Samples are often frozen for preservation until needed for use. It has been a common practice to store fresh dairy manure in the freezer until needed for fly development studies. However, conflicting data have suggested that freezer temperature and duration of manure may impact fly development studies, and it is likely due to the change in microbial comminutes due to the freezer conditions. In this study manure storage conditions were assessed to ascertain how temperatures impact stable fly, Stomoxys calcitrans L., survival to pupation and determine which bacterial populations impacted fly development using massively-parallel sequencing and 16S metagenomic analysis. Stable fly survival to pupation was greater in manure that was stored warm (27˚C) or frozen (-20˚C or -80˚C) for 24 days as compared to fresh manure samples. Refrigeration (4˚C) of the manure for 24 days did not affect fly development and slightly decreased the pupal weights. Over 80 bacterial families were detected by sequencing allowing for a more thorough assessment of changes in bacterial populations. Only minor shifts were observed in bacterial family composition in the manure when refrigerated or frozen for 24 days, but significant population changes were observed when the manure was incubated for 24 days at 27˚C. Since it is the temperature and incubation time that yielded the greatest pupation rate, it is hypothesized that the manure microbial community impacts the growth and development of stable flies. This study has determined suggested freezer conditions for the best storage of manure samples to maintain bacterial diversity and retain the closest bacterial populations to freshly collected manure. Although untouched, aged (20 days) manure is best to use to assess fly development, it is not always feasible in laboratory experimentations. This study demonstrates the importance of preservation techniques on manure samples, which could also confer to storage of other biological specimens that contain resident microbes.
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