Randy, I read directly from the paper, and I just don't see the quote you offered.  I am confused as to what you are reading????

 Here is the section on the queenless/non-laying part of the study, in the journal "Science" by the AAAS....and I copy and paste directly:

"We quantified hygienic behavior and the presence
of a laying queen in treated colonies and
control colonies over the course of our 12-week
experiment. We hypothesized that phenotypic
effects of exposure—if they exist—should manifest
as a function of exposure time (20) (i.e.,
significant treatment by time interactions). We
detected a significant treatment by time interaction
on hygienic behavior [F(1,23) = 14.86,
P = 0.001, N = 34]; the average hygienic behavior
of clothianidin-treated colonies decreased
over time but that of control colonies did not
(Fig. 2B). We observed a similar pattern in the
field in 2014, where exposed colonies near
corn had significantly lower hygienic behavior
relative to unexposed colonies at the end of the
season (Fig. 2C) [F(1,48) = 6.42, P = 0.015, N =
50]. Our study is similar to a recent study that
found an association between chronic exposure
to imidacloprid and reduced hygienic behavior
(21). Our findings indicate that NNIs
impair the honey bee’s social immune system.


We also observed a significant treatment by
time interaction on queenlessness [generalized
linear mixed model (GLMM), z = 2.242, P = 0.025,
N = 54] ...

...whereby the presence of a laying queen
declined over time in the clothianidin-treated
group (Fig. 2D). Strong colonies, like many of our
controls, typically become queenless in midsummer
during swarming season, but then rapidly rear
and sustain a replacement queen. However, that
pattern of queen loss in treated colonies peaked
well after Ontario’s swarming period, and most
treated colonies were not able to rear replacement
queens by the end of our experiment. Our
finding is consistent with a recent study (22) that
documented NNI effects on queen mortality and
reproductive physiology. The association between
chronic clothianidin exposure and queenlessness
is expected to have major consequences on colony
fitness, because colonies that are unable to
rear replacement queens eventually perish (23)."

____

It seems clear to me that there were 54 total colonies in the queenless/non-laying trials...approximately 25 each in the control and test groups (one queen was accidentally squished).  (text separated from the full excerpt, above).

Christina
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