deletions | additions       diff --git a/While_experimental_models_of_infectious__.tex b/While_experimental_models_of_infectious__.tex  index 3d6b308..45b9c45 100644  --- a/While_experimental_models_of_infectious__.tex  +++ b/While_experimental_models_of_infectious__.tex 
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While experimental models of infectious disease historically have been developed around mammalian host species, non-vertebrate hosts continue to gain attention as an alternative approach for studying pathogenic microorganisms \cite{25699030,24392358,23517918,23271509}. In particular, studies using the fruit fly \textit{Drosophila melanogaster} \cite{20865166, 20479082} and Greater Wax Moth \textit{Galleria mellonella} larvae \cite{26155740,25172272,17400503} have significantly advanced our understanding of \textit{F. tularensis} pathogenesis. \textit{D. melanogaster} offers powerful host genetic tools but the small body size of this insect makes delivering an exact dose of bacteria difficult without specialized equipment and training. Moreover, \textit{D. melanogaster} is temperature-restricted and cannot survive at typical mammalian body temperatures, making this host of limited use for analysis of pathogens with temperature-sensitive virulence patterns such as \textit{F. tularensis} \cite{18842136}. In contrast, \textit{G. mellonella} survives well at 37$^{\circ}$C and is large enough for confident dosing with a small-gauge syringe. \textit{G. mellonella} larvae also are readily-available in large quantities from a number of commercial suppliers. However, there also are significant drawbacks to the use of \textit{G. mellonella}. In particular, pupation, the process by which the larvae metamorphesize into adults, typically occurs within a short period of time when the larvae are kept at 37$^{\circ}$C, thereby limiting the experimental window available to researchers. Additionally, immune functions vary widely before, during, and after pupation \cite{19414015, 24006915, 22937093}, bringing into question results from \textit{G. mellonella} that pupate during the experiment.   When working with \textit{G. mellonella} from commercial suppliers, we encountered tremendous shipment-to-shipment variability in the general health status of the larvae. Other groups have observed a similar trend and have addressed it by adding antibiotics to the food? ??food??  \cite{18195031} or setting a mortality threshold in control groups that, when surpassed, allows investigators to discard the results and repeat the experiment with a new batch of insects \cite{23402703,26388863,26379240}. Disatisfied with these options, we began to rear \textit{G. mellonella} in the laboratory so that we could better control their quality. We were surprised to find that, in contrast to larvae purchased from commercial sources, those reared in the lab quickly became encased in silk when transfered from the rearing vessel to a Petri plate for experimental manipulation (Figure 1). In search of an explanation for the behavioral difference between commercially-obtained \textit{G. mellonella} and those that we reared in the lab, we found several on-line forums for hobbyists that described a similar problem. These sites commonly recommended a 10 minutes freeze treatment to destroy the silk gland. Although we were unable to confirm the utility of this method, we presume that commercial suppliers use a similar method to prevent silk production in their insects. If true, this freeze treatment is problematic for pathogenesis studies for two important reasons: (1) it creates a necrotic organ with unknown immunological consequences, which may help explain the variability in health observed for commercially obtained animals; and (2) it eliminates an important part of the lepidopteran immune system \cite{19414015,17981330}. Given the inherent problems with the \textit{G. mellonella} model, we sought to identify another insect host that is simple and inexpensive to rear in the laboratory, survives well at 37$^{\circ}$C for long periods of time, and is large enough to allow inoculation with known doses of bacteria without specialized equipment.