deletions | additions       diff --git a/While_experimental_models_of_infectious__.tex b/While_experimental_models_of_infectious__.tex  index 87b2e4d..bfb1dbe 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, this insect host also requires investigators to accept certain limitations and tradeoffs. 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. Immune function can vary widely before, during, and after pupation \cite{19414015,24006915,22937093}, thus making it difficult to standardize host immunological status in \textit{G. mellonella}. When working with \textit{G. mellonella} from commercial suppliers, we encountered tremendous shipment-to-shipment variability in experimental outcome, presumably due to differences in the general health status of the larvae. Other groups have observed similar trends and have addressed this concern by supplementing the insect meal with antibiotics \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 (\textbf{Figure 1A and 1B}). Others have reported a similar cocoon in laboratory-reared insects and recommend that larvae be mechanically removed from the structure prior to infection \cite{23271509}. However, we found it difficult to perform this procedure without causing physical trauma to the larvae. Moreover, larvae would generally spin a new cocoon within a matter of hours, making it necessary to perform this manipulation each day of the study in order to observe the larvae for mortality. Thus when using laboratory-reared \textit{G. mellonella} larvae, the throughput advantage of an insect model is compromised by this cumbersome procedure. In search of an explanation for this behavioral difference between commercially-obtained \textit{G. mellonella} and those reared in the lab, we found two on-line forums for hobbyists that described the use of a brief freeze treatment to destroy the silk gland /cite{BestBetWormKits,OpenWormFarm}. /cite{BestBetWormKits,OpenBugFarm}.  Although we were unable to confirm that commercial suppliers of \textit{G. mellonella} use this particular method, it is clear that they treat their insects in some way that prevents silk production. While this does aid in handling and improves experimental throughput, it is problematic for pathogenesis studies because the immunological consequences of a necrotic silk gland are unknown. On the one hand, necrosis could activate a generalized immune response \cite{24746817}. Alternatively, the silk gland is a recognized component of the lepidopteran immune system \cite{19414015,17981330} and its loss could hamper functional immune responses. Given these 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.