Supplementary file
Methods
Larval rearing
Wild type honeybees (U.S. stocks) were maintained in the apiary of Arizona
State University, Tempe USA. Two wild-type queens were caged on a wax comb
for 24 h in order to control the time of egg-laying. Newly hatched (12-18 h old)
larvae were subjected to the in vitro feeding protocol, and larvae from both
queens were assigned randomly to the treatments throughout the study. Larvae
were reared in an incubator at 34oC. They were fed twice daily for two
consecutive days with either irs dsRNA (to elicit RNAi) or gfp dsRNA (control).
Thereafter, larvae were maintained on diet without dsRNA until pupation.
Individuals were collected as larvae for mRNA and protein analysis at 72 h
after the first feeding and for protein analysis, additionally, at 96 and 120 h after
the first feeding. Samples were collected for mRNA analysis in order to verify the
success of the knockdown procedure (one time point only), while proteomics
samples were obtained at three different time points to get detailed data on the
molecular response to irs knockdown.
During the study, daily larval mortality was about 5 %, while 50 % of
pupae did not survive until adulthood. Mortality was independent of treatment,
indicating that although honeybee in vitro rearing protocols can be improved,
they are not sensitive to different dsRNA treatments.
Preparation of dsRNA, in vitro feeding regime for larvae
PCR primers fused with T7 promoter sequence (underlined) 5’TAATACGACTCACTATAGGGCGAGCGAACCGGTAGTCGTAAAG-3’ and 5’TAATACGACTCACTATAGGGCGAGCAGTGATCAAACGTGGCTT-3’ (forward
and reverse, respectively) were used for irs dsRNA template synthesis. PCR on
a cloned fragment of the honeybee irs gene (GB11037) resulted in a 583 bp
product (Wang et al. 2010). dsRNA for the green fluorescent protein (GFP) was
synthesized as previously described (Amdam et al. 2006). PCR products were
excised and purified from 1 % low melting agarose gels using Qiaquick Gel
Extraction Kit (Qiagen). Following the manufacturer’s protocol, the AmpliScribe
T7 transcription kit (Epicentre Biotechnologies) was used for production of
dsRNA. Purification of dsRNA was achieved by phenol:chloroform extraction.
Size and purity of the final products were verified by electrophoresis (1 %
agarose gel).
For irs and gfp dsRNA treatment, 250 µg dsRNA was mixed thoroughly
with 1 ml of VS queen diet before the food mixtures were given to the separate
larval groups (Patel et al. 2007).
RNA isolation and qPCR for knockdown verification
Total RNA was isolated using Trizol reagent (Invitrogen) following the procedure
provided by the manufacturer. Thereafter, total RNA was treated with DNaseI
(Ambion) following standard instructions. Following quantification, total RNA was
diluted to 25 ng/µl concentration and 2.0 µl was used as template for quantitative
real-time PCR (qRT-PCR). For knockdown verifications, qRT-PCR was
performed in triplicate using an ABI Prism 7500 (Applied Biosystems), and
analyzed with the comparative CT method (Schmittgen & Livak 2008) with the
highest expression value as reference for relative quantification and actin
(XM_623378) and tubulin (XM_392313) as housekeeper genes. For irs and actin
primers as well as PCR conditions see (Wang et al. 2010).
For tubulin; 5’ GTA CCC GAG CTA ACC CAA CA 3’ and 5’ GCT CGT
CGA CCT CTT TCA TC 3’ were forward and reverse primers, respectively.
Protein extraction
Individual larvae were homogenized in 150 µl of protein extraction buffer (50 mM
tris pH 8.5, 2 % SDS, 5 % beta-mercaptoethanol, 0.15 M NaCl, 30 % glycerol).
Samples were then vortexed vigorously, boiled at 95 °C for 5 min, vortexed
again, and centrifuged for 2 min at 10,000 rcf. The supernatant was subjected to
methanol:chloroform precipitation as described in (Wessel & Flugge 1984).
Protein digestion
Proteins were redissolved in 50 µl of buffer containing 50 mM tris pH 8.5, 6 M
urea, 2 M thiourea, 0.15 M NaCl, 1 mM CaCl2. Consecutively, 150 µl of the same
buffer without urea and thiourea was added, samples were spun at 10,000 rcf for
2 min, and the supernatant was used for analysis. Bradford assay was used to
determine protein concentration (Bradford 1976) and 40 µg/sample was digested
over night at 30 °C with 1 µg of trypsin in digestion buffer (50 mM tris pH 8.5,
0.15 M NaCl, 1 mM CaCl2). Peptide desalting was performed the next day as
described (Rappsilber et al. 2003; Wolschin & Amdam 2007).
LC-MS/MS Analysis
Dried peptides (from 10 μg protein) were dissolved in 5 % acetonitrile, 2 % TFA
and used in a non-targeted LC-MS/MS analysis. Peptides were separated on a
picofrit column (75 µm ID, New objective, Woburn, USA) using a 105 min
gradient ranging from 95 % A (0.1 % formic acid, 99.9 % H2O) to 80 % B (0.1 %
formic acid, 99.9 % acetonitrile) followed by a 15 min equilibration step. Peptides
were eluted from the reversed phase µLC column directly into an LTQ mass
spectrometer (Thermo, San Diego, USA) and the following settings were used:
isolation window: 3 m/z, collision energy: 35, and activation time: 30 ms. MS 2
spectra were recorded for the five most abundant peaks in each MS survey
spectrum. Using the open source search tool OMSSA (version 2.0.0) (Geer et al.
2004) the spectra were matched against an A. mellifera sequence database
retrieved from NCBI (http://www.ncbi.nlm.nih.gov) containing additional trypsin
and keratin sequences. The following filtering criteria were used: 0.8 Da fragment
tolerance, 0.8 Da precursor tolerance, maximum of 2 missed cleavages, only
tryptic sequences allowed, initially 10 possible peptide hits per spectrum reported
then filtered to 1 peptide hit per spectrum, variable modifications: methionine
oxidation, deamidation of N and Q. Acceptance threshold for peptides: e ≤ 0.1. At
least two peptides per protein were required for protein identification.
Quantification for proteins required a spectral count ≥ 3 in 3 or more of the 4
biological replicates from one treatment group. Missing spectral count values
were replaced by 0.1 and individual spectral counts were divided by the total
spectral count to correct for total protein abundance.
Statistics
Normal Q-Q plots and Shapiro Wilk tests were used to test for normality.
Developmental time, ovariole count, irs expression using tubulin as a
housekeeper, and protein values displayed non-normal data distributions. For
reasons of consistency, we opted for a non-parametric analysis with rank sum
tests for all characters in the main manuscript. Fresh weight and irs expression
using actin as a housekeeper did display a normal distribution. F tests revealed
an inequality of variances for fresh weight (F = 0.276, num df = 19, denom df =
19, p-value<7.4e-3) but not for irs expression using actin as a housekeeper (F =
0.6605, num df =11, denom df = 11, p = 0.5029). Welch (fresh weight) and
ordinary (irs expression using actin as a housekeeper) two-sample t-tests
corroborated the significance of the non-parametric results for these characters
(fresh weight (t=-21.177, df=28.747, p<2.2e-16, n=20), irs expression (t=-8.9119,
df=22, p<9.4e-09, n=12)). Corroboration for the irs expression results came from
two separate analyses with n=6 each using tubulin as the housekeeper (nonparametric rank sum tests, p<9.0e-03 and p<2.2e-03).
Statistical analysis on proteomic data was conducted as described
previously using a non-parametric rank sum test and a correction for type 1 error
inflation (Wolschin & Amdam 2007). For hierarchical clustering analysis protein
values were z-transformed and subjected to HCA based on Pearson correlation
and full linkage clustering.
All statistical analyses were conducted using R 2.10.1 or Matlab R 2008b.
Acknowledgements
We thank Zhengping Yi and Lawrence Mandarino for providing access to the MS
facilities, Kim M. Fondrk and Osman Kaftanoglu for apicultural support, Kevin
Flores and Brenda Rascón for comments on the manuscript and Adrian Smith for
help with illustrations.
References
Amdam, G. V., Norberg, K., Page, R. E., Jr., Erber, J. & Scheiner, R. 2006
Downregulation of vitellogenin gene activity increases the gustatory
responsiveness of honey bee workers (apis mellifera). Behav Brain Res.
Bradford, M. M. 1976 A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72, 248-54.
Geer, L. Y., Markey, S. P., Kowalak, J. A., Wagner, L., Xu, M., Maynard, D. M.,
Yang, X., Shi, W. & Bryant, S. H. 2004 Open mass spectrometry search
algorithm. J Proteome Res 3, 958-64.
Patel, A., Fondrk, M. K., Kaftanoglu, O., Emore, C., Hunt, G., Frederick, K. &
Amdam, G. V. 2007 The making of a queen: Tor pathway is a key player
in diphenic caste development. PLoS ONE 2, e509.
Rappsilber, J., Ishihama, Y. & Mann, M. 2003 Stop and go extraction tips for
matrix-assisted laser desorption/ionization, nanoelectrospray, and lc/ms
sample pretreatment in proteomics. Analytical Chemistry 75, 663-670.
Schmittgen, T. D. & Livak, K. J. 2008 Analyzing real-time pcr data by the
comparative c(t) method. Nature protocols 3, 1101-8.
Wang, Y., Mutti, N. S., Ihle, K. E., Siegel, A., Dolezal, A. G., Kaftanoglu, O. &
Amdam, G. V. 2010 Down-regulation of honey bee irs gene biases
behavior toward food rich in protein. PLoS Genet 6, e1000896.
Wessel, D. & Flugge, U. I. 1984 A method for the quantitative recovery of protein
in dilute solution in the presence of detergents and lipids. Anal Biochem
138, 141-3.
Wolschin, F. & Amdam, G. 2007 Plasticity and robustness of protein patterns
during reversible development in the honey bee (apis mellifera). Anal
Bioanal Chem 389, 1095-100.