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\section{Discussion}
\textit{F. tularensis} is a highly-virulent zoonotic pathogen that causes significant morbidity and mortality globally. To facilitate future advances in our understanding of this important bacterium, we sought to develop an improved insect host system that eliminates undesirable biological and logistical trade-offs that accompany other popular host species such as \textit{D. melanogaster} and \textit{G. mellonella}. While insects lack adaptive immune functions, their innate immune systems share similar regulation and effector mechanisms with mammalian innate immune systems \cite{25699030,24392358,23517918,23271509}. Because of this, insects can provide investigators with a host-pathogen interaction system capable of high-throughput that would be either financially or ethically unacceptable in mammals. Importantly, insects also provide scientists at institutions that lack access to mammalian housing facilities an alternative means by which to assess \textit{in vivo} host-pathogen interactions. Finally, insects and other arthropods can be important environmental reservoirs and vectors for numerous zoonotic pathogens, including \textit{F. tularensis}. Thus, insect host systems also aid in illuminating how these microorganisms evade arthropod immune systems during this part of their lifecycle without the necessity of rearing sanguinivorous arthropods in the lab. Here, we sought to identify an experimental host for \textit{F. tularensis} that is (1) readily-available, (2) simple to rear in the laboratory, (3) tolerant of mammalian body temperatures, (4) large enough in size to allow consistent delivery of bacterial inoculations using standard needle-syringe combinations, (5) long-lived with low background mortality, and (6) hardy 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 fruit flies and wax moth larvae, OS cockroaches are readily available. Several strain repositories supply the scientific community with \textit{D. melanogaster} seed stocks and in-house rearing is easily accomplished using well-established protocols \cite{21709308, 17921418}. Unfortunately, \textit{D. melanogaster} does not tolerate incubation at mammalian body temperatures and quantitative infection requires highly specialized equipment. Thus, we did not consider \textit{D. melanogaster} for our studies. In contrast, both \textit{G. mellonella} and \texit{B. dubia} survive at mammalian body temperatures. Critically, both of these hosts are able to mount effective immune responses against non-pathogenic microorganisms while infection by \textit{F. tularensis} results in dose-dependent mortality (\cite{17400503}; \textbf{Figure 1} and \textbf{Table 1}). Thus, \textit{F. tularensis} LVS is able to evade active immune functions and establish a lethal infection in both of these experimental hosts.
Unfortunately, suppliers of wax worms and OS cockroaches are generally focused on non-scientific audiences. Wax worms are a popular choice for fishing bait and both wax worms and OS cockroaches are used as food for captive reptiles. In these markets, easy handling by consumers is critically important. This has led commercial suppliers to inactivate the wax worm silk gland by some unknown procedure (possibly using a freeze treatment as described by hobbyists in online forums \cite{OpenBugFarm, BestBetWormKits}). The physiological and immunological impacts of silk gland dysfunction are unknown, but it is clear that this organ is an import part of the antibacterial response in \textit{G. mellonella} \cite{19414015}. To avoid this serious complication for pathogenesis studies, wax worms can be reared in the laboratory \cite{23271509} but we found it difficult to consistently do so without microbial contamination, a factor that might contribute to unpredictable rates of background mortality in our and others' studies \cite{23402703,26388863,26379240, 18195031}}. In contrast, maintenance of a cockroach breeding colony in the laboratory is simple and straightforward \cite{7966174, 14272467}. Compared to other cockroach species, OS cockroaches are docile and easy to handle. They are relatively slow, remain immobile when placed on their back, do not climb vertical glass or plastic surfaces, and they do not fly. It is unclear if these are characteristics of wild OS cockroaches or if they have been selected during captive breeding. Importantly, a minimal amount of maintenance is required to prevent microbial contamination (and odor) in OS cockroach breeding colonies. As a result, we rarely observe mortality in uninfected control groups of OS cockroaches (\textbf{Figure 1} and \textbf{Table 2}).
Both wax worms and cockroaches can be infected with known doses of microorganisms using needle and syringe combinations \cite{23271509, 22892068}. But unlike wax worms, OS cockroaches can also be infected using sharpened gel-loading pipette tips, which increases the safety and decreases the cost associated with pathogenesis studies in this host. After infection, wax worms can survive at least one subsequent administration of antibiotics \cite{17400503, 23402703}. Here, we established that OS cockroaches can tolerate at least 3 injections following infection without an increase in background mortality (\textit{Table 2}). Importantly, the experimental window available to investigators is substantially different between wax worms and OS cockroaches. 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 adulthood. In contrast, we often observed that considerable fractions (>25\% in some cases, data not shown) of wax moth larvae pupated during a typical 7 day survival experiment. This is troublesome for studies of host-pathogen interactions because wax worm immune responses are known to vary throughout the period leading up to pupation \cite{17198709}. Thus, small differences in individual age may impact the immune status of wax worms. While we did observe that adult male cockroaches were more susceptible to \textit{F. tularensis} (\textbf{Figure 5}), the similarity between mortality in juvenile and adult female cockroaches indicates that small differences in age are unlikely to effect experimental results in this system. Collectively, these differences demonstrate that OS cockroaches offer important improvements compared to wax worms for studies of microbial pathogenesis. Thus, we went on to characterize several relevant factors in this model, including temperature, intracellular versus extracellular growth, and the usefulness of the model for pharmacological screening.
