Effect of Banana Bunchy Top Virus on the Heat Shock Protein Genes of Pentalonia nigronervosa during Temperature Susceptibility and Its Effect on Virus Transmission

Round 1

Reviewer 1 Report

This research intended to understand the influence of temperature susceptibility in banana aphid upon acquisition of BBTV. The authors found reduced tolerance to either low temperature and high temperature stress in Vr (virus+) aphids compared with NVr (virus-) aphids by examining (i) differences of the life cycle time, (ii) mortality rate, (iii) expression of heat shock protein genes, and (iv) transmission rate under different temperature conditions. The data presented in this manuscript is still preliminary and limited, however, the phenomena itself they found, reduction of temperature susceptibility upon viral acquisition, looks interesting, therefore I think it is worth sharing their findings as a publication so to inspire researchers in this field.

 

I would not require additional experiments under current pandemic situation, but instead, to increase the scientific value of this paper, I’d like to recommend the authors to discuss more about the following points:

1. Based on the data, acquisition of the virus made its vector susceptible to either low and high temperature. In that case, I’m wondering if the response is temperature specific or not. What do you think about other abiotic stress?
2. Do you think it is passive effect of virus acquisition (e.g. replication of virus should consume host’s metabolites, may hijack host’s metabolic pathways, etc. which could lower the stress tolerance of the host individuals), or host’s active response to weaken the Vr individuals?
3. What kind of study will be required to improve our understanding of physiological function/mechanism of temperature susceptibility upon viral acquisition? Engineering viral acquisition as well as viral genome sequence along with omics analysis (RNA-Seq, metabolomics) would be interesting to identify molecular factors involved in the interaction between the virus and the vector.
4. How could we make use of the findings to protect Banana species from viral infection? How could your research contribute to develop “ameliorate decision support tools”? Please explain more specifically.

 

The followings are minor comments.

1. I found lots of careless mistakes in the manuscript. Please find and correct all of them. For example,
   1. Put a space accurately (e.g. one space is needed before the opening parenthesis, after comma, no space between number and “h”, the symbol of hour.)
   2. A period was sometimes missed (at least in line 2 and 10 in the introduction section)

 

 

2. Regarding figures, you should use the same font type and size as possible for all figures in your article.
3. The symbol of Celsius seems to be garbled at least in my PDF reader. Please correct it.
4. As for Fig. 1, please correct the followings:
   1. I don’t think you need to add “min” in the label of x-axis since you added the title of the axis along with the unit.
   2. Change “heat shock’ to “cold shock” for low temperature treatments.
   3. It is ambiguous how you calculated the percentage in the y-axis. Did you calculate the ratio by setting the data collected at 25C as 100%? or set the total number of samples as 100%? Please clarify this point in the caption.
   4. I would recommend you to use the same range of y-axis. In the current version, only Fig. 1B uses 0-20% while the others use 0-100%, which is confusing.
   5. Please make sure that you added error bars in Fig. 1C. They are invisible.

 

5. As for Fig. 2, I would suggest to change the title of x-axis to “temperature (°C)”, while the title of y-axis could be “Relative mRNA level of Hsp40/70/90”.

 

6. As for Fig. 3,

 

3. I think Fig. 3A and 3B should be moved to supplemental data because they just support the reliability of the calculation of Fig. 3C and Table 3.
4. In either case, please use white background to make the graph cleaner, as well as use the same font type and size as possible with other figures.
5. Additionally, the style of the legend in Fig. 3 looks different from the others. Please keep the same style.
6. 3C was already described in Table 3. I can’t understand what is the reason for expressing the same data as a violin plot. If you wanted to emphasize the fact that there are two peaks in the distribution of either violin plot, you should explain what is the possible reason for this. Please rethink what kind of table/graph is effective and sufficient to present your data.

 

7. In Table 3, the label BBTD incidence (%) is not correct because the data is not expressed in percentage.

 

 

Author Response

Response to Reviewer’s Comments

 

Dear Editor,

Coauthors and I very much appreciated the encouraging, critical and constructive comments on this manuscript by the reviewer. The comments have been very thorough and useful in improving the manuscript. We strongly believe that the comments and suggestions have increased the scientific value of revised manuscript by many folds. We have taken them fully into account in revision. We are submitting the corrected manuscript with the suggestion incorporated the manuscript.The manuscript has been revised as per the comments given by the reviewer, and our responses to all the comments are as follows:

 

Reviewer #1:

1. Comment 1- Based on the data, acquisition of the virus made its vector susceptible to either low or high temperature. In that case, I’m wondering if the response is temperature specific or not. What do you think about other abiotic stress?

Response-

Considering the agroclimatic situations of India especially of West Bengal, where the maximum average temperature reaches 38-39°C during summer and minimum average temperature 5-8°C during winter, the banana aphid population greatly varies among this temperature. Aquisition of BBTV is greatly influenced by the temperature regimes as it is reported in other papers (Anhalt at al., 2008; Wu et al., 1990). However other abiotic stress also have role to affect the virus transmission(Munster,2020) but its effect on the developmental biology of the vector is not yet scientifically acknowledged anywhere and hence there is further scope of research regarding this aspect.

2. Comment 2- Do you think it is passive effect of virus acquisition (e.g. replication of virus should consume host’s metabolites, may hijack host’s metabolic pathways, etc. which could lower the stress tolerance of the host individuals), or host’s active response to weaken the Vr individuals?

 Response-

Vector-borne pathogens can enhance their transmission by altering the phenotypes of their primary hosts and vectors in ways that influence the outcome of interactions between them. It can be envisaged that there might be evolutionary pressures on pathogens to manipulate plant biology in such a manner that the performance of their insect vector is not significantly degraded. The precise mechanisms facilitating the modifications of vectors undergo upon virus infection remain unanswered at the physiological and molecular levels. It has been studied that circulative plant viruses are retained for a significant part of their cycle in competent vector’s hemolymph. The aphid-transmitted luteoviruses and whitefly transmitted begomoviruses have been shown to bind specifically to GroEL proteins abundantly present in the hemolymph of the vectors (Van den Heuvel et al., 1994; Morin et al., 1999). GroEL proteins are not produced by the insects themselves; instead they are produced by symbiotic bacteria living in the aphids (Buchnera aphidicola) and whiteflies (Carsonella ruddii). However these interactions have not been yet proved and well studied in case of BBTV in association with aphids. Virus acquisition in case of BBTV may result in metabolic upregulation in the vector thus diverting the vectors energy and decreasing stress tolerance and developmental biology.

3. Comment 3- What kind of study will be required to improve our understanding of physiological function/mechanism of temperature susceptibility upon viral acquisition? Engineering viral acquisition as well as viral genome sequence along with omics analysis (RNA-Seq, metabolomics) would be interesting to identify molecular factors involved in the interaction between the virus and the vector.

 Response-

With the advent of new technology now available, an increasingly greater number of studies involve coordinated research that is beginning to assemble a more complete picture of how vectors and viruses have coevolved to facilitate transmission. Transcriptomic sequencing technology has been widely used as a tool to study the interaction between plant virus and host which can lead to an ability to monitor the changes that occur in vector insects associated with specific aspects of virus transmission. Involvement of endosymbionts or other physiological or molecular factors facilitating virus transmission and vector susceptibility can be identified and later can be utilized in genetic engineering for further downstream applications

 

 

 

4. Comment4- How could we make use of the findings to protect Banana species from viral infection? How could your research contribute to develop “ameliorate decision support tools”? Please explain more specifically.

                                                                                                        

  

 

          Response-

      

Since BBTV is quarantine best of worldwide importance it is the main barrier at the quarantine stations during import and export of banana planting materials. However certain decision control system would help to improve the situation during export and import of the planting materials and banana tissue culture industry as well..

• Keeping the planting materials at higher temperature in green house will help to decrease the transfer of the virus through the vector. Since higher temperature drastically reduces banana aphids vector population as a result acquisition and transmission also decreases.
• Temperature can play a big role in the tissue culture banana industry for micro propagation of the virus free plantlets.

  Minor comments.

 

1. Comment - I found lots of careless mistakes in the manuscript. Please find and correct all of them. For example,
1. Put a space accurately (e.g. one space is needed before the opening parenthesis, after comma, no space between number and “h”, the symbol of hour.)

Response- Thank you for your minute observation and valuable comments. We have mentioned it in revised manuscript.

 

1. A period was sometimes missed (at least in line 2 and 10 in the introduction section).

 

Response- Thank you for your minute observation and valuable comments. We have mentioned it in revised manuscript.

 

 

1. Comment- Regarding figures, you should use the same font type and size as possible for all figures in your article

 

Response- Thank you so much for your suggestion. We have made all the possible changes in the said figure.

 

3. Comment - The symbol of Celsius seems to be garbled at least in my PDF reader. Please correct it.

Response-   Thank you so much for your suggestion. We have made all the possible changes

4. Comment - As for Fig. 1, please correct the followings:
   1. I don’t think you need to add “min” in the label of x-axis since you added the title of the axis along with the unit.

Response- Thank you for your valuable observations. All the possible changes have been corrected.

 

2. Change “heat shock’ to “cold shock” for low temperature treatments.

  Response- Thank you so much for your suggestion. We have made the necessary changes in the figure.

 

 

3. It is ambiguous how you calculated the percentage in the y-axis. Did you calculate the ratio by setting the data collected at 25C as 100%? or set the total number of samples as 100%? Please clarify this point in the caption.

 Response- Thank you so much for your comment. We have addressed it during the   revision of the manuscript.

 

4. I would recommend you to use the same range of y-axis. In the current version, only Fig. 1B uses 0-20% while the others use 0-100%, which is confusing.

Response- We have taken reviewer’s comment in full consideration and it will be well
      reflected in the figure.

 

5. Please make sure that you added error bars in Fig. 1C. They are invisible.

 

     Response- Thank you so much for your suggestion. We have made the necessary changes.

 

 

5. Comment- As for Fig. 2, I would suggest to change the title of x-axis to “temperature (°C)”, while the title of y-axis could be “Relative mRNA level of Hsp40/70/90”.

     Response- Thank you so much for your suggestion. We have made the necessary changes.

 

6. Comment- As for Fig. 3,

 

3. I think Fig. 3A and 3B should be moved to supplemental data because they just support the reliability of the calculation of Fig. 3C and Table 3.

 Response- Thank you so much for your suggestion and it will be followed as per your suggestion.

 

4. In either case, please use white background to make the graph cleaner, as well as use the same font type and size as possible with other figures.

 Response- Thank you for the suggestion but the Standard curve and Melting curve  is generated from the Mx3000P qPCR software on the qPCR system which is by default system and hence unfortunately the background colour couldn’t be changed.

 

5. Additionally, the style of the legend in Fig. 3 looks different from the others. Please keep the same style.

Response- Thank you so much for your comments. The mistake is corrected in the revised version.

 

6. 3C was already described in Table 3. I can’t understand what is the reason for expressing the same data as a violin plot. If you wanted to emphasize the fact that there are two peaks in the distribution of either violin plot, you should explain what the possible reason is for this. Please rethink what kind of table/graph is effective and sufficient to present your data.

Response- Thankyou for the suggestion. Keeping the following advice in mind the violin plot will be removed.

 

7. In Table 3, the label BBTD incidence (%) is not correct because the data is not expressed in percentage.

 Response- Thank you so much for your comments. The mistake is corrected in the    
   revised version.

 

Reviewer #2:
 

1. I recommend the authors to address the few comments written in the pdf version of the manuscript prior to further consideration for publication in agronomy.

 Response-

Thank you so much for your valuable comments. All the corrections and valuable suggestions are corrected and sorted in the revised manuscript as mentioned accordingly.

Here are some of the following responses of certain comments which I would like to address.

• Comment: This statement is not true for all the temperatures given the fact that in figure 2, the regulation of hsps in NVr insects is already higher than the tm 25 in these cases: Tm 38 of in all the three hsps, and Tm 5 and 15 of hsp70. Overall, this suggests that thermal change is the main reason for upregulation of hsp genes not the virus acquisition at least in cases mentioned above.

Author’s response:

Thank you for your valuable suggestion. We have modified the line accordingly. It is correctly pointed out that the expression of Hsp70 in non-viruliferous aphids is already higher at 5°C, 15°C and 38°C as compared to the Non-viruliferous ones at 25°C (Control). In case of Hsp40 and Hsp90, the same holds true at 38 °C. Hence suggesting that at normal temperature Hsps play a role in temperature adaptation rather than virus acquisition. However, it is also to be noticed that the transcript level of three Hsps were significantly higher in Vr aphid in comparison to the NVr aphids. Therefore, acquisition of virus by vector aphid might have some role in the increased transcript level of the Hsps. The results are clearly suggestive to the role of Hsps being more specific to vectors response against temperature stress in comparison to virus acquisition or transmission.

 

• Comment:

 

1) In the three figures above, please define numbers below each asteroid in the caption.

 

Author’s response:

Thank you for your valuable suggestion. The definition of the asteroid in the figure is already given in the caption determining the level of their significance.

** indicate significant difference between the two means at a given temperature (P <0.01). *** indicate signifi-cant difference between the two means at a given temperature (P <0.001). **** indicate significant difference between the two means at a given temperature (P <0.0001).

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The current manuscript "Effect of Banana Bunchy Top Virus (BBTV) on the Heat shock protein genes (Hsps) of Pentalonia nigronervosa during temperature susceptibility and its effect on virus transmission" investigated the effects of different temperatures on the biology of viruliferous and non-viruliferous aphid vector (P. nigronervosa). This study highlighted important findings related to increased thermal susceptibility of the vector upon the virus acquisition. Expression level of some homologs of heat shock proteins were inspected and their association to the thermal susceptibility have also been discussed.

I recommend the authors to address the few comments written in the pdf version of the manuscript prior to further consideration for publication in agronomy.

Comments for author File: Comments.pdf

Author Response

1. I recommend the authors to address the few comments written in the pdf version of the manuscript prior to further consideration for publication in agronomy.

 Response-

Thank you so much for your valuable comments. All the corrections and valuable suggestions are corrected and sorted in the revised manuscript as mentioned accordingly.

Here are some of the following responses of certain comments which I would like to address.

• Comment: This statement is not true for all the temperatures given the fact that in figure 2, the regulation of hsps in NVr insects is already higher than the tm 25 in these cases: Tm 38 of in all the three hsps, and Tm 5 and 15 of hsp70. Overall, this suggests that thermal change is the main reason for upregulation of hsp genes not the virus acquisition at least in cases mentioned above.

Author’s response:

Thank you for your valuable suggestion. We have modified the line accordingly. It is correctly pointed out that the expression of Hsp70 in non-viruliferous aphids is already higher at 5°C, 15°C and 38°C as compared to the Non-viruliferous ones at 25°C (Control). In case of Hsp40 and Hsp90, the same holds true at 38 °C. Hence suggesting that at normal temperature Hsps play a role in temperature adaptation rather than virus acquisition. However, it is also to be noticed that the transcript level of three Hsps were significantly higher in Vr aphid in comparison to the NVr aphids. Therefore, acquisition of virus by vector aphid might have some role in the increased transcript level of the Hsps. The results are clearly suggestive to the role of Hsps being more specific to vectors response against temperature stress in comparison to virus acquisition or transmission.

 

• Comment:

 

1) In the three figures above, please define numbers below each asteroid in the caption.

 

Author’s response:

Thank you for your valuable suggestion. The definition of the asteroid in the figure is already given in the caption determining the level of their significance.

** indicate significant difference between the two means at a given temperature (P <0.01). *** indicate signifi-cant difference between the two means at a given temperature (P <0.001). **** indicate significant difference between the two means at a given temperature (P <0.0001).

 

Author Response File: Author Response.pdf