- Background: Queen failure is a persistent problem in beekeeping operations, but in the absence of overt.
- Previously, we suggested candidate protein markers indicating heat-shock in queens.
- Here, we further investigate these heat-shock markers and test new stressors to identify additional candidate protein markers..
- Results: We found that heat-shocking queens for upwards of 1 h at 40 °C was necessary to induce significant changes in the two strongest candidate heat-shock markers, and that relative humidity significantly influenced the degree of activation.
- In blind heat-shock experiments, we tested the efficiency of these markers at assigning queens to their respective treatment groups and found that one marker was sufficient to correctly assign queens 75% of the time.
- Queens that failed in the field had higher expression of both heat-shock and pesticide protein markers, but not cold-shock markers..
- For example, previous research has found that heat-shock, cold-shock, and pesticide exposure all decrease the via- bility of sperm stored in queen spermathecae [4, 5], which would theoretically lead to the same colony-level symptoms: inconsistent brood patterns, poor population.
- If bee- keepers cannot diagnose the root cause of queen failure, they are less equipped to identify the source of stress and are unable to make evidence-based management de- cisions to mitigate or eliminate the stress in the future..
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- Some commer- cial queen suppliers include temperature loggers in their shipments as a quality control measure, but these are typically only large-order, international deliveries, and it is up to the discretion of the supplier.
- found that the mean haz- ard quotient of the cumulative pesticide ‘exposome’ in wax was 2155 [18].
- Those colonies experiencing ‘queen events’ (i.e., the colony was queenless, had queen cells, or a virgin queen) had an average wax HQ of ~ 3500, while queenright colonies had average wax HQ of ~ 1700.
- Finally, we conducted a blind heat-shock trial to test the efficacy of using the top two candidate heat-shock biomarkers to assign queens to their relevant treatment groups.
- To expand our stressor scope, here we also compared cold-shocked queens to controls and in- vestigated the proposed heat-shock markers in more de- tail through an exposure time-course.
- Of the 2094 quantified proteins, 21 were differentially expressed be- tween cold-shocked (2 h at 4 °C) and control queens (n = 5 and 7, respectively), and only three of those were upregulated in the cold-shock group (Fig.
- threonylcarbamoyltransferase) as candidate cold-shock biomarkers..
- The two most significant candidate heat-shock bio- markers we identified in our previous experiment [7].
- were the small heat-shock proteins XP_395659.1 and XP and here we confirm that these two proteins are consistently upregulated across queens from independent origins.
- This HQ is considerably lower than the mean wax HQ in commercial colonies (2155), which was intentional because we expect the transfer of toxins from wax to the queen to be inefficient and we also sought to examine the queen biomarkers resulting from sublethal pesticide exposure.
- The imidacloprid dose is also an equivalent amount to the lowest dose Chaimanee et al.
- Although imidacloprid has a unique mode of action relative to the compounds included in our pesticide treatment mixture, queens exposed to the.
- 2 Effects of heat-shock and cold-shock on spermathecal protein abundance.
- a Protein expression changes induced by cold-shock at 4 °C for 2 h (only significant proteins surviving 5% FDR threshold by the Benjamini-Hochberg method are shown).
- Circle size is proportional to the number of proteins belonging to the GO term.
- c Protein expression changes induced by heat-shock at 40 °C for 2 h.
- e Temporal changes in expression of two previously identified candidate heat-shock biomarkers.
- Despite showing the same trends as the cocktail treatments, only three pro- teins were differentially expressed between the imidaclo- prid and acetone groups, apparently owing to higher variability in the imidacloprid samples (Fig.
- The top two most significantly upregulated proteins in the pesticide cocktail-treated queens were XP catalase, a well-known peroxidase) and XP_392368.1 (cytochrome c oxidase subunit 5a, the final complex in the electron transport chain)..
- To observe proteomic shifts in queens that failed in the field, we solicited samples of healthy (n = 45), imported Table 1 Concentrations and hazard quotients (HQ) for components of the pesticide cocktail.
- The pesticide treatment was a low-dose mixture of compounds (acaricides, fungicides, herbicides, and pesticides) found in the wax of honey bee colonies, blended in field-realistic proportions.
- Failed queens were defined as any queen whose reproductive capacity was not satisfac- tory to the beekeeper (e.g., symptoms include poor lay- ing pattern, poor colony build-up, poor colony strength, and opportunistic diseases).
- Interestingly, the candidate markers, two for heat-shock and two for pesticide exposure, were sig- nificantly upregulated in failed queens relative to con- trols at a global scale, whereas the cold-shock markers were not (Fig.
- Blind heat-shock trial.
- To determine if we could use the heat-shock markers, XP and XP_395659.1, to correctly identify.
- heat-shocked queens despite no knowledge of their treatment group, we performed a blind heat-shock trial (Fig.
- b Protein expression patterns of failed queens ( N = 60), imported queens from Hawaii ( N = 9) and California ( N = 9), and healthy queens ( N = 45).
- Plots show their expression patterns in the experimental stress conditions and the healthy vs.
- failed queens.
- Interestingly, some of these pro- posed markers are also upregulated in queens that failed passively in the field, indicating that they could have practical utility for the apiculture industry..
- In our previous experiment, proposed candidate heat- shock biomarkers by investigating queens which were at least third generation locals, all reared from a single mother colony.
- In the present study, we sourced queens from a commercial supplier in California.
- heat-shock proteins are consistently upregulated in queens of both origins, this suggests that these candidate biomarkers may be part of a broadly conserved heat- shock response.
- Before these candidate biomarkers (for heat stress or otherwise) are widely used, they will need to be validated across a range of queen sources that are representative of the commercial industry..
- XP Heat Heat-shock: Control .
- XP_395659.1 Heat Heat-shock: Control .
- XP Cold Cold-shock: Control .
- XP_395122.1 Cold Cold-shock: Control .
- 5 Expression of candidate heat-shock biomarkers in a blind heat-shock trial.
- Protein expression (normalized LFQ intensity) was normalized to the protein with the lowest variance across all the spermatheca samples (XP_623495.1, V-type proton ATPase catalytic subunit).
- The heat-shock and control data were previously published.
- Queens in the HS group were exposed to 40 °C for 2 h, whereas queens in the control group were held at 27 °C.
- Both cold-shock and heat-shock caused general down- regulation of proteins involved in small molecule metab- olism (e.g., carboxylic acids and amino acids) and oxido- reductase activity, whereas for cold-shock, proteins involved in lipid metabolic processes increased.
- Therefore, we think it is more likely that XP is involved in the downregulation of oxidoreductases and small molecule catabolic enzymes that we observe with cold-shock.
- But, overall, these results are consistent with temperature stress generally causing metabolic dysregulation in the spermatheca, which may contribute to how heat-and cold-shock leads to sperm death..
- however, the expression patterns of the imidacloprid-treated queens were remarkably similar to the cocktail-treated queens.
- It could be that other tissues (e.g., the fat body and hemolymph) are the major sites of detoxification from contact exposures, and the changes observed in the spermathecal fluid proteome are mainly secondary.
- The observation that not only catalase, but also other enzymes contributing to the ‘response to toxic substance’ GO term were upregulated in both the pesticide-treated and the failed queens suggests that failed queens may suffer from increased exposure to en- vironmental toxins, possibly through chronic lifetime contact exposure to pesticides in wax.
- Although catalase was also upregulated in failed queens as well as experimentally pesticide-stressed queens, this does not necessarily mean that the failed queens were pesticide stressed.
- Similarly, although the candidate heat-shock markers were upregulated in failed queens, at this stage we cannot say definitively if this was related to heat.
- Indeed, catalase expression appears to be tightly linked to sperm maintenance, as the enzyme becomes significantly upregulated in the spermatheca during the transition from virgin to mated queen .
- In the blind heat-shock trial, we were surprised that the candidate biomarkers (the small heat-shock proteins (HSPs) XP and XP_39659.1) were not more useful for classifying queens into their respective treatment groups.
- For the test to be practically useful, we aim to explore different methods of data acquisition and normalization to minimize noise, as well as deter- mine the biological limits of marker utility (e.g., how long after heat-shock the marker remains upregulated)..
- Heat-shock, cold-shock, and pesticide exposure induce unique proteomic stress signatures in honey bee queen.
- Some of these protein expression patterns are also apparent in queens that failed in the field, but due to sampling bias, we cannot determine the extent to which these expres- sion patterns are linked to failure, and the extent to which they are linked to age.
- Our blind heat-shock trial shows that markers with similar stress-associated expres- sion patterns can have markedly different predictive power, demonstrating that each marker will need to be individually optimized and validated before they can be used as a reliable diagnostic test..
- Queens for the survey of failed and healthy queens were obtained from local beekeepers and queen breeders in British Columbia, Canada (Kettle Valley Queens, Nic- ola Valley Honey, Wild Antho, Campbells Gold Honey, Heather Meadows Honey Farm, Six Legs Good Apiaries, Wildwood Queens, Cariboo Honey, and Worker Bee Honey Company) between the first week of June 5th and July 10th, 2019.
- ‘Healthy’ queens were all evaluated to have a good laying pattern by the beekeepers (consistent egg distribution, few missed cells, one egg per cell) and were 2 months old.
- For the time-course heat-shock experiment, queens were held in an incubator set to 40 °C and 40% RH for or 2 h (n = 7 each).
- Another group of queens (n = 6) were held at 40 °C for 2 h at 80% RH to test for a hu- midity effect.
- For the cold-shock experiment, queens (n = 5) were held at 4 °C for 2 h, then 30 °C for 2 d.
- Con- trol queens (n = 7) were not exposed to heat, but were held in the same 30 °C incubator for 2 days with the other queens..
- All compounds in the pesticide mixture were purchased as pure technical material (≥95% purity) from Sigma Aldrich (St.
- An additional group of n = 10 queens were untreated.
- Queens for the blind heat-shock experiment were re- moved from active hives near Kutztown, PA and were 1–6 months of age and actively laying.
- Instrument performance tests were performed weekly after routine maintenance by infusing a low concentra- tion tuning mixture (Agilent), diluted 1:5 in Buffer A, plus glu-1-fibrinopeptide (200 pmol/ml), and assessing ion funnel transmission, quadrupole transmission, colli- sion cell dynamic performance and collision cell trans- mission according to the manufacturer’s protocol (Bruker HDX Solution).
- The Apis mellifera reference prote- ome database was downloaded from NCBI on November 18th, 2019 (HAv3.1) and all honey bee virus sequences were added to the fasta file.
- The final search database is also available in the data repository.
- Data for temperature exposure duration, the blind heat- shock trial, pesticide, and cold-shock data has the acces- sion MSV000085041.
- Detailed mass spectrometry data quality metrics are available in the summary files within the MaxQuant output folders, which are included in the MassIVE repositories..
- For the pesticide experi- ments, we used acetone-treated queens as the negative control because we were interested in genes that were influenced by the pesticides specifically, over and above the effect of the solvent.
- However, we still evaluated ex- pression in untreated queens in order to confirm that proteins differentially expressed between acetone and pesticide queens were also differentially expressed be- tween pesticide queens and untreated queens, and in the same direction (representing the more natural condi- tion).
- For this experiment, we relaxed the FDR because at 5% FDR there were not two proteins significantly up- regulated compared to acetone samples which were also identified in the healthy vs.
- HSP: Heat-shock protein.
- We also thank Robyn Underwood for supplying the queens used for the blind heat-shock test, and Heather Higo, Marta Guarna, and Liz Huxter for organizing the BC queen survey..
- JSP conducted the blind heat-shock trial.
- Data for temperature exposure duration, the blind heat-shock trial, pesticide, and cold-shock data is available at ftp://
[email protected]..
- JSP owns a honey bee consulting business.
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