>The move to starting with newly emerged bees was a significant switch made in the 90s.  Before that, it was not part of the published protocols, presumably because most of us 50 years ago were well familiar with the work of researchers such as DeGroot.

It is noted that the following study is included in the analysis of bee longevity. How might the control results in this study have differed if the protocols prior to the 90's had been employed?

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013181

'Cage trials of 1–3 day old newly-emerged bees demonstrated increased mortality in the experimental group fed both N. ceranae and IIV-6 in comparison with the control group (P = 0.0001) and bees fed only N. ceranae (P = 0.04) or only IIV-6 (P = 0.04, Figure 3). As the actual infectious dose of N. ceranae or IIV that occurs in the field is currently unknown, we chose to utilize a relatively low infectious dose for both pathogens in our experiments. As is common in cage bee trials, mortality was observed in the control groups in all four biological replicates. To confirm that the controls likely died from a non-infectious cause, deceased bees from all treatment groups were further screened with MSP. The controls did not have any detectable IIVs, but did show some evidence of Nosema, which was not apparent from PCR analysis of the same samples.'

'Following emergence from brood frames in an incubator, 1–3 day old bees were placed into sterile cardboard cups in a plant growth chamber with controlled temperature (28°C, relative humidity, and light). Using a 10 µl pipette, each bee was inoculated by feeding it a total of 2 µl in sugar water containing one of four treatments. Only bees that ingested the entire inoculum were used. The following treatments were given: 1) Controls = Sugar Water/PBS 1∶1, 2) Nosema ceranae – 2 µl containing 50,000 spores, 3) Virus – 2 µl of 0.25 mg/ml IIV-6 suspension in PBS/Sugar Water 1∶1 (0.25 ug Virus), and 4) N. ceranae + Virus - 2 µl containing 50,000 spores+0.25 µg of virus in PBS/Sugar Water 1∶1.'

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