Cupressaceae Pollen in the City of Évora, South of Portugal: Disruption of the Pollen during Air Transport Facilitates Allergen Exposure

Round 1

Reviewer 1 Report

All my comments are in the text

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

The authors thank the reviewer for the thorough revision of our manuscript and for all the questions and comments that greatly have contributed to improve our manuscript.

We hope to have responded fully to all the questions and comments. Please, see below the answers in detail. Please also bear in mind that some of the line numbers might have been changed in the revised manuscript.

Yours sincerely,

--------------------

Question 1. (Line 66)  As Cupressaceae is gymnosperm, put so as not to confuse

Answer: Agreed. The term “gymnosperm” was added to the sentence: “Typically, anthers of the gymnosperm plants…”

Question 2. (line 128) How has this average been made for the wind direction in degrees?

Answer: The average of the wind direction is obtained applying the arctangent function to the ratio between the mean east-west and the north-south components of wind. The mean east-west and north-south wind components are computed using all the non-zero wind speed samples.. This average is done according to the procedure described in the manual of the data logger used in the weather station (CR100) from Campbell Scientific (https://s.campbellsci.com/documents/br/manuals/cr1000.pdf, last accessed 21/12/2020). This information was added to section 2.3.

Question 3. (line 127) atmospheric pressure ( hPa)

Answer: The authors have corrected the sentence. It now shows “...atmospheric pressure (AtP; hPa)

Question 4. (line 153) Kruskall Wallis is not ANOVA. It is a non-parametric test

Answer: Although this expression could be used, the authors agree that it may be confusing hence the word “ANOVA” has been removed.

Question 5. Nothing is said about the PCA in the discussion. Comment on the discussion or remove it from the rest of the work

Answer: The authors have improved the discussion. It reads as follows (lines 372-376): “In fact, PCA analyses show that pollen disruption, rainfall and relative humidity vary in the same manner while atmospheric pressure, temperature and global solar radiation vary together opposing pollen disruption (Fig. 5 and Tables 3 and 4). These parameters are all part of the first component that explains most of the variability of the data, in agreement with the correlation analyses.“.

Question 6. (line 156). Superscript. 

Answer: Superscript corrected. 

Question 7. (table 1) How have these prevailing directions been calculated?

Answer: Please see the answer above to question 2. The text of section 2.3 was updated to include this information.

Question 8. (lines 183-186) 

Answer: Abbreviations added.

Question 9. (Lines 191-195) Name each graph in figure 3 (a, b, c and d) to avoid lower, midle, etc.

Answer: Done.

Question 10. table 2. include abbreviations in the table

Answer: Done.

Question 11. (Lines 216-217) What is a, b, c, d in the graphs? Does it refer to homogeneous groups? How it is expressed is not understood

Answer: The letters indicate the statistical significance between groups. The authors tried to clarify and changed the sentence in the legend to “The different letters within the graphs indicate the statistical significance at 5% (p<0.05).”

Question 12. (Line 280) include (HPI)

Answer: Abbreviation included.

Question 13. (section 3,4) Some statements in this section should be in the discussion

Answer: The sentences regarding discussion were moved to the section 4.

Question 14. As indicated, include the PCA in the discussion or remove that section of the paper

Answer: Thank you for the remark. Comments on the PCA analysis of its relevance have been included in the discussion, as follows: “In fact, PCA analyses show that pollen disruption, rainfall and relative humidity vary in the same manner while atmospheric pressure, temperature and global solar radiation vary together opposing pollen disruption (Fig. 5 and Tables 3 and 4). These parameters are all part of the first component that explains most of the variability of the data, in agreement with the correlation analyses.”

Reviewer 2 Report

Cupressaceae Pollen in the City of Évora, South of Portugal: Disruption of the Pollen during Air Transport Facilitates Allergen Exposure

General comments

This study represents an important analysis of the influence of meteorological conditions in pollen rupture. This is a very little-known topic which it is very interesting from the clinical point of view as remarked the authors. I strongly encourage the publication of this type of papers as it covers an important gap in the aerobiological knowledge of a very important pollen type in cities. The authors should address before several improvements. The manuscript is well-written and well-structured. However, several references need to be updated with more recent publications. Botanical aspects of the family Cupressaceae exposed in the introduction should be carefully revised for avoiding confusions between native and introduced species. The results are presented in mainly two parts the daily results and the hourly patterns. The authors should connect better both parts of the paper and explain well why were observed opposite results between daily and hourly data.

Specific comments

1. Abstract (line 19): I would say "responsible for late winter and early spring allergic outbreaks". In other regions relevant pollen concentrations of Cupressaceae pollen are registered since January or before.

2. Abstract (line 25): Please, include the meaning of the abbreviations in abstract, or remove if it is not relevant.

3. Abstract (line 31): "Hourly pollen index" perhaps is not the most appropriate term, using better "hourly pollen concentrations" or "intradiurnal pattern".

4. Abstract (lines 32-33): Is a such important episode to be remarked in the abstract? Explain better or remove here.

5. Introduction (lines 40-41): Please cite a reference supporting this statement.

6. Introduction (lines 42-43): As far as I know, only there are Northern and Southern Hemisphere, please, clarify this sentence.

7. Introduction (lines 46-56): The authors should revise carefully this paragraph paying attention to the botanical descriptions and distributions of the species in the Mediterranean region. Particularly, native and allochthonous species should be well distinguished. I encourage the authors to describe first native species represented in most of the Mediterranean areas by the Juniperus genus (San-Miguel-Ayanz et al. 2016), and Tetraclinis in very specific areas. Cupressus species are introduced in West Mediterranean region, only Cupressus sempervirens is native in East Mediterranean coast. It is not clear in your description, where native and cultivated species are mixed. In parks and gardens are often cultivated also the genera Chamaecyparis and Platycladus. The proposal of this paper does not require a very detailed description of chorological aspects on this regard, but differences between native and cultivated species are interesting.

8. Introduction (lines 59-61): According to the previous comment, what Cupressaceae species the authors consider as more important from the allergenic point of view and why?

9. Introduction (lines 63-71): Please, update some references. For example, the influence of the meteorological conditions in pollen release and dispersal is well descripted in Rojo et al. (2015), and several intradiurnal patterns may be consulted in a very recent work (Fernández-Rodríguez et al. 2020). It is very relevant also in this aspect the review of Cresti and Linskens (2000).

10. Introduction (line 71): I think "lixiviation" is not a very appropriate term, used in soils instead of in air. Use better "wash-out effect"

11. Materials and Methods (line 142): What is the meaning of "three-week periods"? Are back trajectories calculated each three weeks?

12. Materials and Methods (lines 142-147): It is interesting here to indicate the meteorological global model used (GDAS, Reanalysis...), pointing out the 3D-resolution of the approach (see methods of Rojo et al. 2019).

13. Materials and Methods (line 163): Why ">5994" of the seasonal pollen integral? Why not the specific value is indicated in this year? Is there a lack of data? Not too relevant for the purpose of this work, although it should be indicated.

14. Results (Figure 2): The differences in different years are clear. Could be that percentage of disruption would be inversely dependent on the pollen amount simply due to the effect of probability? I mean, when higher pollen concentrations were recorded (year 2017) the percentage of disrupted pollen decreased since in large amounts of pollen is more likely find large amounts of not disrupted pollen? Anyway, discuss this fact, although only with two years you do not reject

15. Results (Table 1): Is There such a great difference in precipitation between both seasons?

16. Results (lines 211-212). These lines are not results, it is interpretation and more adequate in the discussion section. The authors should take care of the correlations. Most of the meteorological factors are correlated each other, then the correlations may indicate this relation between factors, and not a real relationship with the pollen disruption. I mean, a significant correlation does not mean causality. In this sense, I do not imagine how temperature and solar radiation could "contribute to the maintenance of pollen integrity". It could simply be that temperature and radiation is negatively correlated with relative humidity.

17. Results (Figure 4): Very interesting results. Yes, in my opinion, these three parameters explained the rate of disruption of pollen.

18. Results (line 249): Was "DPI" defined previously? What is the meaning of Daily Pollen Index, daily concentrations?

19. Results (Table 5): It is very remarkable that the least relevant meteorological parameter for daily pollen disruption (wind speed) is the most relevant meteorological parameter for hourly pollen disruption. How we can explain this? And the relative humidity is not correlated.

20. Results (Figure 7): In methods the hours for calculating back trajectories were not indicated. Then in Figure 7, the authors indicated that trajectories were calculated at 10:00 UTC. Do you think that the wind pattern for one specific hour is enough representative for the entire day. Usually, 3-hourly or 6-hourly trajectories are periodically calculated during the day (Rojo et al. 2019), or explain why 10:00 UTC is used as representative (perhaps is the period for the maximum realease).

21. Results (Figure 8): Legend and axis are not correctly visible in this graph.

22. Discussion (lines 348-353): There are strong evidences of the influence of relative humidity in the rupture of pollen. In addition, the effect of hydration is a key factor in the allergen release as previously has been indicated (Buters et al. 2015; Aloisi et al. 2018). I encourage to the authors to discuss a little more this aspect.

23. Discussion (lines 359-371): In general, the first part of the paper presents very good and clear results, however, the authors should explain better the findings of the intradiurnal patterns and how this is connected with the first part. Opposite results seem to be obtained in relation to the meteorological variables involved. How the authors explain this?

24. Discussion (Figures 7 and 8): How these results may support the findings obtained in the first part of the paper? The authors should connect a little more daily and hourly analyses or explain better why are different.

References of the review:

Aloisi, I., Del Duca, S., De Nuntiis, P., Vega Maray, A.M., Mandrioli, P., Gutiérrez, P., Fernández-González, D., 2018. Behavior of profilins in the atmosphere and in vitro, and their relationship with the performance of airborne pollen. Atmospheric Environment 178, 231–241. https://doi.org/10.1016/j.atmosenv.2018.01.051

Buters, J., Prank, M., Sofiev, M., Pusch, G., Albertini, R., Annesi-Maesano, I., Antunes, C., Behrendt, H., Berger, U., Brandao, R., Celenk, S., Galan, C., Grewling, Ł., Jackowiak, B., Kennedy, R., Rantio-Lehtimäki, A., Reese, G., Sauliene, I., Smith, M., Thibaudon, M., Weber, B., Cecchi, L., 2015. Variation of the group 5 grass pollen allergen content of airborne pollen in relation to geographic location and time in season. Journal of Allergy and Clinical Immunology 136, 87-95.e6. https://doi.org/10.1016/j.jaci.2015.01.049

Cresti, M., Linskens, H.F., 2000. Pollen-allergy as an ecological phenomenon: A review. Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology 134, 341–352. https://doi.org/10.1080/11263500012331350495

Fernández-Rodríguez, S., Maya-Manzano, J.M., Colín, A.M., Pecero-Casimiro, R., Buters, J., Oteros, J., 2020. Understanding hourly patterns of Olea pollen concentrations as tool for the environmental impact assessment. Science of The Total Environment 736, 139363. https://doi.org/10.1016/j.scitotenv.2020.139363

Rojo, J., Rapp, A., Lara, B., Fernández-González, F., Pérez-Badia, R., 2015. Effect of land uses and wind direction on the contribution of local sources to airborne pollen. Science of The Total Environment 538, 672–682. https://doi.org/10.1016/j.scitotenv.2015.08.074

Rojo, J., Serrano-Bravo, M.D., Lara, B., Fernández-González, F., Pérez-Badia, R., 2019. Halo-nitrophilous scrub species and their relationship to the atmospheric concentration of allergenic pollen: case study of the Mediterranean saltbush (Atriplex halimus L., Amaranthaceae). Plant Biosystems 153, 98–107. https://doi.org/10.1080/11263504.2018.1461699

San-Miguel-Ayanz, J., De Rigo, D., Caudullo, G., Houston Durrant, T., Mauri, A. (Eds.), 2016. European Atlas of Forest Tree Species. Publ. Off. EU, Luxembourg.

Author Response

Dear Reviewer,

The authors thank the reviewer for the thorough revision of our manuscript and for all the questions and comments that greatly have contributed to improve our manuscript.

We hope to have responded fully to all the questions and comments. Please, find the detailed answers to the question in the uploaded file. Please also bear in mind that some of the line numbers might have been changed in the revised manuscript.

Yours sincerely,

--------------------------

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Cupressaceae Pollen in the City of Évora, South of Portugal: Disruption of the Pollen during Air Transport Facilitates Allergen Exposure (Round 2)

General comments

The authors have detailed answered all my comments and they have addressed important improvements in this revised version of the manuscript. The authors also include a new supplementary figure as answer for a previous question which is very interesting to support the effect of the meteorological conditions of the year on the disruption rate independently of the pollen concentration. Congratulations. However, in my opinion they should consider several minor points.

last effort to connect both parts

Message masked. Some remarks in abstract and aims.

Specific comments

1. Abstract (lines 19-20): "Despite being considered moderately allergenic...". I do not know, if there is a consensus about this, but maybe it is better to remove this statement, or develop a little more in introduction with references supporting it.

2. Abstract (line 31): The authors missed the global solar radiation in the list of meteorological variables in the lines 25-26, and then several lines below this variable is remarked as relevant factor in the pollen disruption.

3. Abstract (lines 33-35): In the original submission, I felt a disconnection between two main parts of the analysis, the daily analysis of disruption rates and the study of the intradiurnal patterns. This problem was solved in part and relevant explanations were included. However, still in abstract I think that the main findings focused on the influence in disruption rates should be included instead of a merely description of the intradiurnal pattern. Also, the aims of the work should be clearly exposed on this regard in the introduction.

4. Introduction (lines 48-49): The botanical description of the Cupressaceae family in the Mediterranean region has considerably improved. But, still several errors have been included in specific descriptions. To solve this avoiding specific geographical descriptions like lines 48-49). For example, Juniperus communis is widely distributed in Europe but in Spain, this species is mainly distributed in the mid-North of Spain and specific mountains of the South (San-Miguel-Ayanz et al. 2016). Yes, Juniperus oxycedrus subsp. macrocarpa is also an autochthonous species but is not common beyond very specific points of the coast of South of Spain. Then, I recommend describing this in general.

5. Introduction (lines 99-100): In my opinion these lines should be removed from the introduction. It is the result, and here only the aims should be exposed.

6. Results (Figure 7): I had a question about the number of backtrajectories calculated for each day. The authors answered me that hourly trajectories were calculated which is a good approach. But, why all these hourly trajectories are not presented in Figure 7 instead of only one for the 10:00 UTC? It increases the representativeness of these trajectories.

7. Discussion (lines 430-434): The explanation of these paragraph of the discussion clarifies a little the connection between the daily analysis and the study of the intradiurnal pattern. But the authors could clearly indicate that in the case of the intradiurnal analysis, these results were analysed for specific cases (I think four days of episodes) and then to generalize these findings, more extensive researches should be carried out in the future in wider environmental ranges.

References of the review:

San-Miguel-Ayanz, J., De Rigo, D., Caudullo, G., Houston Durrant, T., Mauri, A. (Eds.), 2016. European Atlas of Forest Tree Species. Publ. Off. EU, Luxembourg.

Author Response

Dear Reviewer,

The authors thank you for the reviewer for taking the time to critically revise our manuscript. We have considered all the comments and have revised the manuscript accordingly. Please, find below the detail answers to the comments. Again, we hope to have fully responded to your concerns and thank for the input contributing to further improve our manuscript.

Yours,

Célia Antunes

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Reviewer’s specific comments and answers:

1. Abstract (lines 19-20): "Despite being considered moderately allergenic...". I do not know, if there is a consensus about this, but maybe it is better to remove this statement, or develop a little more in introduction with references supporting it.

Answer: The reviewer is right. The sentence removed and the sentence re-written as follows: “Cupressaceae family includes several species that are widely used as ornamental plants pollinating in late winter-early spring and might be responsible allergic outbreaks. Cupressaceae pollen disruption may favour allergen dissemination, potentiating its allergenicity.”

1. Abstract (line 31): The authors missed the global solar radiation in the list of meteorological variables in the lines 25-26, and then several lines below this variable is remarked as relevant factor in the pollen disruption.

Answer: True. Global solar radiation was included in the list.

1. Abstract (lines 33-35): In the original submission, I felt a disconnection between two main parts of the analysis, the daily analysis of disruption rates and the study of the intradiurnal patterns. This problem was solved in part and relevant explanations were included. However, still in abstract I think that the main findings focused on the influence in disruption rates should be included instead of a merely description of the intradiurnal pattern. Also, the aims of the work should be clearly exposed on this regard in the introduction.

Answer:  The reviewer is correct. The sentence (lines 33.36) is now: “Intra-diurnal pollen pattern peaked between 9:00am-2:00pm, suggesting local origin, confirmed by the back trajectory analysis. Intra-diurnal pollen disruption profile followed hourly pollen pattern and it negatively correlated with AtP, temperature and Global Srad but was uncorrelated with RH.”

1. Introduction (lines 48-49): The botanical description of the Cupressaceae family in the Mediterranean region has considerably improved. But, still several errors have been included in specific descriptions. To solve this avoiding specific geographical descriptions like lines 48-49). For example, Juniperus communis is widely distributed in Europe but in Spain, this species is mainly distributed in the mid-North of Spain and specific mountains of the South (San-Miguel-Ayanz et al. 2016). Yes, Juniperus oxycedrus subsp. macrocarpa is also an autochthonous species but is not common beyond very specific points of the coast of South of Spain. Then, I recommend describing this in general.

Answer: The reviewer has a point. The authors accepted the suggestion and have re-written the paragraphs as follows: “In the Iberian Peninsula, species from the genus Juniperus, form part of the natural vegetation and the scrubland and are considered autochthonous while species from the genera Cupressus, Chamaecyparis and Platycladus, widely used in parks and gardens as barriers against wind and noise, are considered allochthonous [4]. In Portugal, several species from the genera Juniperus and Cupressus are distributed throughout the continental territory, and Cryptomeria, particularly Cryptomeria japonica, is abundant in the Azores islands [5]. Among the several autochthones species from the genus Juniperus, the Juniperus communis L., Juniperus navicularis Gand., Juniperus oxycedrus L. and Juniperus turbinata Guss are well represented [6]. The genus Cupressus, is considered allochthonous in Portugal and is distributed throughout the country; the species Cupressus sempervirens L. is mainly found in urban environments, due to their use as ornamental plants, Cupressus macrocarpa Hartweg is usually found in mountains of Sintra, Buçaco and Geres, and Cupressus lusitanica Miller is found both environments; the distribution of Cupressus arizonica Greene is mostly unknown  [6,7].”

1. Introduction (lines 99-100): In my opinion these lines should be removed from the introduction. It is the result, and here only the aims should be exposed.

Answer: The reviewer is right. The sentence was removed.

1. Results (Figure 7): I had a question about the number of backtrajectories calculated for each day. The authors answered me that hourly trajectories were calculated which is a good approach. But, why all these hourly trajectories are not presented in Figure 7 instead of only one for the 10:00 UTC? It increases the representativeness of these trajectories.

Answer:  The hourly back trajectories calculated for the four days selected under analysis are now included in the supplementary materials. It seems better to include them in this section due to the great amount of space that would take in the manuscript. It is now indicated in the manuscript (sections 2.4 and 3.4) that the hourly back trajectories are included in the supplementary materials (Figures 2 to 4).

1. Discussion (lines 430-434): The explanation of these paragraph of the discussion clarifies a little the connection between the daily analysis and the study of the intradiurnal pattern. But the authors could clearly indicate that in the case of the intradiurnal analysis, these results were analysed for specific cases (I think four days of episodes) and then to generalize these findings, more extensive researches should be carried out in the future in wider environmental ranges.

Answer: The authors accepted the reviewer’s suggestion; The word “four” was introduced to the line 430 and the sentence in the lines 441-444 was changed. It now reads as follows: “Despite the low number of events analysed (4 days), hourly observations revealed a relevant interplay between the key weather factors on airborne pollen integrity loss and a wider research should be carried out in the future to further analyse this phenomenon.”