Randy raised pointed questions about a paper in two posts in the discussion
"Re: Feed 2 ppb Imidacloprid For 2 Weeks"
http://community.lsoft.com/scripts/wa-LSOFTDONATIONS.exe?A2=ind1307&L=BEE-L&
P=R6336
or
http://tinyurl.com/l22m9e2
and
http://community.lsoft.com/scripts/wa-LSOFTDONATIONS.exe?A2=ind1307&L=BEE-L&
P=R2818
or
http://tinyurl.com/kqe2o99
I forwarded them along, and below is the point-by-point response of the
principal author of the study questioned,
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0068191
or
http://tinyurl.com/k4ktced
in which he addresses each of the questions raised.
In light of the responses from Dr. Stoger below, my view remains unchanged -
the findings are valid and conservatively stated. I will repeat - this
paper seems to be the first substantial proof of low-level, sublethal impact
of Imidacloprid on honey bees, and it deserves a very careful read.
================
The mention of “neonicotinoids” frequently provokes Pavlov-like reactions;
data that do not fit with ones “belief system” or financial interests are
reflexively rubbished.
I will however assume that Mr. Oliver’s assessment of our recent paper
(“Transient Exposure to Low Levels of Insecticide Affects Metabolic Networks
of Honeybee Larvae; PLoS ONE 8(7): e68191.”) was not motivated by such
belief systems and interests. Rather, it appears to be just a cavalier
judgment (as easily occurs on discussion boards…) when he states that:
“…this study was so poorly executed that I doubt that we can learn anything
from it”.
Nonetheless, as Mr. Oliver lists various “…questionable aspects of the
study…”, I can clarify these issues.
Mr. Oliver’s main concerns are the confounding factors that could have
influenced our data: diverse variables and environments that individual bees
from different colonies experience (genetic background, age, different
vegetation, pollen, xenobiotics, climate etc.)
For example, the test and control colonies were kept in separate apiaries.
We kept separate apiaries (three hives each) in order to reduce the risk of
honey robbing and therefore cross-contamination of control hives with the
insecticide and vice versa. The two apiaries were separated roughly by a
distance equivalent of the length of 1 ½ football fields (150 meters). Both
apiaries were located in virtually identical small wooded areas (old and
young oak, some hawthorn). Each wooded lot covers an area that approximately
10 cars occupy when they are parked next to each other on a parking lot. The
two apiaries were flanked by the same fields. We think this is an
appropriate setting for a field-like experiment.
It is impossible to establish a 100% controlled environment for experiments
taking place in the field. In fact, naturally occurring variations in
environmental conditions was a premise of our work. The reason is as
follows:
Bees in Albuquerque are exposed to different environments than bees in
Zanesville. Many parties in the USA and around the world claim that
low-levels of a given insecticide cause some kind of effect(s). If this
really is the case, then such effects should be detectable and measurable.
Importantly, such effect(s) should be detectable in the field, despite the
“noise” (different genetic background, age, different vegetation, pollen,
xenobiotics, climate etc.). That is, effects should be measurable
independent of confounding factors.
Thus, the key question of our whole endeavor was this:
“Is it possible to detect any tangible effect(s) or distinct molecular
signature(s) in larvae from hives that have been exposed to field-realistic
levels of imidacloprid?”
The answer to this question: “YES”.
We employed “RNA-Seq”, a highly sensitive method to test for differences in
gene expression levels. This method is successfully used to detect low-dose
effects of xenobiotics in humans (i.e. benzene exposure) and can outperform
clinical chemistry-based tests. Of note, there are certainly more
confounding factors in such human studies than exist in our bee study. We
are of the opinion that methods employed for clinically relevant studies in
humans should be good enough for studies in bees (beekeepers may disagree…).
Our data are sufficiently robust to state that the molecular profiles
identified in larvae from test hives can be attributed to
imidacloprid-exposure. Why do we think this? And why were we confident to
publish these findings?
Out of about 10000 genes present in the honeybee genome, what are the
chances to identify 300 genes with altered activity in larvae from
imidacloprid-exposed hives? More specifically what are the chances that many
of these 300 genes operate in distinct metabolic pathways? To put it mildly,
our analysis indicates that this is not a chance event:
We feel confident that genes involved in detoxification (cytochrome P450s)
are up-regulated and that genes involved in sugar metabolism are down
regulated by this low dose exposure because the methods that we used will
probably underestimate the significance. We had 10843 genes in the honey
bee genome with some type of annotation and 32 of those genes are cytochrome
P450 genes and we can ask what are the odds of randomly taking 107 genes
(equivalent to the number of up regulated genes that we found) and finding
that 9 of these 107 are cytochrome P450s. In a simulation, we can mix 10811
black balls with 32 red balls in a bag and ask if we randomly pick out 107
balls from the bag, how many will be red? In a million simulations, we
found more than 70% of the time we picked up not a single red ball and the
maximum number of red balls picked was 5 (18 times in 1mil simulations). In
10 million simulations, the maximum number of red balls picked was 6 (5
times in 10mil simuations). We would estimate that we would have to make
more than 10 billion picks before we would pull out 9 red balls (Note: odds
of winning the Mega Millions Jackpot in the US is 1 in 200 million). We
probably could have calculated the odds based on there being 15000 to 16000
genes in the bee genome (instead of just using the 10000 genes we could
annotate) and this would push the odds closer to 1 in 100 billion, so we
feel confident this is not random. The odds are very similar for our
findings in the sugar metabolism genes. We also feel confident that these
genes are involved in detoxification as there are more than 1000 published
articles linking cytochrome P450 genes to detoxification in a huge range of
organisms (from yeast to humans) and genes involved in sugar metabolism are
likely to be some of the most studied genes in all of biology and conserved
across all living organisms.
Yes, overall, the numbers were small – yet, the experiments yielded solid
data.
Yes, there are differences between larvae and between hives when we validate
RNA-Seq data – differences in RNA expression levels can exist even between
identical (clonal) cells. Biological data are messy.
What surprises Mr. Oliver “… about this study is that such a strong effect
was claimed for differential gene expression in larvae…”
No! The effect is not strong. The effect is very subtle (but statistically
significant as explained above). The changes in gene expression levels can
be compared to turning a dimmer switch: turning the switch just slightly up
or down causes changes in light intensities that are barely detectable for
the human eye. Similarly, our methods are sensitive enough to detect subtle
changes in gene expression levels. Subtle changes in expression of distinct
gene-networks should not be surprising – physiological adjustments are
characteristic of living organisms when they respond to altered
environments.
We also used a different diagnostic approach – lipid profiling – on a
different set of test and control larvae. The lipid profiling results are
consistent with the above mentioned gene expression analysis. The results
further support our statement that lipid-carbohydrate-mitochondrial
metabolic pathways are affected upon low-level imidacloprid exposure.
Mr. Oliver compares and contrasts our study with that of Cousin et al.
(2013) “Size Changes in Honey Bee Larvae Oenocytes Induced by Exposure to
Paraquat at Very Low Concentrations. PLoS ONE 8(5): e65693.”
The study by Cousin et al. is indeed beautiful and nicely controlled.
Exposing age-defined larvae to a pesticide in the laboratory (incubator) and
pipetting a human-prepared diet directly into the cells harboring the
developing larvae eliminates many variables. As a result, the experimental
setting by Cousin et al. is also somewhat detached from the conditions in
the real world. In contrast, and as emphasized earlier, our aim was to check
if effects are detectable in larvae collected from hives in the field that
had been exposed to low concentrations of a pesticide.
Yes, in the Cousin et al. study “…variables were held to a minimum and the
size of the oenocytes were measured with a ruler...”. In our study, we used
even more sensitive tools - RNA-Seq, quantitative RT-PCR and liquid
chromatography coupled with mass spectrometry (LC-MS) to measure the
relative abundance of RNA and lipids. That is, we measured underlying
factors that likely contribute to the morphological and physiological
changes that Cousin et al. observed in oenocytes.
Cousin et al. conclude from their study that “…Since a model molecule has
proved to be efficient at this contamination level, it seems legitimate to
think that the same phenomenon is likely to take place with other
pesticides….”
Our study indeed provides relevant data to support the statement made by
Cousin et al.
We disagree with Mr. Oliver’s conclusion “…as to whether the results of the
Derecka Imidacloprid study really tell us anything”.
What our study tells is the following:
Rather than doubting, questioning, guessing, proselytizing and speculating
whether or not very low concentrations of the neonicotinoid imidacloprid can
affect worker bee larvae in the real world, we provide very good and
measurable evidence that imidacloprid indeed causes effects.
The most important point is that we identified distinct molecular pathways
that are altered by low levels of imidacloprid.
Therefore, the least our study does is: it provides working hypotheses.
This means that ongoing and future experiments (biochemical assays,
mitochondrial respiratory efficiencies etc) will allow us - and others - to
dissect and assess the impact of imidacloprid and other pesticides on these
distinct molecular pathways, in minute detail, under very controlled
laboratory conditions.
Such studies will either confirm – or disprove - the results of our
field-realistic experiment.
It is a slow and careful process. That is how science works. Doesn’t this
tell us something?
Reinhard Stöger
( School of Biosciences
University of Nottingham
http://www.stoegerlab.com
[log in to unmask] )
***********************************************
The BEE-L mailing list is powered by L-Soft's renowned
LISTSERV(R) list management software. For more information, go to:
http://www.lsoft.com/LISTSERV-powered.html
|
